FREQUENCY OF STAPHYLOCOCCI STRAINS WITH ......LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL....

LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. XLIX(3), 2016, TIMIŞOARA

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FREQUENCY OF STAPHYLOCOCCI STRAINS WITH ANTIBIOTIC RESISTANCE ISOLATED FROM ANIMALS

IULIA BUCUR, CORINA PASCU, D. MITRICĂ MĂRĂCINE, L. FLUERAȘU,

N. CĂTANA

Banat’s University of Agricultural Sciences and Veterinary Medicine ”King Michael I of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Aradului

Street No. 119, Timisoara, Romania E-mail: [email protected]

Summary

Antibiotic resistance is a growing phenomenon with great importance in medicine. It

has as a genetic support the resistance genes found in the bacterial chromosome, the R plasmids, in integrons and transposons. Resistance genes can be transmitted intraspecific and interspecific through mobile genetic elements.

Within the Staphylococcus genus, the multiple antibiotic resistance is present both on coagulase-positive and coagulase-negative species, pathogenic to humans and animals.

The study of resistance patterns provides information on the epidemiological circuit of staphylococci strains from animals to humans that can cause nosocomial infections. The researches aimed the patterns of resistance on a total of 240 staphylococci strains coagulase positive and coagulase negative, using the disk diffusion method against 15 antibiotics that represent the classes of antibiotics used in therapy. The resistance patterns detected ranged from 3.75% on novobiocin to 50.83% on erythromycin.

Key words: multiple antibiotic resistance, resistance patterns, staphylocococci

strains

The phenomenon of antibiotic resistance in bacteria was reported since 1950, after the introduction of sulfonamides and antibiotics in the prophylaxis and therapy of some infectious diseases in animals and humans. In the coming years, this phenomenon has been amplified, due to the use of tetracyclines as growth promoters factors in pigs and poultry. The expansion of antibiotic resistance in Gram positive and Gram negative bacteria, pathogenic to animals and humans, required extensive studies conducted over time (2). Researches showed that antibiotic resistance is genetically encoded, as the resistance genes are present in the bacterial chromosome, R plasmids and

DNA fragments existing in bacterial cytoplasm (transposons, cassette genes, mobile genomic islands). Through these structures, genes encoding antibiotic resistance can be transferred intraspecific and interspecific (1). The phenomenon of antibiotic resistance was reported both in coagulase positive and coagulase negative staphylococci, which are pathogenic for animals and humans. Antibioresistance study, in staphylococci, provides valuable information concerning the animal-human-animal epidemiological circuit of certain

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multiple resistant strains. During the last years, a special attention was given to MRSA (Methicillin Resistant S. aureus) strains, as methicillin resistance is correlated with multiple resistance to other antibiotics. By increasing degree in the last years, MRSA strains are considered at risk of zoonotic importance (1, 2). Researches were made in order to determine the resistance patterns in coagulase positive and coagulase negative staphylococci isolated from animals.

Materials and methods Pathological samples for bacteriological examination were harvested from

a total of 249 of farm and companion animals. Samples were collected with sterile swabs, and then they were inseminated in peptone water, and later incubated for 24 hours at 37°C. For the isolation of staphylococci in pure culture, passages were made by dispersing on Chapman solid medium, and then the plates with this medium were incubated in a thermostat for 24 hours at 37°C. The resulted cultures were bacterioscopic examined and it was appreciated the mannitol fermentation, followed by more tests. Pigmentogenesis, hemolysis, mannitol fermentation, susceptibility to furazolidone and novobiocin and the presence of free coagulase on Baird-Parker medium were appreciated to include the strains in Staphylococcus genus.

The Kirby-Bauer disc diffusion method was used to test the susceptibility to antibiotics, using Mueller-Hinton broth and agar, Petri plates and antibiotic impregnated biodiscs.

The antibiotics used were: Aminocumarine: Novobiocin; Aminoglycosides: Gentamicin, Kanamycin, Tobramycin; Beta-lactams: Ampicillin, Amoxicillin, Ampicillin with Sulbactam, Amoxycillin with Clavulanic acid, Penicillin G; Lincosamides: Lincomycin; Macrolides: Clindamycin, Erythromycin; Nitrofurans: Furazolidone; Quinolone: Ciprofloxacin and Tetracyclines: Tetracycline.

The interpretation of results was made according the recommendations of the Institute for Clinical Laboratory Standards, USA (CLSI-Clinical and Laboratory Standard Institute) taken by the European Committee for Testing the Antibiotic Susceptibility of the European Society of Infectious Diseases and Clinical Microbiology and the strains were classified into three categories: susceptible, intermediate and resistant.

Results and discussions

The strains of staphylococci isolated from healthy animals or with different

diseases showed several resistance patterns, which are presented in Table 1. In order to detect the resistance patterns, 15 antibiotics included in more classes were used, some of which are associated with beta-lactamase inhibitors.


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Table 1 Resistance patterns of staphylococci strains isolated from animals

Crt. no.

Antibiotic

Antibiogram results Total

strains Susceptible Intermediary Resistant

No. % No. % No. %

1. Amoxicillin 128 53.33 51 21.25 61 25.41 240

2. Amoxicillin /

Clavulanic acid 136 56.66 57 23.75 47 19.58 240

3. Ampicillin 101 42.08 62 25.83 76 31.66 240

4. Ampicillin / Sulbactan

154 64.16 46 19.16 40 16.66 240

5. Ciprofloxacin 147 83.52 15 8.52 14 7.95 176

6. Clindamycin 115 47.91 51 21.25 74 30.83 240

7. Erythromycin 87 36.25 31 12.91 122 50.83 240

8. Furazolidone 165 68.75 52 21.66 23 9.58 240

9. Gentamicin 143 59.58 52 21.66 45 18.75 240

10. Kanamycin 152 63.33 34 14.16 54 22.5 240

11. Lincomycin 108 45 39 16.25 93 38.75 240

12. Novobiocin 174 72.5 57 23.75 9 3.75 240

13. Penicillin G 95 39.58 43 17.91 102 42.5 240

14. Tetracycline 112 46.66 39 16.25 89 37.08 240

15. Tobramycin 108 61.36 50 28.4 18 10.22 176

The frequency of antibioresistance strains was studied at a number of 240

strains of staphylococci isolated from healthy animals or with various diseases. In this study, only free coagulase was pursued, along with antibioresistance, because its presence represents a taxonomic criteria that differentiate staphylococci into two groups: positive coagulase and negative coagulase.

Analyzing the results, it appears that resistance patterns had a variable frequency depending on the group of antibiotics to which the test was performed.

The frequency of resistance patterns was minimal (3.75%) to novobiocin and maximum (50.83%) to erythromycin. Intermediate resistant strains frequency ranged between 8.52% (ciprofloxacin) and 28.40% (tobramycin), while susceptible strains ranged from 36.25% (erythromycin) and 83.52% (ciprofloxacin).

A total of 64 strains, isolated from bovines with clinical or sublcinical mastitis, weren’t tested against ciprofloxacin and tobramycin, because there is no veterinary medicinal products for the therapy of mastitis in bovines, comprising these two antibiotics.

Resistance patterns to beta-lactams were monitored using 5 antibiotics from this group, two of which are associated with beta-lactamase inhibitors. Resistance patterns had a frequency between 16.66% and 42.50%. The intermediate strains had a frequency between 17.91% and 25.83%, while the susceptible strains had a frequency between 39.58% and 64.16%. Analyzing the behavior of staphylococcal strains tested against beta-lactams, it is noticed that the frequency of resistant strains to ampicillin/sulbactam was the lowest, and the frequency of susceptible strains was highest to ampicillin/sulbactam.


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To test the resistance patterns to macrolides, two antibiotics from this group were used. The frequency of resistant strains to erythromycin was 50.83% and the frequency of resistant strains to clindamycin was 30.83%. High resistance to erythromycin also indicates the resistance to macrolides with 14 atoms, and resistance to clindamycin indicates a lower resistance to macrolides with 16 atoms. The isolates susceptibility to antibiotic was 36.25% to erythromycin and 47.91% to clindamycin.

The resistance patterns to aminoglycosides more frequently used in the therapy of animals and humans had a frequency of 10.22% (tobramycin) and 22.50% (kanamycin). The frequency of strains with intermediate resistance was between 14.16% and 28.40%, and the frequency of susceptible strains ranged between 59.58% and 63.33%.

The frequency of resistance patterns to antibiotics from the tetracycline group was established using tetracycline. Resistant strains had a frequency of 37.08%, the strains with intermediate resistance had a frequency of 16.25% and 46.66% of the tested strains were susceptible.

In order to determine the resistance patterns to fluorquinolone, the only antibiotic used was ciprofloxacin, because this antibiotic is commonly used in therapy, both in humans and in animals. Resistant strains had a frequency of 7.95%, intermediate resistant strains had a frequency of 8.52% and the susceptible strains had a frequency of 83.52%.

The frequency of resistance patterns to lincosamides has been established, using only lincomycin. Resistant strains had a frequency of 38.75%, the intermediate strains had frequency of 16.25%, while the frequency of susceptible strains was 45%.

Furazolidone was used for rapid differentiation of staphylococci from the micrococci, to which the frequency of resistant strains was 9.58%, and the frequency of susceptible strains was 68.75%.

The resistance pattern to novobiocin was only 3.75%, and the frequency of susceptible strains was 72.50%. Only some species of coagulase-negative staphylococci are resistant to this antibiotic, particularly S. saprophyticus.

The frequency of resistance patterns of coagulase negative and coagulase positive staphylococci isolated from animals, is permanently monitored, because these bacteria can pass easily to humans and produce frequently nosocomial infections. In 2012, Ergün Y. et al., in Turkey, studied the frequency of coagulase negative staphylococci resistance patterns isolated from sheeps with subclinical mastitis. The same author found that the isolates were resistant to penicillin and ampicillin, in a proportion of 42.9%. BlaZ gene was detected in all strains resistant to these beta-lactams. The resistance to tetracycline of the strains was 11.4% and at these strains were detected the genes tetK and tetM. Isolates resistance to erythromycin and other macrolides with 14 atoms was 5.7% and at these strains


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was detected msrA gene. All isolates were susceptible to oxacillin, amoxiclav, cephalothin, gentamicin and enrofloxacin (3). F. Kunz et al., in 2011, studied the resistance patterns of S. aureus strains and coagulase negative staphylococci strains isolated from sheeps and goats with mastitis. Strains of S. aureus were resistant to penicillin (31.3%), ampicillin (29.9%), erythromycin (1.5%) and tetracycline (3%), while coagulase negative staphylococci strains were resistant to penicillin (8.2%), ampicillin (1%), erythromycin (10.6%) and tetracycline (7.7%). The resistance pattern to oxacillin was 4.8% of all isolates (4). In Switzerland, in 2013, Moser A. et al. studied the resistance patterns of coagulase negative staphylococci strains isolated from cows with mastitis. The authors identified a total of 12 species with different resistance patterns. Thus, 39% of the strains were resistant to ampicillin and penicillin, 6% were resistant to amoxiclav, ceftiofur, cephalothin and cefoxitin and 5% of the strains were resistant to erythromycin. Resistance patterns to gentamycin and kanamycin were 1 and 2% (5). J. Rubin et al, in 2011, in Canada, studied the resistance patterns of S. pseduintermedius strains isolated from healthy dogs and with different diseases. The results confirmed a relatively high frequency of resistance patterns to antibiotics used more frequently in dogs. Susceptible strains had a frequency of 46.4%, while resistant strains have a different frequency as follows: 39.9% penicillin, 23.5% tetracycline, 3.3% erythromycin and clindamycin. None of the strains were resistant to oxacillin (6).

The results obtained in this study are similar to the results obtained by the cited teams, thus demonstrating the high frequency of resistance patterns in staphylococci to the antibiotics used in therapy.

Conclusions

The resistance patterns of the studied strains of staphylococci had a

variable frequency according to the used antibiotics. The frequency of resistance patterns to beta-lactams used was between

16.66% and 42.5%, being lower in the beta-lactam associated with beta-lactamase inhibitors.

Resistance patterns to macrolides had a variable frequency being higher to macrolides with 14 atoms and lower to macrolides with 16 atoms.

The resistance patterns to aminoglycosides had a lower frequency, because these antibiotics are used less in the therapy of certain diseases in animals.

Resistance patterns to tetracyclines and lincosamides had a similar frequency, and frequency of these patterns to ciprofloxacin was 7.95%.

The resistance patterns to furazolidone and novobiocin, used to differentiate staphylococci from micrococci and S. saprophyticus, had a low


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frequency, which confirms the importance of these antibiotics in including the isolated strains in Staphylococcus genus.

Acknowledgements

This research work was carried out with the support of the

project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR 2669.

References

1. Aarestrup, F. M., Schwarz, S., Antimicrobial resistance in staphylococci and

streptococci of animal origin, în Antimicrobial resistance in bacteria of animal origin, Ed. ASM Press, Washington, D.C., 187- 212, 2016.

2. Codiţă, Irina, Buiuc, D., Determinarea sensibilităţii la antibiotice: teste calitative, în Tratat de microbiologie clinică, ediţia a II-a, sub redacţia Buiuc D., Neguţ M., Ed. Medicală, Bucureşti, 453-482, 2008.

3. Ergün, Y., Aslantaş, Ö., Kireҫci, E., Öztürk, F., Ceylan, A., Boyar, Y., Antimicrobial susceptibility presence of resistance genes and biofilm formation in coagulase negative staphylococci isolated from subclinical sheep mastitis, Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 2012, 18 (3), 449-456.

4. Kunz, F., Corti, S., Giezendanner, N., Stephan, R., Wittenbrink, M. M., Zweifel, C., Antimicrobial resistance of Staphylococcus aureus and coagulase negative staphylococci isolated from mastitis milk samples from sheep and goats, SAT, Schweizer Archiv für Tierheilkunde, 2011, 153 (2), 63-69.

5. Moser, A., Stephan, R., Ziegler, D., Johler, S., Species distribution and resistance profiles of coagulase-negative staphylococci isolated from bovine mastitis in Switzerland, SAT, Schweizer Archiv für Tierheilkunde, 2013, 155 (6), 333-338.

6. Rubin, J. E., Chirino-Trejo, M., Prevalence sites of colonization and antimicrobial resistance among Staphylococcus pseudintermedius isolated from healthy dogs in Saskatoon, Canada, Journal of Veterinary Diagnostic Investigation, 2011, 23 (2), 351-354.


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RESEARCH CONCERNING THE ASSOCIATION OF REOVIROSIS WITH OTHER INFECTIOUS ENDEMIC DISEASES IN BROILERS

N. CĂTANA¹, VIRGILIA POPA², OANA PETREC¹, IONICA IANCU¹

1Banat’s University of Agricultural Sciences and Veterinary Medicine ”King Michael I of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Aradului

Street No. 119, Timisoara, Romania

2S.N. Institutul Pasteur S.A. Bucharest

E-mail: [email protected]

Summary

Reovirosis has an endemic progress, in broilers farms, alone or in combination with

other infectious diseases due to immunosuppression induced by the avian reovirus. The research was performed in a flock of broilers, in which the reovirosis evolved

with two syndromes characteristic, after the age of 14 days. Subsequently, based on symptoms and pathological lesions were suspected other infectious diseases that occurred at different ages.

The PCR techniques has been used for detect the viruses in several variants. For the isolation of germs was used bacteriological exam.

Avian reovirus was detected in samples of small intestine, liver, proventriculus and spleen taken from the cadavers after the age of 6 days.

Infectious bursal virus (very virulent strains) was detected in the Fabricius bursa, with characteristic lesions after 21 days of age.

Infectious bronchitis virus (strain Qx) was detected in samples of lungs taken from cadavers after the age of 21 days, and Marek's disease virus was detected in samples of liver and spleen taken from cadavers after the age of 25 days .

Key words: avian reovirus, broilers, other avian viruses

Intensive avian culture aims to increase production of meat and eggs. For

this purpose birds are raised on farms with biosecurity, and the systematic application of general measures and immunoprophilaxy, led to the elimination of serious infectious diseases, viral and bacterial infections (5,6). During the time, the degree of resistance of birds has decreased due to: the selection and improvement, intensive technology and stress, global trade with poultry material has contributed to the spread of pathogens infectious, viral and bacterial pathogens that can trigger endemic disease, regarded as infections farm. These diseases are difficult to control and evolves in effective as associated infections (3,4,5).

Due to its frequency and economic importance avian reovirosis being very well studied because immunosuppression induced by avian reovirosis promotes the development of other viruses (2,3,7).

The research has pursued, in broiler farms, the presence of viruses that cause infectious diseases associated followed by significant economic losses.

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Materials and methods

For laboratory investigation were collected fragments of organs (proventriculus, small intestine, liver, pancreas, spleen, lungs and Fabricius bursa) from cadavers broiler of various ages, from more effective, tests for organs they were kept in Ependorf tubes, labeled in temperature of - 80°C. For the detection of the viruses that have circulated in monitored flocks has been used PCR technique in several variants. Viral RNA in ribovirus was detected with RT-PCR and the viral DNA was detected in dezoxiribovirus with PCR method for both variants were used primers recommended by the literature.

This technique was performed in the Laboratory of molecular biology of SN Pasteur Institute S.A. Bucharest.


Using PCR, in variants mentioned, using the appropriate primers four

viruses were detected in monitored flocks and the disease produced by them evolve at different ages. Avian reovirus was detected in organs like proventriculus, small intestine, liver, spleen, pancreas, Fabricius bursa taken from cadavers, aged 6-35 days in monitored flocks. The primers used for the target gene (S4) of the RNA-viral amplicons have generated with 1120 nucleotide base pairs, view in agarose gel. Specific bands were strongly positive light, having a larger amount of nucleic acid. Specific positive bands were strongly positive light, having a higher amount of nucleic acid. The results have demonstrated the presence of this virus in broilers of different ages, confirming the evolution of endemic avian reovirosis as multiple clinical syndromes. Similar results were communicated by the other researchers. They have determined that the frequency of avian reovirus in broilers, it ranges between 62.8% and 100% (2,3). For the detection of avian bursal infection virus was used RT-PCR using primers to detect the gene encoding VP2 protein synthesis, responsible for induction of neutralizing antibursitic antibodies. This virus was detected in Fabricius bursa taken from cadavers broiler after the age of 21 days, in all monitored flocks. The strains detected belonged to the patotype very virulent responsible for producing disease. These strains are signaled more frequently in broiler chicken flocks, as a result of escalating phenomenon explained by the emergence of mutant strains, highly virulent strains, due to vaccine and vaccination routes used (5,6)

For detection of infectious bronchitis virus strains was used RT-PCR, being searched Massachusetts type strains, strains of type QX (CR, FR, 4/91) and IT-type strains of IT02. In samples examined were detected strains H126 and Mac 5, used as vaccines. 4 lung samples were detected 4 strains belonging to pathotype QX.


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By RT-PCR was detected avian coronavirus RNA as well as genes characteristic of this pathotype using amplicon 190 (170-190) bp (1). In some flock of broilers monitored after 20 days ages, the lesions were characteristic of Marek's disease. For the confirmation of the disease, PCR was used to detect the virus that causes purpose of this disease. The viral DNA was detected in liver and spleen of the bodies after the age of 20 days.

Avian Reovirus induce immunosuppression pronounced wich favors the evolution of viruses whose etiological agents are present on farms in some primary or secondary sources.The detected viruses run frequently in broiler flocks being raised in poultry farming, by several collective researchers. The results show the role of immunosuppression induced by avian reovirus, in the onset of endemic viruses that can evolve in broilers, similar to the existing data from literature (2,3,4).

Conclusions

Avian reovirus detection in the examined samples demonstrates the presence of this virus in monitored flocks at different ages due to horizontal and vertical transmission. Immunosuppression induced by avian reovirus favored the emergence and evolution of infectious diseases with endemic evolution.

Using PCR in several variants, were detected very virulent strains of the infectious bursitis virus, vaccine strains, QX strain of infectious bronchitis virus and Marek's disease virus strains.

Acknowledgements



References

1. Amin, O., Valastro, V., Salviato , A., Drago, A., Cattoli ,G., Monne, I.,

Circulation of Qx-like infection bronchitis virus in the Middle East, Vet. Record, 2012, 21, 530-535.

2. Benavente, J., Martinez, Costas, Avian reovirus: structure and biology, Virus Research., 2007, 123, 105-119.

3. Cătana, N., Fodor, Ionica, Botuș, Daniela, Popa, Virgilia, Infecţiile cu reovirusuri la păsări, Magazin Avicol, 2008, 5 (21), 24 – 25.

4. Davis, J.F., Kulkarni, A., Fletcher, O., Reovirus infections in young broiler chickens, Avian Diseases, 2013, 57 (2), 321-325.


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5. Jones, R.C., Reovirus infections, În Diseases of Poultry, 13th Edition, Editor in Chief: David E. Swaine, Ed. Wiley Blackwell, State Avenue, 2013, USA, 351-352.

6. Perianu, T., Tratat de boli infecțioase ale animalelor, Viroze și boli prionice, vol. II, Ed. Universitas, XXI, Iași, 2012

7. Răpuntean, Gh., Răpuntean, S.,Virusologie Veterinară Specială, Ed. Colorama, Cluj-Napoca, 2014.


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RESEARCH ON THE FREQUENCY OF METHICILLIN RESISTANT STRAINS OF STAPHYLOCOCCUS SPP.

N. CĂTANA, IONICA IANCU, IULIA BUCUR, V. HERMAN

Banat’s University of Agricultural Sciences and Veterinary Medicine ”King Michael I

of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Aradului Street No. 119, Timisoara, Romania


Summary

The phenomenon of multiple resistance to antibiotic, is in continuously expanding,

both in coagulase-positive as well as in coagulase-negative staphylococci. A special attention is paid to methicillin-resistant staphylococci, also known as

MRSA staphylococci (Methicillin Resistant Staphylococcus aureus), due to permanent zoonotic risk.

The methicillin resistance in staphylococci is determined by the synthesis of a specific protein (PBP20), encoded by the mecA gene.

Researches were made on a number of 245 strains of staphylococci isolated from animals, 43 coagulase positive strains and 202 coagulase negative strains. The methicillin resistance of these strains was phenotypically researched through the disk diffusion method, using biodiscs with methicillin (5 µg), oxacillin (1 µg) and cefoxitin (30 µg).

The results established a high frequency of the strains resistant to methicillin (32.44%) and oxacillin (41.23%), showing, thus, the existence of MRSA strains of staphylococci, both in farm animals, as well as in pets.

Key words: coagulase positive, coagulase negative, methicillin-resistant

staphylococci strains

Bacterial antibiotic resistance is genetically determined and the resistance genes are present in the bacterial chromosome, the R plasmid and in other mobile genetic elements within the cytoplasm. These genes, coding for antibiotic resistance, are transmitted through several mechanisms between the bacterial cells generating, at all times, strains with multiple resistance to antibiotics (1, 2).

In staphylococci, antibiotic resistance is widespread, multi-resistant strains being isolated from animals and humans, because of the epidemiological circuit animal-human-animal (1, 3).

Particular attention is paid to methicillin resistant staphylococci strains, since resistance to this antibiotic is correlated with the multiple resistance to several or all antibiotics used. Methicillin-resistant staphylococcal strains are considered as high-risk strains of zoonotic importance in human and animal pathology (1, 3).

Methicillin resistance has genetic basis in the staphylococcal chromosome mec boxes (mecA, mecR1 and mecO) and can be type A, type B, type C and type D (2, 7).


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Researches followed the frequency of the phenomenon of methicillin-resistant strains of coagulase positive and coagulase negative staifilococi.

Materials and methods The phenomenon of methicillin resistance was followed in a number of 245

strains of coagulase positive and coagulase negative staphylococci, isolated from healthy animals or with various diseases.

Baird-Parker medium was used to identify coagulase positive and coagulase negative staphylococci.

The Kirby-Bauer disc diffusion method was used in order to test the susceptibility of staphylococci to methicillin (5 µg), oxacillin (1 µg) and cefoxitin (30 µg) using: Mueller-Hinton broth and agar, Petri plates and biodiscs impregnated with the above antibiotics.

The interpretation of results was made according the recommendations of the Institute for Clinical Laboratory Standards, USA (CLSI-Clinical and Laboratory Standard Institute) taken by the European Committee for Testing the Antibiotic Susceptibility of the European Society of Infectious Diseases and Clinical Microbiology and the strains were classified into three categories: susceptible, intermediate and resistant (3).


The obtained results, regarding the resistance to three beta-lactams,

resistant to penicillinase, were different, but confirmed that at coagulase positive and coagulase negative staphylococci, isolated from animals, the resistance patterns to these antibiotics are present. 43 coagulase positive strains and 202 coagulase negative strains were identified by using Baird-Parker medium.

Coagulase positive and coagulase negative staphylococci strains had a different behavior to methicillin, an antibiotic commonly used for detection of methicillin-resistance phenomenon. Thus, 32.24% of the tested strains were resistant to this antibiotic, 45.71% of the tested strains were susceptible, and 22.04% had intermediate resistance.

In order to detect the cross-resistance of staphylococci to penicillins resistant to penicillinase, oxacillin is also recommended, both for its stability and for the reproducibility of results. The obtained results, when using oxacillin, were as follows: 41.22% of strains were resistant, 30.20% of strains were susceptible and 28.57% had an intermediate behavior.

The increased frequency of methicillin-resistant stafiloccoci strains and, in particular, strains of S. aureus subsp. aureus, respectively MRSA strains (Methicillin Resistant S. aureus) determined using the disk diffusion test to cefoxitin, which is more reliable than methicillin and oxacillin (1, 3).


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The obtained results on testing coagulase positive and coagulase negative staphylococci strains to cefoxitin were: 5.47% of strains were resistant, 87.56% of strains were susceptible, and 6.97% had an intermediate behavior.

Analyzing the results, is observed that the frequency of the resistant strains to these three beta-lactams, resistant to penicillinase, was variable, as follows: • the frequency of resistant strains to oxacillin was 1.28 times higher than the frequency of methicillin-resistant strains; • the frequency of resistant strains to oxacillin was 7.54 times higher than the frequency of resistant strains to cefoxitin; • the frequency of methicillin-resistant strains was 5.89 times higher than the frequency of resistant strains to cefoxitin. These results confirm that oxacillin, compared with methicillin, has found a large number of resistant strains. The results, also, confirmed that through the disk diffusion test to cefoxitin, was found a frequency of 5.47% of resistant strains of which, according to the literature, can be considered MRSA strains.

Mendonça E. C. L. et al., in 2012, studied the resistance patterns of Staphylococcus spp. strains isolated from cattle, through phenotypic tests (disc diffusion) and molecular biology. The authors recommend oxacillin to determine the frequency of resistant methicillin strains of Staphylococcus spp., considering this method as the standard method. The authors also recommend PCR for detection of mecA, mecI and mecRI gene, and the blaZ gene. Based on these results, the authors found a close link between disc diffusion method with oxacillin and the presence of the mentioned genes (5).

The results confirm the data in the literature, regarding the frequency of resistance patterns to beta-lactams resistant to penicillinase and, in particular, the frequency and the zoonotic risk of MRSA strains. The fact that, in the last years, are increasingly being isolated MRSA strains from animals and, particularly in pigs, required extensive studies on the circuit of these strains and, also, the use of some phenotypic and genotypic tests to identify them.

Dressler A. E. et al., in 2012, conducted a study on the resistance patterns of 157 strains of staphylococci isolated from pigs, 15.9% being S. aureus strains and the rest, strains of coagulase negative. On the isolated strains by phenotypic tests, the authors found the following frequency of resistance patterns: 8% of strains were resistant to methicillin, 92% of strains were resistant to tetracycline, 56% were resistant to erythromycin and 60% of strains were resistant to clindamycin. On methicillin-resistant strains, mec gene has been detected, encoding the resistance to methicillin. Based on these results, the authors believe that swine are a potential reservoir of MRSA strains with zoonotic risk to humans (4).

In our country, studies on the frequency of MRSA strains in pigs were made, as Decision 2008/55/EC recommends, in particular, setting the frequency of MRSA strains isolated from pigs. There have also been optimized multiplex PCR


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techniques for the detection of mecA and nucA gene present in MRSA strains. (2, 6, 7).

The obtained results from this research completes the existing data in our country on the resistant patterns of coagulase positive and negative staphylococci strains isolated from animals.

Conclusions

Baird-Parker medium allowed the identification of coagulase-positive and

coagulase-negative staphylococci isolated from animals. The obtained results by disk diffusion method on resistance patterns to

three beta-lactams, resistant to penicillinase, revealed a different frequency of resistant strains to these three antibiotics.

The oxacillin disk diffusion test detected a higher frequency of resistant strains compared with the disk diffusion test to methicillin.

Cefoxitin disk diffusion test revealed a frequency of 5.47% of resistant strains, which can be considered MRSA strains.

Acknowledgements



References

7. Aarestrup, F. M., Schwarz, S., Antimicrobial resistance in staphylococci and streptococci of animal origin, în Antimicrobial resistance in bacteria of animal origin, Ed. ASM Press, Washington, D.C., 2006, 187- 212.

8. Bicheru, Monica, Necșulescu, M., Cumpănășoiu, Bianca, Tîrziu, E., Popescu, Diana, Ionescu, Lucia, Dumitrescu, Gabriela, Necșulescu, A., PCR for the identification of methicillin resistant Staphylococcus aureus (MRSA) strains using primers specific for scc mec elements, Lucr. Șt. Med. Vet. Timișoara, 2014, XLVII(2), 5-12.

9. Codiţă, Irina, Buiuc, D., Determinarea sensibilităţii la antibiotice: teste calitative, în Tratat de microbiologie clinică, ediţia a II-a, sub redacţia BUIUC D., NEGUŢ M., Ed. Medicală, Bucureşti, 2008, 453-482.

10. Dressler, A. E., Scheibel, R. P., Wardyn, S., Harper, A. L., Hanson, B. M., Kroeger, J. S., Diekema, D. J., Bender, J. B., Gray, G. C., Smith, T. C., Prevalence, antibiotic resistance and molecular characterisation of Staphylococcus aureus in pigs at agricultural fairs in the USA, Veterinary Record, 2012, 170(19), 495.


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11. Mendonça, E. C. L., Marques, V. F., Melo, D. A., Alencar, T. A., Coelho, I. Da S., Coelho, S. M. O., Souza, M. M. S., Phenogenotypical characterization of antimicrobial resistance in Staphylococcus spp. isolated from bovine mastitis, Pesquisa Veterinária Brasileira, 2012, 32(9), 859-864.

12. Mocuta, N., Chindris V., Stetca, Gh., Prevalența tulpinilor de stafilococ methicilin rezistente în exploatații de porci din Ardeal, Rev. Rom. Med. Vet., 2011, 2, 29-34.

13. Vanghelie, M., Dragnea, C. M., Maftei, D. N., Bria, P. C., Coman A., Optimizarea unui test multiplex PCR pentru detecția genei mecA și identificarea tulpinilor de Staphylococcus aureus, Rev. Rom. Med. Vet., 2012, 3, 143-150.


20

THE PREVALENCE OF POSITIVE ESBL E. COLI AND KLEBSIELLA PNEUMONIAE STRAINS ISOLATED FROM THE

OWNERS OF PETS

ANDREEA-PAULA COZMA, OANA-ALEXANDRA CIOCAN, C. CARP-CĂRARE, ELEONORA GUGUIANU,CRISTINA HORHOGEA,

CRISTINA RÎMBU, M. MAREȘ, M. CARP-CĂRARE

"Ion Ionescu de la Brad" University of Agricultural Sciences and Veterinary Medicine from Iaşi, Faculty of Veterinary Medicine

Mihail Sadoveanu Alley No. 8, 700489, Iaşi, Romania E-mail: [email protected]

Summary

The production of extended-spectrum Beta-lactamase enzymes by Enterobacteriaceae is a threat to public health in the human as well as the veterinary medicine at a global level. The ESBL enzymes are encoded by genes that are located on mobile genetic elements. Consequently, at the microbial level, between people and pets, or within the same species, the horizontal transfer of bacteria bearing ESBL resistance genes can occur. The purpose of this study was to detect the presence of extended-spectrum beta-lactamase producing E. coli and K. pneumoniae strains in pet owners and determine the resistance of isolates to antibiotics. In 2015, 41 faecal samples of human origin were processed. Out of the total of the samples that were analyzed, 17 (41.46%) were positive, the certainty confirmation will be performed through techniques of molecular biology. The phenotypic confirmation of ESBL positive strains was performed in accordance with the protocol recommended by CLSI. The ultimate purpose of the study aims at establishing some resistance genes that are common to pets and owners in order to determine their role in the circuit of the ESBL strains “in-cross” transmission.

Key words: ESBL, enterobacteriaceae, pets, owners

Extended-spectrum beta-lactamase-producing Enterobacteriaceae

represent a growing public health concern. Most of the bacterial pathogens associated with the enteric diseases in humans come from animals and can be passed on directly from animals to humans or from humans to animals, either directly through the close cohabitation between humans and pets or indirectly, through food products, contaminated water or a common reservoir (6,10). These various ways and cotamination sources complicate the epidemiological investigations. The various interactions between animals and humans favour both both the intraspecies and interspecies exchange of resistance genes between the bacterial strains. The wide use of some common antibiotics for treating some bacterial infections both in the human and veterinary medicine favours the emergence of the antibiotic resistance phenomenon.


21

In Romania, the lack of some epidemiological investigations on the issue of antibiotic resistance conducted in the veterinary medicine, influences and affects the evolution of this phenomenon in human medicine. The horizontal transfer of genes and the encoding of extended-spectrum beta-lactamase enzymes on mobile genetic elements facilitates thei easy spreading. In order to get an overview on the phenomenon of antibiotic resistance, a “One health” (3,4) type integrating approach is required both in Romania and at a global level. Applying this integrative concept globally, concept that should use a trans-sectorial interpretation of data, can improve the prevention and control of bacterial infections with zoonotic and antibioresistant strains (2,11).

The purpose of this study was to detect the presence of extended-spectrum beta-lactamase producing strains of E.coli și K. Pneumoniae in the owners of pets and establish the resistance of the isolates to anitbiotics.


In a first stage of our study, faecal samples from clinically healthy pets were collected and processed: from stray dogs, owned dogs and cats, for the isolation, identification and phenotypic confirmation of ESBL positive E.coli and K.pneumoniae bacterial strains.

In 2015, 41 faecal samples were collected and processed from the owners or the veterinary staff that came into contact with the pets mentioned above.

After collection, the faecal samples were sown on broth and incubated at 37°C for 24 h. Due to the reduced number of enteric agents per unit of volume, such seeding is recommended in advance because, in some enterobacterioses, the enrichment of the inoculum through the sub-cultivation on the media that preferentially favour the multiplication of the enteric pathogens, represents a necessary stage (4).

From the liquid medium, 100 μl were taken and sown on the specific chromogenic medium, the Oxoid Brilliance ESBL Agar medium (11), for the isolation of the ESBL positive Enterobacteriaceae. According to the producer’s specifications, Brilliance Esbl Agar contains cefpodoxime, o second-generation cephalosporine in which all the ESBL bacterial strains are resistant. Furthermore, according to the producer’s specifications, the E. coli colonies are blue, while the Klebsiella spp. Colonies are turquoise.

As a result of the screening conducted, the colonies obtained were retransplanted on a neutral medium, TSA, with a view to their identification and taxonomic classification. The MIU, TSI, EMBA and TBX polytrope media were used, the incubation taking place at 37°C, for 24 hours for the first 3 polytrope media, respectively at 44.5°C, for a period of 4 hours for the identification of the biotype 1 Escherichia coli strains.


22

The phenotypic confirmation of ESBL Enterobacteriaceae strains was performed through the combined disc method, method recommended by the CLSI standard (3).

This method involves the use of some discs of simple cephalosporins and some discs of cephalosporins whose effect is intensified by the clavulanic acid, discs spread at distances between 15-30 mm. For the interpretation of the results, the difference between the diameters of the inhibition zones or the relationship betweeen the diameters of the inhibition zones is compared. The increase in the diameter of the inhibition zone greater than 5 mm or equivalent to 50% when the clavulanate is present, indicates the production of ESBL.

The following antibiotics were used: cefpodoxime, cefpodoxime/clavulanic acid, cefotaxime, cefotaxime/clavulanic acid, ceftazidime, ceftazidime/clavulanic acid, cefoxitine, cefpirome and cefepime. The phenotypic confirmation of ESBL strains is sometimes hindered by the existence, within the same bacterial strain, of the resistance genes that encode the ESBL genes and of the AmpC resistance genes. According to the literature, all the Ampc enzyme-producing strains are resistant to the action of cefoxitine and sensitive to the action of the fourth-generation cephalosporins: cefpirome and cefepime (5).

The ATCC 25922 strain of E. coli and ATCC 700603 (blaSHV-18) strain of Klebsiella pneumoniae were used as quality control reference for the test of sensitivity to antibiotics of the ESBL enzyme-producing strains (9).

Fig.1. The phenotypic confirmation of

the isolated ESBL strains; Fig. 2. E.coli bacterial strains on the Oxoid Brilliance ESBL Agar medium


23


After processing the data and the results obtained, the prevalence of the individuals bearing ESBL Enterobacteriaceae was established, this one being itself a statistical indicator (fig. 3).

Fig. 3. Synthesis of the results after processing samples

Out of the 41 processed faecal samples, 17 (41.46%) generated cultures on the Brilliance ESBL Agar medium, 17 presumptive ESBL-producing Enterobacteriaceae strains being obtained. After using the combined disc method, all the strains were phenotypically confirmed as being extended-spectrum beta-lactamase producing strains.

The prevalence of the transfer of the ESBL Enterobacteriaceae to the veterinary staff or the owners of pets that were considered for the study was 41.46%.

As far as the taxonomic classification is concerned, out of the 17 isolated strains, 3 (17.65%) belong to the Klebsiella pneumoniae species, and most of them, 14 (82.35%) belong to the E.coli species (fig. 4).

In 2015, the same study was conducted by the same authors on clinically healthy pets, dogs and cats that came into direct contact with the staff analyzed in the present study. After processing 201 faecal samples, the prevalence of dogs and cats bearing ESBL Enterobacteriaceae was 29.85%.

Although a smaller number of samples of human origin was analyzed, we consider that the higher prevalence of the transfer of the ESBL positive


24

Enterobacteriaceae in humans than in animals, can be explained if we take into consideration the subjects’ medication history, the high consumption and the preferential use of antibiotics presenting risks and sometimes, the lack of a solid hygiene. According to the CARMIN 2013 report, the level of the consumption of antibiotics increased by 4.5% in 2013 compared to 2012 (when it was the second highest consumption of antibiotics per 1,000 inhabitants in Europe) (8).

Fig. 4. The result of the taxonomic classification of the obtained strain

Conclusions

The prevalence of the transfer of ESBL Enterobacteriaceae noticed in this study is 41.46% in owners or the veterinary staff that came into direct contact with the pets.

The prevalence of E.coli strains was 82.35% and of Klebsiella pneumoniae strains was 17.65%.

In Romania, the lack of some epidemiological investigations on the issue of antibiotic resistance in the veterinary medicine influences and affects the evolution of this phenomenon in human medicine.

References

1. Calistri, P., Iannetti, S., Danzetta L., Narcisi, M., Cito, V., Di Sabatino, F., Bruno, D., Sauro, R., Atzeni, F., Carvelli, M., Giovannini, A.A., The components of ‘One World-One Health’ approach. Transbound. Emerg. Dis., 2013, 60 (Suppl. 2), 4–13.

2. Carmena, D., Cardona, G.A., Echinococcosis in wild carnivorous species: epidemiology, genotypic diversity, and implications for veterinary public health. Vet. Parasitol., 2014, 202, 69–94.


25

3. Clinical and Laboratory Standards Institute (CLSI)., 2014, Performance Standards for Antimicrobial Susceptibility Testing.

4. Cozma, A., Ciocan, O. A., Carp-Cărare, C., Carp-Cărare, M., Guguianu, E., Rîmbu C., Mareș M., The prevalence of positive ESBL E. coli and Klebsiella pneumoniae in the dogs in the Iași paddock, Lucrări științifice Medicină Veterinară Timișoara, 2015, Vol. XLVIII.

5. Mirelis, B., A simple phenotypic method for the differentiation between

acquired and chromosomal AmpC beta‐lactamases in E. coli., Enferm. Infecc Microbiol Clin 24, 370-372.

6. Newell, D.G., Koopmans, M., Verhoef, L., Duizer, E., Aidara-Kane, A., Sprong, H., Opsteegh, M., Langelaar, M. Threfall, J. Scheutz, F. van der Giessen, J. Kruse, H., Food-borne diseases – the challenges of 20 years ago still persist while new ones continue to emerge. Int. J. Food Microbiol., 2010, 139 (Suppl. 1), S3–S15.

7. Parmley, E.J., Pintar, K., Majowicz, S., Avery, B., Cook, A., Jokinen, C., Gannon, V., Lapen, D.R., Topp, E., Edge T.A., Gilmour M., Pollari, F. Reid-Smith, R. Irwin, R., A Canadian application of one health: integration of Salmonella data from various Canadian surveillance programs (2005–2010). Foodborne Pathog. Dis., 2013, 10, 747–756.

8. Popescu, G. A., Șerban, R., Report concerning the consumption of antibiotics, the microbial resistance and nosocomial infections in Romania – 2013.

9. Rajkumar Manojkumar Singh, Comparative evaluation of six phenotypic methods for detecting extended-spectrum beta-lactamase-producing Enterobacteriaceae, The Journal of Infection in Developing Countries, 2014, 8(4):408-415.

10. Schlundt, J., Toyofuku, H., Jansen, J., Herbst, S.A., Emerging food-borne zoonoses. Rev. Sci. Tech., 2004, 23, 513–533.

11. Wendt, A., Kreienbrock L., Campe A., Zoonotic disease surveillance – inven- tory of systems integrating human and animal disease information. Zoonoses Public Health, 2014, http://dx.doi.org/10.1111/zph.12120.

12. Willems, E., Cartuyvels, R., Magerman, K., Verhaegen, J., Evaluation of 3 different agar media for rapid detection of extended-spectrum β-lactamase-producing Enterobacteriaceae from surveillance samples, Diagn. Microbiol. Infect.Dis., 2013, 76 (1), 16:9.


26

ANTIBIOTICS SENSITIVITY OF ISOLATED BACTERIA FROM DAIRY COWS WITH CLINICAL ENDOMETRITIS

IOANA CRISTINA CRIVEI

1, ANDREEA PAULA COZMA

1, S. I. BORS

2, ELENA

RUGINOSU2, P. ROȘCA

1, D. DRUGOCIU

1

1“Ion Ionescu de la Brad” University of Agricultural Sciences and Veterinary

Medicine of Iaşi, Faculty of Veterinary Medicine, Mihail Sadoveanu Alley, No. 8, 700489, Iaşi, Romania

2The Research and Development cattle DANCU - IASI SCDCB Holboca village,

No. 9 Sos. Iasi-Ungheni E-mail: [email protected]

Summary

Clinical endometritis is a common cause of infertility in cows, causing delays in uterine involution, low conception rates and an increased number of services per conception. The aim of this study was to isolate and identify the involved bacteria in the development of endometritis in cows, and establishing a primary treatment using antibiotic susceptibility by determining germs sensitivity to antibiotics from different classes. The isolated bacteria were stained Gram, transferred after on solid media, where they showed characteristic colonies and were confirmed by standard biochemical tests. The in-vitro antibiotic sensitivity test for each isolated and identified bacteria was done by disc diffusion method. Out of 24 samples, were isolated and identified 30 bacterial strains: Escherichia coli (40%), Klebsiella spp. (16.7%), Staphylococcus spp. (23.3%), Corynebacterium spp. (13.3%) and Streptococcus spp. (6.7%). This study revealed that both Gram negative and Gram positive strains showed an increased resistance to antibiotics from Beta-lactams Class, Penicillin Subclass. All Gram negative strains were sensitive to Fluoroquinolone Class antibiotics (Enrofloxacin and Flumequine).

Key words: bacteria, cows, endometritis, antibiotic sensitivity.

Clinical endometritis, a common cause of infertility in cows, it occurs

usually during the post-partum period, causing delays in uterine involution, prolongs the time until first oestrus, causing an increased number of services per conception and thus low conception rates.

Uterine diseases, such as metritis and endometritis, are highly prevalent in high-producing dairy cows and lead to economic losses because of decreased milk yield and fertility (7, 9). Clinical endometritis is characterised by the presence of purulent (>50% pus) or mucopurulent (approximately 50% pus, 50% mucus) uterine exudate in the vagina, 21 days or more post-partum and is not accompanied by systemic signs (6, 11).

The uterus of postpartum cows is usually contaminated with a range of bacteria, but this is not consistently associated with clinical disease. Infection implies adherence of pathogenic organisms to the mucosa, colonization or


27

penetration of the epithelium, and/or release of bacterial toxins that lead to establishment of uterine disease (10). The development of uterine disease depends on the immune response of the cow, as well as the species and number (load or challenge) of bacteria (10).

Uterine infections caused by bacteria are treated with antibiotics, but the efficacy of such therapeutic agents needs to be evaluated from time to time due to continuous emergence of drug resistant bacterial strains (15).

The aim of this study was to identify and isolate the involved bacteria in clinical endometritis in cattle and to establish an effective treatment using antibiotic sensitivity by determining germs susceptibility to different classes of antibiotics.

Material and methods

Twenty-four uterine secretion samples were collected from Romanian Black Spotted Breed cows with clinical cases of endometritis. After collection, a sterile nutrient broth was added to each sample and incubated at 37

oC overnight.

After 24 hours, these samples were inoculated by streaking method on Levine Eosin-Methylene Blue Agar Medium (Gram negative bacteria), Chapmann Medium and blood agar (Gram positive bacteria).

The inoculated media were incubated under aerobic conditions at 37oC and

were examined after 24 hours post inoculation. The isolated colonies obtained on LevineEosin-Methylene Blue Agar Medium were further inoculated on MIU Medium Base (motility, urease and indole production), on TSI Agar (Triple Sugar Iron Agar) and Simmons Citrate Agar, in order to establish some minimum biochemical characters and thus to get the taxonomic classification of the isolated strains.

For the isolated colonies obtained on Chapmann Medium and blood agar, were performed Gram-stained smears andbased on their characteristic clusters, microscopicallyobserved, correlated with some minimum biochemical characters (Catalase test and Mannitol Fermentation Test) the taxonomic classification was obtained.

All the isolated strains were tested for in vitro antibiotic sensitivity by disc diffusion method of Kirby and Bauer (1966). All the isolated colonies were inoculated in nutrient broth and incubated for 12 hours at 37

oC.

Using a sterile cotton swab dipped into the bacterial suspension, the surface of the Muller-Hinton agar medium was inoculated with the bacterial suspension.

Antibiotic discs of Penicillin, Amoxicillin, Ampicillin, Oxacillin, Cefoxitin, Cefotaxime, Cefoperazon, Ceftiofur, Erythromycin, Lincospectin, Gentamicin, Kanamycin, Vancomycin, Enrofloxacin, Flumequine, Florfenicol, Tetracycline, Doxycycline, Polymyxin B, Colistin, Chloramphenicol and Trimetrophrim with Sulfometaxazol were placed on the surface of inoculated plates using a disk dispenser for antimicrobial susceptibility (Oxoid) and incubated overnight at 37

oC.


28

Interpretation of the results was performed by measuring the diameter of the inhibition zone for each antibiotic disc. Obtained results were compared and interpreted according to the CLSI standard 2014 (16).


Our bacteriological study of uterine secretion samples, revealed the presence of more aerobic organisms belonging to both Gram positive and Gram negative genera.

Thus, from the total of 30 bacterial strains isolated from the 24 cows with clinical signs of endometritis, more than half of them wereGram negative bacteria (57%) and 43% of strains were Gram positive bacteria (Fig.1).

Fig. 1. Bacterial strains distribution by their type

In our study, the isolated bacterial species from the uterine secretion samples were distributed as following: 12 strains of Escherichia coli (40%), 7 strains of Staphyloccocus spp. (23.3%), 5 strains of Klebiella spp. (16.7), 4 strains of Corynebacterium spp. (13.3%) and only 2 strains of Streptoccocus spp. (6.7%) (Fig. 2).Thus, these results suports Singla et al. (2001) findings, which reported that E.coli is the most commonly isolated bacteria in cows with clinical endometritis.

Mixed infections were found in 85% of cases, while single infections were detected only in 20% of cases.


29

Fig.2. Strains of bacterial species isolated from cows with clinical endometritis Other authors, (2) isolated Staphyloccocus spp., Escherichia coli, Bacillus

spp., Corynebacterium spp., Pseudomonas spp., Proteus spp., Klebiella spp. and Streptoccocus spp. from cows with clinical signs of metritis. Another author (13) reported the isolation of these bacterial strains from uterine discharge and intravaginal washing of cows suffering from endometritis.

Regarding in vitro antibiotic sensitivity of the bacteria isolated species to different antibiotics, we exposed our results in two charts.

Thus, regarding the antibiotics sensitivity for Gram negative bacterial strains, we noticed that all of them were resistant toAmoxycillin/Clavulanic Acid; we also noticed that a high percentage of Gram negative bacterial strains were resistant to Penicillin, Ampicillin, Oxacillin and Vancomycin (Fig. 3).

Increased resistance to class of β-lactam antibiotics is explicable and normal in the current situation ofantibioresistance, knowing that the primary treatment for infectious episodes both in human medicine and veterinary medicine is made with antibiotics from class of β-lactam. All Gram negative bacterial strains were sensitive to antibiotics from class ofFluoroquinolones: Enrofloxacin and Flumequine.

As for the antibiotic sensitivity of Gram positive bacterial strains, we noticed that all the strains were sensitive to antibiotics from class of β-lactam, Cephalosporins subclass: Ceftiofur, Cefoxitime and Cefotaxime (Fig. 4).


30

Fig. 3 Antibiotic sensitivity of Gram negative bacterial isolates from clinical cases of endometritis. AM-Amoxycillin/Clavulanic Acid; FUR- Ceftiofur; VA-Vancomycin; P-

Penicillin; AM- Ampicillin; OX- Oxacillin; FOX-Cefoxitin; CTX-Cefotaxime; E-Erythromycin; FF-Florfenicol; UB-Flumequine; EN- Enrofloxacin; K- Kanamycin;

CN-Gentamicin; LS-Lincospectin; CF- Cefoperazone; DO-Doxycycline; TE- Tetracycline; PB- Polymixin; C- Chloramphenicol; TS- Sulfadiazine/trimethoprim;

CT- Colistin.

Similar to Gram negative strains, we noticed that the isolated Gram positive bacterial strains presented an increased resistance to antibiotics from class of β-lactam, Penicillin subclass: Amoxycillin/Clavulanic Acid, Ampicillin and Penicillin, but also for antibiotics from Macrolides class (Erythromycin) or to antibiotics from Cyclic Peptide class (Colistin) (Fig. 4).

Some of the Gram positive isolated strains showed selective sensitivity for Enrofloxacin.

We also noticed selective resistance to antibiotics from Fluoroquinolones class (Florfenicol and Flumequine) from Aminoglycoside class (Kanamycin, Gentamicin and Lincospectin), Sulfonamides class (Trimethoprim with

Sulfamethoxazole) and Tetracycline class (Doxycycline and Tetracycline).


31

Fig. 4. Antibiotic sensitivity of Gram positive bacterial isolates from clinical cases of endometritis. AM- Amoxycillin/Clavulanic Acid; FUR- Ceftiofur; VA-Vancomycin; P-

Penicillin; AM- Ampicillin; OX- Oxacillin; FOX-Cefoxitin; CTX-Cefotaxime; E- Erythromycin; FF-Florfenicol; UB-Flumequine; EN- Enrofloxacin; K- Kanamycin;

CN-Gentamicin; LS-Lincospectin; CF- Cefoperazone; DO- Doxycycline; TE- Tetracycline; PB-Polymixin; C- Chloramphenicol; TS- Sulfadiazine/trimethoprim;

CT-Colistin. The resistance of these organisms to the commonly used antibiotics, viz.

Penicillin, Nitrofurantoin and Furazolidone may be attributed to large scale and indiscriminate use of these antibiotics over a long period of time (1, 8).

Conclusions

From the present study, we conclude that the endometritis in cattle are mainly caused by organisms belonging to family Enterobacteriaceae (Escherichia coli and Klebsiella spp.) and also by Staphylococcus spp., Coynebacterium and Streptoccocus spp.

From these results it seems that Enrofloxacin and Flumequine, as well as the antibiotics of β-lactam class, Cephalosporins subclass: Ceftiofur, Cefoxitime and Cefotaxime are very effective in treating endometritis in cows.


32

References

1. Arora, A.K., Singh, J., Pangaonka,r G.R., Nanda, A.S., Bacteriological studies on the genital tract in repeat breeder bovines. International Journal of Animal Science, 2000,15: 205-207.

2. Bhat, F. A., Bhattacharyya, H. K., Management of metritis in crossbred cattle of Kashmir using oxytetracycline, cephalexin and prostaglandin

F2ɑ. Indian Journal of Animal Research, 2012, 46(2), 187-189.

3. Kirby, W.M.M., Bauer, A.W., Sherris, J.C., Turck, N., Antibiotic susceptibility testing by a standardized single disc method. American Journal of Clinical Pathology, 1965,45: 493-496.

4. LeBlanc, S.J., Duffield, T.F., Leslie, K.E., Bateman, K.G., Keefe, G.P., Walton JS, Defining and diagnosing postpartumclinicalendometritis and its impact on reproductive performance in dairy cows. J Dairy Sci., 2002; 85, 2223–36.

5. Lee, J.I,. Kim, I.H., Pregnancy loss in dairy cows: the contributing factors, the effects on reproductive performance and the economic impact. J Vet Sci 2007, 8, 283–8.

6. Mohanty, B.C., Mohanty, B.N., Ray, S.K., Mohanty, D.N., Clinical and therapeutic studies of bovine endometritis. Indian Veterinary Journal, 1992,6, 379-380.

7. Potter, T.J., Guitian, J., Fishwick, O.J.G., Sheldon, M., Risk factors for clinical endometritis in postpartum dairy cattle. Theriogenology 2010, 74, 127–34.

8. Sheldon, I.M., Lewis, G.S., LeBlanc, S., Gilbert, R.O., Defining postpartum uterine disease in cattle. Theriogenology, 2006, 65, 1516–1530.

9. Sheldon, I.M., Noake,s D.E., Comparison of three treatments for bovine endometritis. Vet Rec ,1998,142, 575–9.

10. Shweta, S., Thesis submitted to College of Veterinary and Animal Sciences, CSK, M.V.Sc, Himachal Pradesh KrishiVishvavidyalaya, Palampur, India, 2003.

11. Singla, P, Singh, J., Sharma, N.S., Effects of post AI immunotherapy on dynamics uterine microflora and conception in sub-clinical endometritis. Proceedings of XVIIth Annual Convention and National Seminar on Fertility Management of Farm Animals under Adverse Agroclimatic conditions, Jodhpur, Oct. 6-8, 2001pp. 45.

12. Zahid, A., Culture and sensitivity of bacterial growth from exotic cows suffering from endometritis under Pakistani conditions, Pak Vet J, 2004; 24(2): 107-108.

13. http://ncipd.org/control/images/NCIPD_docs/CLSI_M100-S24.pdf


33

ABORTIONS IN SHEEP CAUSED BY THE ASSOCIATION OF TOXOPLASMA GONDII, CHLAMYDOPHILA ABORTUS AND

CAMPYLOBACTER spp. CASE STUDY

M. DANES

1, VIRGILIA POPA

1, G. ENE

3, E.M. CAPLAN

1, M. CULCESCU

1,

DANIELA BOTUS1, ANA DOBRINESCU

1, D. MILITARU

1, S. BARAITAREANU

2,

MARIA RODICA OTELEA2, DOINA DANES

2

1NS Pasteur Institute SA, 333 Giuleşti Street, Bucharest, Romania, 060269

2UASVM Bucharest 3SC Farmavet SA


Summary

At the beginning of the last cold season, in a lot of imported pregnant sheep, from specialized breeds for meat, abortions and calving of non-viable lambs were registered, with a rate of 25 % .The abortion age was find out on all the gestation ages. Laboratory testing by PCR / rPCR, bacteriological and physicochemical techniques revealed the concomitant presence of Toxoplasma gondii, Chlamydophila abortus and Campylobacter spp, and installation of metabolic acidosis. The results for Brucella ovis by ELISA, Brucella melitensis by rose Bengal, Coxiella burnetii and Schmallenberg virus by PCR and Leptospira spp. by MAT were negative.

Key words: Sheep, Toxoplasma gondii, Chlamydophila abortus, Campylobacter

spp, acidosis

Adapting the sheep to the intensive growth achieved by maintaining them

in semi-stabling and / or permanent stabling, accompanied by unilateral feeding, deficiencies in watering and housing, led to the emergence of some pathological conditions of great importance in pregnancy and the perinatal period. Abortion is usually the consequence of a vicious metabolism, because of a physiological overload of gestation, over which abortigenic infectious agents may arise (1, 5, 6, 7). Over the last decades, in Romania, in cases of abortion in sheep, were identified the following abortigenic agents: Salmonella Abortusovis (10-30% abortion, 25% mortality in newborn lambs); Chlamydophila abortus (3-30% abortion and premature calving); Campylobacter fetus (3-28% abortion and non-viable lambs and 0.5 - 4% complications after abortion); Listeria monocytogenes (0.5-8% abortion); Toxoplasma gondii (12% abortion and calving dead lambs); Brucella ovis (sporadic); Brucella melitensis (abortion and placental retention in low proportions); Coxiella burnetii (sporadic abortion but frequent complications after abortion or normal calving); Aspergillus fumigatus; Trueperella (Corynebacterium) pyogenes; Mannheimia haemolytica; Staphylococcus spp .; Streptococcus spp. (2.3). Some of


34

abortigenic infectious agents are subject to regulations of international trade of animals and animal products (7).

Recently, at the beginning of the cold season, in a lot of imported pregnant sheep, from specialized breeds for meat, abortions and calving of non-viable lambs were registered, with a rate of 25%. The abortions occurred at all ages of gestation.

Following the epidemiological survey, complex laboratory exams were performed, real time PCR were developed for detection of Toxoplasma gondii and the types of biological samples were established for the molecular detection tests.


The detection of mycotoxins (fumonisin, ochratoxin, zearalenone,

deoxynivalenol / vomitoxin, total aflatoxins) was performed on samples of pelleted feed specific for sheep, corn, silage, straw, lucerne mixed with chopped straw and dry lucerne.

The percentage of crude protein, the humidity, the percentage of fermentation acids (acetic acid, butyric acid, lactic acid), and the pH value were analyzed in the case of silage administred to pregnant sheep.

For drinking water there were determined the concentrations of nitrites and nitrates.

The blood samples from aborted sheep were biochemical analyzed for the concentration of glucose, uric acid, urea, total proteins, albumin, aspartate aminotransferase/glutamic oxaloacetic transaminase and alanine aminotransferase / glutamic pyruvic transaminase.

Bacteriological analyzes were carried out on samples of pericardial fluid, lung, heart, liver, brain, intestine, mesenteric lymph node and synovial fluid; specific media were inoculated with samples of liver and intestine in order to isolate Clostridium spp and Campylobacter spp.

The organ samples for molecular analyzes from aborted fetus or 7 days age lambs were represented by tissue (25 mg, or 10 mg for the spleen) or fingerprints of tissue harvested on sterile swabs with Dacron (Deltalab 300 263). The swabs were suspended in 1 ml phosphate buffered saline (PBS): 111.23 mM NaCl, 15.64 mM Na2HPO4 * 12H2O, 6.61 mM KH2PO4, pH 7.2 ± 0.2 (Pasteur I.), and 200 ul were used for the DNA extraction. The organ samples or organ fingerprints for Chlamydophila abortus detection were collected from the lung, pericardium, liver, spleen, kidneys, eyes, brain, thymus, placenta, synovial fluid. The organ fingerprint samples for Toxoplasma gondii detection were taken from the lung, pericardium, liver, brain, placenta.

Samples of brain tissue from four aborted sheep foetus were used for the detection of Schmallenberg virus.

Detection of Toxoplasma gondii was performed by three PCR assays: a classic one for hsp70 gene, and two others in real time PCR format with SYBR Green, for B1 gene and gra6 gene respectivelly; the primers sequences and


35

amplicons size were previously published (4). The thermal cyclers used were iCycler (Biorad), for the classic PCR, and Mx3005P (Agilent), for the real-time PCRs. The positive control was Toxoplasma gondii RH, ascitic fluid from a mouse experimentally infected in 2007 and stored at -20°C; 61.2 ng of DNA extract were used in the PCR classic and 1.22 pg in rPCR assays. Nuclease free water (PCR grade, Promega) was used as negative control. The primers concentration was 50 pmol each (hsp70) and 12.5 pmol each (B1, gra6). The final volume for both PCR reactions, classic and rPCR, was 25 ul. Amplification reagent, for classic PCR, was illustra Ready-To-Go RT PCR Beads (GE Healthcare 27-9267-01), and Brilliant QPCR Master Mix (Agilent 600828), for real time PCRs. QIAamp DNA Mini Kit (Qiagen 51306) was used for the DNA extractions, from tissues or liquids, according to the protocols recommended by the manufacturer; the lysis steps were made by the use of TS-100 thermo shaker (Biosan) and the DNA concentration of the obtained extracts was quantified by Quant-it ds DNA HS assay kit (0.2 -100 ng, Q31851, Life Technologies), and Qubit fluorometer (Life Technologies). From examined samples, 1 ul of DNA extract was used in conventional PCR reactions and respectivelly, 2 ul in rPCRs. The cycling program for Hsp70 gene was: 94

oC,

10 min., 1x; 94oC, 1 min + 55°C, 1.5 min. + 72°C, 2.5 min, 30x; 72°C, 10 min., 1x;

4°C, 10 min., 1x. The cycling program for B1 and gra6 genes was: 94oC, 10 min.,

1x; 94oC, 45 sec. + 55°C, 30 sec + 72°C, 45 sec, 50x; 72°C, 10 min + 25°C, 1 min,

1x; at the end of the amplification program, in order to confirm the obtained amplicons, the control by agarose gel electrophoresis TBE1.5x and the dissociation curve (25°C, 30 min, 1x; 95

oC, 1 min + 55

oC, 30 sec + 95

oC, 30 sec,

with data collection during the ramp from 55oC to 95

oC), were performed. The

interpretation of the rPCR results was based on the amplicon size, similar to those of positive control, and Tm values from dissociation curve within the range of ± 4Tm of positive control.

Detection of Chlamydophila abortus was made by LSI VetMAX Chlamydophila abortus CHLAB50 kit (Life Technologies), which is based on real-time amplification of a fragment of ompA gene and confirmation by Taqman probe. The DNA extraction was performed with the commercial kit QIAamp DNA Minikit (Qiagen 51306), according to the protocols recommended by the manufacturer, but with two finally elution steps in AE buffer. The quantification of DNA extracts was performed similar to those mentioned above. Amplifications were performed on Mx3005P (Agilent). The final volume of the reaction was 25ul, and from the extracts were analyzed 5 ul DNA per reaction. The cycling program was: 50

oC, 2 min, 1x;

95oC, 10 min, 1x; 95

oC, 15 sec + 60

oC, 1 min, 50x; 60

oC, 5 min + 25

oC, 1 min, 1x.

The positive control was the one from the kit, EPC (EPCCHLAB-005, FAM: 25 ± 3 Ct, VIC/HEX: No Ct). DNA extract from the Vero cell line (FAM: No Ct, VIC/HEX: 27 ± 3 Ct) was used as positive control for the internal control of the kit (CPI control). NTC negative controls: nuclease free water (PCR grade, Promega). FAM / HEX negative control: AE buffer (kit QIAamp DNA mini kit).


36

The results interpretation for the field samples was: positive, if Ct FAM<45, and Ct VIC <45; negative, if Ct FAM >45, and Ct VIC <45.

QIAamp cador Pathogen Mini Kit (Qiagen) was used for the RNA extraction, according to the manufacturer’s instructions.

Detection of Schmallenberg virus by real-time RT-PCR was performed in accord with the protocol described in virotype SBV RT-PCR Kit Handbook (Qiagen, Germany), on SmartCycler (Cepheid). Briefly, preparation of reaction mix was performed in 25 μL/sample (20 μL master mix + 5 μL sample), and the real-time RT-PCR protocol consisted in one cycle of 45°C - 10 min and one cycle of 95°C - 10 min, followed by 40 cycles of 95°C - 15 s, 56°C - 30 s, 72°C - 30 s. The reaction was valid: the positive controls had a Cq 27.4 and the negative control did not recover an amplification curve.

Serologic tests for Brucella ovis (ELISA), B. melitensis (Rose Bengal), Leptospira spp (MAT), and PCR for Coxiella burnetii were performed by other veterinary diagnostic laboratories (for fee).


The mycotoxins were present in concentrations below the levels allowed

for each category of analyzed feed (unpublished data); however, one can notice that DON was present in a concentration of 4.9 mg / kg in dry lucerne (the allowed limit is of 5 mg / kg). Samples of drinking water were within allowed limits regarding to the concentration of nitrites and nitrates (unpublished data). The silage recorded variations in the concentration of fermentation acids (the absence of lactic acid, compared to 1.4% which is permissible for an appropriate silage, a lower concentration, 0.16 versus allowed limit of 0.7 for acetic acid) and pH values higher than the normal ones for a good quality silage (6.19 versus <4.2). In the case of the sheep which had aborted, the values of blood glucose were within 20.7 and 28.2 mg / dL (reference range: 50-60 mg / dl), urea between 26 and 53 mg / dL (reference range 10-26 mg / dl), AST / GOT between 60 and 107 IU / L (reference range 20-60 IU / L) and the ALT / GPT between 17 and 35 IU / L (reference range 15-44 IU / L ). Hypoglycaemia and hypouremia, together with an increased activity of the AST / GOT led to the diagnosis of gestation toxemia. Glucose correction was made by the administration of polyethylene glycol in feed 20ml / day and animal for 10 days; then the administration of 100% glucose in feed 10 g / animal and day, for 9 days. After this, the levels of blood glucose ranged between 43.2 and 52.9 mg / dl. The uric acid ranged between 1.4 and 1.6 mg / dL and the blood urea recorded values between 50.4 and 52.8 mg / dl. The blood total proteins ranged between 5.3 and 6.2 mg / dL, and albumin between 1.2 and 2.4 g / l. AST / GOT values ranged between 33.2 and 58.2 IU / l, and the ALT / GPT between 6.9 and 7.1 IU / l. The results for B. melitensis, B. ovis, Leptospira spp and Coxiella burnetii were negative.


37

The results of the bacteriological and molecular tests for the presence of Toxoplasma gondii, Chlamydophila abortus and Schmallenberg virus are summarized in Table 1.

Table 1. Results of bacteriological and PCR tests for detection of Toxoplasma gondii, Chlamydophila abortus and Schmallenberg virus in aborted sheep foetus and

non-viable lamb samples

Sample of foetal organ / 7 days age

lamb

Bacteriological (n=46)

T. gondii (Dacron swabs, n=22)

C. abortus (n=31)

SBV (n=4) hsp70

(n=21) B1

(n=21) gra6 (n=4)

tissues (n=14)

Dacron swabs (n=17)

heart Negative ND ND ND ND ND ND

brain Negative Negative Negative ND ND Positive

(n=1) Negative

liver Clostridium spp.

(n=1) Positive

(n=3) Positive

(n=1) ND Negative Negative ND

intestine Campylobacter

spp. (n=2) ND ND ND ND ND ND

synovial fluid Negative ND ND ND Negative ND ND

mesenteric lymph node

Negative ND ND ND ND ND ND

eyes ND ND ND ND ND Negative ND

pericardium Negative Positive

(n=5) Positive

(n=5) Positive

(n=1) Negative

Positive (n=1)

ND

placenta ND Negative Negative ND Negative ND ND

lung Negative Positive

(n=2) Positive

(n=3) Positive

(n=1) Negative Negative ND

kidney ND ND ND ND Positive

(n=1) ND ND

spleen ND ND ND ND Positive

(n=1) Negative ND

tymus ND ND ND ND Negative ND ND

Total positive

3 (46) (6.5%)

10 (21) (47.6%)

9 (21) (42.9%)

2 (4) (50%)

2 (14) (14.3%)

2 (17) (11.8%)

0 (4) (0%)

ND - not determined


38

From a bacteriological point of view, 3 of 46 analyzed samples were positive: 2 abortions (Campylobacter spp, fig. 1) and 1 non-viable lamb (Clostridium spp).

Fig. 1. Sheep aborted fetus of twins gestation. Necrotic hepatitis foci (diam. 0.5

cm). Bacteriological test positive for Campylobacter spp. The samples analyzed for the presence of Schmallenberg virus were negative (fig. 2).

Fig. 2. rPCR for detection of Schmallenberg virus (commercial kit SBV virotype RT-PCR Kit, Qiagen). Amplification curve of the kit positive control. Software

SmartCycler (Cepheid) Toxoplasma gondii (fig.3, fig. 4) was present in about 50% of samples. The genetic tests for the detection of T. gondii applied to the same samples recorded however some discordances: gra6 generated positive results, while the two other tests were negative; similarly, in the case of some of the samples, hsp70 recorded positive results, opposite to B1 for which the same samples were negative. The appropriate organs for the detection of T. gondii proved to be the pericardial fluid, lungs and liver. The minimum appropriate concentration of DNA organ fingerprints


39

was found to be 2 ng / ul, both for hsp70 and gra6 or B1 gene-based amplification reactions.

Fig. 3. rPCR for detection of Toxoplasma gondii B1 gene. Samples of organ fingerprint from aborted sheep. Dissociation curve (Software MxPro QPCR v4.1D,

Agilent). Positive samples from abortions (Tm values in the range of ± 4 Tm positive control).

Fig. 4. Gel - electrophoresis confirmming rPCR for detection of Toxoplasma gondii, B1 gene. Organ samples from aborted sheep (lines 2-6). Samples of lung and

pericardium - rPCR positive (presence of 199 bp specific amplicons, B1gene). Line 8: positive control - Toxoplasma gondii RH; Line 1: negative control - NFW

(Promega); line 10: standard DNA - PCR Marker (Sigma P2993). Chlamydophila abortus (fig. 5) was present in 4 of 31 analyzed samples. The organs of choice for the detection of C. abortus were kidney and spleen. The minimum suitable concentration of DNA in the organ samples for the detection of C. abortus was shown to be of 20 ng / ul. Moreover, the DNA extracts from tissues


40

have been found suitable for the detection of C. abortus, and not the organ fingerprints.

Fig. 5. Gel - electrophoresis confirmming post- real time PCR for detection of Chlamydophila abortus, ompA gene (LSI VetMax CHLAB50 commercial kit, Life

Technologies). Samples of organ and organ fingerprint (lines 4-9; sample RU751-R, line 9, positive C.abortus). Line 11: positive control - EPC. Line 1: negative

control - NFW (Promega). Line 12: standard DNA PCR Marker (Sigma P2993). Lines 2.3 - negative controls Fam / Hex. Line 10 - positive control IPC (Hex: Vero)

The presence of Campylobacter spp was concomitant on the same animal (fetus) with T. gondii and / or C. abortus (unpublished data), as with T. gondii and C. abortus.

Conclusions

The data of chemical, biochemical, toxicological, bacteriological and

molecular investigations obtained during this study concluded to the following: 1. Abortions recorded in the imported herd of gestant sheep were due to a

polifactorial etiology. 2. The metabolic acidosis / dysmetabolic acetonaemia, hypoglicaemia, due

to deficiencies in nutrition and physiological overload of gestation, mainly in twin gestation, was found to be the main cause of abortions recorded in this case study.

3. Among the infectious abortigenic agents, there were mainly highlighted Toxoplasma gondii followed by Chlamydophila abortus and Campylobacter spp., individually or in combination on the same individual.

4. Investigations have revealed the status of "career" of the imported sheep and the need to improve the international trade control and expert system (TRACES).

5. The detection of T. gondii can be performed on the organ fingerprints collected by sterile swabs with Dacron.

Acknowledgements

Part of this study was granted by N.S. Pasteur Institute S.A / 2015 – 2016.


41

References

1. Avram, N., Hurduc, M., Popa, Vasilica, Boaru, N., Bratu, Mihaela, Garofil, D., Voicu, Georgeta, Sheep metabolic disorders and the health of the suckling lambs, Animal production - animal husbandry and veterinary medicine (only into Romanian), 1987, 7, 40–44.

2. Grigore, C., Prevention aspects of enzootic abortions in sheep, Journal of Livestock (only into Romanian), 1972, 5: 69–77.

3. Micu, Eleonora, Andrei, M., Aspects of enzootic abortion in sheep, Journal of Livestock (only into Romanian), 1976, 5, 58–59.

4. Militaru, D., Virgilia, Popa, Stirbu Teofanescu, Beatrice, Belteghi, C., Nica, Irina, Roman, Corina, Pastrama, F., Studies regarding the experimental infection and molecular detection of the Toxoplasma gondii genome, Scientifical Papers Veterinary Medicine, UASVM Timişoara, 2008, 41, 689-699, ISSN:1221-5295, http://www.usab-tm.ro/vol8MV/110_vol8.pdf.

5. Petroseanu, D., Darie, P., Abortions in sheep, Journal of Livestock (only into Romanian), 1981, 12, 34–37.

6. Pop, P., Roth, G., Falca, C., Stana, Letitia, Clinical observations, blood chemistry, and therapy in sheep gestation toxiemia, Journal of Livestock (only into Romanian), 1985, 7, 44–48.

7. *** World Organisation for Animal Health: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 2015, Chapter 2.1.9, Leptospirosis; chapter 2.1.12, Q fever; chapter 2.7.7, Enzootic abortion of ewes (ovine chlamydiosis); chapter 2.7.9, Ovine epididymitis (Brucella ovis); chapter 2.9.10, Toxoplasmosis, http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/ , accessed 04/13/2016.


42

DISTRIBUTION AND ANTIMICROBIAL SUSCEPTIBILITY OF COAGULASE-NEGATIVE STAPHYLOCOCCI ISOLATED FROM

BOVINE MASTITIC MILK – PRELIMINARY STUDY

J. DÉGI, IONICA IANCU, CORINA PASCU

Banat’s University of Agricultural Sciences and Veterinary Medicine ”King Michael I of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Aradului

Street No. 119, Timisoara, Romania E-mail: [email protected]

Summary

Growth and the placing on the market of products of animal origin constitute an important source of income for the farming population. The rational development of this sector is to take place through the development of the veterinary actions aimed at raising the level of public health in the European Union. The protection of human health against diseases and infections directly or indirectly transmissible between animals and man (zoonosis) is of a major importance.

The zoonosis which exists in the phase of primary production must be the subject of a proper control in order to guarantee the achievement of the objectives of the launched by the Community rules. However, in the case of primary production at the origin of the direct supply to the final consumer or trade locally, it is necessary to protect public health. Zoonoses present at the level of primary production must be subject to appropriate supervision to ensure the objectives launched by Community regulations. However, if primary production to the direct supply to the final consumer or to local shops, it is necessary to protect public health in accordance with local veterinary.

Given the economic losses recorded in a farm dairy, domestic type, in Timis county, because of subclinical mastitis, research of this work were directed towards identifying and characterizing coagulase negative staphylococci involved in the etiology of cows mastitis, respectively determination of antibiotic susceptibility. In mastitic milk samples were isolated and identified six species of coagulase-negative staphylococci (S. xylosus, S. epidermidis, S. haemolyticus, S. chromogenes, S. sciuri, S. saprophyticus) in a ratio of 37.38%.

The predominant species of coagulase-negative staphylococci isolated from samples of milk were S. xylosus, S. epidermidis or S. haemolyticus. After testing

staphylococci strains isolated from cows CN mastitis, to antibiotics were identified methicillin-resistant strains and more type of resistance to β-lactams, tetracyclines, macrolides and polymyxin B .

Key words: staphylococi, bovine, mastitis, milk

Mastitis are considered inflammatory processes of the mammary gland of

cattle that produce physicochemical changes, pathological, bacteriological, of glandular tissue and milk. Mastitis is the most costly disease of dairy cows found in its growth (2,10).


43

Growing and marketing of products of animal origin constitute an important source of income for farmers. Rational development of this sector takes place through the development of veterinary measures aimed at raising the level of public health in the European Union. Protection of human health against diseases and infections directly or indirectly transmissible between animals and humans (zoonoses) is of paramount importance (2).

Zoonoses present at the level of primary production must be subject to appropriate supervision to ensure the objectives launched by Community regulations. However, if primary production to the direct supply to the final consumer or to local shops, it is necessary to protect public health in accordance with local veterinary (10, 12).

There is in this case a close relationship between producer and consumer. This production does not have significant effects on the average prevalence of zoonoses in animal populations. The general requirements for sampling and analysis may not be practical or appropriate for producers with very small numbers of animals that are located in regions suffering from special geographical constraints (2, 10, 11, 12).

Given the economic losses recorded in a farm dairy, domestic type, in Timis county, because of subclinical mastitis, research of this work were directed towards identifying and characterizing coagulase negative staphylococci involved in the etiology of mastitis in cows, respectively determination of antibiotic susceptibility.


In this study, a dairy farm in Timis County, with a herd of 35 cows were

conducted indirect tests for diagnosing clinical mastitis and subclinical, using R-Mastitest.

The experiment was conducted over a period of 3 months in the period March-May 2015 were collected 65 samples of milk. All samples were submitted for examination with indirect tests for detecting mastitis (Califormia Mastitis Test). From indirect tests positive cases, were collected 45 milk samples, which were processed for bacteriological exams and antibiotics sensitivity test. Following the results obtained was instituted antibiotic treatment.

The farm is situated in the Izvin, Timis County by arranging a cow barn in the household. Feeding is done manually (fibrous and concentrates) from a single feeding. Missing cows silage rations. Drinking is the constant drinkers. Garbage disposal is done manually, from a platform located about 50 meters from the stall. Milking system is to bottle the milk is collected in a cooling tank located in a room at the end of the barn. Meeting the cooling tank is located comply with hygiene and veterinary regulations.

Antisepsis before milking of the mammary gland consists of washing of breast, wiping with paper and application the P3-Oxyfoam product (manufactured


44

by Ecolab) and after milking using the product P3-Io-Shield (Ecolab). To clean the milking machines and cooling tank using P3-Chlorasept product.

For bacteriological samples of milk were processed in the laboratory research in bacterial infectious diseases from the Department of Infectious Diseases and Preventive Medicine in the Faculty of Veterinary Medicine Timisoara.

Identification of bacterial agents from milk samples collected was only possible if they were isolated in pure culture. Subsequently, it switched to the study of the morphological and biochemical isolated bacterial agents. Therefore, bacteriological samples of milk collected included the following steps:

sampling of milk;

bacterioscopic exams (direct examination of milk samples);

isolation of bacterial agents in pure culture;

typing isolated bacterial agents by establishing morphological characters, cultural and biochemical.

In order to have accurate results from bacteriological point of view, milk samples were collected with special care so that they are less exposed to contamination with bacteria from the environment, as follows:

udder and teats were washed and dry well with paper towels;

in which the test tubes were collected milk samples were sterilized in advance. After closing containers have passed each of the sample number;

at harvest, containers were kept in almost horizontal position to avoid any contamination of milk with impurities from the skin or hair fallen cow examined.

Normally, it is recommended that the sampling of the milk in order to achieve bacteriological examinations to be carried out after the removal of the first streams of the teat channel, in which germs can penetrate from the outside.

It is also important to remember that processing of milk samples is very important time elapsed since the time of harvest until bacteriological examinations.

For a time less than 6 hours, milk samples can be processed immediately. If the time exceeds 6 hours, refrigeration is required, namely the samples

of milk to be transported in refrigerated boxes, but in this case it does not exceed 24 hours and examined.

For bacteriological, keeping milk samples refrigerated more than 24-48 hours, allows multiplication and numerical growth of other microorganisms (psychrophilic) than those considered causal etiological agent.

They were studied to identify the etiologic agents of mastitis only crop monocultures or those who had more than two types of colonies, one of which predominates.

Purification of bacterial etiologic agent was carried out by a single colony bedding, with the aid of a bacteriological loop, and passing it on a selective medium specific for the type of bacterial partially identified.

Later, after the partial identification and purification was passed typing isolated bacterial agents by determining metabolic or biochemical characteristics.


45

Initially, for each culture were studied two types of reactions or assays, which shows the presence of oxidizing or reducing enzymes, oxidase and catalase respectively. These assays distinguish indirectly genera Staphylococcus spp. (which are catalazo positive and oxidazo negative, with the exception of Staphylococcus aureus subsp anaerobius), Streptococcus spp. (which are oxidazo- and catalase-negative) and Micrococcus spp. (which are oxidazo- and catalase-positive).

For taxonomic classification of bacteria isolated from milk tests were used API Staph, products of BioMerieux SA of France.

Antibiotic susceptibility testing Strains of CN staphylococci isolated tested for sensitivity to antibiotics, was

used Kirby-Bauer disc diffusion method. The following antibiotics were used: ampicillin with sulbactan, amoxycillin with clavulanic acid, tetracycline, doxycycline, gentamicin, kanamycin, erythromycin, vancomycin, ciprofloxacin, polymyxin B, novobiocin, rifampicin, pristinamycin, lincomycin, ceftrioxone, cefaxitrizine and cefaclor. By susceptibility testing of staphylococci strains isolated from mastitis milk, it was watched multiple antibiotic sensitivity determination, namely methicillin.


Of all isolates in this study were within the Staphylococcus genus

commonly identified species of coagulase-positive in all 28 strains (62.22%, 28/45) and 14 strains of S. aureus (16%) 8 strains of S. intermedius (12%) and 6 strains of S. hycus (4%). 17 strains were isolated (37.38%, 17/45), which were identified and classified in six species of coagulase-negative, namely: five strains of S. xylosus (20%), four strains of S. epidermidis (16%), 3 strains of S. haemolyticus (12%), 2 strains of S. chromogenes (8%) and 2 strains of S. sciuri (8%), and a strain of S. saprophyticus (4%) .

S. xylosus was the species most commonly isolated (5 strains - 20%) of all staphylococcal isolates (25 strains). Of the 25 staphylococcal strains isolated CNS have frequencies between 4.0 to 20% containment.

CNS of the 17 strains isolated from mastitis cows, the species most frequently isolated were S. xylosus (20%), followed by another 5 species: S. epidermidis (16.0%), S. haemolyticus (12%), S. sciuri respectively S. chromogenes (both with and S. saprophyticus 8 (4%).


46

Table 1 Frequency isolation of CNS in mastitic milk

CN staphylococci species Nr. of

samples/pos sitive for CMT

Positive samples for CNS (no. of isolates)

Nr. % S. xylosus 65/45 5 11.12

Staphylococcus epidermidis 65/45 4 8.89 Staphylococcus

haemolyticus 65/45 3 6.67

Staphylococcus chromogenes

65/45 2 4.45

Staphylococcus sciuri 65/45 2 4.45 Staphylococcus saprophyticus

65/45 1 2.23

Total 65/45 17 37.38

Coagulase-negative staphylococci have been isolated for two cases of

clinical mastitis, in association with coagulase-positive staphylococci. Subclinical mastitis on evolving were coagulase-negative species isolated only by staphylococci.

Bacteriological examination on sheep blood agar 5%, coagulase-negative staphylococci conducted variable haemolysis depending on bacterial species as follows: α haemolysis type, S. chromogenes and S. haemolyticus; haemolysis type β, S. xylosus, S. sciuri; absence of hemolysis to S. epidermidis, S. saprophyticus and some strains of S. xylosus, S. sciuri and S. chromogenes.

Reaction of CNS on hiperclorurat Chapmann agar, varied thus being observed the following reactions: positive reaction to the species S. xylosus (late), S. sciuri (late), S. chromogenes negative reaction species, S. epidermidis and variable reaction the species S. haemolyticus and S. saprophyticus.

Results of antibiotics susceptibility test Regarding behavioral testing to antimicrobials, CN staphylococci presented

the greatest sensitivity to ciprofloxacin, novobiocin, vancomycin, ceftriaxone, cefoxitim, rifampicin, cefaclor, pristinamycin and ampicillin/sulbactan. Staphylococcal strains were sensitive, in varying proportions, to: gentamicin, tetracycline, kanamycin, erythromycin, doxycycline and lincomycin. All tested of CNS strains have been shown to be resistant to polymyxin B. In the case of the staphylococcal strains tested, there was resistance to several groups of antibiotics used to treat mastitis in cattle: β-lactams antibiotics (methicillin), aminoglycosides (gentamicin, kanamycin), macrolides (erythromycin) and tetracyclines (tetracycline, doxycycline). It was noted that the phenomenon of multiple antimicrobial resistance test. In the literature, the multiple antibiotic resistance is defined as the same strains of bacterial resistance to 4 or more antibiotics.


47

Table 2 Test results of antimicrobial sensitivity to CNS

Antimicrobial name (Minimum inhibitory

concentration)

No. of sensitive isolates

S. xylosus (n=5)

S. epidermidis

(n=4)

S. haemolyticus

(n=3)

S. sciuri (n=2)

S. chromogenes

(n=2)

S. saprophyticus

(n=1) Methicillin - ME - 30

µg 5 3 2 2 2 1

Gentamicin - CN - 10 µg

2 2 2 1 2 0

Tetracycline - TE – 30 µg

1 2 1 1 1 0

Ciprofloxacin - CIP - 30 µg

5 4 3 2 2 1

Kanamycin - K - 30 µg

2 1 1 1 1 1

Novobiocin - NV - 30 µg

5 4 3 2 2 1

Doxycycline - DO - 30 µg

3 2 1 1 2 0

Erythromycin - E - 15 µg

2 2 1 2 1 0

Vancomycin - VA - 30 µg

5 4 3 2 2 1

Ceftriaxone - CRO - 30 µg

5 4 3 2 2 1

Cefoxitin - FOX - 10 µg

5 4 3 2 2 1

Polymyxin - PB - 50 IU

0 0 0 0 0 0

Rifampicin - RA - 30 µg

5 4 3 2 2 1

Lincomycin - L - 30 µg

4 2 1 1 1 0

Cefaclor - CEC - 30 µg

5 4 3 2 2 1

Pristinamycin - PT - 15 µg

5 4 3 2 2 1

Ampicillin/sulbactan - SAM - 30 µg

5 4 3 2 2 1

Table 3

Resistance models of coagulase-negative staphylococci isolated from mastitis milk

CN staphylococci species Models of antibiotics resistance (no. of isolates)

S. xylosus CN (3), TE (4), K (3), DO (2), E (3), PB (5), L (1) Staphylococcus epidermidis TE (2), CN (2), L (2), K (3), ME (1)

Staphylococcus haemolyticus TE (1), K (1), E (1), PB (3), ME (1) Staphylococcus chromogenes TE (1), E (1), L (1), K (1), PB (2)

Staphylococcus sciuri TE (1), CN (1), K (1), DO (1), PB (2), L (1) Staphylococcus saprophyticus PB (1), CN (1), TE (1), DO (1), E (1), L (1)


48

Analyzing the results of the table we can see that antibiotics sensibility was variable depending on the group of antibiotics.

If antibiotics: novobiocin, rifampicin, pristinamycin, ciprofloxacin, vancomycin, ceftriaxone, cefoxitin, cefaclor and ampicillin/sulbactan, the number of sensitive strains were 100%, all isolates were sensitive (Table 2). This suggests that the tested strains isolated from mastitic milk to which these antibiotics were not used.

Against used β-lactams (methicillin, ceftriaxone, cefoxitin, cefaclor, ampicillin with sulbactan) antibotics sensitivity was total, except Staphylococcus aureus, which were isolated two resistant strains. Of these methicillin-resistant strain of S. epidermidis and S. haemolyticus one (Table 2 and 3). The strains tested were mostly sensitive to other β-lactams, as a result of previous treatments done correctly.

The phenomenon of antibiotic resistance, β-lactams in the case, is based on genetic determinants of plasmid and chromosomal type, β-lactamases governing synthesis, broad-spectrum, ensuring the resistance. Resistance to methicillin is transmitted by plasmids (R factor) and having a pattern common to other β-lactams. For this reason methicillin-resistant staphylococcal strains are considered particularly zoonotic risk, strains having a complex circuit or human-animal-human (4, 15).

Compared to aminoglycosides (gentamicin, kanamycin), and macrolides (erythromycin and vancomycin), antibiosensibilitatea was different, being maximum to vancomycin (Tables 2 and 3). In the case of gentamicin were isolated 8 strains resistant to kanamycin and 10 strains respectively nine strains resistant to erythromycin (Table 2 and 3).

Most of the strains were resistant to polymyxin B (13 strains), through the use of topically applied preparations containing this antibiotic (Table 3).

Antibiosensibilitatea to tetracyclines (tetracycline, doxycycline) was reduced 19 strains being resistant to this group of antibiotics to which resistance phenomenon is type plasmid and chromosomal (11 strains tetracycline and 8 strains to doxycycline) (Tables 2, 3).

All tested strains were susceptible to ciprofloxacin, because this quinolone drug therapy is used in dairy cows.

The development of resistance staphylococci to different antibiotics, it is a consequence of wasteful use in the treatment of mastitis in cows. Antibiotics used irrationally creates a selective pressure being selected and transmitted genetic determinants of type plasmid and chromosomally. Consequently, the phenomenon of multiple resistance intra- and interspecific that is transmitted. Methicillin resistance of special importance as it can be associated with resistance to β-lactams and other antibiotic groups (7).

After testing staphylococci strains isolated from cows CN mastitis, against 17 antibiotics were identified methicillin-resistant strains and more rezistotipuri, β-lactams to, tetracyclines, macrolides and polymyxin B.


49

The data on methicillin resistance and rezistotipurile identified are similar to the results communicated by other authors on the phenomenon of antibiotic resistance (1, 6, 9, 15, 16, 17).

Data obtained from this study are important in terms of knowledge on CNS involved in mastitis cows, respectively phenomenon of resistance in a bacterial isolates. These data are consistent with the data reported in the literature.

Thus Pyorala and Taponen (10) in a study on the role of CNS, emerging pathogens of mastitis in cows, they concluded that these bacteria are common pathogens of mastitis in many countries, that these infections remain in stage subclinical or manifest clinical signs deleted. These issues lead to persistent infections, the number of somatic cells in milk, but also affecting the quality of milk production decrease possibility. All this has an economic impact by restricting the use in industry. The predominant species in mastitis cows are S. simulans and S. chromogenes.

Jake EL et al. (4) in another study carried out in Egypt, on 884 milk samples from cows, buffaloes, sheep and goats, he has described a frequency of isolation of SNA these milk samples from cases of subclinical mastitis: 16.6%, 59.4%, 50% and 55.6% respectively. Among the species isolated were Staphylococcus xylosus, Staphylococcus cohnii, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus chromogenes, Staphylococcus lentus, Staphylococcus simulans and Staphylococcus lugdunensis.

HOSSEINZADEH and HIS DASTMALCHI (8) in a study conducted in the provinces of East and West Azerbaijan, Iran, isolated 108 strains of CNS in 158 mastitic milk samples. They identified nine different species of CN stafilcoci: 44 Staphylococcus haemolyticus (40.7%), 17 Staphylococcus chromogenes (15.7%), 11 Staphylococcus epidermidis, Staphylococcus warneri and Staphylococcus cohnii (10.2%), 6 Staphylococcus simulans (5.5%), 4 Staphylococcus hominis (3.7%), 3 Staphylococcus capitis (2.7%) and 1 Staphylococcus xylosus (0.9%). S. haemolyticus, S. warneri and S. chromogenes were isolated and clinical mastitis.

In 2010, a study by Brinda et al. (3) identified 10 species of coagulase-negative staphylococci in mastitic milk samples. The predominant species were S. xylosus, S. chromogenes, S. lentus and S. sciuri.

Conclusions

In mastitic milk samples were isolated and identified six species of coagulase-negative staphylococci (S. xylosus, S. epidermidis, S. haemolyticus, S. chromogenes, S. sciuri, S. saprophyticus) in a ratio of 37.38%.

The predominant species of coagulase-negative staphylococci isolated from samples of milk were S. xylosus, S. epidermidis or S. haemolyticus.

Of milk from cows with subclinical mastitis were coagulase-negative species isolated only by staphylococci.


50

After testing staphylococci strains isolated from cows CN mastitis, to antibiotics were identified methicillin-resistant strains and more type of resistance to β-lactams, tetracyclines, macrolides and polymyxin B.

Acknowledgements



References

1. Anaya-Lopez, J.L., Contreras-Guzmân, O.E., Cârabez-Trejo, A.,

Baizabal-Aguirre, V.M., Lopez-Meza, J.E., Valdez-Alarcon, J.J., Ochoa-Zarzosa, A., Invasive potential of bacterial isolates associated with subclinical bovine mastitis, Res. Vet. Sci., 2006, 81, 358-361.

2. Bochniarz, M.W., Szczubial, W.M., Coagulase-negative staphylococci (CNS) as an aetiological factor of mastitis in cows, Polish Journal of Veterinary Sciences, 2010, 16(3), 487–492.

3. Brinda, M., Herman, V., Fodor, I., Phenotypic characterization of coagulase-negative staphylococci isolated from mastitic milk cows, Lucrari Stiinifice Medicina Veterinara Timişoara, 2010, 53(1), 97-101.

4. El-Jakee, J.K., Aref, N.E., Gomaa, A., El-Hariri, M.D., Galal, H.M., Omar, S.A., Samir, A., Emerging of coagulase negative staphylococci as a cause of mastitis in dairy animals: An environmental hazard, International Journal of Veterinary Science and Medicine, 2013, 1:74–78.

5. Feßler, Andrea T., Billerbeck, Carmen, Kadlec, Kristina, Schwarz, S. Identification and characterization of methicillin-resistant coagulase-negative staphylococci from bovine mastitis, J Antimicrob Chemother, 2010, 65: 1576 –1582.

6. Garcia-Migura, L., Hendriksen, R.S., Fraile, L., Aarestrup, F.M., Antimicrobial resistance of zoonotic and commensal bacteria in Europe: The missing link between consumption and resistance in veterinary medicine, Veterinary Microbiology, 2014, 170:1–9.

7. Hameed, K.G.A., Sender, G., Korwin-Kossakowska, A., Public health hazard due to mastitis in dairy cows, Animal Science Papers and Reports, 2007, 25, 2, 73-85.

8. Hosseinzadeh, S., Dastmalchi Saei, H. Staphylococcal species associated with bovine mastitis in the North West of Iran: Emerging of coagulase-negative staphylococci, International Journal of Veterinary Science and Medicine, 2014, 2, 27–34.


51

9. Özkan, Aslantaş, Mehmet Ali Yilmaz, Ebru Şebnem Yilmaz, Cemil Kurekci, Antimicrobial susceptibility pattern and SCCmec types of methicillin-resistant coagulase-negative staphylococci from subclinical bovine mastitis in Hatay, Turkey, Bull Vet Inst Pulawy, 2014, 58, 563-566.

10. Pyorala, S., Taponen, S., Coagulase-negative staphylococci-Emerging mastitis pathogens, Veterinary Microbiology, 2009, 134:3–8.

11. Pyorala, S., Taponen, Suvi, Coagulase-negative staphylococci as cause of bovine mastitis—Not so different from Staphylococcus aureus?, Veterinary Microbiology, 2009, 134:29–36.

12. Schukken, Y.H., Gonzalez, R.N., Tikofsky, Linda L., Schulte, H.F., Santisteban, C.G., Welcome, F.L., Bennett, G.J., Zurakowski, M.J., Zadoks, Ruth N., CNS mastitis: Nothing to worry about?, Veterinary Microbiology, 2009, 134:9–14.

13. Silva, Nathalia C.C., Felipe, F.G., De P. Manzi, Marcela, Gomez-Sanz, Elena, Gomez, Paula, Araujo-Junior, J.P., Langoni, H., Rall, Vera L. M., Torres, Carmen, Characterization of methicillin-resistant coagulase-negative staphylococci in milk from cows with mastitis in Brazil, 2014, Antonie van Leeuwenhoek, 106:227–233.

14. Soares, Lidiane C., Pereira, Ingrid A., Pribul, B.R., Oliva, M.S., Coelho. S.M.O., Souza, Miliane M.S., Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis, Pesq. Vet. Bras., 2012, 32(8):692-696.

15. Supre, K., K. De Meulemeester, L.L., Antimicrobial susceptibility and distribution of inhibition zone diameters of bovine mastitis pathogens in Flanders, Belgium, Veterinary Microbiology, 2012, 171:374–381.

16. Veera Gindonis, Suvi Taponen, Anna-Liisa Myllyniemi, Satu Pyörälä, Suvi Nykäsenoja, Saara Salmenlinna, Laura Lindholm, Merja Rantala, Occurrence and characterization of methicillin-resistant staphylococci from bovine mastitis milk samples in Finland, Acta Veterinaria Scandinavica, 2013, 55:61.

17. Vishnupriya, S., Antony, P.X., Mukhopadhyay, H.K., Pillai, R.M., Thanislass, J., Vivek Srinivas, V.M., Sumanth Kumar, R., Methicillin resistant staphylococci associated with bovine mastitis and their zoonotic importance, Veterinary World, 2014, 7(6): 422-427.


52

THE SEROPREVALENCE OF PRRS SYNDROME IN YOUNG SWINE INTENDED FOR FATTENING

L. FLUERAȘU

1, A. STANCU

1, IULIA BUCUR

1, D. MITRICĂ

1, VIRGILIA POPA

2

1Banat’s University of Agricultural Sciences and Veterinary Medicine ”King Michael I of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Aradului

Street No. 119, Timisoara, Romania 2NS Pasteur Institute SA


Summary

The PRRS syndrome has rapidly expanded in intensive rearing pigs farm, where is producing significant economic losses. The researches followed the seroprevalence of this syndrome in 7 in young swine farms after weaning. There were prelevate blood samples from 20 pigs, at 2.5 youth -3 months of age (R1) and after an interval of 2 months (R2).

PRRS antibodies were detected with INGEZIM PRRS 2.0 1.1.PR2.K.1. kit and

the results have been processed and graphically presented. PRRS antibodies were detected in six farms, youth A swine farm being negative on both tests.

At young swine aged 2.5-3 months seroprevalence was between 27.37% and 100% and R2 seroprevalence was between 51.23% and 100%. The dynamic evolution of this indicator proves the existence of the phenomenon of seroconversion corresponding controlled expansion of the disease in flocks.

Key words: PRRS, seroconversion, antibodies

PRRS is a dominant component of infectious pathology of pigs reared intensively. After reporting this disease in the USA and Europe, followed a rapid expansion and currently endemic disease progresses, is known worldwide (5,7).

The existence of predisposing intrinsic and extrinsic factors favoring development and expansion of this syndrome, and the economic losses produced by it are significant (5,7).

Control of the PRRS are based on general and specific measures, serological surveillence is recommended to follow the frequency in swine farms (2,6).

The research was conducted in seven farms, of youth swine growth, being monitorizated the seroprevalence of the disease, on youth. The presence of specific antibodies was detected at age 2.5 - 3 months and 5.5 to 6 months.


For the detection of specific antibodies were taken 25 blood samples from the jugular confluence of youth swine randomly as follows:

- The collection I-a (R1) at the age of 2.5 to 3 months;


53

- An second collection (R2) at the age of 5.5-6 months. The sera were decanted after expression and individually labeled, in

Eppendorf tubes, being kept in the freezer. Specific antibodies were detected by immunoassay test kit using INGEZIM

PRRS 2.0 1.1.PR2.K.1. This technique is based on indirect variant which allows the detection of antibodies induced by antigen present at ORF 7 both, European and American strains (8).

Results are expressed as a ratio of positivity (S / P) calculated as follows: - Positive sample (S / P≥40) - Negative sample (S / P <40).


Serological examination confirmed the evolution of PRRS syndrome in monitorizated farms and established its seroprevalence. The results were processed and summarized in table 1, being observed the dynamic evolution of geometric mean titers of specific antibodies and frequency of positive samples, depending on the age at which the samples were collected.

Analyzing these results it is observed that PRRS syndrome was confirmed serologically in six farms with young swine of different ages and, in farm A were negative results on both tests.

In young swine monitored the seroprevalence of PRRS syndrome, at the age of 2.5 to 3 months was variable, the frequency of positive samples being between 23.41% and 100%. At the age of 5.5-6 months increased frequency of positive samples being between 33.36% and 100%.

In farms B and E all samples examined were positive, regardless of the age at which young swine were collected.

The results obtained demonstrate the existence of an immune response after infection, in dynamics, highlighted by growing values of the geometric mean titers of specific antibodies based on the age at which young swine sampling was performed.

The phenomenon of seroconversion and increased numbers of positive samples demonstrate the expansion of the PRRS syndrome at young swine as a result of horizontal and vertical transmission of the infection. Through these transmission mechanisms syndrome PRRS is maintained in farms with closed circuit, this being evidenced by epidemiological researches.

In Romania, Stănuică D. et al., have been performed serological studies in primary PRRS outbreaks and they have found that prior to the occurrence of disease, serum samples taken from both sows and piglets of groups were negative, but after the appearance of disease as a result of seroconversion 75.8% of samples from sows and 92.5% of samples of weaned piglets were positive (6).

Maes. D. et al. in 1997, conducted an seroepidemiological investigation in 50 farms for fattening pigs frequency PRRS and found that 96% of control animals


54

were serologically positive demonstrating the spread of the disease in young swine for fattening (3).

Mejia Silva, W., et al. in 2012, investigated 16 farms pigs using serological immunoassay test, for the presence of PRRS. The quote authors found that 50% of farms were investigated serologically positive to the disease (4).

Results provided by the serological study, in swine effective youth, are consistent with the results quoted authors, proving extent of the disease in swine herds and the existence of the phenomenon of seroconversion.

In control of PRRS syndrome seroprevalence syndrome by serological cross contributes to the development of programs on limiting spread of the disease in swine farms.

Establishing seroprevalence by cross serological investigation, contributes to the development of programs on limiting spread of the disease in swine farms (1,2,3).

Table 1

The results of the serologic exam

No FARMS POSITIVE SAMPLES MG

R1 R2 R1 R2

1 A - - - -

2 B 100% 100% 2.95 3.51

3 C 27.26% 36.35% 2.75 3.72

4 D 30.00% 33.36% 2.90 3.82

5 E 100% 100% 3.12 3.85

6 F 41.15% 56.23% 2.85 3.79

7 G 23.41% 46.14% 2.90 3.61

Conclusion

In investigated farms the development of the PRRS syndrome was

confirmed on youth swine monitored; Serological examination revealed the seroconversion phenomenon,

characteristic to active outbreaks of this syndrome, at both youth and sows. Serological examination performed by immunoassay test can be used as a

diagnostic and as a method of epidemio, of PRRS syndrome in swine farms.

Acknowledgements

This research work was carried out with the support of the project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR 2669.


55

References

1. Bierk, M. D., Dee, S. A., Rossow, K. D., - Diagnostic investigation of chronic porcine reproductive and respiratory syndrome virus in a breeding herd of pigs, Vet.Rec., 2001,148, 687-690.

2. Dewey,C., Melnichouk, O., Friendship, R., Hayden, D., Seroepidemiological study of PRRS infection patterns in nursery pigs, in Proceedings vol.1 International Pig Veterinary Society 18th Congres, Hamburg, Germany, 27 iunie-1 iulie, 32, 2004.

3. Maes, D., Descriptive epidemiological aspects of the seroprevalence of five respiratory disease agents in slaughter pigs from fattenings herds, Epidemiol Sante Anim, 1997, 31-32.05.B.19.

4. Meji aSilva, W., Derwin, Calatayud, Denice, Zapata, Armando, Quintero Moreno, Paola, Torres, Miguel, Chango, Seroprevalence of Aujeszky Disease and Porcine Reproductive and Respiratory Sindrome (PRRS) in Pig Farms Located in the Municipality of Mauroa, Falcon State, Veterinary Bulletin, 2012, 82, (9), p. 1132.

5. Stănuică, D., Sindromul de Reproducţie şi Respirator Porcin, Lucrare realizată în cadrul Proiectului „Sprijinirea Serviciilor din Agricultură”, 2005.

6. Stănuică, D., Pancă, C., Caraivan, I., Draghici, D., Investigaţii seroepidemiologice într-un focar de PRRS (Sindromul Reproductiv şi Respirator Porcin) Lucr. Șt., Med.Vet., Timişoara, 1999, Vol XXXII, p.115-130.

7. Zimmerman, J. J., Benfield, D. A., Scott, A. D., Murtaugh, M. P. Stadejek, T., Stevenson, W. G., Torremorell M., Porcine reproductive and respiratory syndrome virus (Porcine arterivirus) in Disease of swine edited by Zimmerman J.J. 10 th edition, Wiley-Blackwell, 2012.

8. ***http://www.ingenasa.eu/index.php?op=vn&nid=24.

https://www.researchgate.net/researcher/2009050694_Derwin_Calatayud

https://www.researchgate.net/researcher/2009049310_Denice_Zapata

https://www.researchgate.net/researcher/2009043378_Armando_Quintero_Moreno

https://www.researchgate.net/researcher/2009043378_Armando_Quintero_Moreno

https://www.researchgate.net/researcher/2009061115_Paola_Torres

https://www.researchgate.net/researcher/2009060891_Miguel_Chango

https://www.researchgate.net/publication/287085591_Seroprevalence_of_Aujeszky_Disease_and_Porcine_Reproductive_and_Respiratory_Sindrome_PRRS_in_Pig_Farms_Located_in_the_Municipality_of_Mauroa_Falcon_State




56

PREVALENCE AND ETIOLOGY OF SUBCLINICAL MASTITIS IN DAIRY BUFFALOES VERSUS DAIRY COWS FROM

TRANSYLVANIA, ROMANIA

R.M. GIUPANĂ, C.G. CERBU, A. VASIU, SILVANA POPESCU, CARMEN DANA ŞANDRU, MIHAELA NICULAE, MARINA SPÎNU

University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca

400372, 3-5 Mănăştur Street, Cluj-Napoca, Romania [email protected]

Summary

The prevalence of clinical mastitis in buffaloes and cows is considered similar by the literature ranging from 8 to 40% and 19.9% to 44.8%, respectively. The percentage is highly variable depending on the region and raising technology. Nevertheless, not much is known about subclinical mastitis agents in buffaloes in Romania. The aim of the study was to comparatively evaluate the prevalence and etiology of mastitis in dairy buffaloes and cows from Transylvania. Additionally, antimicrobial susceptibility profile of microorganisms isolated from milk samples was tested. The research was carried out on a group of buffaloes (n=108) and a group of Romanian Spotted and Red Holstein cows (n= 211). After the physical examination of the mammary gland, R. Mastitest was performed to diagnose subclinical mastitis. Subsequently, a total number of 56 milk samples from the buffaloes, and 28 milk samples from the dairy cows were collected. Classical microbiological methods were used to cultivate the bacteria from the milk samples and the Kirby-Bauer disk diffusion technique was performed to evaluate the sensitivity/resistance to antibiotics commonly used to treat the disease, such as ampicillin, amoxicillin, enrofloxacin, cloxacillin and oxytetracyclin. Antibiotic resistant or highly resistant staphylococci were encountered in almost all milk samples in bovine mastitis (90%) compared to the buffaloes where Streptococcus spp was present in the highest percentage (71.42%). The results indicated that frequent and uncontrolled use of antibiotics against mastitis or other current diseases of both buffaloes and dairy cows led to the development of multi- or total resistance to antibiotics. The highest overall sensitivity for the isolated strains was recorded for amoxicillin in both species.

Key words: buffaloes, dairy cows, subclinical mastitis, antibiotic resistance

Mastitis is a severe disease commonly found in dairy cows and buffaloes in

Romania causing great economic losses due to a reduction in milk yield as well as lowering its nutritive value. Mastitis is characterized by pathological changes in the glandular tissue of the udder and physical, chemical, and bacteriological changes in the milk (10). Due to the involvement of multiple etiological agents, mastitis always represented a challenge to veterinarian all over the globe (11). Depending on the climatic condition, habitat and technology, etiological agents may vary and the involvement of pathogenic bacteria such as Staphylococcus aureus, Streptococcus agalactiae and environmental pathogens such as S. dysgalactiae, S. uberis, Corynebacterium bovis and coagulase negative Staphylococcus, viruses,


57

fungi and protozoa transform mastitis an compilation of most pathogens (2, 5, 8). In spite of advancement in management (1), milk remains an excellent culture medium for the growth of almost all kind of microbes (5). Due to numerous zoonotic agents, such as mycobacteria, Brucella, leptospirae and also agents that cause intoxications, transferred via the milk to consumers, mastitis of various origins represent a threat to human health, thus an important research topic for veterinarians and physicians (4) . This study aimed to compare mastitis outbreaks in buffaloes and dairy cows from Transylvania from etiological and epidemiological points of view. Additionally, antimicrobial susceptibility profile of microorganisms isolated from milk samples was tested.


The study was carried out on a group of buffaloes (n=108) and a group of Romanian Spotted and Red Holstein cows (n= 211), previously diagnosed with subclinical mastitis by using R Mastitest, a local diagnostic kit is based on the principle of Californian Mastitis Test - CMT (9). Mixing the milk sample with an equal amount of the reagent resulted in the dissolution and rupture of cell and nuclear membranes, which lead to the release of DNA molecules, forming a gel. The higher the number of disrupted cells from the milk sample (neutrophiles, macrophages, lymphocytes and epithelial cells) is, the more consistent the gel is.

Approximately 2 ml of milk from each quarter were collected into a plastic paddle that has 4 shallow cups marked A, B, C and D (corresponding to the four mammary quarters). An equal amount of reagent was added to the milk. The paddle was then rotated to mix the contents. In approximately 10 seconds, the score should be read while continuing to rotate the paddle. The reaction disappears within 20 seconds, so that the test must be read quickly. The reagent reacts with the white blood cells and the mixture thickens or gels proportionally to the amount of protective cells present. The results of the test, based on the thickness of the gel, could range from negative (no infection, the mixture remains liquid without precipitation) to intensely positive (mastitis, immediate appearance of the persistent gel consistency, in spite of shaking) (3).

To become accurate and consistent, this test should be performed on animals with a known somatic cell count (SCC). Subsequent to grouping by R-Mastitest, milk was sampled from positive individuals and cultured in a simple broth for 24 hours at 37ºC. Isolated colonies were obtained by transfer from the broth to agar plates. Simultaneously, smears and bacterioscopic examination were performed. Bacteria that showed grouping and shape/size typical for Staphylococcus spp . were transferred to Chapman agar in order to obtain pure cultures, which were then identified by API Staph 20.

Hemolysis of the isolated Streptococcus spp. and Staphylococcus spp. strains was monitored on blood agar. Kirby Bauer disc diffusion method on Muller-Hinton agar was used to investigate the sensitivity/resistance to antibiotics


58

ampicillin, amoxicillin, enrofloxacin, cloxacillin and oxytetracyclin. The results were interpreted by measuring the inhibition areas around the antibiotic discs.

Results and discussion

Bacteria found at the udder level in dairy animals represent their

microbiota, with no harmful influence in the majority of the cases. When environmental factors intervene, including changes in feeding, technological mistakes or changes in the macro and microclimate, these agents, or others aquired from the environment could induce pathological changes and cause mastitis (4-6, 8).

As a result of the microbiological examinations, four different bacterial genera in buffaloes (fig.1) and five in cows (fig.2), respectively, were isolated and identified from all the milk samples. Gram positive bacteria were the most prevalent in both species.

0

10

20

30

40

50

60

70

80

Streptococcus spp.

Staphylococcus spp.

E. coli

Pseudomonas spp.

Fig. 1. Streptococcus spp. showed the highest prevalence in buffalo mastitis


59

0

10

20

30

40

50

60

70

80

90

100

Staphylococcus spp.

Streptococcus

E.coli

Pseudomonas spp.

Corynebacterium spp.

Fig. 2. Staphylococcus spp. showed the highest prevalence in buffalo mastitis

Their distribution was presented figure 1 and 2, indicating the presence of

a number of species, of which the most incriminated is Streptococcus spp. (fig 1) in 71.42% of the cases in buffaloes and Staphylococcus spp. (fig. 2) in 90% of cases in dairy cows. Based on these results, it could be stated that the most frequently isolated bacteria from buffalo or cow milk, in case of subclinical mastitis belonged to the genus Streptococcus or Staphylococcus, respectively.

Gram negative bacteria were also present, but in a much smaller (between 5 and 9%) proportion, while E.coli was present in a higher percentage when compared to other gram-negative agents.

Since mastitis in cows proves to be a severe disease most of the times, antibiotics were used in a non-discriminatory manner to control the bacterial agents. Knowledge about antibiotic resistance trends over time is important to ensure long-term efficacy of the antibiotic and to help guide the veterinarian in selecting the most appropriate antibiotic for treatment of specific mastitis cases.

In the framework of responsible use of antibiotics, monitoring is emphasized as a key tool to detect and follow the emergence of resistance in addition to providing veterinarians data to optimize the treatment. In emergency situations, surveillance data can help provide rational therapy before sensitivity testing, to save the lives of diseased animals and ensure their welfare (6, 7).


60

0

5

10

15

20

25

30

35

amc

amp

enf

cloxa

cillin

ot

mean values in buffaloes

mean values in cows

Fig. 3. The comparative antibiotic sensitivity test results for bacterial strains isolated from buffaloes and dairy cows

In this study, the Kirby-Bauer disk diffusion technique was performed to

evaluate the sensitivity/resistance to antibiotics commonly used to treat the disease in dairy cows and buffaloes in Transylvania. Out of the antibiotics described in methods, the most effective for both cows and buffaloes was amoxicillin. There were differences in terms of resistance to certain antibiotics in cows and buffaloes, the least effective for buffaloes and for cows being oxytetracycline and cloxacillin, respectively.

The results indicated an increased resistance to antibiotics, either as total resistance or resistant colonies, to all tested antibiotics.The antibiotic susceptibility encountered in this study in relation to the retrospective epidemiological survey of antibiotic therapy for the animals showing mastitis could guide the treatment toward obtaining favorable results in treating this disease.

Conclusions

The results indicated that frequent and uncontrolled use of antibiotics

against mastitis or other current diseases of both buffaloes and dairy cows led to the development of multi- or total resistance to antibiotics. The microbial flora isolated from animals with subclinical mastitis could be either a subject for direct selective pressure exerted by the antibiotics used in udder therapy, or subject to the transfer of antibiotic resistance from the environmental microflora.


61

References

1. Amin, A. S., Amouda, R.H.H., Abdel-Ail, A. A., PCR assays for detecting major pathogens of mastitis in milk samples. World J. Dairy Food Sei., 2011, 6: 199-206.

2. Awandkar, S.P., Khode, N.V., Sardar, V.M., Mendhe, M.S., Prevalence and current antibiogram trend of mastitic agents in udder and its vicinity, Maharashtra State, India. Int. J. Daily Sei., 2009,4: 117-122.

3. Brinda, M., Moga-Manzat, R., Brezovan D., Toth, E., Study of correlation between different diagnosis tests in bovine mastitis, Lucrari stiintifice med. vet. Vol. XLII, 2009, Timisoara

4. Hussain, M., Malik, M. A., Fatima, Z., Yousaf, M. R., Participatory surveillance of livestock diseases in Islamabad capital territory. Intern. J.Agri. Biol., 2005,7(4): 567-570.

5. Kumar, A., Rahal, S.K., Dwivedi, Gupta, M.K., Bacterial Prevalence and Antibiotic Resistance Profile from Bovine Mastitis in Mathura, India. Egypt. J. Dairy Sei., 2010,38: 31-34.

6. Minst, K., Märtlbauer, E., Miller, T., Meyer, C., Short communication: Streptococcus species isolated from mastitis milk samples in Germany and their resistance to antimicrobial agents. J Dairy Sci, 2012, 95:6957–62.

7. Persson, Y., Nyman, K.J., Grönlund-Andersson, U., Etiology and antimicrobial susceptibility of udder pathogens from cases of subclinical mastitis in dairy cows in Sweden. Acta Vet Scand 2011; 53, 36.

8. Reyher, K.K., Haine, D., Dohoo, I.R., Revie, C.W., Examining the effect of intramammary infections with minor mastitis pathogens on the acquisition of new intramammary infections with major mastitis pathogens-a systematic review and meta-analysis. J Dairy Sei., 2012, 95: 6483-6502.

9. Sargeant, J.M., Leslie, K.E., Shirley, J.E., Pulkrabek, B., Lim, G.H., Journal of Dairy Science, 2001, 84, 2018-2024

10. Sharma, N., Alternative approach to control intramammary infection in dairy cows- Review. Asian J. Anim. Vet. Adv., 2007, 2(2): 50-62.

11. Vashney, S., Vashney, P., Dash, S.K., Gupta, M.K., Kumar, A., Singh, B., Sharma, A., Antibacterial activity of fruits of Terminelia chebula and Terminalia belerica against mastitis field isolates, Med. Plants, 2012, 4: 167-169.


62

THE CHANGES IN NON-SPECIFIC SYSTEMIC IMMUNITY IN CATTLE WITH SUBCLINICAL MASTITIS

R.M. GIUPANĂ, C.G. CERBU, DIANA OLAH, A. MOSCVICIOV, C. ILAŞCĂ, V.

NEGRUŢIU, MARINA SPÎNU

University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca 400372, 3-5 Mănăştur Street, Cluj-Napoca, Romania


Summary

Bovine mastitis, the most significant and costly disease of dairy herds, has vast effects on farm economics due to reduction in milk production. The bacterial etiology of mastitis exerts its impact on both the local and systemic immune effectors, thus the immune status of the animals can help in predicting the outcome of the disease (Giupana et al., 2015). The investigations aimed to monitor the changes in non-specific systemic humoral and cell-mediated immunity in cattle of different breeds, previously diagnosed with subclinical mastitis (n=10), when compared to a healthy group (n=9). The cattle originated from private households located in NW Transylvania. Milk and blood samples were collected from each animal, and further tested by the use of precipitation techniques to quantify total immune globulin levels and the carbon particle inclusion test to monitor the phagocytosis from whey and blood, respectively. The study revealed that there were no statistically significant differences between the total immunoglobulin present in the serum of the cattle diagnosed with subclinical mastitis (0.31±0.079 optical density units, ODU) when compared to the healthy ones (0.26±0.088 ODU). Values for phagocytosis were significantly higher (p<0.01) in mastitic (2.24±0.92 ODU) versus healthy (4.33±0.71) animals. Moreover, the higher values obtained for phagocytosis in cows with subclinical mastitis suggested a stronger involvement of non-specific cell-mediated rather than humoral systemic immunity in this category.

Key words: subclinical mastitis, total immunoglobulin, phagocytosis

There are numerous factors that influence the productive capacity of dairy

cattle, such as: poor nutrition, unefficient management and various diseases. Mastitis is considered to be the outcome of interaction between various factors in connection with the host, such as pathogens and the environment (5). In spite of advancement in scientific skill and management (2), milk remains an excellent culture medium for the growth of almost all kind of microbes (8). Infectious agents, in particular various species of bacteria, are the most important causative agents of mastitis. When bacterial pathogens intervene at local or general levels, an alteration of qualitative and quantitative traits of the milk are changed (13). Changes could be an indicator for the diagnosis of the disease (7, 16).

During mastitis, it is common to observe an increased number of somatic cells in the milk. Neutrophile migration from the bloodstream to mammary gland (MG) tissue occurs as a response to pro-inflammatory cytokines such as tumor


63

necrosis factor alpha (TNF-a) and interleukin 1 beta (IL-1). Several cytokines might also increase phagocytic bactericidal activity. Some bacteria are able to modulate cytokine production in the MG immune system, which modifies the host innate immune response (6).

The study and complete characterization of the MG innate and acquired immune responses to different etiological agents are necessary to understand in detail the pathophysiology of mastitis, and also to design efficient approaches to the diagnosis, treatment, and control of this disease (15).


The research was carried out on a group of Romanian Spotted and

Holstein cows, aged 3 to 8 years, previously diagnosed with subclinical mastitis. The investigations aimed to monitor the changes in non-specific systemic humoral and cell-mediated immunity in cattle that were previously diagnosed with subclinical mastitis (n=10), when compared to a healthy group (n=9). The cattle originated from private households located in NW Transylvania. Milk and blood samples were collected from each animal, and further tested by the use of precipitation techniques to quantify total immune globulin levels and the carbon particle inclusion test to monitor the phagocytosis from whey and blood, respectively. To monitor the changes in nonspecific systemic humoral in cows diagnosed with subclinical mastitis, the precipitation technique to quantify total immunoglobulins levels from whey was used. Dysproteinemia tests are based on the fact that at a pH=7,4, the electric charge and colloidal stability of gamma globulins are lower than those of serum albumins. One of the easiest because both applicability and is easy to interpret test results of zinc sulphate precipitation (Reagent SERB) (9). The pH of the buffer was adjusted to 7,4. 6.6μl of each serum were mixed with 193,3 μl of a 24%o zinc sulphate solution and stirred on an electric stirrer. The incubation period is of 30 minutes at room temperature. The optical density was measured by a spectrophotometer at a wavelength of λ=475nm, and d=0,5cm against the blank (Reagent SERB).

Phagocytic activity as a measure of the non-specific cell mediated immunity was investigated by the use of the carbon particle inclusion test. For that, blood samples (1.5 ml) from each animal were collected on heparine (50 IU/ml)

then mixed with 20 l of a supernatant of India ink, centrifuged at 6000 rpm for 45 min. From each mixture, aliquotes of 0.15 ml were transferred at moment 0 (when

mixing), then after 35 and 60 min of incubation at 37C, to 2 ml of saline. Finally, all tubes containing the blood + India ink+ saline mixture were centrifuged for 5 minutes at 800 rpm and the supernatants were read spectrophotometrically (λ=535·nm, d=1·cm). Phagocytic activity index was calculated as the difference between the ln of the optical densities of the phagocytosis at 0–35·min and 35–60·min divided by time (25·min).


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The bovine mammary gland is equipped with a non-immune anatomical

barrier, and a plethora of immune-mediated defense mechanisms that include innate and adaptive immune responses (15). The immune system is a network of cells and molecules with specialized roles in defending against infection. Innate (natural) responses occur to the same extent subsequent to each aggression, while the adaptive immune response is enhanced by booster contacts (4). Innate immunity is non-specific while the adaptive immunity stands for highly specific response (3). This variability in host–pathogen interaction is controlled by the genetic background of the host, including innate and adaptive immune responses, particularly the acquired immunological memory, as well as the nature of the microbial pathogen (20). The immune system is responsible for recognizing, resisting, and eliminating health challenges, including pathogens, injuries, parasites, stress and the immune system should react and work fast when necessary so the productive functions of the animal are not impaired (1).

In this study, the changes in non-specific systemic humoral and cell-mediated immunity in cows from different breeds diagnosed with subclinical mastitis, were observed by the use of precipitation techniques and the carbon particle inclusion test. One of the ndicators of the non-specific humoral immune response is the circulating level of immunoglobulins. Subsequent to external influeneces, including bacterial priming provided by the udder infection, changes in immune globulin levels could help in predicting the outcome of the disease (13).

Phagocytosis is defined as a large (≥0.5-μm) particles ingestion by cells and, unlike macropinocytosis, this involves the recognition and binding of “non-self” by the receptors located on the cell surface (4, 6). Blood phagocytes possess the ability to ingest not only bacteria but also inert particles. The result of the carbon particle inclusion test provides important information regarding the functional capacity of circulating phagocytes (11).

The total levels of Ig in mastitic cows were somewhat increased, but not supported statistically when compared to those observed in health cows (Fig. 1). This increase is explicable by the non-specific short term response to the bacterial stimulation exerted at the udder level, by the infecting bacteria. Similarly, these values are close to the physiological ones.


65

0.31

0.26 Ig ODU(m)

Ig ODU(h)

healthy cows

mastitic cows

Fig. 1.The total IgG levels mastitic (m) versus healthy(h) cows (ODU)

The host immune system is also adaptive and has a large arsenal to

control or eliminate microbial threat. It is widely accepted that individual susceptibility within a given species differs towards the same bacterial pathogen. Neutrophil recruitment to the mammary gland to facilitate bacterial clearance through phagocytosis, production of reactive oxygen species, antibacterial peptides, such as lactoferrin and β-lactoglobulin, and defensins, result in increased somatic cell count (16, 27).

Fig.2. Dynamics of the systemic phagocytic activity levels in mastitic and healthy

animals (ln)

ln


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Phagocytosis contributes to the first line of defense against infection. It also plays a key role in the initiation of the adaptive immune response (3, 10). In this study, the values of the phagocytic activity were significantly higher (p<0.01) in mastitic (2.24±0.92 units) versus healthy (4.33±0.71 units) animals. The speed of the phagocytosis was increased in healthy animals versus the mastitic ones.

Conclusions

The higher values obtained for phagocytosis but slightly increased levels of

Ig in cows with subclinical mastitis, suggested a stronger involvement of non-specific cell-mediated rather than humoral systemic immunity for this category.

References

1. Abbas, A. K., A. H. Lichtman, Cellular and Molecular Immunology, 6th

edition. Saunders, Philadelphia, PA. 2007, AcademicPres, Cluj-Napoca, 2006

2. Amin, A. S., Amouda, R.H.H., Abdel-Ail, A. A.. PCR assays for detecting major pathogens of mastitis in milk samples, World J. Dairy Food Sci., 2011, 6: 199-206.

3. Borghesi, L, Milcarek, C., Innate versus adaptive immunity: a paradigm past its prime? Cancer Res., 2007, 67:3989–93,doi:10.1158/0008-5472.CAN-07- 0182.

4. Delves, Peter J., Roitt, Ivan The immune system: first of two parts. New England Journal of Medicine, 2000, 343 (1). pp. 37-49. ISSN 0028-4793.

5. Hussain, M., Malik, M. A., Fatima, Z., Yousaf, M. R., Participatory surveillance of livestock diseases in Islamabad capital territory. Intern. J. Agri. Biol., 2005, 7(4): 567-570.

6. Janeway, Jr CA, Medzhitov, R. Innate immune recognition, Annu Rev Immunol, 2002, 20:197e216.

7. Jiusheng, W., Yuehuan, L., Songhua, H., Jiyong, Z., Development of a rapid PCR test for identification of Streptococcus agalactiae in milk samples collected on filter paper disks. Asian-Aust, J. Anim. Sei., 2008, 21, 124-130.

8. Kumar, A., Rahal, S.K., Dwivedi, Gupta, M.K., Bacterial Prevalence and Antibiotic Resistance Profile from Bovine Mastitis in Mathura, India. Egypt. J. Dairy Sei., 2010, 38: 31-34.

9. Oviedo-Boyso, J., Valdez-Alarcón, J.J., Cajero-Juárez, M., Ochoa-Zarzosa, A., López-Meza, J.E., Bravo-Patiño, A., Baizabal-Aguirre, V.M., Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis, J. Infect., 2007, 54(4):399-409.

10. Flannagan, R. S., Jaumouill´e, V., Grinstein, S., The Cell Biology of

Phagocytosis Annu. Rev. Pathol. Mech. Dis. 2012, 7, 61–98.

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11. Rindt, Iulia Krisztina, Evaluarea calitatilor terapeutice si imunostimulatoare ale unor produse apicole,Teza de doctorat, Facultatea de Medicina Veterinara Cluj Napoca, 2011.

12. Rindt, Iulia, Şandru, Carmen Dana, Brudaşcă, Gh. F., Niculae, Mihaela, Cadar, D., Ungvari, A., Spînu, Marina, Antibacterial Activity of Different Propolis Concentrations Against Staphylococcus Aureus Stains Isolated from bovine Mastitis, Lucrări ştiinţifice Medicină veterinară, Iaşi, 2009, vol. 52, p. 1096-1098.

13. Roitt, I. M., Delves, P. J., Essential Immunology, Blackwell Science, USA, 2001.

14. Sharma, H., Maiti, S.K., Sharma, K.K., Prevalence, etiology and antibiogram of microorganisms associated with sub-clinical mastitis in buffaloes in durg, chhattisgarh state, Int. J. Dairy Sei., 2007, 2: 145-151.

15. Sordillo, L.M., Shafer-Weaver, K., DeRosa, D., Immunobiology of the mammary gland, J Dairy Sci, 1997.

16. Syring, C., Boss, R., Reist, M., M. Bodmer, Hummerjohann, J., Gehrig, P., Graber, H.U., Bovine mastitis: The diagnostic properties of a PCR- based assay to monitor the Staphylococcus aureus genotype? status of a herd, using bulk tank milk, J. Dany Sei., 2012, 95: 3674-3682.

17. Wellnitz, O., Bruckmaier, R.M., The innate immune response of the bovine mammary gland to bacterial infection, Vet J, 2012, 192:148–52. doi:10.1016/j.tvjl. 2011.09.013.


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THE PREVALENCE OF GASTROINTESTINAL PARASITES IN RED FOXES (VULPES VULPES) IN BIHOR COUNTY

F. Ş. HORA, NARCISA MEDERLE, M. S. ILIE, MIRELA IMRE, CORINA BADEA,

GH. DARABUŞ

Banat's University of Agricultural Sciences and Veterinary Medicine “King Michael I” Timisoara, Faculty of Veterinary Medicine, Department of Parasitology, 300645,

Calea Aradului, Timişoara, Romania E-mail: [email protected]

Summary

During the period December 2014 – February 2015, 42 dead foxes (Vulpes vulpes)

from 19 hunting sites belonging to Bihor County were necropsied. Out of 42 dead foxes, 25 were males and 17 females. Faeces samples and gastrointestinal mass from all dead foxes were collected and examined macro- and microscopically to identify the endoparasites. Carefully each section of the digestive tract, the mucosa, the content and then the gastrointestinal mass were examined macroscopically, by successive washes method. The gastrointestinal content and also each section of the digestive tract, previously washed, were microscopically examined by stereomicroscope. Subsequently the faeces samples were examined by flotation method (Willis). The endoparasites were identified in 27 of 42 samples and the overall prevalence was 67.28%. Referring to the identified parasites prevalence the parasitism with: Eimeria spp. was found in seven samples (16.16%), Alaria alata in five samples (11.90%), Mezocestoides lineatus in 13 samples (30.95%), Taenia pisiformis in eight samples (19.04%), not identified cestodes in six samples (14.28%), Toxocara canis in 15 samples (35.71%), Pterygodermatites affinis in three samples (7.14%), Ancylostoma spp, in eight samples (19.04%) and with Trichocephalus vulpis in nine samples (21.42%).

Key words: parasites, prevalence, red fox, gastrointestinal.

The fox’s body (Vulpes vulpes) resembles that of a dog, but the difference

is in its tail. The fox tail is long and bushy with white tip and reddish coat. She is carnivorous, feeding on a wide range of small animals she can catch, but also she can be omnivorous. Foxes are widely found throughout the world (6,19), having an important role in the transmission of diseases and also some important zoonoses (10,12,17).

They are host for many gastro-intestinal parasites species which can be harmful to humans and animals (domestic and wild) (12).

It is well known that performance drops in gastro-intestinal parasitic infestations in domestic animals and wildlife worldwide. The main symptoms of parasitic diseases are: loss of appetite, impaired reproduction, apathy and dull hair. However, most studies have focused on the parasitological investigating of domestic animals and recently it was found that parasitic infestations are common and important also in wild animals, which could serve as a potential reservoir of parasites.

https://ro.wikipedia.org/wiki/Vulpe


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The aim of this study was to identify the prevalence of gastro-intestinal parasite infestations in foxes (Vulpes vulpes) from the hunting sites belonging to Bihor County.


1.1. Description of the area from which the samples belong Bihor County is in north-western Romania bordered by Apuseni Mountains in

the eastern side of the County and in western side by Tisa Plain. In north is bordered by Satu-Mare County, in east with Salaj and Cluj County, in south-east with Alba County, in south with Arad County and in west with Hungary. The County area covers mountains (24%), hills (32%) and plain (44%). The rivers from this County belonging to the Cris hydrographic basin: Barcau with Ierul, Crisul Repede, Crisul Negru and its tributaries. The climate is temperate-continental with oceanic and humid influences (18).

Fig. 1. The area of origin for foxes samples (20).

1.2. Animals under study, parasitological examination and epidemiological

investigation During the period December 2014 – February 2015, 42 dead foxes (Vulpes

vulpes) from 19 hunting sites belonging to Bihor County were necropsied. Out of 42 dead foxes, 25 were males and 17 females, with the age ranged from two to five years old.

The dead foxes samples were from 19 hunting sites: Vărşand (three samples), Cetariu (three samples), Pădurea verde (two samples), Marginea (two samples), Mişca (two samples), Paleu (two samples), Giriş (two samples), Oşand (one sample), Cociuba (two samples), Lunca (two samples), Corbesti (two samples), Lunca Sprie (two samples), Grănceşti (two samples), Cuiesd (three samples), Curăţele (two samples), Roşia (two samples), Finiş (three samples), Cusuiuş (two samples), Biharia (three samples).


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The foxes were killed by shooting and transported to DSV (Sanitary Veterinary Directorate) Bihor for the effectiveness of vaccination against rabies been determined. The gastro-intestinal mass in the Clinique of Parasitology and parasitic diseases of the Veterinary Medicine Faculty was examined.

For parasites determination, the gastrointestinal mass from each dead fox was collected.

Macroscopic examination on digestive tract sections was performed, the stomach and the intestines were opened, with scissors longitudinal sections were made and carefully the gastro-intestinal content was examined. After removing the contents of the digestive tract, successive washes method for the stomach and intestines was performed and carefully the sediment was examined.

The adult parasites found in the gastro-intestinal mass were collected, washed in saline solution 0.9%, to eliminate the dirt and then examined by stereomicroscope for identification. For preservation the parasites in 70% ethanol were maintained. The stomach and intestines, with the content removed, were also by stereomicroscope examined.

Faeces samples from the gastro-intestinal mass of dead foxes were collected and by flotation (Willis) and successive washes methods were examined.

Parasites species identification in accordance with the determination keys for trematodes, cestodes and nematodes from the scientific literature was performed (1, 9, 4, 3, 5, 7, 8, 11, 13,14).


After analyzing samples from a macroscopic viewpoint the stomach and

intestines, showed abundant mucus, microbleeds and individual or balls of adults of Toxocara canis, Trichocephalus vulpis, Pterygodermatites affinis, Ancylostoma spp., Taenia pisiformis, Mesocestoides lineatus, Alaria alata were identified.

For the examination of the faeces Willis metod was performed and eggs of Eimeria spp., Toxocara canis, Ancylostoma spp. and Trichocephalus vulpis were identified.

In this study the parasitism with protozoa, trematodes, nematodes and cestodes was identified in 27 (67.28%) of 42 dead foxes from Bihor County. In 15 (35.71%) of 42 dead foxes examined any gastro-intestinal parasitism was not identified.

Overall the prevalence of parasitism with Eimeria spp. was 16.16%, Alaria alata 11.90%, Taenia pisiformis 19.04%, Mezocestoides linetatus 30.95%, not identified cestodes 14.28%, Toxocara canis 35.71%, Trichocephalus vulpis 21.42%, Ancylostoma spp., 19.04% and Pterygodermatites affinis 7.14%,


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0 5 10 15 20 25 30 35 40

Eimeria spp.

Alaria alata

Taenia pisiformis

Mesocestoides lineatus

Cestode nd.

Toxocara canis

Trichocephalus vulpis

Ancylostoma spp.

P. affinis

7

5

8

13

6

15

9

8

3

16

12

19

31

14

36

21

19

7

Fig. 2. Prevalence of gastrointestinal parasites in foxes in Bihor County

Out of 42 examined samples of dead foxes, 25 were males and 17

females. 18 of the 25 males were positive (72%), in four samples was found Eimeria

spp., (16%), in three samples Alaria alata (12%), in 11 samples Toxocara canis (44%), in five samples Trichocephalus vulpis (20%), in three Pterygodermatites affinis (12%), in five samples Ancylostoma spp.,(20%), in five samples Taenia pisiformis (20%), in eight samples Mesocestoides lineatus (32%) and four samples with not identified cestodes (16%) were parasitized.

9 of the 17 females were positive (52.94%), three samples with Eimeria spp. (17.64%), three with Alaria alata (17.64%), four with Toxocara canis (23.59%), four with Trichocephalus vulpis (23.59), any sample with Pterygodermatites affinis, three samples with Ancylostoma spp. (17.64%), three with Taenia pisiformis (17.64%), five samples with Mesocestoides lineatus (29.41%), two samples with not identified cestodes (11.74%) were parasitized.

Using the t-test test (Stat Pac Inc.) statistically the data was analyzed. Differences statistically significant when p values were less than 0.05 (p ≤ 0.05) were considered.

The prevalence and intensity for gender group (males and females) were calculated. The prevalence was not significantly different (p = 0.1928) between the number of positive male (18/25, 72%) and female foxes positive (9/17, 52.94%).

There were no statistically significant differences between gender/sexes and level of infestation.

Vergles Rataj et al. in a study conducted in Slovenia on 428 foxes showed 93.2% positivity in examined samples, with a prevalence of 38.3% for Toxocara


72

canis, 4.2% for Pterygodermatites affinis, 0.7% for Trichuris vulpis, 27.6% for Mesocestoides sp. and 2.1% for T. pisiformis. (16).

In West Australia, Dybing et al. in 2013 were performed a study on 147 foxes from 14 cities and showed a prevalence of 14.9% for Toxocara canis (6).

Stuart P. et al. (2013) conducted in Ireland on faeces samples from foxes a study which showed the prevalence for Toxocara canis 20% and 4 % for Trichuris vulpis (15).

A study by Barabasi et al. during the period 2007-2010, on 561 foxes in 15 counties in Romania reported the prevalence for nematode parasitism (91.4%), followed by cestodes (90.7%) and trematodes (15%). They have found 17 intestinal helminth species: Alaria alata, Dipylidium caninum, Echinococcus multilocularis, Mesocestoides lineatus, Taenia polyacantha, T. hydatigena, T. multiceps, T. pisiformis, T. serialis, T., Taenia T. crassiceps, T. ovis, Ancylostoma caninum, Uncinaria stenocephala, Toxascaris leonina, Toxocara canis and Trichuris vulpis (2).

Conclusions

The results of this study highlights the importance and spread of gastro-

intestinal parasites in foxes in Romania, confirmed earlier by researchers in other countries.

Foxes in Western Romania are simultaneously parasitized by protozoa, trematodes, cestodes and nematodes.

The prevalence of gastrointestinal parasitosis in foxes (Vulpes vulpes) in Bihor County was 67.28%.

The most frequent parasitism was with the nematode Toxocara canis 35.71%, while the least common parasitism was with Pterigodermatites affinis 7.14%.

The gender was not a risk factor that we should take in consideration.

Acknowledgements

This research work was carried out with the support of the project Research, education and services development in the veterinary medicine and innovative technologies areas for RO 05, code SMIS-CSNR 2669.

References

1. Anderson, R.C., Nematodes parasites of vertebrates 2nd

Edition: their development and transmission, CAB International - CABI Publishing, 2000.

2. Barabási, Sikó, Fok, E., Gubányi, A., Mészáros, F., Cozma V., Helminth fauna of the small intestine in the Europe red fox, Vulpes vulpes with notes on the morphological identification of Echinococcus multilocularis, Sci. Parasitol., 2000.11 (3) 141–151.


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3. Dărăbuş, Gh., Oprescu, I., Morariu, S., Mederle, Narcisa, Ilie, M.S. Ghid practic în bolile parazitare, Ed. Mirton, Timişoara 2013.

4. Dărăbuş, Gh., Oprescu, I., Morariu, S., Mederle, Narcisa. Parazitologie şi boli parazitare, Ed. Mirton, Timişoara 2006.

5. Dunn, A.M. Veterinary Helminthology. Ed. W. Heinemann Medical Books Ltd. London, second ed. 1978.

6. Dybing, N.A., Fleming, PA., Adam,s P.J., Environmental conditions predict helminth prevalence in red foxes in Western Australia. Int J Parasitol Parasites Wildl. 2013. 2:165-72.

7. Euzeby, J., Les maladies vermineuses des animaux domestiques et leurs incidences sur la pathologie humaine. Tome II. Maladies dues aux plathelminthes. Deuxieme fascicule: Trematodes, livre 1. Ed. Vigot Freres, Paris. 1971.

8. Euzeby, J., Les maladies vermineuses des animaux domestiques. Tome I, Fasc. 2, Ed. VigotFreres, Paris. 1963.

9. Gibson, I.D., Jones, A., Bray, R.A. Keys to the trematoda, Vol. I, Ed. CAB International. 2002.

10. Henderson, W., Pathogens in vertebrate pests in Australia, Invasive Animals, Cooperative Research Centre, Canberra. 2009.

11. Khalil L.F., Jones A., Bray R.A., Keys to the Cestode Parasites of Vertebrates, CAB International University Press, Wallingford, UK. 1994.

12. Ilie, M. S., Imre, K., M. Imre, I. D. Sorescu, I. Hotea, S. Andrei, F.Ş. Hora, C. Badea, S. Morariu, GH. Dărăbuș. The prevalence of gastrointestinal parasites in red foxes (Vulpes vulpes) from western Romania – preliminary study. International Conference "Agriculture for Life, Life for Agriculture" Section 4, Veterinary Medicine, Bucharest. Book of Abstracts 2015. 82.

13. Skryabin, K.I., Key to Parasitic Nematodes, vol. 4, Camallanata, Rhabditata, Tylenchata, Trichocephalata, Dioctophymata and distribution of parasitic nematodes in different host, Ed. E J. Brill, 1992.

14. Skryabin, K.I., Key to Parasitic Nematodes, vol. 3, Strongylata, Ed. E J. Brill. 1992.

15. Stuart, P., Golden, O., Zintl, A., de Waal, T, Mulcahy, G., McCarthy, E., Lawton, C., A coprological survey of parasites of wild carnivores in Ireland. Parasitol Res. 2013. 112(10):3587-93.

16. Vergles Rataj, A., Posedi, J., Zele, D., Vengust, G., Intestinal parasites of

the red fox (Vulpes vulpes) in Slovenia. Acta Vet Hung. 2013. 61(4):454-62. 17. Wolfe, A., Hogan, S., Maguire, D., Fitzpatrick, C., Vaughan, L., Wall, D.,

Hayden, T.J., Mulcahy, G., Red foxes (Vulpes vulpes) in Ireland as hosts for parasites of potential zoonotic and veterinary significance, Vet. Rec., 2001.149, 759–763.

18. ***http://www.turismbihor.info/. 19. ***https://ro.wikipedia.org/wiki/Vulpe. 20. ***https://ro.wikipedia.org/wiki/Jude%C8%9Bul_Bihor.

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http://www.ncbi.nlm.nih.gov/pubmed?term=Adams%20PJ%5BAuthor%5D&cauthor=true&cauthor_uid=24533331

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OBSERVATIONS UPON INFECTION WITH MYCOPLASMA GALLISEPTICUM IN JAPANESE QUAIL (COTURNIX COTURNIX

JAPONICA)

IONICA IANCU, J. DEGI, CORINA PASCU, V. HERMAN, N. CATANA

Banat University of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Calea

Aradului 119, Timişoara, Romania E-mail: [email protected]

Summary

This paper presents an episode of micoplasmosis with M. gallisepticum in quails

aged 28 days. The objectives of this study were to diagnose in a flock of 1,400 quail with clinical respiratory, infraorbital sinus inflammation, mucous nasal cavities, conjunctival sacs and air sacs. The cumulative mortality losses expressed, has recorded maximal values in the fourth weeks when it was 2.07% and respectively 2.29% in the fifth weeks. Serological examination performed by agglutination reaction on the slide, confirmed infection with M.gallisepticum in the flock of Japanese quail.

Key words: M. gallisepticum, micoplasmosis, Japanese quail

Micoplasma gallisepticum produce respiratory infections in chickens,

turkeys and other poultry, but has been isolated and the pheasants, chukar partridge, peafowl, bobwhite quail and japanese quail (3).

This is the most pathogenic and economically significant mycoplasmal pathogen of poultry.


The episode was reported in a flock of 1,400 Japanese quail from a wild

game farm in Timis. To confirm the diagnosis, were performed epidemiological surveys, clinical, anatomic-pathological, bacteriological and serological exams.

Epidemiological, clinical and pathological exams were made in farm and laboratory investigation were performed in the Department of Infectious Diseases FMV. Timisoara.

For the epidemiological survey, the main monitored parameter followed was cumulative mortality.

Clinical examination. After 28 days of age clinical examination of flock was performed daily, because starting with this age were reported first symptoms of disease. Mainly, some symptoms were traced to denote a change in the condition of the quail. No determinations were made on the microclimate parameters.


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Anatomopathological examination was weekly performed and dailly when mortality increased, necropsies were performed aiming to observation the specific lesions on corpses quail.

Bacteriological examination was performed from bone marrow and trachea. Serological examination was carried out on 30 blood samples taken from

axillary vein, both from quail with clinical signs as well as from the quail clinically healthy. Sampling of blood was performed at 28 days and 42 days, and after expression of the serum was stored in the freezer in order to be further processed all in the same conditions.The sera were tested by rapid agglutination reaction slide for the detection of antibodies to M gallisepticum, Mycoplasma gallisepticum using Flockscreen kit produced by X-OVO Limited.


Epidemiological examination. During the examination of the farm, there

were several species of birds: pheasants, partridges, quail and chicken meat. Feed have been qualitatively and quantitatively in accordance with their age; hygiene conditions in the halls were inadequate, the main parameters (humidity, drafts, gas) exceeded the permissible limits, with the mention that we have not performed specially determinations concerning the microclimate parameters. Feeding and watering hall was done manually. The quails were vaccinated against Newcastle disease.

The losses of mortality, expressed by cumulative mortality, were within the parameters of the technology until the fourth week, when it started to grow, registering peaks in the fifth week.

Evolution of cumulative mortality was calculated weekly and is presented in Table 1.

Table 1 The evolution of cumulative mortality in Japanese quails

Week No corpses %

I 8 0.57

II 18 1.29

III 22 1.57

IV 29 2.07

V 32 2.29

VI 13 0.93

Total 122 8.71

The cumulative mortality losses expressed, has recorded maximal values

in the fourth weeks when it was 2.07% and respectively 2.29% in the fifth weeks. In the literature, the cumulative mortality in a flock of 75 000 Japanese

quail, bred for egg production, was reported as having values of 5.7% per day (2). At clinical examination we observed that the symptoms appeared after 4

weeks. The quails presented shortness of breath, loss appetite, birds refuse ti


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move, seromucous nasal discharge, sneezing, tracheal rales, infraorbital sinusitis and conjunctivitis. The birds did not move to drink water and to feed, so that led to gradual weight loss.

Murakami et al. observed following clinical quail signs: swelling of the facial skin and eyelids, increased lacrimation, congestion of conjunctival vessels and respiratory rales (2).

After the anatomopathological examination of quail carcasses were performed, it could be observed following lesions: infraorbital sinus inflammation, mucous nasal cavities, conjunctival sacs and air sacs.

Some authors mentioned that in natural infection of quails, partridges and pheasants, the most common lessions are conjunctivitis and sinusitis in sinus infraorbital unilateral or bilateral (5).

Other authors observed the presence of the following lessions: gelatinous or caseous exsudate in sinuses, airsacculitis, and yolk peritonitis (1, 2, 3).

Bacteriological exam results were negative. Isolation of the pathogen was not possible because from case history information it was known that this quails recived Enrofloxacin for 5 days, in drinking water.

Serological examination provided the following results. All serum samples examined were positive for antigen M gallisepticum. The reaction was positive immediately when serum was put in contact with the antigen. Given the fact that all examined sera, collected both from quail with clinical signs as well as from the clinically healthy, show that morbidity studied herd tends towards 100%. Rapid slide agglutination test can be used successfully to confirm the diagnosis of mycoplasmosis, given that isolation of etiologic agent is quite difficult.

Suheyla Türkyilmaz et al. in a comparative study between different serological examinations performed, reported that the rapid agglutination reaction sesibility has a 40% and a specificity of 90% (4).

Following the imposition Tylozin treatment in drinking water, the percentage of morbidity and mortality has reduced in the studied flock of quail.

Conclusions

The cumulative mortality has recorded maximal values in the IV th week to

2.07% and V th of the week, respectively 2.29%. Specific clinical symptoms of mycoplasmosis, in the flock of quail, occurred

after the age of 28 days. Bacteriological examination did not allow the isolation of M gallisepticum

because quails received treatment with Enrofloxacin in drinking water. Serological examination performed by agglutination reaction on the slide,

showed antibodies against M.gallisepticum, which confirms the existence of the infection in the flock.


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Acknowledgements


References

1. Kleven, S.H. Micoplasmosis, in Disease of Poultry, 12th Edition, Editor in

Chief SAIF, Y.M., Iowa State Press, a Blackwell Publishing Company, Ames Iowa, 2008.

2. Murakami, S., Miyama, M., Ogawa, A., Shimada, J., Nakane, T. Occurrence of conjunctivitis, sinusitis and upper region tracheitis in Japanese quail (Coturnix coturnix japonica), possibly caused by Mycoplasma gallisepticum accompanied by Cryptosporidium sp. infection, Avian Pathology, 2002, 31, 4, 363-370

3. Raviv, Ziv, Ley, D. H. Mycoplasma gallisepticum Infection in Diseases of poultry. – 13th ed. / editor-in-chief, David E. Swayne; associate editors, John R. Glisson, 2013.

4. Turkyilmaz, Suheyla, Coven, Fethiye, Eskiizmirliler Seza, Detection of Antibodies Produced in Quails (Coturnix coturnix japonica) against Mycoplasma gallisepticum with Different Serological Tests, Turk. J. Vet. Anim. Sci., 2007, 31, (4), 267-270.

5. http://www.cfsph.iastate.edu/Factsheets/pdfs/avian_mycoplasmosis_mycoplasma_gallisepticum.pdf.


78

PREVALENCE OF ANGIOSTRONGYLUS VASORUM INFESTATION IN DOGS FROM WESTERN ROMANIA -

PRELIMINARY RESULTS

M. S. ILIE, MIRELA IMRE, K. IMRE, F.Ș. HORA, GH. DĂRĂBUȘ, A. OLARIU JURCA, IRINA PATRAȘ, DENISA IONELA SORESCU

Banat’s University of Agricultural Sciences and Veterinary Medicine „King Michael I

of Romania” from Timisoara, 119 Aradului Street, 300645, Timisoara, Romania, Phone: 0256277190


Summary

Angiostrongylus vasorum is potentially fatal metastrongyloid parasitic nematode

that infects dogs. The aim of this study was to investigate the prevalence of A. vasorum in dogs from western part of Romania. A total of 115 dog fecal samples were examined for the presence of first stage larvae of A. vasorum by Baermann test. Six of the examined dogs were infested, and overall prevalence of A. vasorum infestation was 5.21 %. This is the first report of Angiostrongylus vasorum infestation in canine population in Romania. The results highlight the emerging nature of parasitism with A. vasorum in dog in this area of the world and induce mandatory extension of research.

Key words: Angiostrongylus vasorum, prevalence, dogs, Baermann test.

Angiostrongylus vasorum (Nemotoda, Stongylida, Metastrongyloidea) also

known as French heartworm is a nematode parasite of the pulmonary arteries and heart of wild and domestic canids. Canine Angiostrongylosis is a gastropod borne helminthic infection considered as an emergent disease in Europe and Canada (2, 3, 4, 7). Infestation with Angiostrongylus vasorum is usually characterised by progressively worsening signs of respiratory and/or cardiac disease, manifested clinically by verminous pneumonia in dogs with gagging, coughing and dyspnoea, depression, weight loss, exercise intolerance and haemorrhage with a lethal outcome in severe cases (1, 10). The aim of this study was to investigate the prevalence of A. vasorum in dogs from western part of Romania.


Fecal samples of 115 dogs from western part of Romania were collected

from dogs attending Universitary Veterinary Clinics for different reasons and were examined for the presence of first stage larvae of A. vasorum by Baermann test.

Five grams of faeces were subjected to a sieve (600-700 µm) and placed in a Baermann apparatus filled with warm water (aprox. 30°C) until of the faecal sample was immersed in water. The apparatus was left at room temperature for 24 h and the clamp was carefully opened. First four drops were collected on a slide,


79

covered with a cover slip and examined at microscope (100 and 400 X magnification).

First larvae of A. vasorum were identified on the basis of morphological findings at the tail according to Traversa, et al., (2010) (8).

At the time of samples collection data regarding age, gender and lifestyle of animals were recorded. The data were statistically analyzed using GraphPad Software Inc.® (QuickCalcs, 2016). Differences were considered statistically significant when p values were less than 0.05.


Six of the examined dogs were infested, and overall prevalence of A.

vasorum infestation was 5.21 % (Table 1).

Table 1 Sinoptic of positive samples resulting from epidemiological investigation of Angiostrongylus vasorum infection in 115 dogs studied, in western Romania

Epidemiological data Angiostrongylus vasorum

Baermann test Age n= %

≤ 3 years (n=46) 1 2.17 > 3 to 6 years (n=43) 3 6.97

≥ 6 years (n= 26) 2 7.69 Gender

Female (n=48) 3 6.25 Male (n=67) 3 4.47

Breed Pure (n=83) 4 4.81

Mixed (n=32) 2 6.25 Life style (Owner)

With (n=103) 5 4.85 Without (n=12) 1 8.33

Total 6/115 5.21

n=– positive dogs examined; % - prevalence obtained

Dogs subjected to the study were aged between 3 months and 11 years

old. Animals under study were represented by 48 females and 67 males

belonging to 30 different breeds, 32 mix breed and 83 pure breed. Regarding lifestyle 12 (10.4%) were stray dogs and 103 (89.6%) were with

owner. The animals were distributed into three age groups, namely under three

years (n=46), between three and six years (n=43) and over six years (n=26).


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Prevalence of Angiostrongylus vasorum infestation reported at age groups was 2.17%, 6.97% and 7.69% respectively.

Regarding of animal gender prevalence was 6.25% and 4.47% for female and male respectively.

Among the pure breed dogs 4.81%, while 6.25% from mixed breed were positive at parasitologic exam.

Dogs with owner were infested in percent of 4.85 while dogs whitout owner were 8.33% positive.

To the authors’ knowledge, this is the first study performed in Romania to assess A. vasorum prevalence in dogs.

No significant differences were discovered among the epidemiological factors studied and prevalence.

Prevalence or single report for Angiostrongylus vasorum in some European countries was noted by Traversa et al. (8) when data varied between 0.3 and 7.4%.

Majoros et al. (5) reported during the course of a helminthological survey of the dogs of Baranya County, Hungary Angiostrongylus vasorum infection was detected in two asymptomatic dogs.

Van Doorn et al. (9) noted that A. vasorum larvae were identified in the samples of four dogs (0.8%).

According to Morgan et al. (6), year, month, breed, sex, and whether were not significant predictors of positive tests.

Conclusions

This is the first report of Angiostrongylus vasorum infestation in canine

population in Romania. The results highlight the emerging nature of parasitism with A. vasorum in

dog in this area of the world and induce mandatory extension of research.

Acknowledgements

This study is published under the frame of European Social Fund, Human Resources Development Operational Programme 2007-2013, project No. POSDRU/159/1.5/S/132765 and this research work was carried out with the support of the project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR 2669.


81

References

1. Ansell Brendan, R. E., Schnyder, Manuela, Deplazes, Peter, Korhonen Pasi, K., Young, N.D., Hall, R. S., Mangiola, S., Boag, P. R., Hofmann, A., Sternberg, P.W., Jex, A.R., Robin, B., GasserInsights into the immuno-molecular biology of Angiostrongylus vasorum through transcriptomics-Prospects for new interventions, Biotechnology Advances, 2010, 31, 1486–1500.

2. Houpin, E., McCarthy, G., Ferrand, M., Waal, T. De, O’Neill, E.J., Zintl, A. Comparison of three methods for the detection of Angiostrongylus vasorum in the final host, Veterinary Parasitology, 2016, 220, 54–58.

3. Jolly, Sandra, Poncelet, L., Lempereur, Laetitia, Caron, Y., Bayrou, Calixte, Cassart, D., Grimmd, F., Lossona, B., First report of a fatal autochthonous canine Angiostrongylus vasorum infection in Belgium, Parasitology International, 2015, 64,97–99.

4. Kistler, Whitney M., Brown, J. D., Allison, A. B., Nemeth, Nicole M., Yabsley, M.J. First report of Angiostrongylus vasorum and Hepatozoon from a red fox (Vulpes vulpes) from West Virginia, USA, Veterinary Parasitology, 2014, 200, 216– 220.

5. Majoros, G., Fukár Orsolya, Farkas, R., Autochtonous infection of dogs and slugs with Angiostrongylus vasorum in Hungary, Veterinary Parasitology, 2010, 174, 351–354.

6. Morgan, E.R., Jefferies, R., van Otterdijka, L., McEniry, R.B., Allen, F., Shaw, M., Bakewella, S.E., Angiostrongylus vasorum infection in dogs: Presentation and risk factors, Veterinary Parasitology, 2010, 173, 255–261.

7. Mozzer, L.R., Lima, W.S. Gallus gallus domesticus: Paratenic host of Angiostrongylusvasorum Veterinary Parasitology, 2015, 207, 81–84.

8. Traversa, D., Di Cesare, A., Conboy, G., Canine and feline cardiopulmonaryparasitic nematodes in Europe: emerging and underestimated. ParasitesVectors, 2010, 3, 62.

9. van Doorn, Deborah C.K., van de Sande, Angelique H., Nijsse, E. R., Eysker, M., Ploeger, H. W., Autochthonous Angiostrongylus vasorum infection in dogs in The Netherlands, Veterinary Parasitology, 2009, 162, 163–166

10. Willesen, J. L.. Jensen, A. L., Kristensen, Annemarie T., Koch J., Haematological and biochemical changes in dogs naturally infected with Angiostrongylus vasorum before and after treatment, The Veterinary Journal, 2009, 180, 106–111.


82

TESTING THE MICROFILARICIDAL EFFECTIVENESS OF SOME DRUG FORMULATIONS IN TREATMENT OF DOGS NATURALY

INFESTED WITH DIROFILARIA REPENS

M. S. ILIE, MIRELA IMRE, IONELA HOTEA, I. OPRESCU, NARCISA MEDERLE, S. MORARIU, GH. DĂRĂBUȘ, K. IMRE

Banat’s University of Agricultural Sciences and Veterinary Medicine „King Michael I

of Romania” from Timisoara, 119, Calea Aradului, 300645, Timisoara, Romania, Phone: 0256277190


Summary

The filarial nematode Dirofilaria (Nochtiella) repens is the etiologic agent of canine

and feline subcutaneous dirofilariosis, and mosquito-borne disease that can affect humans. Epidemiological surveillance studies lately have shown a continuous increase in the

prevalence of subcutaneous dirofilariosis, both in our country and in the European countries. The study was conducted to test the microfilaricidal efficacy (curative) and safety in

case of infestation with D. repens of two licensed drug formulations for the treatment of

various endo - and ectoparazitosis in dogs (imidacloprid 10% / moxidectin 2.5% spot-on and ivermectin injection) after a single administration and monitoring of their effect for 90 days.

The study included a total of 32 dogs of different ages, races and both sexes with circulating microfilariae of D. repens.

The samples were analysed for the microfilariae presence by Knott modified test, interspecific differentiation was made on the basis of the morphological characters.

The identification of species was confirmed by diagnostic multiplex-PCR method to yield amplicons of approximately 300 base pairs from the gene amplification of a specific region of 12S rDNA.

The results of the study confirm the effect of the combination of imidacloprid - moxidectin on microfilariae of D. repens, demonstrating a high degree of efficiency (microfilaricidal effect) of 100% for 30 days after administration, and 83.3% during days 30 and 90 post-treatment.

Regarding the microfilaricidal effect on D. repens of ivermectin a degree of efficacy of 100% was observed in the first three weeks post therapy, 75.0% on day 30 from the start of treatment and 12.5% on day 60 post administration.

Key words: Dirofilaria repens, dogs, therapy, effectiveness

Nematode Dirofilaria (Nochtiella) repens is the ethiologic agent of subcutaneous dirofilariasis in dogs and cats. The development of sexually mature worms in the subcutaneous tissue of the definitive hosts can take from six to eight months, whereupon adults release microfilariae (larvae) in peripheral blood. Epidemiological surveillance studies lately have shown a continuous increase in the prevalence of heartworm subcutaneous, both in terms of our country and in the European countries.


83

Recital to minimize symptoms in dogs infected and reduce the risk of transmission to humans, treatment of infection with D. repens is considered under parasitological pilot control. Therefore, the most reliable method of control is considered removing subcutaneous circulating microfilariae in infected dogs to avoid transmission to vectors during blood outlets. In this sense, lately has been designed a series of injectable drug formulations or "spot-on" with microfilaricidal and adulticide effect, which are based on active substances from ivermectin group or macrocyclic lactones (1, 4).

Most often, the choice of the drug by a veterinarian or owners is based on analysis of "cost - benefit" and on the principle of the use of active substances with a minimal number of administrations and a long term effect.

The study was conducted to test the microfilaricidal effectiveness (curative) and safety in case of infestation with D. repens, of two licensed drug formulations for the treatment of various endo - and ectoparazitoze in dogs after a single administration and monitoring their effect for 90 days.


The study included a total of 32 dogs of different ages, breeds and both

sexes with circulating microfilariae of Dirofilaria repens. The animals in the study were part of the street dogs category (n = 21) brought by the Faculty of Veterinary Medicine for the sterilization campaign organized in the city of Timisoara; dogs housed in shelters canine group (n = 9); and dogs with owners (n = 3) breed under different conditions that were presented for routine consult to the University Veterinary Clinics.

On the first day of the investigation with the consent of each owner, managers and veterinarians from kennels and based on the principles of ethics and animal welfare, from each dog in the study were collected blood samples by puncturing the forearm vein in sterile vacutainer with acid - ethylene - diamine - tetraacetic acid (EDTA).

The samples were analysed through the modified Knott's test for highlighting the microfilariae, the differentiation being made on the basis of inter-specific morphological traits (3). In each case the level of microfilaremia was stirred for 1.0 ml of blood with EDTA. Larvae of D. repens, with size between 300 and 400 micrometres occurred in optical microscopy, cephalic part rounded and the distal part in umbrella handle shaped. Also, in all cases, the identification of species was confirmed by multiplex-PCR method, obtaining amplicons of approximately 327 base pairs trough the amplification of a specific region of 12S rDNA gene.

The dogs included in the study were randomly assigned into three groups. Group I included 12 mixed-breed dogs with age between 8 months to 10

years old, of different sexes (seven males and five females) and weight between 8.0 and 32.0 kg. The animals in this group were treated once with a combination of imidacloprid 10% / moxidectin 2.5% spot-on (Advocate®, Bayer) in dose of 10 mg


84

moxidectin imidacloprid and 2.5 mg per kg body weight according to manufacturer's recommendations.

Group II included eight dogs from one breed (Beagle) and seven crossbreed, with age between 1 to 12 years old, of different sexes (3 females and 5 males) and weigh between 10 and 30 kg. To this group it was administered a single injection of ivermectin, one time (Dufamec®, DutchFarm) in dose of 0.2 mg / kg according to the manufacturer's recommendations.

Group III or control group consisted of 12 dogs placebo-treated with a mixture of distilled water and sugar spot - on. The dogs in this group were crossbreed with ages between 2 to 8 years old, of different sexes (five females and three males) and weight between 5 and 20 kg.

On zero day of the study, each animal joined the experiment, and depending on the group which belonged, were administered the drug which was being tested. Overall, the study lasted for 90 days for group I and III and 60 days for group II. After treatment, in order to quantify the microfilaricidal effectiveness of the substances tested it were collected individual blood samples again in the morning of days 7, 14, 21, 30, 60 and 90 of the experiment.

These days the biological material was again taken to be tested for evidence of microfilariae by direct exam between slides and modified Knott's test. To confirm the diagnosis, samples with microfilariae, were examined by PCR.

During the study the dogs general condition has been monitored, especially in the first 4 hours after the drug administration. It has pursued, especially side effects occurrence. Also a basic criterion in the design of the study was introducing animals with microfilariae which were not treated with any of the substances tested prior three months.

The evaluation criterion of the microfilaricidal effect of drug substances was quantified by calculating the degree of effectiveness (GE) (13). GE was represented by the percentage of free microfilariae dogs in the days of the investigation within three months of observation from the seventh day of drug administration. GE was calculated for each study group using the following formula: GE% = [(percentage positive dogs in the control group - the percentage of positive dogs in the study group) / percentage of positive dogs in the control group] x 100.

Statistical analysis was performed using Statistical Calculator (StatPac® Inc., Bloomington, MN, USA). Differences between drug efficacy on days between groups were analysed using TETS - t nonparametric between percents and were considered significant when p ≤ 0.05.


The results of microfilaremia evaluation in dogs from Group I of study,

treated with a combination of imidacloprid 10% / 2.5% moxidectin spot-on (Advocate®, Bayer), each days of the investigation are presented in the following. Before treatment all 12 dogs included in the experiment shown microfilaremia


85

values of 30 to 4000 microfilariae / ml of blood. On days 7, 14, 21 and 30 after drug administration, were negative for microfilariae. Instead, two months after the treatment a dog presented microfilariae of D. repens in peripheral blood (75 MF / ml) by Modified Knott's test, this case being considered as a relapse. Continuing with the same dog, the presence of microfilariae was maintained and the next date of investigation (day 90 with 540 MF / ml). In all cases, the presence of microfilariae was molecularly confirmed. During all days of investigation at dogs from the control group microfilaremia was kept observing slight variations on their number. Therefore, the degree of effectiveness of imidacloprid 10% / 2.5% moxidectin (Advocate®, Bayer) combination administered as spot-on formulation, in the natural infection with D. repens, was calculated as 100% for up to 59 days after administration, and 83.3% on days 60 and 90 post administration.

Microfilaricidal effect of the combination imidacloprid 10% / moxidectin 2.5% on D. repens larvae has been tested by other authors. Like our study, Traversa et al. (2011) (6) in a study conducted on 18 dogs which were tested for the effect of a single administration of microfilaricidal Advocate® (Bayer), reported a level of 88.9% efficacy of the product associated with an intensity effectiveness 99.6 %, on a monitoring period of six months. Athough from all tested dogs, 16 were found negative for microfilariae throughout the study. Two dogs became positive for microfilariae after at least 60 or 90 days from treatment.

In another investigation Paran Svobodová (2011) (5) evaluated the microfilaricidal effect in subcutaneous Dirofilariasis of the imidacloprid - moxidectin combination spot-on on 11 dogs. Treatment was administered monthly for four months, with examination after each application, after which the presence of microfilariae was watched another six months. All dogs became negative for microfilariae after the second administration. This state was maintained for eight more months.

The research conducted by Fok et al. (2010) (2) aimed the elimination of microfilariae by D. repens in a number of 44 dogs who received one treatment with the product Advocate® (Bayer) spot-on by various schemes. Thus, five dogs received the drug that was administered once per month for three months, at 22 dogs once per month for six months and 17 dogs each two weeks for 6 months. Microfilaremia quantification was performed once per month and six months after the last administration. Two weeks after the first treatment, 38 of 44 dogs were microfilaremic negative. Four weeks after the initial treatment, one dog still showed a reduced rate of microfilaremia. After the second administration all treated dogs have become free of microfilariae. This status was maintained for a further six months period. These data demonstrates that the combination of imidacloprid - with application of moxidectin spot - on successfully eliminates long-term microfilariae of D. repens. Moreover, it has been hypothesized that this product has a 100% adulticides effect, based on the supposition that there were no reported microfilariae in the peripheral blood for six months after the last administration. The results of microfilaremiae evaluations in dogs (n = 8) from group II of study, which


86

received a single administration of injectable ivermectin (Dufamec®, DutchFarm) in dose of 0.2 mg / kg per each day of investigation are presented in the following. Before the treatment the eight dogs included in the experiment had microfilaremiei values from 100 to 11,000 microfilariae / ml of blood. On days 7, 14 and 21 after drug administration, the dogs were found negative for microfilariae. Instead, one month after treatment two dogs had microfilariae of D. repens in peripheral blood (200 MF / ml respectively 320 MF / ml) by Modified Knott's test, this case being considered as a relapse. Further, on day 60 of the investigation in seven from eight dogs was found the presence in the peripheral blood of microfilariae, microfilaremia ranging from 150 to 2,500 MF / ml.

In all cases, the presence of microfilariae was confirmed molecularly. During all days of investigation at dogs in the control group microfilaremia was kept observing slight variations on their number.

Therefore, the degree of efficacy of ivermectin injectable (Dufamec®, DutchFarm) as subcutaneous drug formulation in the natural infection with D. repens, was calculated as 100% for up to 21 days from the administration, 75.0 % on day 30 from the start of treatment and 12.5% on day 60 post administration.

In literature, studies with similar design are rare and for this reason have not been made comparisons with other results.

Conclusions

The results of the study confirm the effect of the combination imidacloprid -

moxidectin against microfilariae of D. repens, demonstrating a high degree of efficiency at the same time (microfilaricidal effect) 100% for 30 days after administration, and 83.3% between days 30 and 90 post-treatment.

The study results also confirm indirectly that a single treatment with a combination of imidacloprid - moxidectin does not guarantee a 100% adulticide effect, as demonstrated by the presence of microfilariae in the peripheral blood of a dog from a total of 12.

The results of the study on the microfilaricidal effect on larvae of D. repens of ivermectin showed a degree of efficiency of 100% in the first three weeks after administration, 75.0% in day 30 after the start of the treatment and 12.5% at day 60 post administration.

In summer period, the cost benefit analysis on the microfilaricidal effect of D. repens parasitism between two tested drugs recommended formula based on ivermectin as most advantageous combination than imidacloprid - moxidectin, with administration every three weeks.


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Acknowledgements

This study is published under the frame of European Social Fund, Human Resources Development Operational Programme 2007-2013, project No. POSDRU/159/1.5/S/132765 and this research work was carried out with the support of the project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR 2669.

References

1. Cancrini, G, Tassi, P, Coluzzi, M., Ivermectin against larval stages of

Dirofilaria repens in dogs. Parassitologia, 1989 ;31 (2-3): 177-82. 2. Fok, É., Jacsó, O., Szebeni, Zs., Győrffy, A., Sükösd, L., Lukács, Z.,

Schaper, R., Elimination of Dirofilaria (syn. Nochtiella) repens microfilariae in dogs with monthly treatments of moxidectin 2.5%/imidacloprid 10% (Advocate®, Bayer) spot-on. Parasitol. Res., 2011, 106, 1141–1149.

3. Genchi, C., Guerrero, J.J., Mccall, W., Venco, L., Epidemiology and prevention of Dirofilaria infections in dogs and cats, Mappe Parassitologiche, Ed. Rolando, Italia, 2007.

4. Mccall, J.W., Genchi, C., Kramer, Lh., Guerrero, J., Venco, L., Heartworm disease in animals and humans. Adv. Parasitol., 2008, 66,193 – 285.

5. Paran, R.D., Svobodová, V., Effect of therapy by using Advocate spot-on combination (Imidacloprid 10% and Moxidectin 2.5%) on subcutaneous dirofilariosis in dogs. Vet. Med. Int., 2011, Vol. 2011 Article ID 482746.

6. Traversa, D., Asteb, G., Di Cesarea,A., Paoletti, B., Di Tommaso, Moreno, Di Giulioc, E., Pampurinid, F., Tunesid, C., Boarib, A., Efficacy of a single administration of a spot-on solution containing imidacloprid 10%/moxidectin 2.5% in eliminating Dirofilaria repens microfilariae in naturally infected dogs. Vet. Parasitol., 2011, 179, 107 – 112.


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MINIREVIEW OF THE EUROPEAN SURFACE WATER DISTRIBUTION OF G. DUODENALIS GENOTYPES:

FROM 2005 TO NOWADAYS

K. IMRE, CLAUDIA SALA, ADRIANA MORAR, M.S. ILIE, MIRELA IMRE, S. HORA, CORINA BADEA, G. DĂRĂBUŞ


of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Aradului Street No. 119, Timisoara, Romania E-mail: [email protected]

Summary

The current work summarizes the results of epidemiological investigations aimed to detect Giardia spp. in surface water samples in different European countries. Mainly, results of molecular characterizations of the obtained Giardia isolates according to the tested water

type, and prevalence values obtained through microscopy (IFAT) and molecular biology (PCR) methods and results of genotyping are presented. A total of 14 scientific papers retrieved in the biomedical literature, providing from 10 countries, were selected and processed. The results showed that all tested water types (sewage, waste, drinking, recreational and river) can harbor viable infected forms of Giardia cysts and the frequency of isolation rate of the parasite have been markedly varied within countries and regions. The dominance of zoonotic assemblages A and B, beside a low number of assemblages E, underlined that human and livestock wastewater can represent the major pollutants of surface waters. Likewise, the lack of the pets and wildlife adapted assemblages such as C or D suggest that these animals plays a minor or neglected role in the contamination of surface waters. The awareness of the relevance of monitoring the surface water contamination with Giardia cysts by water and public health authorities using molecular tools, plays a crucial

role in the comprehensive understanding of circulation of this waterborne parasite in the natural environment beside correct evaluation of the risk that is poses for humans and animals. Key words: surface water, Giardia, Europe

General considerations

Species of the genus Giardia are recognized as flagellated ubiquitous

protozoan parasites. They are commonly diagnosed at gastrointestinal level, as one of the most common causes of protozoan diarrhea in many vertebrate species, including humans. Out of at least six species of the genus (G. agilis, G. ardeae, G. duodenalis, G. microti, G. muris, G. psittaci), only Giardia duodenalis (synonym with G. intestinalis and G. lamblia), which contains eight distinct genotypic assemblages from A to H, distributed in different hosts, infect humans (8). Epidemiological surveys showed that the assemblages A and B have been predominantly isolated from the human infections, and also have been detected in


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a considerable number of water samples. These two zoonotic assemblages differs from each other by as more then as 20% at the DNA sequence level. The other assemblages like C and D have been tipically identified in dogs; or the assemblages E, F and G have been found in pigs, cats and rodents, respectively (8, 16). The disease it causes, giardiosis, can occur asymptomatic or symptomatic as consequence of oral ingestion of Giardia contaminated food or water. The most common symptoms enrolled chronic diarrhea, malabsorption, weight loss, weakness and fatigue (6).

Generally, water is considered an excellent environment for the survaillance and spreading of the transmissible stages of the parasite, namely cysts (8, 16). Surface water supplies may easely become contaminated with infected stages of the parasites providing from human wastewaters, livestock, wildlife and aquatic birds (12). Consequently, this aspect, in regions or countries (i.e. Belgium) where a much part of drinking water is obtained through treatment of surface water, there are a constant threat on drinking water safety (7).

For the greater understanding of the epidemiology of Giardia infections, the using of molecular biologic methods, and especially the genotyping, is considered to be the most reliable tool in attempts to differentiate between animal and human origin cysts. In order to obtain a more comprehensive understanding of Giardia cysts circulation in European surface waters, the present review aimed to process and incorporates representative studies available in the biomedical literature focused on the detection of G. duodenalis and conducted in as many countries.

European surface water spreading of G. duodenalis and his assemblages

Several studies investigated the presence of Giardia spp., as major public

health concern, in different surface water types of Europe. Results of epidemiological investigations aimed to detect and molecular characterize of the obtained Giardia isolates, conducted in the last decade at the European countries level, are summarized in the Table 1.

In a Belgian study conducted by Ehsan et al. (7), a total of 53 microscopy-positive water samples, providing from monthly collected raw and purified drinking water at four catchment sites, during a period of two years, were molecularly processed. Out of them, 38 (71.7%) were successfully sequenced showing a high genetic diversity. The identified genotypes were G. duodenalis A-I (in 9 samples), G. duodenalis A-II (n=8), G. duodenalis E (n=10), G. duodenalis B-IV (n=2) and G. duodenalis B-IV-like (n=1), respectively. No cysts were detected in purified water samples. Based on the high contamination rate of raw surface water samples (92 % positivity), the study results suggests that water contamination is mainly due by runoff from farms and fields during agricultural activities than human waste contamination (7).


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Table 1 Frequency of isolation and diversity of G. duodenalis assemblages in

different tested water types from the European countries level

Country

Type of the tested water

Processed / molecularly confirmed Giardia

positive samples (%)

Genotypes of G. duodenalis

References

Belgium

raw and purified drinking water

drinking

4§(*53)/38 (71.7) genotype A-I (9)

genotype A-II (8) genotipu B-IV (2) genotype B-IV (1) genotype E (10)

(7)

Finland river, residual 71/20 (28.2) genotype B (3) (10)

Hungary drinking, sewage, recreational 36(*24)/13 (36)

genotype A (7), genotype B (1), genotype A + B (4)

(14)

Italy

filtered, fountain 42(*11)/4 (9.5) genotype A genotype B (13)

effluent 42(*23)/23 (14.3)

genotype A (15), genotype B (2),

(9)

Luxembourg

recreational, drinking

91(*26)/7 (7.8) genotype A, genotype B

(11)

Poland 5 natural water bodies

5§/4 (80) genotype B (4) (1)

Portugal Untreated surface 175(*67)/9 (5.1) genotype A-I (12)

Norway sewage

40/34 (85%) genotype A (32) genotype B - (3)

(15)

Spain (Galicia)

waste influent

12§/12

genotypes A-I (2) genotype A-II (3) genotype A-I + E (5) genotype A-II + E (2)

(4) waste effluent

12§/12

genotype A-I (2) genotype A-II (3) genotype A-I + E (5) genotype A-II + E (2)

drinking influent 16

§/16

genotype A-II (1) genotype A-I + E (2) genotype A-II + E (3) (5)

drinking effluent 16

§/16

genotype A-I + E (1) genotype A-II (2)

river

29§/17 (58.6)

genotypes A-I (2) genotype A-II (4) genotype A-I + E (4) genotype A-II + E (5) genotype E (2)

(3)

waste 12

§/3 (25%) genotype A-II (5)

genotype A-I + E (2) genotype A-I + A II (1) genotype A-II + E (2)

(2) drinking 12

1/12 (100%)

untreated surface

127(*76)/56 (44.0) genotypes A-I (15) genotype A-II (5) genotype E (21)

(6) drinking

127(*58)/43 (33.8) genotypes A-I (14) genotype A-II (4) genotype E (16)

Legend: * - microscopically positive samples; § – the number meaning investigated

sampling points and not processed samples


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In order to assess the Giardia contamination level of the Vantaa river basin in Finland, Hänninen et al. (10) carried out investigations at four wastewater treatment plants, in downstream river water samples and in river mussels (A. piscinalis). The results showed that all enrolled sampling sites were commonly contaminated by a low numbers of cysts. Thus, influent samples of wastewater plants (n=12) were positive for Giardia cysts by IF microscopy, and six of these were successfully PCR amplified. In the downstream river water samples two (2/20) were Giardia cysts positive using both detection methods. Regarding the accumulation of cysts in A. piscinalis positive results using microscopy were reported for 10 out from the 16 depuration samples and four tissue samples. At the same time, positive results were reported in 5 PCR processed samples. The identified low genetic diversity of Giardia isolates consists in the presence of the only one genotype, namely group B (10).

In Hungary, of the 36 analyzed water samples collected from different water sources (drinking water, sewage treatment plants, recreation area of the Lake Balaton) and various geographic areas, 24 (67%; 10 raw, 10 sewage 4 surface and 1 beach water samples) were Giardia positive by IFT and 13 (36%; 5 raw, 7 sewage and 1 surface) by PCR, respectively. Successfully sequencing of twelve isolates showed the presence of G. lamblia assemblage A in seven samples, and assemblage B in one samples, respectively. In four cases the simultaneous detection of the two assemblages has been recorded (14).

The presence of Giardia cysts in filtered water before and after plant overhaul and in well water has been recorded in Italy by Lonigro et al. (13). The overall registered prevalence value through IF and PCR was 26.2% (11/42) and 19.0% (4/21), respectively, and the sequencing procedure showed the presence of G. duodenalis assemblage A (n=3) and B (n=1). In another study conducted in the same country by Giangaspero et al. (9), 23 out from 42 water samples, including water channel outlets from wastewater treatment plants and from the water channel inlets into the Lagoon, were Giardia positive through IF as well as PCR. Successfully genotyping of 17 G. duodenalis isolates showing the dominance of assemblage A (n=15) beside assemblage B (n=2). Similar distribution of G. duodenalis subtypes has been reported in Norway by Robertson et al. (15) processing 40 different water samples from sewage treatment plants. The results showed a positivity of 85% (34/40) for G. duodenalis, and out from positive samples 32 (89%) were considered to contain Giardia cysts from assemblage A (with genotypes A3 -17 samples and genotype A2 - 10 samples) and three samples from assemblage B (genotype B3) (15).

Two-year monitoring of the occurrence of G. duodenalis in Luxembourg showed a positivity of 41.3% (26/63) in recreational water samples collected from three sites and the absence of the parasite in drinking water samples (n=28) using standardized microscopy technique. The qPCR technique analysis of the microscopically positive samples showed positive results in only seven (27%) matrix, but without capacity to distinguish the isolates at genotype level (11).


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The analysis of five water bodies in Poland showed contamination with G. duodenalis in four catchments, with identification of three variants of the assemblage B (1). The occurrence of Giardia cysts has been monitored in Portugal by Lobo et al. (12) in a total of 175 water samples including raw surface and ground (n=69), treated (n=67)and finished water (n=39) samples, meaning 106 treated and 69 untreated samples. Overall, 67 (38.3%) samples were Giardia positive by IFA, and cysts were detected in 58.0% (40/69) and 25.6% (27/106), of raw and treated water samples, respectively (12). Subtyping has been successfully done in only one sample and the identified genotype was G. duodenalis subtype A1.

Several studies conducted in Galicia, North-Western of Spain, confirmed the widely occurrence of G. duodenalis in different type of water, beside remarkable genetic diversity of the identified genotypes. All available scientific papers have been achieved by a research team coordinated by Dr. Castro-Hermida. In a study conducted during a period of one year the presence of G. duodenalis in the influent and final effluent of sixteen drinking water treatment plants, situated in the hydrographic basin of Galicia, has been evaluated. The results showed that Giardia cysts were present in both influent and effluent of all tested plants. The identified assemblages of G. duodenalis were A-I, A-II and E, alone or under different combination forms (Table 1), highlighting the necessity to develop adequate countermeasures for the safety of drinking water (5). In another study, the contribution of the treated wastewater to the contamination of recreational areas and fluvial beaches with G. duodenalis in North-Western of Spain has been investigated. The parasite was detected throughout the year, in all influent and effluent samples, from all treatment plants and the identified assemblages were A-I (n=2, 16%), A-II (n=3, 25%), A-I+E (n=5, 41 %,) and A-II+E (n=2, 16%). The results pointed out a considerable risk of contamination of water courses in the screened region (4). In the same region, evaluation of the health risk for humans and animals has been carried out in the main basin of river Tambre, from 29 sampling points including recreational areas and the mouth of secondary rivers, processing 116 water samples. G. duodenalis cysts were microscopically detected in 78 (67.2%) samples from all sampling points. Molecular identification of the parasite using DNA sequencing has been successfully achieved in 17 points (58.6%) and revealed the presence of the following assemblages: A-I (n=2, 11.8 %), A-II (n=4, 23.5 %), A-I + E (n=4, 23.5 %), A-II + E (n=5, 29.4 %) and E (n=2, 11.8 %), respectively (3). Similarly, the water environment dispersal of the same assemblages under different combination forms (Table 1) in 12 drinking and wastewater treatment plants has been recorded in two areas (coastal and inland) of Galicia. In the coastal area the presence of G. duodenalis cysts was recorded in influent and effluent samples from 3/12 (25 %) of drinking water and 12/12 of wastewater treatment plants (2). Recently, the widespread water occurrence of G. duodenalis cysts, and the largely ineffective treatment in reducing/inactivating of this pathogen in drinking water destined for animal and human consumption, has been demonstrated in another study (6). Thus, in untreated water samples G.


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duodenalis was detected in 76 samples (59.8%) by IFAT and in 56 samples (44.0%) by PCR. In treated drinking water the pathogen detection has been successfully done in 58 samples (45.6%) by IFAT and in 43 samples (33.8%) by PCR. The assemblages A-I, A-II, E of G. duodenalis were recorded without any combination forms (see Table 1).

Conclusions

The overview of the European surface water contamination with Giardia

cysts pointed out that all tested water types can harbor viable infected forms of the parasite and the frequency of isolation rate has been varied markedly between countries and within regions. Genetic characterization of the isolates showed that mainly the assemblages A and B, known to be infective for humans with zoonotic character, beside a low number of assemblages E with predominantly animal origins, has been present. These results have greatly contributed to the understanding of molecular epidemiology of water origin Giardia infections, highlighted that the human and livestock wastewater can represent the major pollutants of surface waters being a threat for safety of drinking water. Furthermore, the lack of the pets and wildlife adapted assemblages such as C or D suggest that these animals plays a minor or neglected role in the contamination of different surface water types. Last but not least, the awareness of the relevance of monitoring the surface water contamination with Giardia cysts or other zoonotic parasites (i.e. Cryptosporidium, Toxoplasma) by water and public health authorities using molecular tools, plays a crucial role in the comprehensive understanding of circulation of waterborne parasites in the natural environment beside correct evaluation of the risk that is poses for humans and animals.

Acknowledgements

This work was supported by a grant of the Romanian National Authority for

Scientific Research and Innovation, CNCS – UEFISCDI, project number PN-II-RU-TE-2014-4-1300.

References

1. Adamska, M., Molecular characterization of Cryptosporidium and Giardia

occurring in natural water bodies in Poland. Parasitol. Res., 2014, 114, 687-692.

2. Castro-Hermida, J.A., García-Presedo, I., Almeida, A., González-Warleta, M., Correiada Costa, J.M., Mezo, M., Cryptosporidium spp. and Giardia duodenalis in two areas of Galicia (NW Spain). Sci. Total. Environ., 2011, 409, 2451–2459.


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3. Castro-Hermida, J.A., García-Presedo, I., Almeida, A., González-Warleta, M., Correiada Costa, J.M., Mezo, M., Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: a health risk for humans and animals. Water Res., 2009, 43, 4133–4142.

4. Castro-Hermida, J.A., García-Presedo, I., Almeida, A., González-Warleta M., Correiada Costa, J.M., Mezo, M., Contribution of treated wastewater to the contamination of recreational river areas with Cryptosporidium spp. and Giardia duodenalis. Water Res., 2008, 42, 3528–3533.

5. Castro-Hermida, J.A., García-Presedo, I., Almeida, A., González-Warleta, M., Correiada Costa, J.M., Mezo, M., Presence of Cryptosporidium spp. and Giardia duodenalis through drinking water. Sci. Total. Environ., 2008, 405, 45-53.

6. Castro-Hermida, J.A., González-Warleta, M., Mezo, M., Cryptosporidium spp. and Giardia duodenalis as pathogenic contaminants of water in Galicia, Spain: The need for safe drinking water. Int J Hyg Environ Health, 2015, 218,132-138.

7. Ehsan, A., Geurden, T., Casaert, S., Paulussen, J., De Coster, L., Schoemaker, T., Chalmers, R., Grit G., Vercruysse, J., Claerebout, E., Occurrence and potential health risk of Cryptosporidium and Giardia in different water catchments in Belgium. Environ. Monit. Assess., 2015, 187, 2, 6.

8. Feng, Y., Xiao, L., Zoonotic potential and molecular epidemiology of Giardiaspecies and giardiasis. Clin. Microbiol. Rev., 2011, 24, 110–140.

9. Giangaspero, A., Cirillo, R., Lacasella, V., Lonigro, A., Marangi, M., Cavallo, P., Berrilli, F., Di Cave, D., Brandonisio, O., Giardia and Cryptosporidium in inflowing water and harvested shellfish in a Lagoon in Southern Italy. Parasitol. Int., 2009, 58, 12-17.

10. Hänninen, M.L., Hörman, A., Rimhanen-Finne, R., Vahtera, H., Malmberg, S., Herve, S., Lahti, K., Monitoring of Cryptosporidium and Giardia in the Vantaa river basin, southern Finland. Int. J. Hyg. Environ. Health, 2005, 208, 163-171.

11. Helmi, K., Skraber S., Burnet, J.B., Leblanc, L., Hoffmann, L., Cauchie, H.M., Two-year monitoring of Cryptosporidium parvum and Giardia lamblia occurrence in a recreational and drinking water reservoir using standard microscopic and molecular biology techniques. Environ. Monit. Assess., 2011, 179, 163-175.

12. Lobo, M.L., Xiao, L., Antunes, F., Matos, O., Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal. Lett. Appl. Microbiol., 2009, 48, 732–737.

13. Lonigro, A., Pollice, A., Spinelli, R., Berrilli, F., Di Cave, D., D’orazi, C., Cavallo P., Brandonisio O., Giardia cysts and Cryptosporidium oocysts in membrane- filtered municipal wastewater used for irrigation. Appl. Environ. Microbiol., 2006, 72, 7916-7918.


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14. Plutzer, J., Karanis, P., Domokos, K., Törökné, A., Detection and characterization of Giardia and Cryptosporidium in Hungarian raw, surface and sewage water samples by IFT, PCR and sequence analysis of the SSUrRNA and GDH genes. Int. J. Hyg .Environ. Health, 2008, 211, 524-533.

15. Robertson, L.J., Hermansen, L., Gjerde, B.K., Ocurrence of Cryptosporidium oocysts and Giardia cysts in sewage in Norway. Appl. Environ. Microbiol., 2006, 72, 5297–5303.

16. Thompson, R.C.A., Ash, A., Molecular epidemiology of Giardia and Cryptosporidium infections. Inf. Gen. Evol., 2016, In press.


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ATTEMPTING TO SOLVE THE PARASITISM WITH DERMANISSUS GALINAE IN LAYING HENS

C. LĂZĂRESCU, CRISTINA GAŞPAR, I. ȚIBRU

Banat’s University of Agricultural Science and Veterinary Medicine Timisoara “King Michael I

of Romania”, Faculty of Veterinary Medicine, 300645, Calea Aradului, No 119, Timisoara, Romania


Summary

The aim of this study was to evaluate the efficacy of saturated vegetable fats incorporated in a soap, on a population of Dermanyssus gallinae (De Geer, 1778) (Acari: Dermanyssidae) (the poultry red mite), based on the property of the fat to obstruate the respiratory stigmas of the mites. It was appraised also the effect of the soap on the laying hens.

The impact on birds was assessed in the farm, by spraying them with a 3% concentration solution and the efficacy of the product on the mites has been tested in the

laboratory, using three solutions of different concentrations (2.5%, 5%, 7.5%).

In the farm, it was found that the soap didn’t had negative effects on the birds health, behavior and production, but in those which weren’t sprayed, it was found a decrease in egg production.

After the tests taken place into the laboratory, we have observed that the number of mites has decreased proportionally with increasing the concentration of the solutions used. Key words: Dermanyssus gallinae, saturated vegetable fats

The infestation with Dermanyssus gallinae (De Geer, 1778), is considered

to be one of the most important health and welfare problems in intensive farming of poultry and especially for laying hens (5), that is also generating large economic losses worldwide (4). These losses are generated by decreasing in laying percentage by 10 to 15%, thickening of the eggshell (3), decreased grow rates and increased mortality, by the toxic action, mechanical-irritative action, by producing anemia (7) and especially by the inoculating action: Salmonella spp., Spirocheta spp.(12, 6), Pasteurella spp., Yersinia spp. and also some viruses (6).

A characteristic of red mite infestation is that once installed in a flock, it is almost impossible to be eradicated (5). The most important factors in maintaining the parasitism in the farms, are represented by: the great resistance of adults to starvation (in the absence of the birds, they can survive hungry until 34-36 weeks) (10, 6), the resistance to acaricides, the lack of new acaricides and the restriction of using certain chemicals on food animals (1, 11).Therefore, alternative methods for the control of D. gallinae are urgently needed (2).

One of these alternative methods is based on the repellent and persistent toxic effects of some herbal extracts, especially of essential oils, against the poultry red mite (9).


97

Another method suggests the use of some entomopathogenic fungal strains (ex. Beauveria bassiana) isolated from naturally infected hosts, against Dermanyssus gallinae (8).

The purpose of this paper was to evaluate the efficacy of a detersive surfactant, containing specific soap fats, on a Dermanyssus gallinae population, by obstruating their respiratory stigmas. It was also aimed the impact of the product on laying hens.


The researches were done both in the laboratory and in a holding of laying hens from Timiș County, populated with 51.000 birds. The housing has 100 m lenght and the battery cages are arranged on five lines of seven levels each. In every cage are 50 hens.

It was used a detersive surfactant, obtained by saponification of vegetal saturated fatty acids with potassium hydroxide, to prepare three solutions of different concentrations (2.5%, 5%, 7.5%), using potable water. In the farm, for each spraying, there was used 1 l of 3% concentration solution, prepared with water from the local source.

There were prepared three large glass vessels with water. In each of them, was placed another smaller glass vessel, each containing one end of a wing with feathers, disarticulated at humero-radio-ulnar joint and one trap (piece of cardboard) containing mites.The large vessels were covered with gauze bandage in three layers in order to allow the parasites to breathe, but to prevent breaking out.

The solution was applied by fine spraying on the piece of wing, as well as on the traps, following a slight moistening of the feathers and insisting on pieces of cardboard. There have been applied three sprayings at intervals of 48 hours, with a volume of 30-35 ml from each of the three solutions taken into study.

After 24 hours from each spraying, above every small vessel was placed a piece of paper and before the next spraying, was pursued the presence of the mites on each sheet. After 7 days, the final evaluation on the efficacy of the product was made by counting the mites on each sheet. On every piece of paper, were marked randomly 10 squares of 1 cm side and there were counted the mites found in each perimeter.

In the farm, the birds on 2 levels (II, III) from one line were sprayed every 48 hours, three times.


In the laboratory, before each administration of the solution, it was monitored the presence of the mites on the covering paper sheets and has been found that as the concentration increases, the number of the mites drops. Also, on


98

each of the three sheets there has been found a decreased number of mites, as the number of administrations has increased.

There has been performed the arithmetic mean of the mites counted on each sheet and the following results were obtained:

1. Concentration 2.5% - 9,1 mites/cm2,

2. Concentration 5% - 3 mites/cm2,

3. Concentration 7.5% - 0,7 mites/cm2.

Conclusions

In laying hens from the holding, there haven’t been observed negative

effects on health, behavior and production, after performing three administrations of solution at a concentration of 3%, every 48 hours.

In laying hens that haven’t been sprayed, it was found a decrease in egg production.

After the tests taken place into the laboratory, it has been observed a decreasing in the number of mites proportionally with the increment of the concentrations of the solutions.

References

1. Beugnet, F., Chauve, C., Gauthey, M., Beert, L., Resistance of the red poultrymite to pyrethroids in France. Vet. Rec., 1997, 140, 577–579.

2. Birkett, M.A., Al Abassi, S., Kröber, T., Chamberlain, K., Hooper, A.M., Guerin, P.A.,Pettersson, J., Pickett, J.A., Slade, R., Wadhams, L.J.,Antiectoparasiticactivity of the gum resin, gum haggar, from the East African plant, Commiphoraholtziana. Phytochemistry, 2008, 69, 1710–1715.

3. Boch, J., Supperer, R., Veterinärmedizinische Parasitologie, Verlang Paul Parey, Berlin, 1992.

4. Bruneau, A., Denburg, A., Chauve, C., Zenner, L., First in vitro cycle of thechicken mite, Dermanyssus gallinae (De Geer, 1778), utilizing an artificialfeeding device. Parasitology, 2001, 123, 583–589.

5. Chauve, C., The poultry red mite Dermanyssus gallinae (De Geer: 1778):current situation and future prospects for control. Vet. Parasitol.,1998, 79, 239–245.

6. Cosoroabă, I., Parazitologie veterinară, Ed. Mirton, Timișoara, 2000. 7. Dărăbuș, Gh., Morariu, S., Oprescu, I., Mederle, Narcisa, Parazitologie și

boli parazitare, Ed. Mirton, Timișoara, 2006. 8. Immediato, D.,Camarda, A.,Iatta, Roberta, Puttilli, Maria Rita, Nascimento

Ramos, R. A., Di Paola, G., Giangaspero, Annunziata, Otranto, D., Cafarchia, Claudia, Laboratory evaluation of a native strain of Beauveria bassiana for controlling Dermanyssus gallinae (De Geer, 1778) (Acari: Dermanyssidae). Vet. Parasitol., 2015, 212, 478–482.


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9. Nechita, I. S., Poirel, M. T.,Cozma, V.,Zenner, L., The repellent and persistent toxic effects of essential oils against the poultry red mite, Dermanyssus gallinae. Vet. Parasitol., 2015, 214, 348–352.

10. Nordenfors, H., Hoglund, J., Uggla, A., Effects of temperature and humidity on oviposition, molting, and longevity of Dermanyssus gallinae (Acari: Dermanyssidae). J. Med. Entomol., 1999, 36, 68–72.

11. Sparagano, A., Pavlicevic, A., Murano, T., Lamarda, A., Prevalence and keyfigures for the poultry red mite Dermanyssus gallinae infections in poultry farmsystems. Exp. Appl. Acarol., 2009, 48, 3–10.

12. Sparagano, O.A.E., George, D.R., Harrington, D.W.J., Giangaspero, A., Significance and control of the poultry red mite, Dermanyssus gallinae. Annu.Rev. Entomol., 2014, 59, 447–466.


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ANTIMICROBIAL PROTOCOLS IN BOVINE RESPIRATORY DISEASE COMPLEX – A REVIEW

C.I. MATES, MARINA SPINU, CARMEN DANA SANDRU, EMOKE PALL,

CATALINA NICULAE, MIHAELA NICULAE

University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, 400372, Calea Mănăștur Street no 3-5, Cluj-Napoca, Romania


Summary

Bovine respiratory disease (BRD) is described as one of the most important causes of morbidity and mortality, leading to significant economic losses in cattle industry.

Given the major role of the bacterial pathogens within BRD polyfactorial etiology, the use of antimicrobial drugs has always represented the cornerstone of the therapeutical protocols although the clinical efficacy may be questionable. Also, evidence of an increased antimicrobial resistance characterising bacterial strains such as Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis and Arcanobacterium pyogenes isolated from lung tissues is pointed out by the great majority of retrospective studies, with several authors reporting discrepancy between the in vitro susceptibility testing results and the clinical response towards the antimicrobials used under field conditions.

The paper was aimed to perform a critical review of the literature, with data collected from both scientific articles and clinical reports or guidelines. This review lists the main antimicrobial groups (broad spectrum penicillins, tetracyclines, macrolides, fluoroquinolones, cephalosporines), their recommended uses of the antimicrobials for BRD control - therapeutic and preventive (metaphylactic and prophylactic), the categories based on the duration of the active tissue concentration and effect duration (daily regimen compared to long-acting or single- shot formulations), together with the particularities regarding the posology (on arrival administration, first days after the arrival) and withdraw period. Also, it underlines the significance of the responsable and prudent use of the antimicrobials.

Key words: bovine respiratory disease, antimicrobial drugs, antimicrobial

resistance

Bovine respiratory disease (BRD) is regarded worldwide as the major

cause of clinical disease and death in feedlot cattle (5,8,9,12,13,18,20). In case of USA feedlots, BRD accounts for 70–80% of all feedlot morbidity and 40–50% of all mortality (9), with similar data reported also by European countries and Australia (8,13). Given the importance, a multitude of studies was conducted to establish the main causes and the predisponding conditions associated with this pathology and currently a multifactorial etiology is accepted (5,12,20). Viruses and bacteria play an important role along with management conditions (8,14,20). A prospective longitudinal study performed in a population of Australian feedlot cattle indicated the role of several factors related to feedlot management and risk of BRD


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occurrence; pen water troughs accessed by animals in adjoining pens and animals added to pens over multiple days were indicated as conditions with increased risk of BRD (8). Since bacterial pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni) are associated with BRD (20), the antimicrobials represent a fundamental tool for the control, but also prevention of this pathology (13).

The paper was aimed to perform a critical review of the literature, with data collected from both scientific articles and clinical reports or guidelines.

Although the clinical efficacy may be sometime questionable, the use of antimicrobial agents in BRD is a common practice, and appears to include more active substances than for any other disease of cattle (5,7). Developed over a period of approximately twenty years, currently the list of these products includes numerous commercial formulas with indications for treatment and metaphylaxis of BRD based on the broad spectrum covering Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and/or Mycoplasma spp. and also bacterial pathogens responsible for mastitis, enteritis, arthritis. The main antimicrobial groups are represented by broad spectrum penicillins, tetracyclines, macrolides, fluoroquinolones, cephalosporins (7,19).

Also, it is important to mention that these antimicrobials are considered for both treatment and methaphylaxis of BRD, and several criteria are essential when selecting both the active substances and the commercial products. Among them, the following are often listed: in vitro susceptibility of the isolated bacteria(s), benefit cost-ratio, meat or milk withdrawal time, the route of administration, frequency of administration (single or multiple injections).

For the therapeutic use of antimicrobials in BRD, most protocols mention oxytetracyclines and spectinomycin as first choice, florfenicol and macrolides (tilmicosin, tulathromycin, spiramycin, tylosin) as second choice, leaving fluoroquinolones (enrofloxacin, danofloxacin, marbofloxacin) and third (ceftiofur) and fourth (cefquinome) generation cephalosporins as the last choice (2,4,7,13,19).

The methaphylactic use of antimicrobials is a management practice in stocker and feedlot production systems aimed to decrease the negative impact of BRD on cattle health and performance (1,15). While the aim and the benefits are clearly stated, there is no consensus regarding the possible relationship with the antimicrobial resistance augmentation (20). There are several protocols adapted to distinct production and age categories and also numerous factors to be considered when selecting the suitable protocol, such as: weight (age) of the cattle, distance traveled, environmental conditions during transportation, previous health history, visual inspection of the cattle at arrival (1,15).

The following table compiles the most important antibiotic based products recommended for BRD therapy with detailed information regarding the doses, route of administration and the withdrawal period (table 1).


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Table 1 Antimicrobial commercialy available recommended in case of BRD

Antimicrobial group

Active substance

Indication Route

Dose

Withdrawal time

Broad spectrum penicillin

Amoxicillin Gram - bacteria Pasteurella spp

IM/PO 10-15 mg/kg

meat 21 days milk 3 days

cephalosporins

Cefquinome P. multocida

M. haemolitica IM

2.5 mg/kg

meat 5 days milk 1 day

Ceftiofur M. haemolytica

Histophilus somni SQ/IM

3 mg/kg


fluoroquinolones

Danofloxacin M. haemolytica

P. multocida Histophilus somni

SQ 6

mg/kg meat 8 days milk 4 days

Enrofloxacin Klebsiella pneumoniae,

Haemophilus spp., Pasteurella spp

SQ/IV 2,5-5 mg/kg


Marbofloxacin P. multocida,

M. haemolytica Histophilus somni

IM/IV/SQ 2

mg/kg meat 4 days milk 1 day

fenicols Florfenicol M. haemolytica,


IM/SQ 20

mg/kg

meat 30 days for IM, 44 days for SQ

aminoglicosides

Gentamicin Susceptible bacteria IM/IV 4


Spectinomycin Pasteurella spp

Mycoplasma spp. IM

15 mg/kg

21 days

tetracyclines Oxytetracycline Pasteurella spp

Mycoplasma spp. IM/IV

20 mg/kg

meat 21 days for IM, 14 days for IV

milk 7 days for IM, 72 hours for IV

macrolides

Tilmicosin M. haemolitica P. multocida

SQ 10

mg/kg meat 60 days

Tilosin Mycoplasma spp. IM 5-10


Gamithromycin M. haemolytica,


SQ 6

mg/kg meat 64 days

Tulathromycin

M. haemolytica, P. multocida

Histophilus somni Mycoplasma bovis

SQ 2.5

mg/kg meat 22 days

Tildipirosin M. haemolytica,


SQ 4

mg/kg meat 47 days


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Differences in the antimicrobial spectrum (targetting the primary bacterial pathogens: Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni and/or Mycoplasma bovis), therapeutic dose, mode of administration and withdrawal period can be noticed. With respect to posology, as opposed to the first developed products, the newer ones belong to a different therapeutic concept - Single Injection described for new formulations of fluoroquinolones (marbofloxacin 100mg/ml, administered at a single dose of 8 mg/kg body weight IM and enrofloxacin 100 mg/ml, administered at a single dose of 7.5 mg/kg body weight SQ) and also for new generations of macrolides (tulatromycin and gamytromycin).These agents have significantly improved the outcomes of BRD prevention and treatment in feedlots by offering superior efficacy or convenience compared with older agents; among such advantages, the following aspects are to be taken into consideration: rapid absorption from the injection site, extensive distribution to tissue, and slow elimination, thus ensuring high, prolonged drug concentration at the site of infection - the lungs (4,7) and long acting or single dose administration (less time consuming for the vet and less stressful for the animals).

The in vivo efficacy is sometimes questioned, but several factors related to the therapy (the best moment to initiate it, the antimicrobial of choice, the duration of the treatment, the route of administration), the animal (severity of the clinical signs, other health issues and stress factors influence) and the bacterial strains (susceptibility, multidrug resistance, virulence factors) influence the outcome. In this last regard, widespread antimicrobial resistance to the primary therapeutic antimicrobial agents was investigated for most of the isolates associated with BRD, triggering an intense search for new molecules (20). Newer antimicrobials (ceftiofur, tilmicosin, tulathromycin, florfenicol, enrofloxacin, and danofloxacin) are preferred also due to the single shot formulation, but recent reports suggest the development of the antimicrobial resistance and the need for new products (2,5). The extent of the antimicrobial resistance is not clearly and homogeneously determined since the surveillance is not mandatory in this case, but several studies underlined the potential for emergence and dissemination of resistant clones for Mycoplasma bovis and Pasteurella spp. (3,10,14,20). Certain particularities related to Mycoplasma bovis complex pathology in cattle - chronic, debilitating health issues with respiratory signs, mastitis, otitis media, arthritis, and reproductive disease and the often reduced response to treatment – appear to be furthermore complicated by the augmentation of the minimum inhibitory concentrations for many of the commercially available antimicrobials reported for many of the current M. bovis European isolates (14,17). A retrospective survey of records from a single diagnostic laboratory pointed out for M. haemolytica pan-resistant isolates (resistant to 5 or more antimicrobials), with annual variations (over 5% isolates in 2009 and more than 35% in 2011. Significant antimicrobial coresistance patterns were reported for oxytetracycline and tilmicosin (12). All bovine strains of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, isolated from 2000 to 2009 proved to be susceptible to ceftiofur, 90% or more of M.


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haemolytica and H. somni isolates were susceptible to florfenicol, while susceptibility among P. multocida was 79% or greater, but less than 50% of the isolates tested were susceptible to tetracycline (17). Data provided by distinct studies may be contradictory when comparing the level and the traits of antimicrobial resistance. For isolates of M. haemolytica recovered from 29% of cattle (1,596/5,498), 13.1% at arrival and 19.8% at second sampling, the antimicrobial resistance analyzed using multivariable regression was characterized as rare, although nearly half of cattle received parenterally antimicrobial drugs, mostly as metaphylactic treatment at arrival (16). Similarly, 34/53 (64%) isolates of Histophilus somni obtained from clinically affected cattle in Queensland and New South Wales, Australia were susceptible to all antimicrobial agents tested (ceftiofur, enrofloxacin, florfenicol, tetracycline, tilmicosin and tulathromycin); the authors reported intermediate susceptibility to tulathromycin in 12 isolates, tilmicosin in seven isolates and resistance to tilmicosin in one isolate (6). A more recent review on the current status of antimicrobial resistance in pathogens associated with bovine respiratory disease (BRD) in beef cattle mentioned not only a relatively reduced number of studies evaluating the susceptibility of bacterial pathogens, but also fluctuating levels that should be further investigated, long-term monitored and analyzed based on standardized susceptibility test methods and BRD-specific interpretive criteria (3,11,13).

New antimicrobials not just as new molecules, but also as more efficient protocols are needed for the treatment of BRD (2, 20). A recent in vivo study indicated the efficacy of nasal instillation of a nitric oxide releasing solution (NORS) on Angus-cross heifers arrived at the feedlot. Based on the results reported as non-inferior to a parenteral injection of tilmicosin, a macrolide antibiotic, and also considering the nasal administration of NORS as a more rational control strategy compared to parental injection of antibiotics to prevent BRD in high-risk cattle, the authors concluded on the potential of nitric oxide, a molecule produced in most mammalian cells, with bactericidal and virucidal properties, as a valuable molecule that requires further studies (2).

Conclusions

The decision to initiate the use of an antibiotic should be done only by a

veterinarian that after consultation on the spot and based on an established diagnosis (results of bacterial culture and antimicrobial susceptibility testing) can determine the optimal treatment protocol for the given situation.

The use of antibiotics should be indicated by a veterinarian and should be carried on under its direct control as a responsible action aimed to limit the emergence and spread of the multidrug resistant bacteria.


105

References

1. Babcock, A.H, White, B.J., Dritz, S.S., Thomson, D.U., Renter, D.G., Feedlot health and performance effects associated with the timing of respiratory disease treatment, J Anim Sci, 2009, 87(1),314-27.

2. Crepieux, T., Miller, C., Regev-Shoshani, G., Schaefer, A., Dorin, C., Alexander, T., Timsit, E., Randomized, non-inferiority trial comparing a nitric oxide releasing solution with a macrolide antibiotic for control of bovine respiratory disease in beef feedlot calves at high-risk of developing respiratory tract disease, Res Vet Sci, 2016, 105, 216-21.

3. DeDonder, K.D., Apley, M.D., A literature review of antimicrobial resistance in Pathogens associated with bovine respiratory disease, Anim Health Res Rev, 2015, 16(2), 125-34.

4. Evans, N.A., Tulathromycin: an overview of a new triamilide antibiotic for livestock respiratory disease, Vet Ther, 2005, 6(2),83-95.

5. Fulton, R.W., Bovine respiratory disease research (1983-2009), Anim Health Res Rev, 2009,10(2),131-9.

6. Goldspink, L.K., Mollinger, J.L., Barnes, T.S., Groves, M., Mahony, T.J., Gibson, J.S., Antimicrobial susceptibility of Histophilus somni isolated from clinically affected cattle in Australia, Vet J., 2015, 203(2), 239-43.

7. Guardabassi, L., Jensen, L.B., Hilde, Kruse, Guide to Antimicrobial Use in Animals, Ed. Wiley-Blackwell, 2008.

8. Hay, K.E., Morton, J.M., Clements, A.C., Mahony, T.J., Barnes, T.S., Associations between feedlot management practices and bovine respiratory disease in Australian feedlotcattle, Prev Vet Med, 2016, 128, 23-32.

9. Hilton, W.M, BRD in 2014: where have we been, where are we now, and where do we want to go? Animal Health Research Reviews, 2014,15,120-122.

10. Klima, C.L., Zaheer, R., Cook, S.R., Booker, C.W., Hendrick, S., Alexander, T.W., McAllister, T.A., Pathogens of bovine respiratory disease in North American feedlots conferring multidrug resistance via integrative conjugative elements, J Clin Microbiol, 2014, 52(2),438-48.

11. Lubbers, B., Using individual animal susceptibility test results in bovine practice, Vet Clin North Am Food Anim Pract, 2015, 31(1),163-74.

12. Lubbers, B.V., Hanzlicek, G.A., Antimicrobial multidrug resistance and coresistance patterns of Mannheimia haemolytica isolated from bovine respiratory disease cases-a three-year (2009-2011) retrospective analysis, J Vet Diagn Invest, 2013, 25(3), 413-7.

13. Lubbers, B.V., Turnidge, J., Antimicrobial susceptibility testing for bovine respiratory disease: getting more from diagnostic results, Vet J., 2015, 203(2),149-54


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14. Lysnyansky, I,, Ayling, R.D., Mycoplasma bovis: Mechanisms of Resistance and Trends in Antimicrobial Susceptibility, Front Microbiol, 2016, 27,7:595.

15. Nickell, J.S., White, B.J., Metaphylactic antimicrobial therapy for bovine respiratory disease in stocker and feedlot cattle, Vet Clin North Am Food Anim Pract, 2010, 26(2),285-301.

16. Noyes, N.R., Benedict, K.M., Gow, S.P., Booker, C.W., Hannon, S.J., McAllister, T.A., Morley, P.S., Mannheimia haemolytica in feedlot cattle: prevalence of recovery and associations with antimicrobial use, resistance, and health outcomes, J Vet Intern Med, 2015, 29(2),705-13.

17. Portis, E., Lindeman, C., Johansen, L., Stoltman, G., A ten-year (2000-2009) study of antimicrobial susceptibility of bacteria that cause bovine respiratory disease complex--Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni--in the United States and Canada, J Vet Diagn Invest, 2012, 24(5), 932-44.

18. Snowder, G.D., Van Vleck, L.D., Cundiff, L.V., Bennett, G.L., Bovine respiratory disease in feedlot cattle: environmental, genetic, and economic factors, J Anim Sci, 2006, 84(8),1999-2008.

19. Sweeney, M.T., Brumbaugh, G.W., Watts, J.L., In vitro activities of tulathromycin and ceftiofur combined with other antimicrobial agents using bovine Pasteurella multocida and Mannheimia haemolytica isolates, Vet Ther, 2008, 9(3), 212-22.

20. Watts, J.L., Sweeney, M.T., Antimicrobial resistance in bovine respiratory disease pathogens: measures, trends, and impact on efficacy, Vet Clin North Am Food Anim Pract, 2010, 26(1),79-88.


107

VACCINATION PROTOCOLS IN BOVINE RESPIRATORY DISEASE COMPLEX – A REVIEW

C.I. MATES, MARINA SPINU, EMOKE PALL, MIHAELA NICULAE

University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary

Medicine, 400372, Calea Mănăștur Street no 3-5, Cluj-Napoca, Romania E-mail: [email protected]

Summary

Vaccination represents one of the key measures listed along with biosecurity and predisponding factors avoidance for the bovine respiratory disease (BRD) control and prevention. Currently, several types of commercial vaccines are available on the market and also both in vitro and in vivo scientific studies are conducted in order to develop new products aimed to ensure a better protection level in bovine susceptible population.

This paper summarises all these products with detalied references: trade name, vaccine type (conventional/deleted or marker vaccines, modified-live/inactivated, monovalent, multiple - valences) vaccinal strains, vaccination regimen, intranasal/injectable), advantages and disadvantages of their use based on the determined level of local and specific humoral immune response (antibody titer). Parammeters such as the occurence and the intensity of the clinical disease, morbidity and mortality percentages, inflammatory response, viremia, viral shedding, feed intake, metabolic response are also listed to evaluate the efficacy of above mentioned vaccines.

The main principles to be consider when establishing the vaccination programmes adapted to the farm conditions (epidemiology of the region, risk factors, farm history, animals’s immune status, age, production category) are mentioned.

Key words: bovine respiratory disease, vaccine, immune protection

Vaccination represents one of the key measures listed along with

biosecurity and predisponding factors avoidance for the bovine respiratory disease (BRD) control and prevention (1,2,3). Valued as an effective tool in a feedlot's animal health management (1,3), specific prevention of BRD targets the main etiological agents responsable for this pathology – viruses: bovine herpes-virus type-1 (BHV-1 or IBRV), parainfluenza type-3 virus (PI-3V), bovine respiratory syncytial virus (BRSV), bovine viral-diarrhoea virus type 1 (BVDV) and bacteria: Mannheimia haemolytica (M. haemolytica) and Histophilus somni (H. somni).

Currently, several types of commercial vaccines are available on the market and also both in vitro and in vivo scientific studies are conducted in order to develop new products (5,7,11,12) aimed to ensure a better protection level in bovine susceptible population and to limit the losses associated with increased morbidity and mortality, carcasses condamnation and antimicrobial treatment.

Bovine Respiratory Disease is the results of a complex, multifactorial interaction between several respiratory pathogens (viruses and bacteria) that are able to exhibit pathogenic modifications depending on host animal susceptibility


108

that is negatively influenced by multiple stress factors (1,14). The presence and diversity of BRD infectious agents and also the important role of one or combination of stressors were demonstrated in cattle populations, therefore an integrated BRD prevention strategy should not be focused only on the vaccination. Some researchers clearly stated that the administration of vaccines against BRD agents may help reduce the incidence of BRD but is unlikely to eliminate the condition (1,2).

This paper summarises all these products with detalied references: trade name, vaccine type (conventional/deleted or marker vaccines, modified-live/inactivated, monovalent, multiple - valences) vaccinal strains, vaccination regimen, intranasal/injectable), advantages and disadvantages of their use based on the determined level of local and specific humoral immune response (antibody titer). Parammeters such as the occurence and the intensity of the clinical disease, morbidity and mortality percentages, inflammatory response, viremia, viral shedding, feed intake, metabolic response are also listed to evaluate the efficacy of above mentioned vaccines.The main principles to be consider when establishing the vaccination programmes adapted to the farm conditions (epidemiology of the region, risk factors, farm history, animals’s immune status, age, production category) are mentioned.

Having the starting point the importance of infectious agents in case of BRD development, the specific prevention measures target the main etiological agents –viruses and bacteria, with vaccines developed based on strains of bovine herpes-virus type-1 (BHV-1 or IBRV), parainfluenza type-3 virus (PI-3V), bovine respiratory syncytial virus (BRSV), bovine viral-diarrhoea virus type 1 (BVDV), Mannheimia haemolytica (M. haemolytica) and Histophilus somni (H. somni). Most of the currently recommended commercial vaccines include at least one of these pathogens, but the great majority offer protection against multiple organisms. In this regard, the trend indicates the use of multiple valences vaccines for a superior protection compared to monovalent ones (5,7,8,12). Such products are designed as both modified live (ML) and killed (K) or inactivated vaccines and several studies provided the documented information regarding the protective value based on the evaluation of vaccinated groups (5,6,7,8,10,11,12).The effectiveness of these vaccines is assessed in terms of production parameters at farm level (increased body weight, morbidity and mortality percentages, quality of carcasses), as well as in terms of the cost-benefit (10). A study performed on 3882 calves at ultra-high risk of developing undifferentiated fever (UF)/bovine respiratory disease (BRD) indicated the opportunity of using a live attenuated vaccine containing IBRV, BVDV type I and II, and toxoids derived from Mannheimia haemolytica and Pasteurella multocida compared to another live attenuated vaccine containing only BRSV and PI3V (10). Another study conducted in Western Canada on 5163 calves divided into two groups - one batch who received live attenuated monovalent against IBRV and a group that was vaccinated with live attenuated 4 valences - IBRV, PI3V, BVDV and BSRV recommended a quadrivalent vaccine because the group that


109

received the vaccine showed higher values for average daily gain, carcass weight and carcasses grades (8). Similar protective effect was suggested for a quadrivalent vaccine used to vaccinate calves between 2 and 9 months of age that were challenged experimentally with one of the viruses, bovine herpes-virus-1 (BHV-1), parainfluenza type-3 virus (PI(3)V), bovine viral-diarrhoea virus type 1 (BVDV), or bovine respiratory syncytial virus (BRSV) and were then kept under observation for at least 2 weeks to determine the occurrence and the severity of clinical signs, the antibody titer and the viral shedding (7). Enhanced antibody response to all four viruses post-challenge, with the reduction of the amount or duration (or both) of virus shedding in the BHV-1, PI(3)V, BVDV and BRSV studies and with milder clinical signs in the BHV-1 (nasal discharge, and rectal temperature) and the PI(3)V studies (abnormal respiration, and depression) were reported for the vaccinated calves (7). Quadrivalent vaccines may determine a longer duration of immunity as pointed out by the results of a study on calves between 7 weeks and 6 months of age (5,6).

Either these vaccines are monovalent or polyvalent, they all serve as the antigenic stimulus that triggers the immune system response. The expected response is the antigen specific protection of vaccinated individuals based on the production of specific antibodies - humoral-mediated immunity; some vaccines induce also a specific cellular – mediated immunity. Thus, this active specific protection involves the immune system integrity and functionality. Unfortunately, several stress factors are responsible for immunosuppression, with the immune system inability to respond promptly and effectively to vaccination. In the case of cattle and especially young cattle, the most important elements with negative influence on the immune system are actually some farming and breeding practices and operations - weaning, transportation, extreme variations of temperature, crowding, poor ventilation, lack of feed and water (1,2). The importance of these factors should not be minimized when establishing the vaccination protocols (14).

In addition, compared efficacy of modified live /killed vaccines is also under debate, with both advantages and disadvantages described for both types (table 1).

Table 2 compiles the commercial vaccines currently available in Romania. The vaccination protocols may include both modified live or killed vaccines, and monovalent or multivalent vaccines. As presented in the table, these products protection covers the most important viral pathogens - BHV-1, PI-3V, BVDV and BRSV and one bacterium - Mannheimia haemolytica.

With the current information, no universal protocol can be suggested and further investigation are needed to develop not only better products, but also better strategies related to predisponding factors control. The vaccination programmes should be adapted to the farm conditions (epidemiology of the region, risk factors, farm history, animals’s immune status, age, production category).


110

Table 1 Advantages and disadvantages of vaccines (13)

Advantages Disadvantages

Killed vaccines

No interference with the gestation Requires more than one dose (more boosters)

Inactivated virulence Immune response is milder and not all immune response phases are triggered

No viral shedding

No immunosuppression Hypersensitivity reactions

Modified live vaccines

Longer duration of the immunity – less doses and boosters

the vaccine may induce the disease

Stronger immunity (humoral and local mucosal immunity)

viral shedding

Injection or intra nasal administration May interfere with the gestation

May induce immunosuppression

Table 2 Commercial vaccines currently available in Romania

Trade name Company

Agents

BVDV IBRV

(BHV-1) BRSV PI-3V

Mannheimia haemolytica

BioBos IBR Marker inact. BIOVETA x

Bovibio Respi 4 BIOVETA x x x x

BioBos Respi 2 intranazal BIOVETA x x

BioBos Respi 3 BIOVETA x x x

Hiprabovis 4 HIPRA x x x x

Hiprabovis Ibr Marker Live HIPRA x

Bovilis IBR Marker inac MSD x

Bovilis Bovipast RSP MSD x x x

Bovilis BVD MSD x

Rispoval IBR-Marker ZOETIS x

Rispoval 3-BRSV-Pi3-BVD ZOETIS x x x

Rispoval IBR-Marker vivum ZOETIS x

Rispoval RS+PI3 Intranazal ZOETIS x x

CattleMaster TM

4 ZOETIS x x x x


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References 1. Bowland, S.L., Shewen, P.E., Bovine respiratory disease: commercial

vaccines currently available in Canada, Can Vet J., 2000, 41(1),33-48. 2. Cusack, P.M., McMeniman, N., Lean, I.J., The medicine and epidemiology

of bovine respiratory disease in feedlots, Aust Vet J, 2003, 81(8),480-7. 3. Hill, K.L., Hunsaker, B.D., Townsend, H.G., van Drunen Littel-van den

Hurk, S., Griebel. P.J., Mucosal immune response in newborn Holstein calves that had maternally derived antibodies and were vaccinated with an intranasal multivalent modified-live virus vaccine, J Am Vet Med Assoc, 2012, 240(10),1231-40

4. Larson, R.L,, Step, D.L., Evidence-based effectiveness of vaccination against Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni in feedlot cattle for mitigating the incidence and effect of bovine respiratory disease complex, Vet Clin North Am Food Anim Pract, 2012, 28(1),97-106.

5. Peters, A.R., Thevasagayam, S.J., Wiseman, A., Salt, J.S., Duration of immunity of a quadrivalent vaccine against respiratory diseases caused by BHV-1, PI3V, BVDV, and BRSV in experimentally infected calves, Prev Vet Med, 2004, 66(1-4), 63-77.

6. Richeson, J.T., Beck, P.A., Gadberry, M.S., Gunter, S.A., Hess, T.W., Hubbell, D.S. 3rd, Jones, C., Effects of on-arrival versus delayed modified live virus vaccination on health, performance, and serum infectious bovine rhinotracheitis titers of newly received beef calves, J Anim Sci, 2008, 86(4),999-1005.

7. Salt, J.S., Thevasagayam, S.J., Wiseman, A., Peters, A.R., Efficacy of a quadrivalent vaccine against respiratory diseases caused by BHV-1, PI3V, BVDV and BRSV in experimentally infected calves, Vet J, 2007,174(3),616-26.

8. Schunicht, O.C., Booker, C.W., Jim, G.K., Guichon, P.T., Wildman, B.K., Hill, B.W., Comparison of a multivalent viral vaccine program versus a univalent viral vaccine program on animal health, feedlot performance, and carcass characteristics of feedlot calves, Can Vet J, 2003, 44(1),43-50.

9. Stilwell, G., Matos, M., Carolino, N., Lima, M.S., Effect of a quadrivalent vaccine against respiratory virus on the incidence of respiratory disease in weaned beef calves, Prev Vet Med, 2008, 85(3-4),151-7.

10. Wildman, B.K., Perrett, T., Abutarbush, S.M., Guichon. P.T., Pittman, T.J., Booker, C.W., Schunicht, O.C., Fenton, R.K., Jim, G.K., A comparison of 2 vaccination programs in feedlot calves at ultra-high risk of developing undifferentiated fever/bovine respiratory disease, Can Vet J, 2008, 49(5),463-72.

11. Windeyer, M.C., Leslie, K.E., Godden, S.M., Hodgins, D.C., Lissemore, K.D., LeBlanc, S.J., Association of bovine respiratory disease or vaccination


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with serologic response in dairy heifer calves up to three months of age, Am J Vet Res, 2015, 76(3),239-45.

12. Xue, W., Ellis, J., Mattick, D., Smith, L., Brady, R., Trigo, E., Immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types 1 and 2, infectious bovine rhinotracheitis virus, bovine parainfluenza-3 virus, and bovine respiratory syncytial virus when administered intranasally in young calves, Vaccine, 2010, 28(22),3784-92.

13. ***http://articles.extension.org/sites/default/files/Effective%20Use%20of%20Vaccinations%20on%20Cow%20Calf%20Operations%20to%20Reduce%20Incidence%20of%20BRD.pdf.

14. ***http://169.237.28.91/BRDComplex/files/presentations/BRDC%20Chase%201-31.pdf.


113

PRELIMINARY RESEARCH ON THE PREVALENCE OF TOXOPLASMA GONDII INFECTION IN WISENTS (BISON

BONASUS) FROM ROMANIAN ARMENIS-PLOPU RESERVE

S. MORARIU1, A. BULACU

2, GH. DĂRĂBUȘ

1, NARCISA MEDERLE

1, M.S. ILIE

1,

C. BADEA1, GH. CIOBAN

3, MIRELA IMRE

1

1Banat’s University of Agricultural Science and Veterinary Medicine, “King Mihai I of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Calea Aradului,

no 119, Timisoara, Romania 2 WWF România 3 DSVSA Arad


Summary

The wisent (Bison bonasus) or European bison is a Eurasian species of bison.

Nowadays, the wisent is downgraded to a vulnerable species status. In this respect, Romania tries hardly to preserve this species in several natural reserves from Neamț, Buzău or Hunedoara counties. During May 2014, 18 wisents from different European countries (9 from Sweden, 4 from Germany, 2 from Italy, 2 from Belgium and 1 from Romania) were moved to the new reservation Armeniș-Plopu from Caraș-Severin County, south-western Romania, in a wooded area. Blood samples were collected when animals were loaded in trucks prior transportation and preserved at 4ºC up to destination. Samples were examined by ID Screen

® Toxoplasmosis indirect test kit (ID VET, France). Four samples (22.22%)

were positive for T. gondii. The OD for positive samples varied between 0.474 and 2.237. Key words: wisents, Toxoplasma gondii, prevalence, Romania.

The European bison (Bison bonasus), known also as wisent, is a species

of bison spread in Ancient times in Asia and Europe. Today the wisent has an extremely vulnerable status, being almost in extinction. Because of their dramatic history, current populations are highly related with important consequences on the health status of these herbivorous mammals. Viruses, bacteria and parasites are the main causes that threaten their health (2; 3).

First investigations on the parasitic status of wisents were carried out at the beginning of the 20th century (7). These investigations also continues nowadays, till now 88 species of parasites being identified in wisents, mainly in Poland, because there are the largest free living populations (1).

In Romania the reserves from Neamt, Brasov, Dambovita and Hunedoara counties are well known, the first being the Hateg-Slivut one, which started out in 1958 with only 2 specimens brought from Poland. The last reserve of Romania was founded in Armenis-Plopu from Caras-Severin County (5).

The aim of this study was to investigate the presence of antibodies against Toxoplasma gondii in the serum samples from wisents of Armenis-Plopu reserve.


114

Materials and methods Blood samples were collected from 18 European bison before boarding in

the origin country of each specimen. Samples were kept at 4ºC till they were processed in the parasitic diseases lab of Faculty of Veterinary Medicine Timișoara. Wisents originated from 5 European countries: 9 from Sweden, 4 from Germany, 2 from Italy, 2 from Belgium and 1 from Romania.

Serum from the blood samples was collected in vials and preserved by freezing till processing in the parasitic diseases lab of the Faculty of Veterinary Medicine Timisoara. Serum samples were examined by ID Screen

® Toxoplasmosis

indirect test kit (ID VET, France).

Results and discussions Results are presented in Table 1.

Table 1 Presence of anti-Toxoplasma antibodies in wisents from Armeniș-Plopu reserve

Crt. no.

Name Sex Age Country Result

1. Egomundis F 2012 Germany (HardeHausen)

-

2. Eggemadel F 2011 Germany (HardeHausen)

-

3. Spomenka F 2009 Germany (Springe) -

4. Spiderwoman F 2011 Germany (Springe) -

5. BBB029 M 2013 Italy -

6. BBB028 M 2012 Italy -

7. Swan F 2012 Sweden (Kolmarden)

-

8. Isabell F 2012 Sweden (Kolmarden)

-

9. Birk M 2010 Sweden (Kolmarden)

+

10. Sussie F 2012 Sweden (Avesta) +

11. Isa F 2004 Sweden (Avesta) +

12. Hilda F 2012 Sweden (Avesta) -

13. Mildred F 2009 Sweden (Avesta) -

14. Terese F 2012 Sweden (Avesta) -

15. Sabina F 2013 Sweden (Avesta) +

16. Zwitcher M 2013 Belgium -

17. Zwanda F 2012 Belgium -

18. Romanita F 2008 Romania -


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Four samples (22.22%) were positive for T. gondii. The OD for positive samples varied between 0.474 and 2.237.

All positive samples (100%) came from Sweden, from animals purchased from 2 reserves: Kolmarden and Avesta. When compared, only 33.33% of the infected wisents were from Kolmarden, while 50% of them came from Avesta. So, the prevalence of anti-Toxoplasma antibodies in wisents from Sweden was of 44.44%. The infection was present mainly in females (3F/1M), in animals of various ages. But it is difficult to explain why only these wisents were infected by Toxoplasma, because no information concerning their history was available.

The data from literature are scanty, so it is quite hard to make a comparison. However, in Poland, Majevska et al. (4) identified a prevalence of 40.5%, much higher than that reported by us in this study. Also, in a study conducted on zoo animals from Czech and Slovak zoos, all 4 wisents investigated were positive, with titers ranging from 1:160 to 1:2560 (6).

Conclusions

Toxoplasma gondii infection was identified in 22.22% of wisents from

Armenis-Plopu reserve. Only wisents from Sweden were infected and the age had no influence on

the receptivity.

Acknowledgments This research work was carried out with the support of the


References

1. Karbowiak, G., Demiaszkiewicz, A.W., Pyziel, A.M., Wita, I., Moskwa, B.,

Werszko, J., Bień, J., Goździk, K., Lachowicz, J., Cabaj, W., The parasitic fauna of the European bison (Bison bonasus) (Linnaeus, 1758) and their impact on the conservation. Part 1. The summarising list of parasites noted, Acta Parasitol, 2014, 59, 3, 363–371.

2. Kita, J., Anusz, K., Infectious diseases in Bison bonasus in the years 1910–1988. In: (Eds J. Kita and K. Anusz) Health threats for the European bison particularly in freeroaming populations in Poland, SGGW Publishers, Warsaw, 2006.

3. Krasinska, M., Krasinski, Z.A., European Bison: The Nature Monograph, Mammal Research Institute Polish Academy of Sciences, Białowieza, Poland, 2007.


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4. Majewska, A.C., Werner, A., Cabaj, J., Moskwa, B., The first report of Toxoplasma gondii antibodies in free-living European bison (Bison bonasus bonasus Linnaeus), Folia Parasitol, 2014, 61, 1, 18-20.

5. Morariu, S., Bulacu, A., Dărăbuș, Gh., Mederle, N., Mihăilă, C., Nechiti, R., Brîncoveanu, A., Badea, C., Prevalence of internal parasites in wisents (Bison bonasus bonasus) from two Romanian reservations - preliminary results, Lucr Șt Med Vet Iași, 2014, 57, 1-2, 170-173.

6. Sedlak, K., Bartova, E., Seroprevalences of antibodies to Neospora caninum and Toxoplasma gondii in zoo animals, Vet Parasitol, 2006, 136, 223-231.

7. Wroblewski, K., Żubr Puszczy Białowieskiej: monografia, Wydawnictwo Polskie, Poznań, 1927.


117

ZOONOTIC POTENTIAL OF STAPHYLOCOCCUS PSEUDINTERMEDIUS – A REVIEW

MIHAELA NICULAE, CATALINA NICULAE, C.I. MATES, EMOKE PALL, R. AL.

POP, MARINA SPINU University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary

Medicine, 400372, Calea Mănăștur Street no 3-5, Cluj-Napoca, Romania E-mail: [email protected]

Summary

The role of Staphylococcus pseudintermedius in small animal’s pathology has been updated over the years, with the current scientific data underlying the complex ability to develop multiple antimicrobial resistance in case of this opportunistic bacterium responsible for skin and soft tissues infections in dogs. It belongs to the coagulase-positive group and it is one of the most frequently isolated Staphylococcus spp. in dogs presenting pyoderma and external otitis, pathologies well known for the poor response to treatment and the recidivant course. Canine skin infections are often regarded as challanges, with the results of the in vitro susceptibility testing indicating the lack of efficacy of β-lactams, erythromycin,

gentamicin, ciprofloxacin, chloramphenicol, clindamycin, oxytetracycline, and tetracycline, and thus few antimicrobial options. Great interest is focused at present on the zoonotic risk of methicillin-resistant S. pseudintermedius (MRSP) isolates and its veterinary-hospital-associated epidemiology. Carrier state was also described for both methicillin susceptible and resistant strains. This review documents the ecology, the classical microbiological and molecular characteristics, and clinical importance of the bacterium with dual activity – both commensal and pathogenic microorganism and emphasizes the emergence of distinct clones of MRSP with notable antimicrobial resistance patterns, such as the inducible clindamycin (iCLI) Staphylococcus pseudintermedius or the European clone (ST71 MRSP). In conjunction with the scientific proof of the increasing antimicrobial resistance level, the study highlights the need for antimicrobial resistance systematic screening and surveillance to be considered for guidelines of rational use of antimicrobials drugs. As for the impact on human health, several reports confirmed Staphylococcus pseudintermedius infections in people and suggested the zoonotic risk of MRSP strains in veterinary facilities (hospitals, clinics) and in the community.

Key words: Staphylococcus pseudintermedius, multi-drug antimicrobial resistance,

zoonotic risk

Staphylococcus pseudintermedius (SP) is a bacterium belonging to Staphylococcus genus and currently the type species of S. intermedius group (SIG) that also includes S. intermedius and Staphylococcus delphini (25). Nowadays, taxonomy distinguishes two species - Staphylococcus pseudintermedius and S. intermedius that can be both isolated from dogs and most authors agree that some scientific data reported between 1976 - 2005 may actually reffer to Staphylococcus pseudintermedius (1,2,11,21,22,25). Classic microbiology does not allow the two species differentiation (1,7,11,25), only by the use of a batch of molecular methods


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such as pulse field gel electrophoresis (PFGE), ribotyping, PCR restriction fragment length polymorphism (PCR-RFLP) and multilocus sequence typing (MLST) (1,7,11,25).The molecular techniques are also valuable tools to draw a distinction between SP and other species of canine origin, most often the case of Staphylococcus schleiferi subspecies coagulans and Staphylococcus aureus (25). The strains matching the characteristics of SIG group should be evaluated based on genomic assays and if not possible to be reported as SP (25). Following the isolation and identification of SP strains, molecular characterization is required also to investigate methicillin-resistant Staphylococcus pseudintermedius (MRSP) in terms of antimicrobial resistance genes (mecA, erm(B), Inu(A), dfrG, ND, tet(M), tet(K)) and virulence factors (biofilm formation assay, exfoliative toxin and Panton-Valentine leukocidin); these protocols include sensitive techniques such as pulse field gel electrophoresis (PFGE), staphylococcal chromosomal cassette mec (SCCmec) typing, and multilocus sequence typing (MLST) (1,7,11,25). In order to detect methicillin resistance in staphylococci, the initial evaluation can be performed using disk diffusion or broth microdilution to screen the susceptibility towards methicilin (oxacillin or cefoxitin, and MIC≥ 0.5 mg/L; R≤ 17 mm according to M31-A4 issued by VAST) (25). Higher sensitivity is guaranteed by the mecA and SCCmec detection by PCR protocols (1,6,11,25,27,31); mecA gene is considered responsible for the methicillin resistance and it is located on a mobile element of bacterial chromosome – staphylococcal chromosomal cassette (SCCmec). Clonal distribution and diversity of MRSP is established by PFGE and MLST (25). All these techniques cannot be considered at this moment routine methods, but the MRSP detection and surveillance is needed due to increasing number of reports suggesting or demonstrating two key elements: the zoonotic potential and the role of potential reservoir for multidrug antimicrobial resistance (5,6,7,11,12,20,26). A review listing 202 scientific papers published from 1980 to 2013 underlined the importance of unitary laboratory antimicrobial susceptibility testing and interpretation criteria and of systematic surveillance either at the country-level or at a larger scale across countries e.g. EU level (17).

In fact, the role of Staphylococcus pseudintermedius in small animal’s pathology has been updated over the years, with the current scientific data underlying the complex ability to develop multiple antimicrobial resistance in case of this opportunistic bacterium responsible for skin and soft tissues infections in dogs (2,4,10,16,17,22,24,27,28). As a commensal bacteria of skin and mucosal flora, SP can be isolated from samples collected from nares, mouth, pharynx, forehead and anal area of healthy dogs and cats (2,5,6,7,9,10,18,25). It belongs to the coagulase-positive group and it is the most frequently isolated Staphylococcus spp. in dogs presenting pyoderma and external otitis, pathologies well known for the poor response to treatment and the recidivant course (24,27,28). The pathogenic potential of SP has a recognized significance in veterinary dermatology, not only because of the induced skin inflammation, but mostly due to the lack of in vitro and in vivo efficacy observed for routine antimicrobials. Canine skin infections


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are often regarded as challanges, with the results of the in vitro susceptibility testing indicating the lack of efficacy of β-lactams, erythromycin, gentamicin, ciprofloxacin, chloramphenicol, clindamycin, oxytetracycline, and tetracycline, and thus few antimicrobial options (1,2,16,17,28). A group of Japanese researchers reported genotypic and phenotypic differences when comparing MRSP strains isolated from dogs and cats presenting dermatitis. Based on the results of molecular assessment, strains were included into five groups, with ST71 and ST26 being the two most prevalent, and displayed multiple resistance patterns (78% of the isolates were resistant or intermediate to twelve or more antibiotics commonly used in veterinary medicine)(1). In this regard, relatively few longitudinal studies of the impact of routine antimicrobial therapy on methicillin-resistant staphylococci emergence or resolution of resistance are published (2), but it is expected that the long term antimicrobial therapy of canine skin infections to increase the antimicrobial resistance level as reported in case of MRSA in people (15).

The antimicrobial treatment of MRSP pyoderma allows the recovery of the animals, but is not able to eliminate the carrier state. In fact, MRSP colonization persists after resolution of the inflammatory modifications (2).

Methicillin resistance is an important aspect when described and confirmed in Staphylococcus pseudintermedius and Staphylococcus aureus that cannot be neglected given the significant consequences for both animal and public health.

Great interest is also focused at present on the zoonotic risk of methicillin-resistant S. pseudintermedius (MRSP) isolates and its veterinary-hospital-associated epidemiology. Carriage (carrier state) in case of both methicillin susceptible and resistant strains is investigated in connection with the possible spread (human exposure – owners, veterinary staff and environmental contamination – veterinary hospitals, shelters, households, kennels) (3,5,6,8,12,13,16,18,19,20,21,23,26).

The MRSP epidemiology needs more studies since not only the contaminated environment, but also dogs owners may represent the source of reinfection with MRSP. In a case report referring to a dog presenting a long-term inflammatory skin disorder associated with MRSP, the same strain was isolated and confirmed from both the owner and the dog (27). Based on the results of several molecular assays, the strain was characterized as a multidrug-resistant MRSP belonging to sequence type (ST) 71, spa type (t)05, harbouring SCCmecIII as well as the genes encoding LukI (leukotoxin encoding operon) and SIET (Staphylococcus intermedius-exfoliative toxin)(27).

Based on the above mentioned literature data, the zoonotic potential of Staphylococcus pseudintermedius should not be minimized and further studies are required to evaluate and monitor the epidemiology related to the public health risks and companion animals as sources for human exposure, with a permanent focus on the antimicrobial resistance patterns fluctuation.


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of methicillin-resistant Staphylococcus pseudintermedius isolated from dogs and cats, Microbiol Immunol, 2013, 57(7), 496-501.

2. Beck, K.M., Waisglass, S.E., Dick, H.L., Weese, JS., Prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) from skin and carriage sites of dogs after treatment of their meticillin-resistant or meticillin-sensitive staphylococcal pyoderma, Vet Dermatol., 2012, 23(4), 369-75, e66-7.

3. Bergström, A., Gustafsson, C., Leander, M., Fredriksson, M., Grönlund, U., Trowald-Wigh, G., Occurrence of methicillin-resistant Staphylococci in surgically treated dogs and the environment in a Swedish animal hospital, J Small Anim Pract., 2012, 53(7), 404-10.

4. Bryan, J., Frank, L.A., Rohrbach, B.W., Burgette, L.J., Cain, C.L., Bemis, D.A., Treatment outcome of dogs with meticillin-resistant and meticillin-susceptible Staphylococcus pseudintermedius pyoderma, Vet Dermatol, 2012, 23(4), 361-8, e65.

5. Epstein, C.R., Yam, W.C., Peiris, J.S., Epstein, R.J., .Methicillin-resistant commensal staphylococci in healthy dogs as a potential zoonotic reservoir for community-acquired antibiotic resistance, Infect Genet Evol., 2009, 9(2), 283-5.

6. Frank, L.A., Kania, S.A., Kirzeder, E.M., Eberlein, L.C., Bemis, D.A., Risk of colonization or gene transfer to owners of dogs with meticillin-resistant Staphylococcus pseudintermedius, Vet Dermatol, 2009, 20(5-6), 496-501.

7. Garbacz, K., Żarnowska, S., Piechowicz, L., Haras, K., Staphylococci isolated from carriage sites and infected sites of dogs as a reservoir of multidrug resistance and methicillin resistance, Curr Microbiol, 2013, 66(2), 169-73.

8. Gingrich, E.N., Kurt, T., Hyatt, D.R., Lappin, M.R., Ruch-Gallie, R., Prevalence of methicillin-resistant staphylococci in northern Colorado shelter animals, J Vet Diagn Invest, 2011, ;23(5), 947-50.

9. Gómez-Sanz, E., Torres, C., Lozano, C., Sáenz, Y., Zarazaga, M., Detection and characterization of methicillin-resistant Staphylococcus pseudintermedius in healthy dogs in La Rioja, Spain, Comp Immunol Microbiol Infect Dis, 2011, 34(5), 447-53.

10. Griffeth, G.C., Morris, D.O., Abraham, J.L., Shofer, F.S., Rankin, S.C., Screening for skin carriage of methicillin-resistant coagulase-positive staphylococci and Staphylococcus schleiferi in dogs with healthy and inflamed skin, Vet Dermatol, 2008, 19(3), 142-9.

11. Ishihara, K., Koizumi, A., Saito, M., Muramatsu, Y., Tamura, Y., Detection of methicillin-resistant Staphylococcus pseudintermedius ST169 and novel































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ST354 SCCmec II-III isolates related to the worldwide ST71 clone, Epidemiol Infect, 2016, 144(2), 434-42.

12. Ishihara, K., Shimokubo, N., Sakagami, A., Ueno, H., Muramatsu, Y., Kadosawa, T., Yanagisawa, C., Hanaki, H., Nakajima, C., Suzuki, Y., Tamura, Y., Occurrence and molecular characteristics of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius in an academic veterinary hospital, Appl Environ Microbiol, 2010, 76(15), 5165-74.

13. Laarhoven, L.M., de Heus, P., van Luijn, J., Duim, B., Wagenaar, J.A., van Duijkeren, E., Longitudinal study on methicillin-resistant Staphylococcus pseudintermedius in households, PLoS One, 2011, 6(11), e27788.

14. Lehner, G., Linek, M., Bond, R., Lloyd, D.H., Prenger-Berninghoff, E., Thom, N., Straube, I., Verheyen, K., Loeffler, A., Case-control risk factor study of methicillin-resistant Staphylococcus pseudintermedius (MRSP) infection in dogs and cats in Germany, Vet Microbiol, 2014, 10;168(1), 154-60.

15. Loeffler, A., Lloyd, D.H., Companion animals, a reservoir for methicillin-resistant Staphylococcus aureus in the community?, Epidemiol Infect, 2010, 138(5), 595-605.

16. Maluping, R.P., Paul, N.C., Moodley, A., Antimicrobial susceptibility of methicillin-resistant Staphylococcus pseudintermedius isolated from veterinary clinical cases in the UK, Br J Biomed Sci, 2014, 71(2), 55-7.

17. Moodley, A., Damborg, P., Nielsen, S.S., Antimicrobial resistance in methicillin susceptible and methicillin resistant Staphylococcus pseudintermedius of canine origin, literature review from 1980 to 2013, Vet Microbiol, 2014, 171(3-4), 337-41.

18. Morris, D.O., Boston, R.C., O'Shea, K., Rankin, S.C., The prevalence of carriage of meticillin-resistant staphylococci by veterinary dermatology practice staff and their respective pets, Vet Dermatol, 2010, 21(4), 400-7.

19. Nienhoff, U., Kadlec, K., Chaberny, I.F., Verspohl, J., Gerlach, G.F., Kreienbrock, L., Schwarz, S., Simon, D., Nolte, I., Methicillin-resistant Staphylococcus pseudintermedius among dogs admitted to a small animal hospital, Vet Microbiol, 2011, 12;150(1-2), 191-7.

20. Paul, N.C., Moodley, A., Ghibaudo, G., Guardabassi, L., Carriage of methicillin-resistant Staphylococcus pseudintermedius in small animal veterinarians, indirect evidence of zoonotic transmission, Zoonoses Public Health, 2011, 58(8), 533-9.

21. Rota, A., Milani, C., Corrò, M., Drigo, I., Börjesson, S., Misuse of antimicrobials and selection of methicillin-resistant Staphylococcus pseudintermedius strains in breeding kennels, genetic characterization of bacteria after a two-year interval, Reprod Domest Anim, 2013, 48(1), 1-6.

22. Ruscher, C., Lübke-Becker, A., Wleklinski, C.G., Soba, A., Wieler, L.H., Walther, B., Prevalence of Methicillin-resistant Staphylococcus





























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pseudintermedius isolated from clinical samples of companion animals and equidaes, Vet Microbiol, 2009, 136(1-2), 197-201.

23. Sasaki, T., Kikuchi, K., Tanaka, Y., Takahashi, N., Kamata, S., Hiramatsu, K., Methicillin-resistant Staphylococcus pseudintermedius in a veterinary teaching hospital., J Clin Microbiol, 2007, 45(4), 1118-25.

24. Siak, M., Burrows, A.K., Coombs, G.W., Khazandi, M., Abraham, S., Norris, J.M., Weese, J.S., Trott, D.J., Characterization of meticillin-resistant and meticillin-susceptible isolates of Staphylococcus pseudintermedius from cases of canine pyoderma in Australia, J Med Microbiol, 2014,63(Pt 9), 1228-33.

25. van Duijkeren, E., Catry, B., Greko, C., Moreno, M.A., Pomba, M.C., Pyörälä, S., Ruzauskas, M., Sanders, P., Threlfall, E.J., Torren-Edo, J., Törneke, K., Scientific Advisory Group on Antimicrobials (SAGAM).Review on methicillin-resistant Staphylococcus pseudintermedius, J Antimicrob Chemother, 2011,66(12), 2705-14.

26. van Duijkeren, E., Kamphuis, M., van der Mije, I.C., Laarhoven, L.M., Duim, B., Wagenaar, J.A., Houwers, D.J., Transmission of methicillin-resistant Staphylococcus pseudintermedius between infected dogs and cats and contact pets, humans and the environment in households and veterinary clinics, Vet Microbiol, 2011,150(3-4), 338-43.

27. Vincze, S., Paasch, A., Walther, B., Ruscher, C., Lübke-Becker, A., Wieler, L.H., Barbara, K., Multidrug- and methicillin resistant Staphylococcus pseudintermedius as a cause of canine pyoderma, a case report, Berl Munch Tierarztl Wochenschr, 2010,123(9-10), 353-8.

28. Wang, Y., Yang, J., Logue, C.M., Liu, K., Cao, X., Zhang, W., Shen, J., Wu, C., Methicillin-resistant Staphylococcus pseudintermedius isolated from canine pyoderma in North China, J Appl Microbiol,. 2012, 112(4), 623-30.

29. Weese, J.S., Faires, M.C., Frank, L.A., Reynolds, L.M., Battisti, A, Factors associated with methicillin-resistant versus methicillin-susceptible Staphylococcus pseudintermedius infection in dogs, J Am Vet Med Assoc, 2012, 240(12), 1450-5.

30. Weese, J.S., van Duijkeren, E., Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in veterinary medicine,Vet Microbiol, 2010,140(3-4),418-29.

31. Windahl, U., Reimegård, E., Holst, B.S., Egenvall, A., Fernström, L., Fredriksson, M., Trowald-Wigh, G., Andersson, U.G., Carriage of methicillin-resistant Staphylococcus pseudintermedius in dogs--a longitudinal study, BMC Vet Res, 2012, 23,8, 34.




















http://www.ncbi.nlm.nih.gov/pubmed/?term=van%20Duijkeren%20E%5BAuthor%5D&cauthor=true&cauthor_uid=19246166

http://www.ncbi.nlm.nih.gov/pubmed/19246166?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pubmed/19246166?dopt=Abstract





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USING OF PAPPENHEIM STAIN FOR HIGHLIGHTING THE REOVIRUS PATHOLOGICAL LESIONS

OANA PETREC, N. CĂTANA, ALINA GHIȘE, A. STANCU


of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Aradului Street No. 119, Timisoara, Romania E-mail: [email protected]

Summary

Avian reovirus infection is prevalent in intensive poultry farming, especially in

broilers, wich evolve with many anatomoclinical forms, pathological signs being corelated with clinical signs. The researches was carried out in a flock of broilers, where reovirosis debuted from the first week of life. The necropsic exam was biweekly performed on a total of 96 cadavers from which samples were taken for microscopic examination. The presence of reovirosis was confirmed by RT-PCR and ELISA test.

At the necropsied chickens were found following macroscopic lesions characteristic for reovirosis: proventriculus, catarrhal enteritis, arthritis- tenosynovitis, unilateral and bilateral necrosis of the femoral head, malabsorption syndrome, pericardial effusion and ascites. To highlight the histopathological lesions was utilized Pappenheim staining method.

Histological lesions in proventriculus, small intestine, spleen and liver, highlighted by this method plead for a general and local infectious process.

Key words: avian reovirus, broilers chickens, Pappenheim staining method;

Avian reovirosis is an endemic disease that develops in chickens and

turkeys, with several syndromes, the most common being malabsorption syndrome and arthritis-tenosynovitis. Macroscopic and microscopic pathological lesions are different and generally more and better expressed in malabsorption syndrome, and in other syndromes are better expressed only gross pathological lesions(1,3,4)

The researches were made in order to elucidate many aspects of the pathological lesions of reovirosis in broilers through the study of histological lesions of the digestive tract, annexes organs and lymphoid organs using Pappenheim staining method.

The disease causes significant economic damage, represented by mortality, poor feed conversion and uneven growth, decreased production of meat and eggs, confiscations in slaughterhouse and immunosuppression induced by reovirus (1,2,3).


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Necropsy was performed biweekly on a total of 96 broiler corpses, from the age of 6 days to 41 days. From chickens with characteristic lesions for avian reovirosis were collected organ samples: proventriculus, small intestine, spleen and liver for microscopic examination. The samples of organs were processed by the classical method, and for colouring was used Pappenheim staining method.


Microscopic examination was performed in order to highlight the

histopathological lesions present in several tissues and organs that have tropism for avian reovirus.

It was carried out only in the organs samples taken from the broilers corpses with pathological lesions characteristic for malabsorption syndrome, which have not been complicated with bacterial infections associated. By using the dye method Pappenheim have been observed histopathological lesions in the proventriculus, small intestine, spleen and liver. In proventriculus were highlighted lesions which denotes affection of the glands and papillae proventriculare and the absence of demarcation between the esophagus and the organ. In histological sections are highlighted inflammatory lesions, necrotic and interstitial lymphoid hyperplasia. In provetricular glands are presents atrophy, dystrophy and necrosis accompanied by denudation papillae. It has also been well highlighted lining of the stroma and massive congestion of fibroconjunctive stroma between the proventricular glands (Fig.1). These lesions are attributed to multiplication of avian reovirus in the structure of this organ. The presence of lesions in proventricular glands pleads for the disruption of digestion consecutive to hiposecretion of HCl and pepsinogen.Macroscopic and microscopic pathological lesions from proventriculus have been reported by several collective research, that awarded these lesions to avian reovirus infection (2,3,5).

In small intestine was present catarrhal enteritis, characterized by inflammatory and infiltrative histopathological lesions. In the sections of intestinal villous were observed: desquamation of villi as a result of enterocytes necrosis,unequal villous with rare lymphocytes, submucosal infiltration with small lymphocytes, histiocytes, lymphoblasts and rare plasma cell (Fig.2). The lesions observed in the small intestine pleads for inflammatory lesions, successive to multiplication of avian reovirus consecutive multiplication in the intestinal mucosa structure. The presence of these microscopic lesions disrupt the normal digestion, resulting the reduced feed conversion.


125

Fig.1. Proventriculus – histological section (Col.Pappenheim, ob x20)

Fig.2. Small intestine - histological section (Col.Pappenheim, ob x10)

In spleen, Pappenheim staining revealed the histopathological lesions wich

pleads for the postinfectious immune response: numerous lymphoid nodules with a


126

medium crown, reticulohistiocytary proliferation and a pronounced dilatation vessels with numerous splenocytes in spleen lumen. Were highlighted the primary splenic follicles (active) and secondary (by activation) looking hypochromic (Fig.3).

Fig. 3. Spleen - histological section (Col.Pappenheim, ob x10)

In the liver, histopathological lesions represented by the edema of Disse space, the tumefaction of hepatocytes and vacuolar dystrophy, wich pleads for critical disruption of the function of this organ and lymphocyte infiltration and Kupffer cell hyperplasia pleads for a massive viral multiplication at the level of this organ (Fig.4).The microscopic lesions of lymphoid organs were reported frequently by several researchers, in avian reovirosis, regardless of evolutive syndromes (2,3,5).

The presence of histological lesions in the proventriculus, small intestine and liver, evidence the impaired of digestion process followed by reduced feed conversion and the emergence and evolution of malabsorption syndrome. The avian reovirosis diagnosis was certainty established in samples examined from the flock of broilers subject to monitoring, using RT- PCR test.


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Fig.4. Liver - histological section (Col.Pappenheim, ob x20)

Conclusions

Microscopic examination conducted by Pappenheim staining method showed histopathological lesions in the digestive tract and lymphoid organs. Proventriculus and histological lesions in the small intestine pleads for alteration digestion followed by reduced feed conversion and splenic lesions that suggest the postinfectious immune response.

The research conducted in broiler flock completed and contoured the pathologic lesions emerged of avian reovirosis based on which disease can be suspected and monitored in routine diagnosis.

Acknowledgements



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References

1. Cătana, N., Fodor, Ionica, Botuș, Daniela, Popa, Virgilia, Infecţiile cu reovirusuri la păsări, Magazin Avicol,2008, 5 (21), 24 – 25.

2. Davis, J.F., Kulkarni, A., Fletcher, O., Reovirus infections in young broiler chickens, Avian Diseases, 2013,57 (2), 321-325.

3. Jones, R.C., Reovirus infections, În Diseases of Poultry, 13th Edition, Editor in Chief: David E. Swaine, Ed. Wiley Blackwell, State Avenue,2013, USA, 351-352.

4. Perianu, T., Tratat de boli infecțioase ale animalelor, Viroze și boli prionice, vol. II, Ed. Universitas, XXI, Iași, 2012.

5. Rajesh, S., Agrawald, D.K., Chauhan, R.S., Pathomorphological changes in tissues of stunted chickens experimentally infected with avian reovirus, Indian Journal of Veterinary Pathology, 2005, 29, 1, Izatnagar, 1-3.


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REPEATED VACCINATION ENHANCES THE IN OVO TRANSMISSION OF Ig Y AND Ig M IN LAYERS

MARINA SPÎNU

1, GH.F.

BRUDAŞCĂ

1, LILIANA CRIŞAN

2, EMOKE PALL

1,

V. NEGRUŢIU1, V. TUDOR

3

1University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 400372, 3-5 Mănăştur Street, Cluj-Napoca, Romania

2DSVSA Maramureş

3MSD Animal Health, România


Summary

Multiple vaccinations in layers enhance the in ovo transmission of subsequently produced total immunglobulins (Ig) and thus, conditions the resistance against diseases at hatching in chickens.

The experiment investigated the distribution of IgY and IgM both in the in serum and components of the egg (white and yolk) in repeatedly antigenically stimulated Anak hens (n=20). The hens were subjected to vaccination according to the technology and epidemiological pressure on the farm. Total Ig and IgY levels and immune complexes were quantified in optical density units (ODU) by 24%o zinc sulphate and 4.2% polyethilene glycol precipitation tests, respectively. IgM concentrations were calculated.

The total levels of Ig were the highest in the yolk (0.198±0.060 ODU), while those in the serum and egg white were similar. Similarly, the higherst value for IgY was recorded in the yolk (0.195±0.047) while IgM was significantly lower (p<0.001, 0.003±0.036). There was a statistically significant difference at various levels (p<0.05-p<0.001) between the IgY and IgM complexes (conventional units) both in the serum and egg components (serum IgY 111±10.7, IgM 75±14.6, egg yolk IgY 538.0±283, IgM 194±89, egg white IgY 71.02 ± 56.00, IgM 8.03±5.44).

These data underline the simultaneous transmission and concentration of IgY, free or in a conjugated form mainly through the yolk, in repeatedly vaccinated hens.

Key words: in ovo immunity, hens, vaccination

The avian egg contains protective antibodies originating from the blood

stream of the hen, therefore the immune globulins with high specificity will also be transferred to the chick embrio. The increased interest allocated to antibodies synthesis in farmed birds (chicken, ducks, etc.) supported by numerous experiments in the field (14) is based on the availability of antigen-specific yolk antibodies for numerous applications in the medical and research fields, including in areas such as diagnostics and proteomics. According to some of the researchers, one of the most important uses of these antibodies, designated as IgY given the morphological differences versus IgG of mammals, would be its use for preventive or therapeutical passive immunization of humans or animals against microbial diseases (8). Instead of using mammals for the obtainment of


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hyperimmune sera, antigenically stimulating hens and collecting their eggs for the isolation of the IgY seems a much more welfare preserving method. Similarly, egg yolk contains a high level of IgY readily available by fast and also simple isolation methods. As a plus, IgY does not react with the rheumatoid factor or the mammalian complement. Thus, IgY proves to be a perfect candidate for use as a polyclonal antibody, in various immunological diagnostic methods or immunotherapy (6). The experiment investigated the distribution of IgY and IgM both in the in serum and components of the egg (white and yolk) of conventionally raised Anak hens.


Birds and eggs. Anak hens (n=20) raised for eggs production on a conventional layer farm, were repeatedly antigenically stimulated, by being subjected vaccination according to the technology and epidemiological pressure on the farm. The protocol included vaccinations against Marek’s, Newcastle and Gumboro diseases and infectious bronchitis. The eggs were sampled during the 47

th week of the birds and preserved at +4°C before processing. The extraction of total IgY from egg yolk was performed according to the

precipitation method described by Pauly et al. (2011)(13). Circulating immune complex (CIC) measurements. The measurement of

circulating immune complexes allows an evaluation of the level of molecular clearance capacity of the body, at a particular moment. Blood samples collected from rodents were allowed to clot (30 min at 37C), and sera were separated by centrifugation (3,000 rpm, 10 min). The samples were kept at -20C until tested. A 4.2% polyethylene glycol (PEG) 6000 solution in borate buffer was used as the precipitating agent, while buffer-treated samples served as controls for borate-

induced precipitation. For each sample, 3.3-l aliquots of the sera/egg yolk/egg

white were mixed with 196.7 l borate buffer or PEG solution in parallel wells. The immune complexes precipitate in 60 min at 22–23C. Spectrophotometrical quantification of the precipitates was done at a wavelength of 450 nm in the test plate (d=0.5 cm; multichannel spectrophotometer SUMAL PE2, Karl Zeiss, Jena). CIC concentrations, expressed in optical density units (ODU), were calculated by subtracting the value of the control serum+buffer) from that of the PEG precipitate (Khokhlova et al., 2004).

Immunoglobulin measurements. Total immunoglobulins, known as opsonins, play an important role in innate immunity. Concentrations as low as 24 mg/l of heavy metal salts precipitate the immunoglobulin, affecting their colloidal

stability and electric charge. Volumes of 6.6 l of the sera/egg yolk/egg white were

diluted in 193.4 l of a 0.024% barbital-buffer zinc sulfate solution, and allowed to precipitate for 30 min at room temperature (22–23C). The levels of total immunoglobulins were quantified in optical density units (ODU) after


131

spectrophotometrical readings (k=475 nm, d=0.5 cm; multichannel spectrophotometer SUMAL PE2, Karl Zeiss, Jena).

Bothe methods were applied to untreated and 2ME treated sera/egg yolk/egg white. 2-mercapto-ethanol was used as an agent to inactivate IgM.

Total Ig, IgY, IgM and CIC concentrations were expressed in conventional units and Vernes degrees, by multiplying the ODU values with 10

2 for the immune

globulins and 103 for CIC. The data were statically processed and the different

values obtained for the tested variants were analysed using t-Student test.


IgY occurs throughout the vertebrate classes Amphibia, Reptilia and Aves, and probably in the subclass Dipnoi (16).

As a counterpart of IgG, IgY represents the major antibody produced by chickens involved in antimicrobial protection. Only IgM, IgA and IgY were well described in chickens, while proven IgD and IgE were only suggested. The amount of IgY is considerably higher in the egg yolk than in the blood stream (100 mg/egg yolk versus 5–7 mg/ml of serum)(1, 4)

Chicken immune system is similar to that of mammals therefore using chicken for the obtainment of specific antibodies brings great benefit in terms of welfare of the immunized animals, egg collection being much simpler than invasive blood sampling (15). This also meets the recommendations of the European Centre for the Validation of Alternative Methods (ECVAM), which specify that yolk antibodies should be used instead of mammalian antibodies for animal welfare reasons (14).

The IgY antibodies are harvested from the egg yolk and the amount obtained by immunizing birds i.e., with mammalian proteins, is substantially increased due to their phylogenetic distance. The IgY antibody activity spectrum is broader, since they recognize the same protein in a number of mammalian species. A supplementary advantage of obtaining IgY is the yield of antibodies: when compared to that of IgG antibodies obtained in mammals, 200 mg of IgG can be obtained monthly, with approximately 5% constituting the specific antibody, while in chickens the amount is of 1500 mg of IgY each month, and between 2 and 10% is the specific IgY (14, 15).

The concentration of IgY in egg yolk varies greatly between different bird species and even between chicken lines, thus 100- 200mg of total IgY can be extracted from a single egg with IgM and IgA which reach concentrations of 0.15 and 0.7mg/ml into the white eggs (12).

A wide variety of antigens including proteins, peptides, lipid hormones and carbohydrate components from viruses, bacteria, fungi, plants and animals were used to produce chicken antibodies (14). Immunoassays to detect Schistosoma japonicum or Pythium insidiosum antigen were developed and used in various ELISA tests among others (12). Quick and inexpensive production of a new generation of antibodies could also link the fields of genomics and proteomics (3).


132

75

111

194

538

8.03

71

0

100

200

300

400

500

600

u

n

i

t

s

Serum Egg yolk Egg w hite

IgM

IgY

Fig. 1. Circulating immune complexes levels in serum of vaccinated hens compared to those present in the egg components (conventional units)

19.8

6

19.5

4.7

0.33.6

0

5

10

15

20

25

30

Vern

es d

egre

es

Ig total IgY IgM

Stdev

Aver

Fig. 2. Total Ig levels in the the egg yolk of vaccinated hens (Vernes degrees)

IgY was suggested as a potential therapeutic agent in certain animal

species such as swine and chickens. In swine, experiments indicated its efficacy in


133

controlling diarrhea diseases (10) while in chickens it was advised in controlling Salmonella infections (2, 9), therefore, IgY could be probably the best alternative to antibiotics and the resultant reduction in antibiotic use in the livestock industry (9, 10). Recent experiments described in ruminants the control ruminal fermentations and modulation of the normal gut microflora by hen egg yolk antibodies (11).In addition, the immunisation of chickens or dugs using DNA constructs was also reported (3). To determine the synergistic effect of IgY with other alternative therapeutic strategies including probiotics and plant extracts could represent a future research direction (5).

Considering the benefits of IgY technology in polyclonal antibody production and the universal application of these antibodies in research and medicine, IgY technology could play an increasing role in research, diagnostics and immune therapy (17).

The amounts of CIC were highly significantly (p<0.001) increased in the egg yolk as opposed to those in the serum (Fig. 1). Both IgY complexes in the serum, egg white and egg yolk were elevated, when compared to IgM complexes, but only the difference between the egg youlk and serum or egg white were significantly different (p<0.05), while the comparison of serum and egg white values were not statistically supported.

The results of this experiment indicated, similarly to the literature, a significantly (p<0.05) higher yield of IgY in the egg yolk when compared to the serum (16.9±21.6 Vernes degrees) subsequently to the conventional vaccination procedures. The amount of IgM was highly significantly (p<0.001) lower, than that of IgY content, supporting the adequate protection against microbial diseases transferred to the offspring (Fig. 2).

Both methods used to quantify total Ig levels and CIC concentrations proved to be efficient and allowed the statistical interpretation of the data and allowed an estimate of the protective value of the eggs obtained from conventional hens.

References

1. Abouzid, K., Ndeboko, B., Durantel, S., Jamard, C., Zoulim, F., Buronfosse, T., Cova, L. Genetic vaccination for production of DNA-designed antibodies specific to Hepadnavirus envelope proteins Vaccine 2006, 24, 4615–4617.

2. Chalghoumi, R., Beckers, Y., Portetelle, D., Théwis, A., Hen egg yolk antibodies (IgY), production and use for passive immunization against bacterial enteric infections in chicken: a review Biotechnol. Agron. Soc. Environ. 2009 13(2), 295-308.

3. Cova, L., DNA-designed avian IgY antibodies: novel tools for research, diagnostics and therapy Journal of Clinical Virology 2005, 34, Suppl. 1, S70

S74.


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4. Dias da Silva, W., Tambourgi D.V., IgY: a promising antibody for use in immunodiagnostic and in immunotherapy, Vet Immunol Immunopathol., 2010, 135(3-4), 173-80.

5. Diraviyam, T., Zhao, B., Wang, Y., Schade, R., Michael, A., Zhang, X. Effect of Chicken Egg Yolk Antibodies (IgY) against Diarrhea in Domesticated Animals: A Systematic Review and Meta-Analysis. PLoS ONE, 2014, 9(5).

6. Gao, J., Zhou, Y.C., Huang, Y.F., Purification and clinical application of egg yolk immunoglobulins Zhonghua Nan Ke Xue. 2008;14(2):166-70.

7. Khokhlova, I.S., Spinu, M., Krasnov, B.R., Degen, A.A., Immune responses to fleas in two rodent species differing in natural prevalence of infestation and diversity of flea assemblages, Parasitol Res , 2004, 94, 304–311.

8. Kovacs-Nolan, J., Mine, Y., Egg Yolk Antibodies for Passive Immunity, Annual Review of Food Science and Technology, 2012, Vol. 3, 163-182.

9. Li, X., Nakano, T., Sunwoo, H. H., Paek, B. H., Chae, H. S., Sim, J. S., Effects of Egg and Yolk Weights on Yolk Antibody (IgY) Production in Laying Chickens Poultry Science, 1998, 77, 266–270.

10. Li, X., Wang, L., Zhen, Y., Li, S., Xu, Y. Chicken egg yolk antibodies (IgY) as non-antibiotic production enhancers for use in swine production: a review Journal of Animal Science and Biotechnology, 2015, 6, 40.

11. Marcq, C., Théwis, A., Portetelle, D., Beckers, Y., Refinement of the production of antigen-specific hen egg yolk antibodies (IgY) intended for passive dietary immunization in animals. A review. Biotechnol. Agron. Soc. Environ. 2013, 17(3), 483-493.

12. Munhoz, L. S., D’Ávila Vargas, G., Fischer, G., Lima, M., Esteves, P. A., Hübner S. O.. Avian IgY antibodies: characteristics and applications in immunodiagnostic, Ciência Rural, Santa Maria, 2014, v.44, n.1, p.153-160.

13. Pauly, D., Chacana, P.A., Calzado, E.G., Brembs, B., Schade, R. IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation. J. Vis. Exp. 2011 (51), e3084, doi:10.3791/3084.

14. Schade, R., Calzado, E.G., Sarmiento, R., Chacana, P.A., Porankiewicz-Asplund, J., Terzolo, H.R. Chicken Egg Yolk Antibodies (Igy-Technology): A Review Of Progress In Production And Use In Research And Human And Veterinary Medicine. Altern Lab Anim. 2005, 33(2), 129-54.

15. Tini, M., Jewell, U.R., Camenisch, G., Chilov, D., Gassmann, M. Generation and application of chicken egg-yolk antibodies Comparative Biochemistry and Physiology Part A 131, 2002, 569–574.

16. Warr, G.W., Magor, K.E., Higgins, D.A., IgY: clues to the origins of modern antibodies, Immunology today, 1995, vol.16, no.8, 392-398.

17. Zhang, W.-W., The use of gene-specific IgY antibodies for drug target discovery, Drug Discovery Today, 2003, Vol. 8, No. 8, 364-371.


135

THE DOSE-EFFECT RELATIONSHIP FOR BACTERICIDAL ACTIVITY OF THE WHOLE AND HEAT INACTIVATED OVINE

SERUM

MARINA SPÎNU, NOEMI VARGA, GH.F. BRUDAŞCĂ

University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary

Medicine, 3-5 Mănăştur street, 400372, Cluj-Napoca, Romania E-mail: [email protected]

Summary

The experiment aimed to establish the correlation and mathematical characteristics of dose –bactericidal capacity relationship for whole and temperature inactivated sheep sera, against G- and G+ bacteria involved in veterinary pathology.

26 blood samples were collected from adult Transylvanian Merino sheep, allowed to clot and separated by centrifugation. Whole or inactivated (56°C, 30 min) sera were diluted to 1/5, 1/25, 1/125, 1/625. Collection strains of Pseudomonas (P. pyocianea), Seratia (S. marcescens), Staphylococcus (S. aureus) and Listeria (L. monocytogenes) were used as test bacteria. The inoculum consisted of a 24 hour culture, diluted in distilled water to the density of 0.5 on McFarland scale. The incubated serum treated cultures were inactivated by formaline and read spectrophotometrically. Regression lines describing bacterial growth, as well as correlation coefficients of the dilution – effect relation – were defined for the tested bacteria and sera.

The function of bacterial growth in serum treated cultures (y=a+bx), was characterized for the whole sera by a indices of 0.024 (Listeria) to 0.0183 (Seratia) and b indices of 0.137 (Staphylococcus) to 0.722 (Seratia). For heat inactivated sera these indices were much lower (a from 0.0195 to 0.0313 and b from 0.071 to 0.332). Correlation coefficients were statistically supported (p<0.05- p<0.001) only for Seratia (both types of sera), Pseudomonas and Staphylococcus (treated serum).

Key words: sheep, serum, bactericidal effect, culture

Disease resistance is greatly influenced by an adequate passive

immunization just after birth. Thus, macromolecule uptake is severely reduced in response to premature birth and when macromolecules are to be absorbed from diets other than species-specific colostrum (11). The majority of strains (57 = 87.6%) proved 'delayed serum-sensitive' (DSS); 4 strains (6.2%) were 'promptly serum-sensitive' (PSS), whereas 4 strains (6.2%) resisted (NSS) complement-mediated killing by human serum. Species differences among fresh sera from sheep, rabbits and guinea pigs, as contrasted with fresh human serum, were noted. In general, PSS ('promptly serum-sensitive') strains of S. marcescens were killed in a slightly delayed fashion; DSS ('delayed serum-sensitive') strains of S. marcescens were killed in an unpredictable kinetic pattern by sheep and rabbit sera, whereas fresh guinea pig serum entirely failed to kill selected DSS isolates of S. marcescens (13). It was concluded that rabbit immunoglobulins of the IgG class


136

accounted for the observed in vitro antagonism of the bactericidal activity of fresh human serum against DSS and PSS strains of S. marcescens (12).

The aim of this study was to provide the picture of the dynamics of some blood parameters in the native Zerasca sheep breed during the peripartum period. Results showed a significant influence of the peripartum and the deviation from the normal range on many parameters (5). Serum antibodies killed efficiently B. ovis in vitro in the presence of either guinea pig or ovine serum (2). No effect of anti-Hp-IgG antibody in human serum on the growth of Hp was found (P>0.05). Human serum or fetal calf serum may be used for Helicobacter pilori culture where sheep blood is not available. Bactericidal activity against Helicobacter pilori in human serum is mediated by complement (3). The study of the sensitivity of F. tularensis to the bactericidal action of normal serum (NS) revealed that all virulent cultures were resistant, while avirulent cultures were highly sensitive to NS. The antibacterial activity of NS, with respect to avirulent strains, was determined by the system of the complement, activated following the classical route. Only complete S-LPS characteristic of virulent strains was capable of preventing the sorption of components and the assembly of the complement on the surface of bacteria, while defective forms of LPS of avirulent strains did not protect serosensitive receptors from interaction with the complement (10). This study aimed to evaluate the bactericidal capacity of the normal and heat inactivated serum and the dose-activity linear relationship, defining the regression lines.


The sera were collected from 26 healthy adult Transylvanian Merino

sheep, were allowed to clot, separated by centrifugation (2500 rpm for 10 min) and

kept at -20C until testing. Both whole and heat inactivated sera were used. The inactivation was carried out on a water bath at 56°C for 30 min. The sera were diluted to 1/5, 1/25, 1/125, 1/625 and used against a collection of field bacterial strains of Pseudomonas (P. pyocianea), Seratia (S. marcescens), Staphylococcus (S. aureus) and Listeria (L. monocytogenes). For this, 4 ml of simple broth were placed into two series of glass tubes. The first tube in each series was treated with either 1 ml of heat inactivated or 1 ml of whole serum. Afterwards, 1 ml of the mixture was transferred to the second tube, and so on, discarding the amount taken from the last tube. Positive control series were treated with saline instead of serum. All tubes were inseminated with an inoculum of a 24 hour culture, diluted in distilled water to the density of 0.5 on McFarland scale. Two series of tubes, for whole and heat inactivated sera were performed for each bacterial species. Subsequent to incubation of all series for 24 h at 37°C, the cultures were inactivated by formalin and read spectrophotometrically. The optical densities read for individual samples were statically processed and analysed for significance using t-Student test. Regression lines describing bacterial growth, as well as correlation


137

coefficients for dilution – effect relation were defined for the tested bacteria and sera.


The bactericidal and opsonic activities of normal sheep serum against 9

smooth and 4 rough strains of gram-negative bacteria were measured. Three smooth strains - Escherichia coli 3662, Salmonella typhimurium, and Salmonella gallinarum - were resistant to the bactericidal action of normal sheep serum with or without complement (8). Cattle and sheep sera had no lytic effect on 36.9-40.1% of pathogenic Leptospira strains, but other pathogenic strains, as well as saprophytes, were lysed by these sera. L. pomona and L. grippotyphosa exhibited high resistance to cattle serum, the latter being also resistant to sheep serum (1).

It was found that the bactericidal activity of the blood in newborn lambs was dependent on the amount of the immunoglobulins resorbed and their specificity (4).The bactericidal activity of blood, plasma, and serum from sheep was measured with 3 strains of Escherichia coli. Plasma showed a more intense bactericidal activity than blood or serum for strains O78:K80(B) and Lilly. It is suggested that the target antigen of the bactericidal antibody is not the somatic antigen, but a rough antigen internal to the O polysaccharide (9). The combination of therapeutically achievable concentrations of polymyxin B (range 5 to 1.25 mug/ml) and fresh, but not heat-inactivated human serum was found to exert an accelerated, additive effect against 9 of 10 'delayed serum-sensitive' isolates of S. marcescens, an organism that is characterized by intrinsic resistance against polymyxins (14). Since the principal defense mechanism against gram-negative bacteria is their destruction by complement in the presence of specific antibody (7), the bactericidal action of serum is one of the mechanisms protecting higher organisms from infection by gram-negative bacteria.

Table 1

Functions defined by calculating the coefficients based on optical density values read for the bacteria treated with whole and heat inactivated sera

Genus Whole serum Heat inactivated serum

Pseudomonas pyocyanea

y=0.705 x +0.011 y=0.800x +0.031

Seratia marcescens y=0.722 x +0.018 y=0.940x +0.024

Micrococcus luteus y=0.068 x +0.004 y=0.071x +0.004

Staphylococcus aureus

y=0.137x -0.003 y=0.332x +0.013

Listeria monocytogenes

y=0.385 x -0.024 y=0.281x -0.020


138

Complement (C) activation by the classical or alternative pathways plays a main role in this protective action. The bactericidal activity of normal newborn sera against some gram-negative bacteria is less efficient than that of normal adult sera. The reason for this may be connected with the damaged alternative and classical pathways of C activation and with the decreased levels of third -C3 and ninth -C9 factors of C system. A physiological deficiency of IgM antibodies may contribute also to the diminished bactericidal activity of newborn sera. Impaired activity of sera favors the susceptibility of neonates to bactericidal infection (6).

The equation y = ax + b is referred to as the slope-intercept form of a linear equation. In this form, the variable is x, and y, is the value of the function. It also has two coefficients, a and b. In this instance, the fact that the values of y depend on the values of x is an expression of the functional relationship between them. The linear equation is expressing the equality of values of the dependent variable y with the functional values of the linear function f(x) = ax + b, in other words y = f(x) for this particular linear function f (15).

In the case of sheep sera and the tested bacterial strains the coefficients defining the linear regression slope were higher for both normal and heat inactivated sera in the case of Gram negative bacteria and statistically significantly (p<0.001) lower in Gram positive bacteria. This stands for differentiation of serum components by antibacterial activity based on the type of bacteria subjected to serum treatment.

Heat inactivated serum induced a higher turbidity of the treated samples, signifying increased bacterial growth and lower inhibitory activity, respectively. Accordingly, the coefficients were higher.

Conclusions

In case of normal and heat inactivated sheep sera, the parameters defining the regression slope are dependant on the treatment the serum was subjected to and also the bacterial species subject to serum activity.

References

1. Ananina, Iu.V., Chernukha, Iu.G., Bactericidal activity of normal sera from various animal species against pathogenic and saprophytic leptospires, Zh Mikrobiol Epidemiol Immunobiol. 1983,(6), 33-7.

2. Estein, S.M., Cheves, P.C., Fiorentino, M.A., Cassataro, J., Paolicchi, F.A., Bowden, R.A., Immunogenicity of recombinant Omp31 from Brucella melitensis in rams and serum bactericidal activity against B. ovis. Vet Microbiol. 2004, 102(3-4), 203-13.

3. Fan, X.G., Li, T.G., Harry, H.X. Effect of human serum on the growth of Helicobacter pylori, Hunan Yi Ke Da Xue Xue Bao. 2000, 28, 25(4), 371-2.


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4. Georgiev, S., Resoprtion of colostric immunoglobulins and serum bactericidal activity of lambs during ontogeny. Vet Med Nauki. 1979, 16(1), 59-65.

5. Giuliotti, L., Lai, O., Cavallina, R., Alfieri, L., Rabusca, G., Benvenuti, M.N., Changes in physiological and immunological parameters during the peripartum period in Zerasca sheep. Pol J Vet Sci. 2014, 17(4), 737-9.

6. Jankowski, S., The role of complement and antibodies in the impaired bactericidal activity of neonatal sera against gram-negative bacteria. Acta Microbiol Pol. 1995, 44(1), 5-14.

7. Marr, J.J., Spilberg, I., A mechanism for decreased resistance to infection by gram-negative organisms during acute alcoholic intoxication. J Lab Clin Med. 1975, 86(2), 253-8.

8. Mittal, K.R, Ingram, D.G., Factors involved in bactericidal activity of sheep serum. Am J Vet Res. 1975, 36(08), 1183-7.

9. Mittal, K.R., Ingram, D.G., Bactericidal and opsonic activities of normal sheep serum against gram-negative bacteria. Am J Vet Res. 1975, 36 (08), 1189-93.

10. Pavlovich, N.V., Sorokin, V.M., Blagorodova, N.S. The resistance of Francisella tularensis to the bactericidal action of normal serum as a criterion for evaluating the virulence of the bacterium, Zh Mikrobiol Epidemiol Immunobiol. 1996, (1), 7-10.

11. Sangild, P.T., Uptake of colostral immunoglobulins by the compromised newborn farm animal. Acta Vet Scand Suppl. 2003, 98, 105-22.

12. Traub, W.H. Susceptibility of Serratia marcescens to human serum, antagonism of serum bactericidal activity by IgG immunoglobulins of homologous rabbit anti-O sera. Zentralbl Bakteriol A. 1980, 246(1), 26-49.

13. Traub, W.H., Fukushima, P.I., Mammalian serum susceptibility of Serratia marcescens, detection of three human serum susceptibility categories. Zentralbl Bakteriol Orig A., 1979, 245(3), 301-11.

14. Traub, W.H., Kleber I. Studies on the additive effect of polymyxin B and the bactericidal activity of human serum against Serratia marcescens. Chemotherapy., 1975, 21(3-4), 189-204.

15. https, //en.wikipedia.org/wiki/Linear_function_(calculus).


140

THE CHARACTERIZATION OF SOME STRAINS OF STAPHYLOCOCCUS ISOLATED FROM DOGS FROM MEHEDINTI

COUNTY, RURAL AREA

V.R. VOICHIŢOIU, J. DÉGI, CORINA PASCU, V. HERMAN


of Romania” from Timisoara, Faculty of Veterinary Medicine, 300645, Aradului Street No. 119, Timisoara, Romania


Summary

In the paper are described the characteristics of some strains of staphylococcus, in

order to investigate the prevalence of staphylococcus isolated from dogs, from four villages in the south-west part of Romania. We also tested the sensibility of these microorganisms to some of the most commonly used antiobiotics by local doctors.

The dogs from which we obtained all the samples were both adult and also puppies, of both gender. We have to mention that we didn’t know the history of previous antibiotic treatment for the animals selected for this study.

The samples were identified and labeled as to source, gender of the dog and also the anatomical area from where they were harvested. This way we were able to obtain 214 samples from different anatomical sites such as nose, ears, lips, neck, tail, legs and back skin.

After growth, staphylococcus isolates were identified according to their characteristics. 183 samples were positive for staphylococcus, being isolated both positive and coagulase-negative species. The most common species that was isolated was S. pseudintermedius. Key words: staphylococci, dogs, countryside, Romania

Staphylococci are considered to be ubiquitous, round shaped, gram-

positive, and arranged in irregular piles, with variations in size. Because their affinity for dermal tissue, pathogenic staphylococci can cause skin infections, but they can also invade other tissues or organs, producing systemic diseases. Coagulase-positive staphylococci (CoPS), Staphylococcus aureus, S. intermedius, S. pseudintermedius, S. delphini and S. schleiferi subsp. coagulans, are the most common cause of staph infections in dogs (1, 2, 5).

The aim of this research was: - to establish the frequency of S. intermedius strains isolated from dogs in

rural areas; - to determinate the frequency of other species of staphylococci isolated

from these dogs; - to characterize phenotypically isolates; - to identify strains of methicillin resistant.


141


Samples were obtained from 214 dogs, males and females, from April 2015 to February 2016. The dogs were from four villages near Drobeta-Turnu Severin, which were submitted to the national program of microchipping.

We included in this study dogs of all ages, both sexes and without having any information about their history of antibiotic treatment prior to sampling.

Isolates were subcultured on blood agar 5% and were incubated at 37°C for about 24 hours in aerobicall conditions. Suspected staphylococcal isolates were identified based on colony characteristics, pigment production, Gram stain and presence of hemolysis. We also used Chapman selective agar.

To identify the prevalence and antibiotic susceptibility of pathogenic staphylococci, investigations were carried out on 214 dogs from 4 different rural areas.

The evaluation of antibiotic susceptibility of 20 strains was performed by a disc-diffusion test. We used 18 antimicrobian agents belonging to several classes of antibiotics.


Results obtained at bacteriological examinations are shown in Table 1.

Table 1

The total number of strains of staphylococci isolated according to the anatomical areas

Anatomycal

areas

S. pseudintermedius

S. aureus

S. intermedius

Positive samples/total number

of samples taken from each region

External auditory canal

45 13 21 79/93

Perianal region

17 1 3 21/34

Skin with pyoderma

lesions 68 4 11 83/87

Total 130 18 35 183/214

Out of 214 samples taken from dogs, there were isolated and identified 183

staphilococci strains (3, 6, 9). The results of antibiotic susceptibility testing of staphylococci strains

isolated from clinically healthy dogs are shown in Table 2.


142

Table 2 The results of antibiotic resistance of 20 strains of staphylococcus isolated

from clinically healthy dogs

No. Antimicrobials Result (no of strains)

Resistant Intermediate Sensitive

1 Polymyxin B 8 6 8

2 Tetraciclyn 2 7 11

3 Gentamicin 5 2 3

4 Meticilyne 7 3 10

5 Novobiocin - 1 9

6 Cefaclor 4 6 10

7 Doxycycline 2 4 14

8 Erythromycin 4 1 15

9 Vancomycin 9 2 9

10 Ceftriaxone 1 9 10

11 Cefoxitin 3 7 10

12 Ciprofloxacin 4 8 8

13 Rimfapicin - 2 18

14 Lincomycin 4 3 13

15 Ampicillin 1 6 13

16 Pristinamycin 1 5 15

17 Amoxicilline 2 - 18

18 Kanamycin 3 2 15

In our survey, the distribution of resistant strains was as it follows: - 8 strains were resistant to polymyxin B; - 7 strains were resistant to meticilyne; - 9 were resistant to vancomycin; - 4 strains were resistant to lyncomicine and also the same numbers of

strains were resistant to cefaclor, erythromycin, ciprofloxacin (table 2). As you can see in table 2, there were also other antimicrobians to whom

our strains presented resistance but the numbers were smaller. The data on meticilyne resistance and type of resistance identified are

similar to the results communicated by other authors on the phenomenon of resistance to antibiotics (1, 2, 4, 5, 7).

Also the results that we obtained this year are similar to those that we obtained last year, when we did the same research on some dogs from another county (8).


143

Conclusions Out of 214 dogs from rural areas there were isolated a total of 183

staphylococci strains. In our study we found out that S.pseudintermedius was the most common strain, 130 from 183.

As a result of the test, in which we observed the resistance of the isolated staphylococci to 18 antimicrobians, there were identified seven methicillin-resistant strains and several type of resistance, to β-lactams, tetracyclines, macrolides, aminoglycosides. Ten of the isolated strains were meticilyne sensitive.

Acknowledgements This research work was carried out with the support of the


References

1. Cătană, N., Necșulescu, M., Herman, V., Rămneanțu, M., Șandor, A., Prevalenţa infecţiei cu Staphylococcus intermedius la câinii cu otită, Lucr. Şt. Al XXV-lea simpozion, Cluj-Napoca, “Actualităţi în creşterea şi patologia animalelor domestice”, Risoprint, Cluj-Napoca, 1999, 378.

2. Chrobak D (2011) Antibiotic resistance of canine Staphylococcus intermedius group (SIG) - practical implications. Pol J Vet Sci 14(2):213-218.

3. Duran, N., Ozer, B., Duran, G.G., Onlen, Y., Demir, C. Antibiotic resistance genes & susceptibility patterns in staphylococci. The Indian Journal of Medical Research. 2012, 135(3), 389-396.

4. Euzeby, J.P., List of Prokaryotic names with Standing in Nomenclature Genus Staphylococcus, http://www.bacterio.cict.fr/s/staphylococcus.html (11.03.2015).

5. Loeffler, A., Pfeiffer, D.U., Lindsay, J.A., Soares-Magalhaes, R., Lloyd, D.H., Lack of transmission of methicillin-resistant Saphylococcus aureus (MRSA) between apparently healthy dogs in a rescue kennel, Veterinary Microbiology, 2010, 141, 1/2, 178-181.

6. Miyamoto, T., Ishii, H. Antimicrobial drug susceptibility of methicillin-resistant Saphylococcus intermedius group isolated freom dogs and cats, Journal of the Japan Veterinary Medical Association, 2009, 62, 11, 882-885.

7. Moga Mânzat, R., Boli infecţioase ale animalelor, Bacterioze, Ed. Brumar, Timişoara, 2001.

8. Voichiţoiu, V. R., Dégi, J., Pascu, Corina., Herman, V., The characterization of some strains of Staphylococcus isolated from dogs from

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http://www.cabdirect.org/search.html?q=au%3A%22Voichit%CC%A7oiu%2C+V.+R.%22

http://www.cabdirect.org/search.html?q=au%3A%22D%C3%A9gi%2C+J.%22

http://www.cabdirect.org/search.html?q=au%3A%22Pascu%2C+C.%22

http://www.cabdirect.org/search.html?q=au%3A%22Herman%2C+V.%22


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9. Wang, Y., Methicillin-resistant Staphylococcus pseudintermedius isolated from canine pyoderma in North China. J Appl Microbiol. 2012, 112(4):623-630.

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145

AUTHORS INDEX

B Badea Corina – 68, 88, 113 Baraitareanu S. – 33 Bors S. I. – 26 Botus Daniela – 33 Brudaşcă Gh.F. – 129, 135 Bucur Iulia – 5, 15, 52 Bulacu A. – 113 C Caplan

E.M. – 33

Carp-Cărare C. – 20 Carp-Cărare M. – 20 Cătana N. – 5, 11, 15, 74, 123 Cerbu C.G. – 56, 62 Cioban Gh. – 113 Ciocan Oana-Alexandra – 20 Cozma Andreea-Paula – 20, 26 Crişan Liliana – 129 Crivei Ioana Cristina – 26 Culcescu M. – 33 D Danes Doina – 33 Danes M. – 33 Darabuş Gh. – 68, 78, 82, 88, 113 Dégi J. – 42, 74, 140 Dobrinescu Ana – 33 Drugociu D. – 26 E Ene G. – 33 F Fluerașu L. – 5, 52

G Gaşpar Cristina – 96 Ghișe Alina – 123 Giupană R.M. – 56, 62 Guguianu Eleonora – 20 H Herman V. – 15, 74, 140 Hora F. Ş. – 68, 78, 88 Horhogea Cristina – 20 Hotea Ionela – 82 I Iancu Ionica – 11, 15, 42, 74 Ilaşcă C. – 62 Ilie M. S. – 68, 78, 82, 88, 113 Imre K. – 78, 82, 88 Imre Mirela – 68, 78, 82, 88, 113 L Lăzărescu C. – 96 M Mareș M. – 20 Mates C.I. – 100, 107, 117 Mederle Narcisa – 68, 82, 113 Militaru D. – 33 Mitrică Mărăcine D. – 5, 52 Morar Adriana – 88 Morariu S. – 82, 113 Moscviciov A. – 62 N Negruţiu V. – 62, 129 Niculae Catalina – 100, 117 Niculae Mihaela – 56, 100, 107, 117


146

O Olah Diana – 62 Olariu Jurca A. – 78 Oprescu I. – 82 Otelea Maria Rodica – 33 P Pall Emoke – 100, 107, 117, 129 Pascu Corina – 5, 42, 74, 140 Patraș Irina – 78 Petrec Oana – 11, 123 Pop R. Al. – 117 Popa Virgilia – 11, 33, 52 Popescu Silvana – 56 R Rîmbu Cristina – 20 Roșca P. – 26 Ruginosu Elena – 26

S Sala Claudia – 88 Sorescu Denisa Ionela – 78 Spînu Marina – 56, 62, 100, 107, 117,

129, 135 Stancu A. – 52, 123 Ș Şandru Carmen Dana – 56, 100 T Tudor V. – 129 Ț Țibru I. – 96 V Varga Noemi – 135 Vasiu A. – 56 Voichiţoiu V.R. – 140


147

CONTENT

BUCUR IULIA, CORINA PASCU, D. MITRICĂ MĂRĂCINE, L. FLUERAȘU, N. CĂTANA

Frequency of staphylococci strains with antibiotic resistance isolated from animals

5

CĂTANA N., VIRGILIA POPA, OANA PETREC, IONICA IANCU

Research concerning the association of reovirosis with other infectious endemic diseases in broilers

11

CĂTANA N., IONICA IANCU, IULIA BUCUR, V. HERMAN

Research on the frequency of methicillin resistant strains of Staphylococcus spp.

15

COZMA ANDREEA-PAULA, OANA-ALEXANDRA CIOCAN, C. CARP-CĂRARE, ELEONORA GUGUIANU,CRISTINA HORHOGEA, CRISTINA RÎMBU, M. MAREȘ, M. CARP-CĂRARE

The prevalence of positive ESBL E. coli and Klebsiella pneumoniae strains isolated from the owners of pets

20

CRIVEI IOANA CRISTINA, ANDREEA PAULA COZMA, S. I. BORS, ELENA RUGINOSU, P. ROȘCA, D. DRUGOCIU

Antibiotics sensitivity of isolated bacteria from dairy cows with clinical endometritis

26

DANES M., VIRGILIA POPA,

G. ENE, E.M. CAPLAN,

M. CULCESCU, DANIELA

BOTUS, ANA DOBRINESCU,

D. MILITARU, S. BARAITAREANU,

MARIA

RODICA OTELEA, DOINA DANES

Abortions in sheep caused by the association of Toxoplasma gondii, Chlamydophila abortus and Campylobacter spp. - Case Study

33

DÉGI J., IONICA IANCU, CORINA PASCU

Distribution and antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitic milk – preliminary study

42


148

FLUERAȘU L., A. STANCU, IULIA BUCUR, D. MITRICĂ, VIRGILIA POPA

The seroprevalence of PRRS syndrome in young swine intended for fattening

52

GIUPANĂ R.M., C.G. CERBU, A. VASIU, SILVANA POPESCU, CARMEN DANA ŞANDRU, MIHAELA NICULAE, MARINA SPÎNU

Prevalence and etiology of subclinical mastitis in dairy buffaloes versus dairy cows from Transylvania, Romania

56

GIUPANĂ R.M., C.G. CERBU, DIANA OLAH, A. MOSCVICIOV, C. ILAŞCĂ, V. NEGRUŢIU, MARINA SPÎNU

The changes in non-specific systemic immunity in cattle with subclinical mastitis

62

HORA F. Ş., NARCISA MEDERLE, M. S. ILIE, MIRELA IMRE, CORINA BADEA, GH. DARABUŞ

The prevalence of gastrointestinal parasites in red foxes (Vulpes vulpes) in Bihor County

68

IANCU IONICA, J. DEGI, CORINA PASCU, V. HERMAN, N. CATANA

Observations upon infection with Mycoplasma gallisepticum in Japanese quail (Coturnix coturnix japonica)

74

ILIE M. S., MIRELA IMRE, K. IMRE, F.Ș. HORA, GH. DĂRĂBUȘ, A. OLARIU JURCA, IRINA PATRAȘ, DENISA IONELA SORESCU

Prevalence of Angiostrongylus vasorum infestation in dogs from Western Romania - preliminary results

78

ILIE M. S., MIRELA IMRE, IONELA HOTEA, I. OPRESCU, NARCISA MEDERLE, S. MORARIU, GH. DĂRĂBUȘ, K. IMRE

Testing the microfilaricidal effectiveness of some drug formulations in treatment of dogs naturaly infested with Drofilaria repens

82

IMRE K., CLAUDIA SALA, ADRIANA MORAR, M.S. ILIE, MIRELA IMRE, S. HORA, CORINA BADEA, G. DĂRĂBUŞ

Minireview of the European surface water distribution of G. duodenalis genotypes: from 2005 to nowadays

88


149

LĂZĂRESCU C., CRISTINA GAŞPAR, I. ȚIBRU

Attempting to solve the parasitism with Dermanissus galinae in laying hens

96

MATES C.I., MARINA SPINU, CARMEN DANA SANDRU, EMOKE PALL, CATALINA NICULAE, MIHAELA NICULAE

Antimicrobial protocols in bovine respiratory disease complex – A review

100

MATES C.I., MARINA SPINU, EMOKE PALL, MIHAELA NICULAE

Vaccination protocols in bovine respiratory disease complex – A review

107

MORARIU S., A. BULACU, GH. DĂRĂBUȘ, NARCISA MEDERLE, M.S. ILIE, C. BADEA, GH. CIOBAN, MIRELA IMRE

Preliminary research on the prevalence of Toxoplasma gondii infection in wisents (Bison bonasus) from Romanian Armenis-Plopu reserve

113

NICULAE MIHAELA, CATALINA NICULAE, C.I. MATES, EMOKE PALL, R. AL. POP, MARINA SPINU

Zoonotic potential of Staphylococcus pseudintermedius – A review

117

PETREC OANA, N. CĂTANA, ALINA GHIȘE, A. STANCU

Using of pappenheim stain for highlighting the reovirus pathological lesions

123

SPÎNU MARINA, GH.F. BRUDAŞCĂ, LILIANA CRIŞAN, EMOKE PALL, V. NEGRUŢIU

, V. TUDOR

Repeated vaccination enhances the in ovo transmission of Ig Y and Ig M in layers

129

SPÎNU MARINA, NOEMI VARGA, GH.F. BRUDAŞCĂ

The dose-effect relationship for bactericidal activity of the whole and heat inactivated ovine serum

135

VOICHIŢOIU V.R., J. DÉGI, CORINA PASCU, V. HERMAN

The characterization of some strains of Staphylococcus isolated from dogs from Mehedinti County, rural area

140

AUTHORS INDEX 145

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