Pathological and molecular diagnosis of rabies in ... · Pathological and molecular diagnosis of...

Pathological and molecular diagnosisof rabies in clinically suspected food animalsusing different diagnostic tests

SUMMARYIntroduction - Rabies is a fatal viral disease of the nervous system caused by a RNA virus, which belongs to the genus lyssavi-rus of the family Rhabdoviridae. Rabies can affect all mammals including humans. It is a serious veterinary and public healthproblem in Jordan and many other countries around the world. Therefore, early diagnosis with highly sensitive and specific te-sts will reduce unnecessary prophylaxis and treatment.Aim - The aim of this present study was to diagnose rabies in the clinically suspected cows, donkeys, horses and goats in Jor-dan by using different diagnostic tests and to compare the results of these tests.Materials and methods - During the years 2012-2013, a total of 11 brain samples were collected from different food animalspecies (5 cows, 3 donkeys, 1 horse and 2 goats) that were provided by the Vaccine and Sera Department / Al-Basheer centralhospital in Amman /Jordan. Clinically, rabies was suspected in these animals. These brain tissues were examined by fluore-scent antibody test (FAT), histopathology, immunohistochemistry (IHC) and reverse transcriptase polymerase chain reaction(RT-PCR).Results and discussion - The results showed that 55%, 45%, 82% and 91% of 11 brain tissues were positive for rabies by FAT,histopathology, IHC and RT-PCR respectively. The results of 5 animals out of 11 (45.5%) were consistent in all diagnostic te-sts where 4 (80%) of them were rabies positive. Two cases were rabies negative by FAT and proven to be rabies positive by theother tests. None of the examined clinically rabies cases was only detected by FAT. No significant difference was found whencomparing between any two diagnostic tests.Conclusions - Although FAT is considered the primary standard confirming test that is used to distinguish rabies encephalitisfrom other viral encephalitidis, these results do emphasize the importance of conducting more than FAT to diagnose rabies inanimals.

KEY WORDSFood animals, rabies, fluorescent antibody test, histopathology, immunohistochemistry, reverse transcriptase polymerase chainreaction.

W.M. HANANEH1, I.M. NASSIR1, M.M.K. ABABNEH2, N.Q. HAILAT1, C.C. BROWN3

1 Pathology Laboratory, Department of Pathology and Public Health2 Department of Basic Medical Veterinary Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, P. O. Box, 3030, Irbid 22110, Jordan.

3 Department of Pathology, College of Veterinary Medicine, University of Georgia, 501 D.W. Brooks Drive,Athens, GA 30602-7388, USA

INTRODUCTION

Rabies is a neuroinvasive viral disease that causes encephali-tis and death to human beings and mammals. The disease iscaused by lyssavirus genus, which belongs to Rhabdoviridaefamily. The genus has seven genotypes: Rabies virus, Lagosbat virus, Makola virus, Duvenhage virus, European bat lys-savirus 1 and 2 and Australian bat lyssavirus genotypes1.There are differences in the ability to infect the host, and dif-ferences in spreading through the host body. Rabies virus ge-notypes are divided to two phylogroups: phylogroup 1 andphylogroup 2. Phylogroup 1 contains genotype 1, 4, 5, 6 and7 while phylogroup 2 contains Lagos bat virus and Mokolavirus. Each phylogroup differs in its biological properties,pathogenicity, induction of apoptosis and recognition of re-

ceptors. The genotypes in phylogroup 1 are more pathogenicthan the genotypes in phylogroup 2.Rabies is a major problem to public health since three billionpeople in Asia and Africa are threatened by this disease2. Itsimportance came from the fact that Rabies is fatal to bothhuman and animals and for this reason a World Rabies Daywas established by CDC, OIE and WHO at 2007. Throu-ghout the world, 40,000 to 100,000 human deaths were rela-ted to rabies3.In Jordan, a retrospective epidemiological study was carriedout investigating the trend of rabies and animal bites from2000 to 2007. This study showed that Rabies was increasedfrom 1 case in 2003 to 50 cases in 2007 [dogs (56.52%), cat-tle (23.21%) sheep (7.6%), and goats (6.6%)]4. In anotherepidemiological study carried by Al Qudah et al.,5 showedthat stray dogs were the most common rabid animals and re-presented 45.12% of rabid cases. The same author stated thatdomestic animals were also affected: cattle 19.51%, donkeys4.87%, sheep, and goats 3.65%.

W.M. Hananeh et al. Large Animal Review 2015; 21: 243-250 243

Autore per la corrispondenza:W.M. Hananeh ([email protected]).

Hananeh_imp:ok 20-06-2016 11:32 Pagina 243

244 Pathological and molecular diagnosis of rabies in clinically suspected food animals using different diagnostic tests

Different diagnostic methods were developed and reco-gnized internationally for rabies diagnosis either in ani-mals or in human. In animals, WHO6 recommended manydiagnostic methods for detection of rabies. The fluore-scent antibody test is considered the gold standard for ra-bies diagnosis because it is rapid, sensitive and accurate.However, other methods could be used to diagnose rabies.These methods include: histopathology, virus antigen de-tection, mouse inoculation technique, and virus cultiva-tion. The aim of this research was to diagnose rabies usingbrains from the clinically suspected food animals via dif-ferent diagnostic tests (FAT, histopathology, IHC and RT-PCR) in Jordan.

MATERIALS AND METHODS

Sample CollectionEleven different brain tissue samples were provided by theVaccine and Sera Department / Al-Basheer central hospitalin Amman/Jordan. These brain samples were collected fromdifferent food animal species: cows (5 cases), donkeys (3 ca-ses), goats (2 cases) and horse (1 case). These animals wereclinically suspected for rabies. Half or part of the submittedbrain tissues samples were fixed in 10% formalin after beingexamined by FAT.

Diagnostic testsFluorescent Antibody Test - The FAT was carriedout in the Vaccine and Sera Department as it considered thereference diagnostic laboratory for rabies in Jordan. Theprocedure was conducted according to Bingham and vander Merwe, 2002. In summary, touch impression were madefrom three parts (cerebrum, cerebellum and brain stem)and fixed at -20 °C in acetone for 30 minute. After thatconjugate polyclonal antibody (fluorescein isothiocyanate)was applied and incubated for 30 minute at 37 °C the slideswere washed and glycerol drops were applied. Finally, theslides were covered and results were recorded under flore-scent microscope.

Histopathology - The formalin fixed brain tissue sam-ples were transported to the Veterinary Pathology Labora-tory at the Department of Veterinary Pathology and PublicHealth / Faculty of Veterinary Medicine at Jordan Universityof Science and Technology (JUST). The brain tissues weretrimmed and routinely processed in an automatic histoki-nette machine. The tissues routinely embedded and 4-5 µmtissue sections were made and stained for histopathologicalexamination with the ordinary Hematoxylin and Eosin stain(H&E).

Immunohistochemistry - The brain tissue samples,from paraffin-wax embedded blocks, 3-4 µm tissue sectionswere cut and mounted on coated slides. The slides were de-paraffinized by heating at 70 °C for 10 min, then by xylenefor 40 min and air dried. The slides were quenched by endo-genous peroxidase 3% H2O2 for 10 min and then were rinsedunder running water for 10 min. The slides were exposed tocitrate buffer 1 X solution and heat-induced epitope retrievalwas performed in a microwave for 4, 3, 3 min periods fol-

lowed by washing with phosphate buffered saline with tween20 (PBST) for 5 minutes. Protein block (Biogenex, USA) wasadded to the slides for 5 min followed by washing with PBSTfor 2 min. Primary rabies polyclonal antibody (Millipore,USA) was diluted in TBST according to manufacturer’s in-struction and applied to the slides and incubated for 2 h at37 °C. The antibody solution was washed twice by PBST for2 min. Biotinylated secondary antibody (Abcam, UK), dilu-ted in TBST according to manufacturer’s instruction, wasadded to each slide and incubated for 3 h at 37 °C. After in-cubation, secondary antibody solution was washed twice byPBST for 5 min each. ABC reagents were prepared accordingto manufacturer’s instruction 30 min prior to use. ABC rea-gents were added to the slides and incubated in humidchamber for 1h at 37 °C then washed twice by PBST for 5min each. DAB solution was added for each slide until colorwas developed. The slides then immersed in dH2O followedby staining with Meyer’s hematoxylin. Finally the slides weredehydrated routinely and prepared for evaluation.The immunohistochemically stained tissue sections wereexamined by light microscope and the IHC reactivity wererecorded.

RNA extraction - RNA extraction was carried out fromformalin fixed brain tissues according to the manufacturer’sinstructions (IQeasy, Plus Viral DNA/RNA Extraction Kit -Intron Biotechnology, Korea). The extraction technique waspreviously described by Faizee et al. (2012). At the end, 2-5µl of eluted solution was used for RT-PCR. However, at thebeginning, formalin was removed from the brain tissuesamples by treating them with xylene then with absoluteethanol alcohol for 5 min each for two times. After that pho-sphate buffer saline was added to the treated brain tissuesand were homogenized by a homogenizer. After homogeni-zation, the samples were centrifuged to remove un-lysed tis-sue particles.

Reverse Transcriptase-Polymerase ChainReaction - RT-PCR was carried out as previously de-scribed by Faizee et al.7. In summary, 4 µl of template RNAand 16 µl of DNase/RNase-free water was added into theRT-PCR premix tube according to manufacturer’s instruc-tion (VeTeK™ RV Detection Kit, Intron Biotechnology, Ko-rea). Two µl of positive control and 18 µl of RNase-free wa-ter was added into a RT-PCR premix tube for monitoringof amplification and easy interpretation (VeTeK™ RV De-tection Kit, Intron Biotechnology, Korea). The followingthermocycling program was used: 45 ºC for 30 min (Re-verse transcription step) and 94 ºC for 5 min (Inactivationof reverse transcriptase enzymes) 1 cycle, 94 ºC for 30 se-cond (Denaturation), 50 ºC for 30 seconds (Annealing),and 72 ºC for 40 seconds (Extension), and 72 ºC for 5 min(Final extension) 1 cycle.

Detection of Amplified Products - Detection ofthe amplified products was carried out using agarose gelelectrophoresis. A 1.5% of agarose gel was prepared andthen loaded with 7 µl of PCR product and 7 µl of positi-ve control. Electrophoresis was run for 30 minute /100volt. Then results were recorded under Ultra violet trans-illuminator.



RESULTS

Fluorescent Antibody TestRabies virus replicate inside infected cells cytoplasm as ovalinclusions bodies, contains mainly Nucleoprotein or N pro-tein, it appears as green bright apple particles by FAT. Elevenbrain tissue samples were included in this study; all were te-sted for FAT in the vaccine and sera department / Al BasheerCentral Hospital, Amman / Jordan. Six (55%) out of elevensubmitted animal brain samples were positive in FAT. Table1 summarizes the FAT results in different animal species.

HistopathologyPresence of Negri bodies within the affected neural cells wasconsidered positive regardless of the other lesions. However,absence of Negri bodies with presence of nonsuppurative in-flammation was considered a suspected case. Out of elevenexamined brain samples, 5 (45%) of them were consideredpositive. Table 2 summarizes histopathological results in diffe-rent animal species. In general, throughout the examined sec-tions in various animals the histopathological findings consi-sted mainly of variable degrees of non-suppurative menin-goencephalitis. The blood vessels within the parenchyma andmeninges were cuffed with one layer or more of mononuclearcells, primarily lymphocytes (Figure 1). Purkinje cells and

neuronal necrosis with or without Negri bodies were seen (Fi-gure 2). The Negri bodies appeared as single or multiple, eosi-nophilic intracytoplasmic inclusions. Multiple glial cell aggre-gates (babe’s nodules) were scattered throughout the affectedsections. Intravascular leukocytes sequestration was evident.

ImmunohistochemistryEleven brain tissue samples were examined by immunohi-stochemistry test. The positive signals appeared as variablysized rounded brownish inclusions within the cytoplasm ofthe neurons and Purkinje cells. The signal was stronger in thePurkinje cells as well as their dendrites (Figure 3). Nine cases(82%) out of 11 showed positive signals in the examined tis-sues. Table 3 summarizes the results of IHC.

Reverse Transcriptase-PolymeraseChain Reaction In RT-PCR examinations, the examined samples which hada band of approximately 263 bp was considered positiveand the samples which did not show a band at the same lo-cation of the positive control band was considered negative

Table 1 - Shows the results of FAT in different food animal species.

Animals Results

Cow (1) –

Donkey (2) +

Goat (3) +

Cow (4) +

Cow (5) +

Donkey (6) –

Donkey (7) –

Goat (8) –

Horse (9) +

Cow (10) +

Cow (11) –

Table 2 - Shows the results of histopathology in different food ani-mal species.

Animals Results

Cow (1) –

Donkey (2) –

Goat (3) +

Cow (4) +

Cow (5) –

Donkey (6) –

Donkey (7) +

Goat (8) –

Horse (9) +

Cow (10) +

Cow (11) –

Figure 1 - Cerebellum; Cow. The blood vessels within the paren-chyma and meninges were cuffed with one layer or more of mono-nuclear cells, primarily lymphocytes (arrows). H&E. 4X.

Figure 2 - Cerebellum; Cow. Purkinje cell necrosis (*) and onecontains more than one eosinophilic intracytoplasmic Negri bodieswere seen (Arrow). H&E. 40X.



consequential damage of any type ofdisease introduction or novel emer-gence are to maintain diagnostic ca-pabilities at specialty labs and to as-sess threats posed by outbreaks inthe other countries”. Moreover, earlydiagnosis with highly sensitive andspecific tests will reduce unnecessaryprophylaxis and treatment. In thepresent study, four different diagno-stic tests were used to diagnose ra-bies in the clinically suspected cows,donkeys, horses and goats in Jordan,during the years 2012-2013. Thebrain tissue samples were examinedby using FAT, histopathology, IHC,RT-PCR.Florescent Antibody Test is conside-red a golden test, recommended byWHO and OIE, because it has highspecificity and sensitivity for rabiesdiagnosis. It is a primary standard

confirming test that is used to distinguish rabies encephalitisfrom other viral encephalitis diseases9. It can be used on fro-zen, fresh or formalin fixed brain tissue samples10. Whitifieldet al.11 validated the use of FAT for routine diagnosis of ra-

Figure 3 - Cerebellum; bovine. A) Brownish IHC signal within the Purkinje cells and their den-drites. 4X. B) Inset higher magnification of A showed multiple variably sized round, strongly po-sitive inclusion bodies within the cytoplasm of Purkinje cells and their dendrites. IHC, labeledstreptavidin biotin (LSAB) method with Mayer’s hematoxylin counterstain. 40X.

Table 3 - Shows the results of IHC in different food animal species.

Animals Results

Cow (1) +

Donkey (2) +

Goat (3) +

Cow (4) +

Cow (5) +

Donkey (6) +

Donkey (7) +

Goat (8) –

Horse (9) +

Cow (10) +

Cow (11) –

A BA B

(Figure 4). Out of 11 examined brain tissue samples, 10(91%) were positive. Table 4 summarizes the results of theRT-PCR in eleven different animals.

In this study, four different diagnostic techniques were used.The summary of the different technique results was presen-ted in Table 5. The table showed that the results of five ani-mals out of 11 (45.5%) were consistent in all diagnostic testswhere four of them (80%) were rabies positive. Histopatho-logy was the least sensitive diagnostic test to diagnose rabieswhile RT-PCR was the most sensitive. No significant diffe-rence was found when comparing any two diagnostic tests. Itis worth mentioning that one case [donkey (7)] was negati-ve only by FAT and only two cases (cow 1 and cow 5) whereonly negative by histopathology.

DISCUSSION

Hanlon and Child8 wrote, “It is of paramount importance tokeep in mind that the first essentials in reducing the risk and

Table 4 - Shows the results of RT-PCR in different food animalspecies.

Animals Results

Cow (1) +

Donkey (2) +

Goat (3) +

Cow (4) +

Cow (5) +

Donkey (6) +

Donkey (7) +

Goat (8) +

Horse (9) +

Cow (10) +

Cow (11) –

Figure 4 - Agarose Gel Electrophoresis of RT-PCR Amplified Ra-bies Virus. The Figure Shows a Single Band of 263 bp RNA frag-ment. L: 50 bp size ladder, N: Negative control, P: Positive controls,1: Cow number 1, 2: Donkey 2.



bies in formalin-fixed brain tissues when fresh tissue was notavailable for testing.Rabies virus antigen appears as oval green bright particles,vary in their sizes. The results of this test are obtainedwithin two hours by well-trained laboratory staff however;the test needs florescent microscope12. Bingham, J. and VanDer Merwe, M.13 conducted a study to determine the mostreliable regions of the brain where rabies antigen was foundand to make recommendations for sampling of brains fromdifferent animal species for FAT. The authors found that thecerebellum, hippocampus and different parts of the cere-brum were negative in, respectively, 4.5, 4.9 and 3.9-11.1%of positive brains while thalamus was positive in examinedbrains.In this current study, FAT revealed 6 (55%) positive out of11 examined brain tissue samples from different animalspecies. In a previous study conducted by Faizee et al. 2012,out of 27 brain tissue samples examined by FAT from diffe-rent animal species, 21 (77.77%) were rabies positive. FATsensitivity depends on the expertise diagnostic staff and thequality of the submitted brain samples in addition to theconjugate used in the procedure, Bansal, K. et al.14. Inanother study carried out by Zimmer et al.15, FAT detected98% positivity of 187 brain tissue sample from clinicallysuspected animals of different species. It has been reportedthat false positive results can occur due the presence ofcross-reaction of rabies with other viral encephalitideswhen using FAT to detect rabies16 which indicate that thesensitivity of the FAT does not reach 100%.Histopathological techniques were used as a routine techni-que for rabies diagnosis. Some histopathology techniqueswere very rapid like Seller’s stain while others are long tech-niques like Mann’s stain and H and E stain on paraffin-em-bedded tissues. The pathognomonic lesions of rabies are en-cephalitis with Negri bodies12. Negri bodies appeared asround or oval eosinophilic intracytoplasmic inclusion bodieswith basophilic granules, in the Purkinje cells of the cerebel-lum, neurons of brain stem, cerebrum and medulla. Presen-ce of Negri bodies is pathognomonic of rabies. However,they were not seen in every single case of rabies. Failing todemonstrate Negri bodies does not rule out rabies becauseNegri bodies are absent in 20%-60% of rabies cases9. Moreo-ver, intracytoplasmic inclusion bodies were seen in thebrains of non-rabid animals17;18,19. Mann’s stain, Geimsa andSeller can differentiate inclusion bodies of rabies from otherintracellular inclusion bodies. In addition to the presence ofNegri bodies, variable degrees of non suppurative encephali-tis that is characterized by lymphocytic perivascular cuffscould be found in different areas of the affected brain. In ourstudy Negri bodies were seen in 5 (45%) of clinically suspec-ted animals. A higher percentage of Negri bodies (59%) we-

re reported by Faizee et al.7. The differences in detection be-cause Negri bodies are not consistently present in every sin-gle case of rabies9.Immunochemistry is the identification or localization of anantigen in histological tissue section via specific antibody.Immunochemistry in the last two decades becomes the mo-st important test for localization of antigen in fixed tissuesamples using labeled antibody. Paraffin embedded sectionswere used in IHC staining. Immunohistochemistry techni-que in formalin fixed tissue samples has been used to inve-stigate rabies virus pathogenesis in humans as well as ani-mals. In this study, 9 (82%) out of 11 examined brain tissueswere positive via IHC. It detected all the positive sampleeither those detected by FAT or those detected by histo-pathology. It was highly sensitive in detecting rabies in clini-cally suspected animals in which FAT and histopathology fai-led to detect rabies antigen. IHC is sensitive as florescent an-tibody test or equals to FAT in rabies diagnosis12. It is requi-red skilled diagnostic personnel. It is more sensitive than hi-stopathology and Seller stain for rabies diagnosis. The anti-bodies and all reagents are expensive and some of them arecarcinogenic or toxic.Reverse transcriptase-polymerase chain reaction amplifyRNA in skin biopsy and saliva, serum, cerebral spinal fluidand brain tissue samples20. The same authors found that thesensitivity and specificity of RT-PCR were 100% comparedto 83.3% of FAT and the authors stated that RT-PCR can beused as a confirmatory test.RT-PCR, reduced the time required for diagnosis and re-sults are obtained in few hours. It detects rabies virus anti-gen from highly decomposed tissue unsuitable for FAT andhistopathology diagnosis. The test is an important rapidsensitive diagnostic method, which has been used to detectthe virus in saliva of a patient who never had been vaccina-ted against rabies21.This method could overtake FAT as the primary test for ra-bies diagnosis because RT-PCR is successful for sequencingof rabies virus genome by using one or more specific pri-mers. It is also used to distinguish between rabies virus anddifferent related lyssaviruses by using specific primer for ea-ch genotype12. Moreover, it detects the rabies antigen earlierthan the other diagnostic tests20. However false positive re-sults were reported to occur in RT-PCR22. It was not recom-mended for routine postmortem diagnosis of rabies due tohigh false positive and false negative results23.When a comparison where done between the results of FATand the result of the histology, no significant difference wasfound (P value > 0.05). Similar results were seen when com-parison between FAT and IHC was conducted. Furthermore,no statistical significant difference was found when compa-ring between any two different tests. These insignificant dif-

Table 5 - Summarizes the test results of FAT, Histopathology, IHC, and RT-PCR in different food animal species.

Cow (1) Donkey (2) Goat (3) Cow (4) Cow (5) Donkey (6) Donkey (7) Goat (8) Horse (9) Cow (10) Cow (11)

FAT – + + + + – – – + + –

Histopathology – – + + – – + – + + –

IHC + + + + + + + – + + –

RT-PCR + + + + + + + + + + –

TestAnimals



ferences might be explained because of the small number ofsamples tested although variation in the results were present.

CONCLUSION

In Jordan, Rabies is more frequently occurred in cows morethan any other food animals. In the present study, althoughthere was no significant difference between the different te-sts to diagnose rabies, FAT alone should not be rely upon todiagnose rabies. At least one more confirmatory test shouldbe carried out to confirm FAT results. RT-PCR is a good can-didate but false positive results might be of concern.

ACKNOWLEDGMENTS

We thank the Veterinary Pathology Laboratories at JordanUniversity of Science and Technology for supporting this re-search Work.

References

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