Fasciola

Some Diagnostic Studies On Fasciola Infestation Among Cattle And Buffalo

لالصابة التشخيصية الدراسات لالصابة بعض التشخيصية الدراسات بعضوالجاموس االبقار في والجاموس بالفاشيوال االبقار في بالفاشيوال

Presented by:

Vet: Seham Fawzy HassanBVSCS (2009)

Taxonomy

Kingdom : AnimaliaPhylum : PlatyhelminthesClass : TrematodaSubclass : Digenea Order : EchinostomifomisFamily : FasioloidaGenus : FasciolaSpecies : hepatic

gigantica

Morphology They are flat leaf-like ,

grayish brown in color

Fasciola hepatica

Fasciola gigantica

they may reach up to 7.5 cm

length.

Averaging 30 mm in length and 13 mm in width

Life cycle

Fascioliosis

Fascioliosis is one of the world wide parasitic disease common in ruminants (sheep, goat, cattle, buffaloes, camels) ,

swine, horses, donkeys and rabbits caused by Trematoda called Fasciola spp characterized by liver damage, animal poor

condition, decrease in productivity beside it has zoonotic importance.

EPIDEMILOGY OF FASCIOLIOSIS

1 -The occurrence

Management

The occurance differed according to:

SeasonSpecies

AgeSex

Zoonotic importance

In a study made by Basem Refat (2013):

he found that the residence distribution of human fascioliosis in Assuit Governorate was 8.33 % and in New Valley Governorate was

8.70% .

Economic importanceThe economic loss represented by:

•Condemnation or down-grading of affected livers at abattoirs.•Suboptimal live weight gain and reduced production and/or quality of

meat and milk in cattle with chronic liver fluke infections .•Cost of prophylactic or therapeutic anthelmintic treatment .

•Animal welfare issues implications .•Risk of zoonotic infection.

Death of animals acutely infected with large numbers of flukes (unusual in cattle) .

The annual loss in the world due to fascioliasis is 3.2 billion dollars.In Egypt losses in meat and milk due to fascioliasis was 30% per year= one milliard pound (according to issue of June 1998 of the General Organization of Veterinary Services, Ministry of Agriculture).

Pathogenesis Infection occur through ingestion of the encysted metacercaria on green fodders.

The prehepatic stage: the metacercaria excysted in the lumen of

intestine .Then cercaria begin it’s journey in the abdominal cavity, newelly exysted juvenile penetrate the intestinal mucosa and found in the abdominal cavity, then begin it’s journey to the liver.

Flukes can be carry on to penetrate other organs as the lung ,diaphram ,fetus in pregnant animal

The hepatic stages

During migeration of the fluke through the liver parenchyma it causes arteritis ,inflammatory reaction ,fiberosis of the parenchyma.

The flukes concenterated at the venteral aspect of the liver .

The bile duct thickened due to hypertrophy and fiberosis of the wall, Ca depositition starts at the wall of the bile duct.

Stenosis or compelete obstruction of biliary ducts .

Clinical signs

Acute type I fascioliasis Occurs when the animal ingests more than 5000 metacercariae, which may lead to its sudden death, especially sheep and goats without showing any previous

clinical signs. Acute type II fascioliasis: Infection occurs by the ingestion of 1000-5000 metacercariae. the animal dies and showing signs of pallor, loss of condition and ascites.

Subacute fascioliasis: occurs due to the ingestion of 800-1000 metacercariae. The animal becomes weak, anemic and weight loss may occur resulting in death of the animal.

Chronic fascioliasis occurs when 200-800 metacercariae are ingested. Chronic Fasciolasis is prolonged and does not have clear key symptoms except for gradual weight loss, pallor of mucous membranes, ventral oedema and wool break.

Loss of condition Lethargy Anaemia Bottle jaw

Sub-optimal growth ratesjaundice

•Diarrhoea •Metabolic disease in dairy cows •Reduced milk yield in dairy cows

•Reduced fertility •Signs are exacerbated by poor nutrition or gastro-

intestinal parasitism

Effect of fascioliosis on blood components

1-Anaemia: which accepted that is hemorrhagic anemia.

2-Plasma protein: decrease in albumin concentration partly due to decrease the rate of synthesis and increase expansion of plasma volume.

3-Immunoglobulin: increase in immunoglobulin synthesis including IgG1, IgG2, IgM, IgE.

4-Dramatic increase in eosinophils.

5-Billirubinemia:due to presence of adult flukes in the bile duct which affect production and flow of the bile.

Postmortem lesions-Emaciated, anemic, edematous and/or icteric carcass due to

liver damage.-Liver enlargement with bumpy, raised and/or depressed

areas, dark blue to black discolorations, hardness in consistence.

- -Hemorrhagic tracts of migratory immature flukes in the liver in an acute infection.

-Black parasitic material (excrement) in the liver . -Cirrhotic effect on the liver (scarred surface) .

-Enlarged, thickened and calcified bile ducts

Diagnosis

1-coprological diagnosis.

2-serological diagnosis.

The prevalenceIn a study made by Sahar M. Selim And Azza Hassan Mohamed (2008) on 362 sheep in Menoufiya Governorate he found that the prevalence of fasciola infection was 65.7% with ELISA and 42.3% by fecal examination

Coprological examination

Coprological examinations involve examining the feces of animals to identify the parasite eggs.

Coprological examination

Detection of fasciola egg is simple and confirmatory but it is not useful diagnostic tool at low levels of adult fluke burden.

Also it cannot detect infection at the prepatent period, because eggs are found in feces when the fluke are already matured usually between (10-14 weeks of infection) by this time major damages to the liver parenchyma, cause hemorrhages and damages to the liver may have already occurred

due to fasciola entering to liver .

Coprological diagnosisFalse negative:

1(Egg deposition of the parasite is irregular.2-the incubation phase of the infection is shorter than that of

the prepatent period.3-clinical findings of the disease may appear long before egg

can be found in feces.4-in case of obstruction of the billiary ducts.

False positive:In case of death of the worm the egg laying persist for 21 days after death of the worm.

Serological diagnosis

Immunological examinations, such as indirect immunofloursence , ELISA, and complement fixation test are methods of identifying different kinds of parasites by detecting the presence of their antigens

or within the parasite itself .These diagnostic methods are used in conjunction with coprological examinations for more specific identification of different parasite species in fecal samples.

Serological method have been developed as an alternative

approach to fecal egg detection ,which can test large number of sera at the same time and also

detect infection earlier than fecal egg examination there are evidences to show that serodiagnosis can detect the presence of infection as early as 2 weeks after infection.

during the chronic phase, in cases with low-level or

sporadic production of eggs ; in cases of ectopic infection, in which eggs are not found in stool; and after treatment, to assess the response



False positive:

In case of cross reaction with other trematods.

False negative:

1-In case of aged animals.

2-In case of animals with exhausted immune system

Aim of the work

1 -Studying the prevalence of animal fascioliasis among cattle and buffaloes in Assiut Governorate through coprological examination and two different serological methods (Agar Gel Diffusion Test and ELISA). 2- comparing between the specificity, sensitivity, accuracy of each method. 3- Rerecording the prevalence of fascioliasis in cattle and buffaloes in Assuit in relation to species (cattle or buffaloes),season, age and sex.

Protocol of workProtocol of workProtocol of workProtocol of work

1-source of the sample

• Live animals from different farms and households (feces and blood samples) , slaughtered animals from different slaughter houses.

Sample collection and handling

• From each live animal (from different farms and different household animals) blood and fecal sample will be taken after clinical examination and infected slaughtered aniamls recording it and taking the fasciola worms from infected livers for antigen preparation.

1-Faecal samples

About 10-20 gm feces from each cattle and buffaloes will be collected for

parasitological examination directly from the rectum of each animal before

slaughtering and freshly defecated feces will be put in plastic bags with gloved

hand.

2-Blood samples:

10 ml of whole blood samples will be taken from jugular vein of each cattle and buffaloes to serum tubes and be allowed to clot. Sera separating by centrifugation at 3000 rpm for 15 minutes after being keeping it in the refrigerator overnight. Sera will be kept at (-20) c until used.

3-Adult worms

Routine postmortem examination of each slaughtered animal will be carried out to check the presence of fasciola, then the adult worms

collected for antigen preparation .

Fasciola antigen1-somatic antigen:

It is an antigen located in the body of the worm.

2-secretory and execretory antigen:a) Proteases: which used by the worm in migration through host tissue, acquisitation of nutrition , evasion of the host immune response .

b) Fluke hemoglobin: which is involved in aerobic respiration.

c) Proline : amino acid used in proliferation of the epithelium of the bile ducts.

d) Tegumental execretion :which produced due to osmoregulation .

1 -Macroscopic examination:The physical characters of the feces studied (color, consistency, presence of blood /or mucus).

2 -Microscopic examinationUsing sedimentation technique

Fecal examination

Egg of fasciola

The eggs are yellow-brown and operculated with a thickening of the shell at the poles; their dimensions are 130 to

145 µm by 70 µm to 90 µm.

operculum


1-Antigen preparation.

2-ELISA.

3-Agar Gel Immuno-diffusion.

1-Antigen preparation.Preparation of somatic antigen:

Antigen preparation from adult fasciola worms according to (J.B. W. J. Cornelissen et al., 1992)

1-Adult flukes (10 g), collected from the bile ducts of infected slaughtered bovine.

2 -washing 3-4 times at room temperature for 1 h in 0.01 M phosphate-buffered saline (PBS) (pH 7.0).

3-The flukes will be homogenized and the ground flukes will be extracted overnight in 100 ml of a mixture of phosphate buffered saline with 100 U/ml Na penicillin, 0.25 mg/ml streptomycin, 100 U/ml nystatin at 4 c.


5-The suspension will be centrifuged at 10,000 x g for 20 min at 4°C. The supernatant filtered and stored at -70 °C until used.

6-The protein concenteration of antigens will be determined.

2-ELISA

ELISA1-Coating ELISA Plates :(Voller et al., 1977)

Coating is achieved through passive adsorption of the antigen to the assay microplate . This process occurs though hydrophobic interactions between the micro titer plate and fasciola antigen .

1-The most common method for coating plates involves adding a 0.1-10 μg/ml solution of protein dissolved in an alkaline buffer such as phosphate-buffered saline (pH 7.4) or carbonate-bicarbonate buffer (pH 9.4) but the technique applied for fasciola antigen the concenteration

of the antigen is 0.5-2 microgram/ml .

N.B. The buffer used for dilution contains no other proteins that might compete with the fasciola antigen for attachment to the micro titer plate.

2-Then washing the plate 3 times with washing buffer .

3-The plate wells will be blocked with 5% bovine serum albumin (200 microliter in each well) for blocking all the spaces to prevent adsorbtion of any protein other than fasciola antigens

4 -incubation of the coated plate 1 hour at 37 c. then the plate will be washed 3 times with washing buffer.

ELISA2 -Addition of the serum :

100 microliter of diluted serum samples (1:50) with phosphate buffer serum (PBS) pH 7.2 will be added in each well and incubated 30 minutes at room temperature .Then washing the plate 5 times with washing buffer.

3 -Addition of the conjugate:

Dilution of enzyme conjugate to (1: 500) with dilution sample (P.B.S).Then 100 microliter of diluted enzyme conjugate will be transferred to each well and incubation for 1 hour at 37 c . After incubation, plate will be washed and 5 times with washing buffer.

ELISA4 -Addition of the substrate

Washing the plate and adding the 3,3,5,5 Tetramethyl benzidine dihydrochloride (TMB) in each well, then incubate the plate in the dark for 30 min at room temperature.

5-Stopping the reaction

The color reaction stopped with 50 microliter 0.1 M sulphoric acid per well.

6-Reading the result

Color changes will be measured in ELISA reader at 450 nm filter.

Adult worms of fasciola sp. Will be collected from the bile ducts of infected cattle and buffaloes. The worms will be washed with physiological saline and storing it at – 20 c until examination.

The antigen will be prepared by homogenizing 0.1 g of each adult fluke for 30 min in 5 ml of physiological saline. the emulsion will be frezed and thawed twice and centrifuged at 5000 rpm for 30 min. the supernatant of each emulsion used as the antigen.The protein concenteration of antigens will be determined.


Preparation of somatic antigen: acc to linh et al., (2003)

2-Agar gel Immunodiffusion

The passive diffusion of soluble antigen and it’s specific antibody towords each other to make precepitation line between each other

Agar gel Immunodiffusion

E) Stastical analysis:

The data obtained will be tabulated in relation to difference in species, sex, age, locality, season and the sensitivity, specificty, accuracy of the methods of diagnosis.

Control

1-Antifasciolia drugs usage (Rafoxanide ,tricalbendazole, albendazole).

2-Snail control (Niclosamide) .3-Vaccination

4-Managemental control

Refrences Ash, L. R. and Orihel, T. C. (1987): Parasites: A Guide to Laboratory Procedures and

Identification. American Society of Clinical Pathologists Press, Chicago. pp. 4-30.Hansen, J. and Perry, B., (1994): The Epidemiology, Diagnosis and Control of Helminth Parasites of Ruminants, in Africa, A Hand Book, International Laboratory for Research on Animal Diseases (ILRAD) Nairobi, Kenya, pp. 25-45.

J.B. W. J. Cornelissen , W. A. de Leeuw& P. J. van der Heijden (1992):comparison of an indirect haemagglutination assay and an Elisa for diagnosing Fasciola hepatica in experimentally and naturally infected sheep,Veterinary Quarterly, 14:4, 152-156.

Linh, B. K.; Thuy ,D. T.; My, L. N.; Sasaki, O. and Yoshihara, S. (2003):"Application

of Agar Gel Diffusion Test to the Diagnosis of Fascioliosis in the Cattle and Buffaloes in the

Red River Delta of Vietnam." J. A. R. Q.37 (3): 201-205.

Soulsby, E.J.L. (1982):Helminths, Arthropods and Protozoa of Domesticated Animals. 7th

ed. London, England. Bailliere Tindall, pp: 42-50, 800- 809.

Voller, A., Bidwell, D. E., and Bartlett, A. (1977a): "The Enzyme Linked Immunosorbent

Assay (ELISA). "pp. 24-26. Flow-line Puplications,Guernsey.

Fasciola

Health & Medicine

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