A total of 7339 fox, dog, cat, cattle, sheep and other mammalian brains were tested to rabies virus antigen. Results of fluorescent antibody test (FAT) were evaluated by two microscopists. Doubtful and discordant results in FAT were analyzed again in virus isolation test (VIT) using mouse neuroblastoma (NA) cell line. Twenty-eight brain samples were determined as doubtful, while 9 brain samples were determined to be positive by one microscopist and negative by the other. Samples which were shown as doubtful and discordant in FAT were retested in VIT. Seventeen of these were positive in the VIT.Na prisutnost antigena virusa bjesnoće bilo je pretraženo ukupno 7339 uzoraka mozgova lisica, mačaka, goveda, ovaca i drugih vrsta životinja. Rezultate izravnog imunofluorescentnog testa vrednovala su dva stručnjaka. Uzorci koji su u testu imunofluorescencije davali sumnjive rezultate, ili se rezultati stručnjaka nisu slagali bili su pretraženi izdvajanjem virusa na kulturi stanica. Rabljena je stanična linija neuroblastoma miševa. Dvadeset osam uzoraka bilo je proglašeno sumnjivim na bjesnoću. Rezultat prvog mikroskopskog pregleda u devet uzoraka bio je pozitivan, a drugog negativan. Od ukupno 37 uzoraka, koji su bili ponovno pretraženi izdvajanjem virusa, 17 je bilo pozitivnih na bjesnoću

Darja Barlič-Maganja

Jože Grom

Marjeta Štrancar

Peter Hostnik

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65Doubtful and discordant results in fluorescentantibody test for rabies diagnosingPeter Hostnik*, Marjeta Štrancar, Darja Barlič-Maganja,and Jože GromInstitute for Microbiology, Veterinary Faculty, University of Ljubljana, Ljubljana, SloveniaHOSTNIK, P., M. ŠTRANCAR, D. BARLIČ-MAGANJA, J. GROM:Doubtful and discordant results in fluorescent antibody test for rabiesdiagnosing. Vet. arhiv 71, 65-73, 2001.ABSTRACTA total of 7339 fox, dog, cat, cattle, sheep and other mammalian brains were tested to rabiesvirus antigen. Results of fluorescent antibody test (FAT) were evaluated by two microscopists. Doubtfuland discordant results in FAT were analyzed again in virus isolation test (VIT) using mouseneuroblastoma (NA) cell line. Twenty-eight brain samples were determined as doubtful, while 9 brainsamples were determined to be positive by one microscopist and negative by the other. Samples whichwere shown as doubtful and discordant in FAT were retested in VIT. Seventeen of these were positivein the VIT.Key words: rabies, immunofluorescence, cell cultureIntroductionRabies being an infectious zoonotic disease, it is still considered themost feared disease present on all five continents. Two epidemiologicalforms of rabies are known: urban rabies, where dogs are responsible forthe maintenance and in many cases for the transmission of the disease toman. The other, wildlife (sylvatic) form of rabies spreads among wildanimals. In Central European countries the main vector for sylvatic rabiesis the red fox (Vulpes vulpes). In Slovenia, where urban rabies is under* Contact address:Doc. Dr. Peter Hostnik, DVM, Institute for Microbiology, Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia;Phone: +386 61 4779 178, Fax: +386 61 4779 542, E-mail: Peter.Hostnik@vf.uni-lj.siISSN 0372-5480Printed in CroatiaVETERINARSKI ARHIV  71 (2), 65-73, 200166control through an effective dog vaccination programme, the rabies diseaseis still present among wildlife. Wild animals, particularly rabid foxes, couldbe a potential source of rabies infection to non-vaccinated domestic animals,and directly or indirectly to humans (PASTORET et al., 1985). The disease iscaused by the Lyssa virus, which is a member of the family Rhabdoviridae,genus Lyssavirus. The virion is enveloped, bullet shaped, lipid-containingand consists of a single-stranded negative sense RNA (TORDO and POCH,1988; VAN REGENMORTEL et al., 2000). This neurotropic virus is pathogenicand deadly to all warm-blooded animals, including humans. The clinicalsign of the disease is characterized by viral encephalitis (CHARLTON, 1991).Diagnosing rabies in animals is most important mainly in cases wherethe examined animal has bitten, harmed or been in any contact with humansprior to death. In cases where a rabies-positive animal is discovered, postexposure or follow-up rabies treatment for humans is essential. A sensitiveand specific diagnostic test must be used. The mouse inoculation test (MIT),traditionally known as the “golden” method in diagnosing rabies, is usedto confirm results of other techniques (KOPROWSKI, 1996). The majordifficulty with this method is a two- to three-week waiting period whilethe test mice are observed. The fluorescent antibody test (FAT) is the mostroutinely employed method and recommended by WHO for detecting viralantigens in brain samples. The most important factors for a reliable resultare: specific rabies immunoglobulin conjugate, a sophisticated microscopeand trained technicians (ANONYMOUS, 1992). The correlation between bothtests is good. However, FAT false-negative results at a level of 0.2% aredescribed (HEMACHUDHA, 1989). The virus isolation test (VIT) on cellcultures has replaced the MIT in many laboratories as the test is moresensitive, easy to perform, less time consuming and more humane (WEBSTERand CASEY, 1996). The ELISA test is also employed routinely usingcommercially available kits (Diagnostics Pasteur), which can be especiallyuseful for epidemiological studies (ANONYMOUS, 1992) Also, the polymerasechain reaction test (PCR) is the most commonly used method forepidemiological studies (WHITBY et al., 1997).The first rabid fox in Slovenia was discovered in 1973 near theHungarian border. This was followed by intensive fox hunting but whichfailed to halt the spread of the disease. The most explosive outbreakP. Hostnik et al.: Doubtful and discordant results in fluorescent antibody test for rabies diagnosingVet. arhiv 71  (2),  65-73, 200167occurred in 1981 in the north-western part of Slovenia near the borderswith Austria and Italy. The occurrence rate increased and decreased inregular cycles. Vaccination against rabies is mandatory for all dogs andfor farm animals that pasture every year; vaccination of other animals ison a voluntary basis (BIDOVEC et al., 1993).In our laboratory the diagnosis of rabies in animals were earlier carriedout by Negri body detection with use of Seller’s stain, but it has beenaccomplished by use of the fluorescent antibody test since 1973. Samplesof the hippocampus (horn of Amon) were stained in both tests. Tissuespecimens with doubtful results on microscopic examination, or a specimenobtained from an animal that had bit a man, were all submitted for mouseinoculation test. The mice were observed for 4 weeks.Post-exposure vaccination of humans following an animal bite orscratch is frequently based upon the results of laboratory diagnosis. False-negative results in IF test are not common but can occur (GRIFFIN, 1984;RUDD and TRIMARCHI, 1987), so WHO (ANONYMOUS, 1992) recommendsmouse inoculation or the virus isolation test on cell cultures in suspiciouscases or antemortem diagnosis in animals which bite or scratch man. RUDDand TRIMARCHI (1987) showed that the virus isolation test on cell cultures isequal to MIT in sensitivity, but most importantly it shortens the test period;in MIT the test takes 10-30 days and the virus isolation test takes 2-5 days.This paper describes our retrospective study of 7339 brain samplesusing the IF test and analysis results of 37 samples, when variance betweenthe results of two observers, or where unclear results were obtained. Thevirus isolation test on a neuroblastoma cell culture was used as aconfirmation test.Materials and methodsDuring our study period 7339 brain samples from different animalspecies were analyzed to rabies virus antigen by the FAT. The FAT wasperformed by the technique of LEPINE and  ATANASIU (1996). Two impressionsof Amon’s horn and two impressions of cerebellum tissue were made andfixed for 10 minutes in cold acetone. The fluorescent anti-rabies conjugate(anti-rabies serum fluorescein-conjugated, Sanofi, Diagnostics Pasteur,Marnes l-al Coquette, France) was used for staining the impressions. AfterP. Hostnik et al.: Doubtful and discordant results in fluorescent antibody test for rabies diagnosingVet. arhiv 71  (2), 65-73, 200168incubation (30 minutes at 37 °C) the slides are washed in a phosphatebuffer (PBS), pH 7.2, dried, covered with buffered glycerol (pH 7.4) andcoverslip and examined. The two specialists evaluated the results with aepifluorescence microscope Opton axioscop (×400). FAT results for brainsample slides were graded on a reading scale, from negative to 3+ positive.The samples with bright green fluorescence particles were graded as 3+,dull fluorescence was graded 2+, and samples with dim fluorescenceparticles were graded as 1+. Slides with only a few fluorescence particles,and the slides with discordants in FAT results between both specialists,were declared as doubtful. The samples were diagnosed as negative whenthe fluorescence was not evident to both microscopers.Brain samples were determined to be FAT positive by one mocroscopistand negative by the other (9 samples), and samples determined as doubtful(28 samples) were retested in the virus isolation test using mouseneuroblastoma cell line, clone NA, obtained by P. Schuster fromImpfstoffwerk, Dessau, Germany. The cell cultures were used at passages90 to 100. Brain samples from 37 animals (15 foxes, 13 cats, 6 dogs, 2cows and 1 sheep) were mashed and pestled until a paste was formed.They were re-suspended in a cell culture medium (minimal essentialmedium, MEM, Gibco) to a final dilution of 1:10. The homogenate wascentrifuged at 2.000 x g for 15 minutes and the collected supernatant wasfiltered through a 0.45 Millipore filter. Two-fold dilutions of samples weremade. Each sample dilution was inoculated onto 24-hour-old  NA cellcultures growing at 37 °C in 96-well cell culture microplate (Nunc) andon NA cell cultures growing at 37°C in tubes (Nunc). It continued toadsorbtion for 0.5 - 1 hour at 37 °C, the inoculate was discarded, Eagle’sMEM added, supplemented with 5% foetal calf serum (0.05ml/well) andincubated. After a 72-hour incubation period at 37 °C in a humid chamberwith 5% carbon dioxide (CO2), the medium in wells was discarded andcells were fixed for 20 minutes in 85% acetone at –20 °C and stained withanti-rabies conjugate (Sanofi Pasteur, France). The microplates werewashed in PBS, dried and examined under a fluorescent microscope (Zeiss,Axiovert 25, Germany).Fluorescent inclusions could be found in the infected cell’s cytoplasm.If results were negative the cells in the tubes were trypsinized. One part ofP. Hostnik et al.: Doubtful and discordant results in fluorescent antibody test for rabies diagnosingVet. arhiv 71  (2),  65-73, 200169suspended cells were added in cell culture microplate wells for testing therabies antigen and a second part into new tubes, and the  passage of sampleswas carried out.ResultsDuring the study period 28 brain samples were determined to be FATdoubtful by both microscopists; 9 brain samples were determined to bepositive by one microscopist and negative by the other microscopist. Inthis study period 15 (0.27%) of 5523 foxes, 6 (1.25%) of 478 dogs, 13(1.97%) of 659 cats, 2 (2.56%) of 78 cattle and 1 (5.25%) of 19 sheepwere determined as doubtful (Table 1).  Of thirty-seven samples withdoubtful or discordant results in FAT, 17 were positive and 20 were negativein VIT test. During retesting of samples in VIT, 3 fox, 3 dog, 10 cat and 1sheep samples were declared positive.Evaluation of the virus isolation test is simple as the fluorescentinclusions in the NA cell infected with rabies virus are clearly visible.Seventeen of the 37 samples (3/15 foxes, 10/13 cats, 3/6 dogs, and 1/1sheep) declared as doubtful in FAT test became positive in virus isolationtest. Fourteen samples were positive after 72 hours in the initial inoculation;only three samples became positive in first passage, and none in the secondpassage (Table 1).P. Hostnik et al.: Doubtful and discordant results in fluorescent antibody test for rabies diagnosingVet. arhiv 71  (2), 65-73, 2001Species  N o of sam ple  FA T   V irus isolation test     N egative Positive (passage) Fox 15 +/- 12 3 (2/1/0)* C attle 2 +/- 2 0 (0/0/0) D og 6 +/- 3 3 (2/1/0) C at 13 +/- 3 10 (9/1/0) Sheep 1 +/- 0 1 (1/0/0) T otal 37  20 17 (14/3/0)  Table 1. Suspicious results of 37 samples in FAT retested in the virus isolation test*(2/1/0) - positive in initial inoculation/positive in first passage/positive in second passage70Discussion Rabies virus infection has persisted in Slovenian wildlife since 1973.The red fox (Vulpes vulpes) is the predominant host to the virus and is themain vector and reservoir of the disease. Rabies in Slovenia amongdomestic animals has been most often diagnosed in cats (HOSTNIK et al.,1999). Not surprisingly, as cats are not vaccinated against rabies regularlyand in rural areas they quite often come into contact with rabid foxes.Rabies virus antigen is usually and routinely shown in brain tissues usingantigen detection or virus isolation techniques. The histopathologicaltechnique for detecting eosinophilic cytoplasmic inclusions known as Negribodies is not an accurate method for rabies diagnosis, (LEPINE and ATANASIU,1996) as it gave 10-30% false negative results. In many laboratories theimmunofluorescence test has been used for the rapid detection of the virusantigen and is a good method for the sensitive and specific detection ofantigens (ANONYMOUS, 1992; TEPSUMETHANON et al., 1997) and is therecommended (but not the prescribed) test by WHO and O.I.E.(Office international des Epizooties, 1996)The WHO recommendation is to perform mouse inoculation test orVIS on samples that gave FAT negative results, if an animal had had contactwith man. Rabies virus can also be isolated and later determined using thevirus isolation test (WIKTOR, 1996) or mouse inoculation test (KOPROWSKI,1996). KULONEN et al. (1991) reported about 6901 specimens, which were inFAT test negative, except 32, all of them being positive in VIT. Mouseneuroblastoma cell line is more sensitive for the field rabies virus strainthan other cell cultures. (UMOH and BLENDEN, 1983; RUDD and TRIMARCHI,1987). IWASAKI and CLARK (1977) demonstrated that street rabies virus ismore invasive on an NA cell line than on other lines.The results of this present study confirm that the FAT is a simpletechnique, but evaluation of results can be also subjective. Many factorshave influenced an objective reading of results. Nowadays, satisfactoryequipment (mainly U.V. microscopes in European countries) cannot impedethe decision. A very important factor is the immunofluorescent conjugate.The strong background of the visible field can cause errors in evaluationof results, and small particles may not be seen. The results of microscopicexaminations were graded using four stages: strong positive, weak positive,P. Hostnik et al.: Doubtful and discordant results in fluorescent antibody test for rabies diagnosingVet. arhiv 71  (2),  65-73, 200171doubtful, and negative. The final results in 37 samples of the 7339 testedwere not conclusive and a second test had to be performed.  In these cases,only small atypical grains, or only a few grains, were found. Thedecomposed and bacterially contaminated brain tissues were declared asuseless for use in the FAT. “Weak” positive samples and especially samplesgiving doubtful results in FAT also have to undergo a second test in virusisolation test. A relatively high incidence of FAT doubtful results (13/659,1.97%) was found in cats. The immunofluorescence grains in brain tissuefrom cats are smaller than in foxes and is less evident in cat tissue. It isconcluded that of 13 cats declared as doubtful in FAT, 10 were positive inVIT, while in foxes of 15 doubtful in FAT only 3 were positive in VIT.Another explanation could be that the cat samples were fresh, while theage of fox samples was often unknown. It is possible that the rabies virusin fox samples was inactivated, which resulted in a high grade of negativeresults in VIT, which have been determined as doubtful in FAT. Our resultsalso indicate that FAT must be read by two specialists. Furthermore, asingle inoculation without passage on neuroblastoma cells is not sufficientfor the sensitive detection of rabies virus.References:ANONYMOUS (1992): Expert committee on rabies. 8th report. WHO Technical reportseries, No 824. Genova, World Health Organization, pp.7-8.ANONYMOUS (1996): OIE Manual of standards for diagnostic tests and vaccines 3rd  ed.,Paris, pp. 207-218.BIDOVEC, A., Z. ŽELEZNIK, A. TOMAŠIČ (1993): Uporaba peroralne vakcinacije zapreprečevanje širjenja stekline v Sloveniji. Zbornik. Prvi slovenski veterinarski kongres,Portorož, 18-20 nov., pp. 465-471.CHARLTON, K. M. (1991): The pathogenesis of rabies. In: Rabies. (Campbell, J. B., K.M. Charlton, Eds.) Boston, Kluwer Academic Publisher.  pp. 101-150.GRIFFIN, C.W. (1984): Performance evaluation critique. Fluorescent rabies antibody test1983-1984. Centers for Disease Control, Atlanta,  pp. 2-4,HEMACHUDHA, T. (1989): Rabies. In: Handbook of clinial neurology. Viral diseases.(Vinken, P. J., Ed.) New York, Elsevier Science Publishing, pp. 383-404.HOSTNIK, P., A. BIDOVEC, J. GROM (1999): The decrease of rabies cases establishedin Slovenia. Vet. nov. 25, 301-308.IWASAKI, Y., H. F. CLARK (1977): Rabies virus infection in mouse neuroblastoma cell.Lab. Invest. 36, 578-584P. Hostnik et al.: Doubtful and discordant results in fluorescent antibody test for rabies diagnosingVet. arhiv 71  (2), 65-73, 200172KOPROWSKI, H. (1996): The mouse inoculation test. In: Laboratory techniques in rabies.(Meslin, F. X., M. M. Kaplan, H. Koprowski, Eds.), 4th ed. Genova, WHO, pp. 80-87.KULONEN, K., E. NEUVONEN, M. TOPI (1991): Methods for detecting artic rabiesvirus in dilution series of field samples. J. Virol. Meth. 32, 149-155.LEPINE, P., A. ATANASIU (1996): Histopathological diagnosis. In: Laboratory techniquesin rabies. (Meslin, F. X., M. M. Kaplan, H. Koprowski, Eds.), 4th ed. Genova, WHO,pp. 66-79.PASTORET, P., B. BOLUANGER, B. BROCHIESR (1985): The rabies situation in Europe.In: The Veterinary Annual 35. (Mary-Elizabeth Raw, T. J. Parkinson, Eds.), BlackwellSciences,  pp. 1-17.RUDD, J. R., C. V. TRIMARCHI (1987): Comparison of sensitivity of BHK-21 and murineneuroblastoma cells in the isolation of street strain rabies virus. J. Clin. Microb. 25,1456-1458.TEPSUMETHANON, V.,  B. LUMLERTDACH, C. MITMOONPITAK, R. FAGEN, H.WILDE  (1997): Fluorescent antibody test for rabies: prospective study of 8987 brains.Clin. Infect. Dis. 5, 1459-1461.TORDO,  N., O. POCH (1988): Structure of rabies virus. In: Rabies. (Campbell, J.B., K.M. Charlton, Eds.). Boston, Kluwer Academic Publishers, pp. 25-45.UMOH, K., C. BLENDEN (1983): Comparison of primary skunk brain and kidney andraccon kidney cells with established cell lines for isolation and propagation of streetrabies virus. Infect. Immun. 41, 1370-1372. VAN REGENMORTEL, M. H. V., C. M. FAUQUET, D. H. L. BISHOP, E .B. CARSTENS,M. K. ESTES, S. M. LEMON, J. MANILOFF, M. A. MAYO, D. J. McGEOCH, C. R.PRINGLE, R. B. WICKLER: Virus Taxonomy: Classification and Nomenclature ofViruses. Seventh Report of the International Committee on Taxonomy of Viruses,edited by Virology Division, International Union of Microbiological Societies,Academic Press, San Diego, 2000, pp. 563-583.WEBSTER, W. A., G. A. CASEY (1996): In: Laboratory techniques in rabies. (Meslin, F.X., M. M. Kaplan, H. Koprowski, Eds.), 4th ed., WHO, Genova,  pp. 96-104.WHITBY, J. E., P. R. HEATON, H. E. WHITBY, E. OSULLIVAN, P. JOHNSTONE (1997):Rapid detection of rabies and rabies related viruses by RT-PCR and enzyme-linkedimmunosorbent assay. J. Virol. Meth. 69, 63-72.WIKTOR, T. L. (1996): Tissue culture method. In: Laboratory techniques in rabies (Meslin,F. X., M. M. Kaplan, H. Koprowski, Eds.), 4th ed., WHO, Genova, pp. 101-123.Received: 17 April 2000Accepted: 20 April 2001P. Hostnik et al.: Doubtful and discordant results in fluorescent antibody test for rabies diagnosingVet. arhiv 71  (2),  65-73, 200173HOSTNIK, P., M. ŠTRANCAR, D. BARLIČ-MAGANJA, J. GROM: Sumnjivi inepodudarni rezultati u dijagnostici bjesnoće testom imuno-fluorescencije. Vet.arhiv 71, 65-73, 2001.SAŽETAKNa prisutnost antigena virusa bjesnoće bilo je pretraženo ukupno 7339 uzoraka mozgovalisica, mačaka, goveda, ovaca i drugih vrsta životinja. Rezultate izravnog imunofluorescentnogtesta vrednovala su dva stručnjaka. Uzorci koji su u testu imunofluorescencije davali sumnjiverezultate, ili se rezultati stručnjaka nisu slagali bili su pretraženi izdvajanjem virusa na kulturistanica. Rabljena je stanična linija neuroblastoma miševa. Dvadeset osam uzoraka bilo jeproglašeno sumnjivim na bjesnoću. Rezultat prvog mikroskopskog pregleda u devet uzoraka bioje pozitivan, a drugog negativan. Od ukupno 37 uzoraka, koji su bili ponovno pretraženiizdvajanjem virusa, 17 je bilo pozitivnih na bjesnoću.Ključne riječi: bjesnoća, izravna imunofluorescencija, stanična kulturaP. Hostnik et al.: Doubtful and discordant results in fluorescent antibody test for rabies diagnosingVet. arhiv 71  (2), 65-73, 2001

Sumnjivi i nepodudarni rezultati u dijagnostici bjesnoće testom imuno-fluorescencije.

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