BENHA VETERINARY MEDICAL JOURNAL, VOL. 23, NO. 1, JUNE 2012: 123- 130

BENHA UNIVERSITY BENHA VETERINARY MEDICAL JOURNAL FACULTY OF VETERINARY MEDICINE

PREVALENCE OF BOVINE VIRAL DIARRHEA VIRUS (BVDV) IN CATTLE

FROM SOME GOVERNORATES IN EGYPT. El-Bagoury G.F.a, El-Habbaa A.S.a, Nawal M.A.b and Khadr K.A.c aVirology Dept., Fac. Vet. Med., Benha University, Benha, bAnimal Health Research Institute (AHRI), Dokki- c Giza, General Organization for Veterinary Medicine (GOVS), Dokki-Giza, Egypt.

A B S T R A C T

Diagnosis of the BVDV infection among suspected and apparently healthy cattle at Kaluobia, Giza, Menofeia and Gharbia governorates was done by detection of prevalence of BVD antibodies. A total number of 151/151(100%) and 97/151 (62.25%) of examined sera were positive for BVD antibodies using serum neutralization test (SNT) and competitive immunoenzymatic assay (cIEA), respectively. Examined sera with cIEA detected antibodies against BVDV non structral proteins P80/P125. Detection of BVDV in buffy coat samples using antigen capture ELISA showed that 22/151(14.56%) of the samples were positive for BVDV. Isolation and biotyping of suspected BVDV from buffy coat on MDBK cell line showed that 19/22 of ELISA positive buffy coat samples were cytopathogenic BVDV biotype (cpBVDV) while only 3/22 samples were CPE negative suggesting a non- cytopathogenic BVDV (ncpBVDV) biotype. Inoculated cell culture with no CPE were subjected to IFAT and IPMA using specific antisera against BVDV revealed positive results indicating presence of non-cytopathogenic strain of BVDV. It was concluded that cIEA detected antibodies against non- structural proteins P80/P125 has many advantages over SNT being for rapid diagnosis of BVDV. However, diagnosis must be confirmed with isolation, biotyping and identification of BVDV using suitable sensitive and specific methods as ELISA, IFAT and IPMA.

KEY WORDS: BVDV, ELISA, SNT.

(BVMJ-23 [1]: 123-130, 2012)

1. I N T R O D U C T I O N

ovine viral diarrhea virus (BVDV) is virus-infected cells, cytopathic viruses an enveloped, single-stranded, RNA produce p80 protein that not observed in virus genus Pestivirus, family cells infected with non-cytopathic BVD Flaviviridae [18]. BVD is viruses [6]. BVDV is a world-wide B characterized with depression, fever, distributed virus of cattle of all ages diarrhea, drop in milk yield, loss of causing infection range from subclinical to appetite and temporary leucopenia [14]. highly fatal condition called mucosal There are two antigenically distinct disease [13]. genotypes of BVDV (types 1 and 2) and In Egypt, the disease was continually each of them occurs in two forms diagnosed by [7, 15-17]. The prevalence of including non-cytopathogenic and BVD has been mainly reported on the cytopathogenic according to whether/or basis of the detection of antibody against not it produces visible change in cell BVDV [5]. However, diagnosis of BVDV cultures [21]. The 2 biotypes of BVD virus through demonstration of BVD are not distinguishable serologically; neutralizing antibodies needs further however, in addition to nonstructural viral explanation. protein (pl25) that is present in all BVD

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Diagnosis of BVDV can be done by a 2.3. Madin-Darby Bovine Kidney (MDBK) variety of serological techniques as Cell Line: detection of BVD antibodies using MDBK cell line was used for isolation of Enzyme linked immuno-sorbent assay the virus. It was supplied by Virology (ELISA) [11], SNT [10] or detection of department at Animal Health Research viral antigen using Serum neutralization Institute (AHRI), Dokki, Giza. Isolation of test (SNT), Immuno-fluorescent technique the virus was done with positive samples (IF), and Enzyme linked immuno-sorbent showed the characteristic cytopathic effect assay (ELISA) [17]. So, the aims of our (CPE) of BVDV that denotes article are screening of BVD neutralizing cytopathogenic biotype of BVDV [9]. antibodies in cattle from different governorates in Egypt using SNT, 2.4. Reference BVDV: detection of BVD antibodies against non- Egyptian field strain of BVDV Iman strain structural viral proteins in sera from (cytopathic) obtained from rinder pest like infected animals and apparently healthy disease department, Veterinary Serum and persistently infected (PI) cattle using Vaccine Research Institute (VSVRI), ELISA that is confirmed with trials for Abbasia, Cairo, Egypt. It was propagated isolation, biotyping and identification of in MDBK cell line and used for detection the virus using indirect fluorescent assay of BVD neutralizing antibodies using SNT. (IFAT) and immunoperoxidase monolayer assay (IPMA). 2.5. Reference antisera: Reference antisera against Egyptian field 2. MATERIAL AND METHODS strain of BVDV used in this study was obtained from Virology department at 2.1. Animals: Animal Health Research Institute (AHRI), A total number of 151 cattle over 6 months Dokki, Giza. of age suspected for BVDV infection showing signs of respiratory disorders, mucosal ulcers in mouth and diarrhea and 2.6. Serum neutralization test (SNT): their apparently healthy contacts from It was conducted for detection of specific different governorates (Kaluobia, Giza, BVDV neutralizing antibodies in cattle Menofeia and Gharbia). serum samples [12].

2.7. Competitive Immunoenzymatic assay 2.2. Samples: (cIEA): Two blood samples were collected from It was used for the specific detection of the jugular vein of each examined animal. antibodies to BVDV in cattle sera samples. A sample set was collected in clean dry BVDV P80/P125 was used as the coated centrifuge tubes, left to clot, centrifuged at antigen in addition with supplying with 1500x g for 20 minutes for separation of BVDV specific monoclonal antibodies serum which kept at – 20 °C until used (peroxidase conjugated) in INGEZIM for detection of BVD antibodies using BVD Compac kit. SNT and competitive ELISA; Another sample set was collected on EDTA as 2.8. Antigen capture ELISA: anticoagulant to separate buffy coat A total number of 151 buffy coat samples samples that used for direct detection of were investigated for BVDV using antigen BVDV using antigen capture ELISA and capture ELISA of commercial ELISA cell culture inoculation. BVD/MD antigen mix screening kit from INSTITUTE POURQUIER, France, according to manufacturer description.

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in 97/151(62.25%) by cIEA in tested sera 2.9. Indirect Fluorescent Antibody from 151cattle at different localities in Technique (IFAT): Kaluobia, Giza, Menofeia and Gharbia It was carried out on the inoculated cell governorates, as shown in table (2). cultures without CPE to detect ncp BVDV biotype [4]. Three blind passages of the Table 2 Detection of BVDV antibodies in tested samples were done on the cattle sera using antibody competitive ELISA: inoculated cell cultures. If there was no Governorate Examined Positive sera sera evidence of any CPE, the third passage Number Percent was conducted on MDBK tubes. Thus, Kaluobia 54 39 72.22% tube cultures including flying cover slips Giza 29 18 62.06% were used to detect ncpBVDV by indirect Menofeia 24 8 33.33% IFAT technique. Anti-bovine IgG Gharbia 44 32 72.73% Conjugate with fluorescein isothiocynate Total 151 97 62.25% (developed in rabbits and supplied by *cELISA detect antibodies against P80/P125 non- Sigma) was obtained for IFAT. structural protein of BVDV in cattle sera samples.

Detection of BVDV in buffy coat Samples 2.10. Immuno-peroxidase monolayer assay using antigene capture ELISA: (IPMA): The total number of buffy coat samples It was carried out on the inoculated cell (151) suspected for BVDV that were cultures without CPE to detect ncpBVDV collected from cattle at different were [17]. identified serologically using ELISA (10 from Kaluobia, 3 from Giza, 3 from 3. RESULTS Menofeia and 6 from Gharbia,). It was found that 22 (14.56%) buffy coat samples Detection of antibodies against BVDV in were positive as induced change in colour cattle sera using Serum neutralization test of substrate by ELISA as reveled in table (SNT): (3). From all tested obtained from cattle at Table 3 Number of positive Buffy coat different localities in Kaluobia, Giza, samples using antigen capture ELISA: Menofeia and Gharbia governorates (151), Governorate Examined Positive buffy coat specific BVD antibodies were detected by samples samples SNT (100%) as shown in table (1). Number Percent Kaluobia 54 10 18.5% Table 1 Detection of BVDV antibodies in Giza 29 3 10.34% cattle sera using SNT: Menofeia 24 3 12.5% Governorate Examined Positive sera Gharbia 44 6 13.63% sera Number Percent Total 151 22 14.56% Kaluobia 54 54 100% Giza 29 29 100% Isolation and biotyping of suspected Menofeia 24 24 100% BVDV from buffy coat on MDBK cell: Gharbia 44 44 100% All of the 22 BVDV positive buffy coat Total 151 151 100% samples using ELISA were inoculated in MDBK cell line. It was found that 19 Detection of antibodies against BVDV in samples (8 from Kaluobia, 3 from Giza, 3 cattle sera using Competitive from Menofeia and 5 from Gharbia) Immunoenzymatic assay (cIEA): induced cytopathic effect by 3 blind serial Specific antibodies against P80/P125 non- passages in MDBK cell culture suggesting structural protein of BVDV were detected a cytopathogenic biotype of BVDV as

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El-Bagoury et al. (2012) shown in table (4). Only 3 samples were infected cell culture was characterized by CPE negative suggesting a non- cell rounding, cell aggregation, cytopathogenic biotype of BVDV. The vacuolations followed by cellular darkness CPE of suspected BVDV isolates in and cluster formation.

Table 4 Isolation and biotyping of BVDV from positive buffy coat samples on MDBK cell line then detection with IFAT and IPMA: Positive samples Examined Governorate Antigen capture Isolation on MDBK cell line samples IFAT IPMA ELISA CPE No CPE Kaluobia 54 10 8 2 8 2 Giza 29 3 3 0 3 0 Menofeia 24 3 3 0 3 0 Gharbia 44 6 5 1 5 1 Total 151 22 19 3 19 3 * Positive result represented by clear apple green emission on MDBK under fluorescent microscope using IFAT. * Positive result represented by clear brownish color on MDBK using IPMA.

Identification of bovine viral diarrhea virus (BVDV) by isolation on MDBK, identification by indirect fluorescent assay (IFAT) and immunoperoxidase monolayer assay (IPMA). Photo (1) Showed normal MDBK cell line (Control) with no morphological changes examined under inverted microscope. Photo (2) Extensive cytoplasmic vacuolization as the morphological changes in response to suspected cp BVDV infection examined under inverted microscope. Photo (3) negative IFAT on inoculated MBDK cell line examined under fluorescent microscope. Photo (4) showed apple green intracytoplasmic flourescence as positive IFAT on inoculated MBDK cell line examined under fluorescent microscope. Photo (5), negative IPMA on inoculated MBDK cell line examined under inverted microscope. Photo (6) showed clear brownish color as positive IPMA on inoculated MBDK cell line examined under inverted microscope

126 Prevalence of bovine viral diarrhea virus (BVDV)

The CPE was clear between the 3rd and system dysfunction in cattle throughout the 5th day post inoculation increased world [8]. gradually till 70-80 % of sheet was Rapid, cheap and accurate tool for completely detached. These findings were diagnosis of BVD virus and their demonstrated in photo (2) as compared antibodies is very important to prevent, with normal control monolayer control and eradicate the persistently demonstrated in photo (1). infected animals, so diagnosis of BVDV in cattle and buffaloes was practiced via Serological identification using Indirect detection of specific anti-BVD-antibodies Fluorescent Antibody Technique (IFAT) in sera by ELISA and SNT as well as and Immuno Peroxidase Monolayer Assay detection of BVDV antigen by using of (IPMA) in inoculated MDBK cell line for antigen capture ELISA commercial kits [7], detection of BVDV. FAT [17] and IPMA [19]. A total of 22 suspected BVDV samples The results of this study showed that, out from cattle buffy coat (10 from Kaluobia, of 151 tested blood samples from cattle 3 from Giza, 3 from Menofeia and 6 from over 6 months of age at different localities Gharbia) were inoculated in MDBK cell in Kaluobia, Giza, Menofeia and Gharbia line. It was found that 3 samples 2 from governorates; 151 (100 %) and 97 Kaluobia and one from Gharbia) did not (62.25%) were positive for anti-BVD induce any cytopathic effect by 3 blind antibodies using SNT and cIEA techniques, serial passages in MDBK cell culture as respectively. SNT showed that all shown in table (4). examined animals showing suspected Inoculated cell culture not showing CPE symptoms and their apparently contact were subjected to IFAT using specific cattle were all positive for BVDV antisera against BVDV revealed antibodies. Serum neutralizing antibody characteristic clear specific titers for positive screened sera in cattle intracytoplasmic yellowish green against BVDV were ranged between 4 and fluorescence indicating presence of non- 64, 122/151 serum samples (80.8 %) were cytopathogenic strain of BVDV. These positive in titer range (8-64) while 101/151 findings were demonstrated in photo (4) as serum samples (66.8 %) were positive in a compared with normal control monolayer titer (4-16). These results agree to some demonstrated in photo (3). extent with those obtained by Hanaa et al. The normal inoculated cell culture [10]. subjected to IPMA using specific antisera The highest detection rate was found in against BVDV revealed characteristic clear cattle at Gharbia (72.73%) and Kaluobia brownish color indicating presence of non- (72.22%) governorates followed by Giza cytopathogenic strain of BVDV. These (62.06%) and Menofeia (33.33%) findings were demonstrated in photo (6) as governorates using cIEA technique. These compared with normal control monolayer results were in agreement with results of demonstrated in photo (5). [7], who found that 58.61 % and 65.5 % were positive for anti-BVD antibodies in 4. DISCUSSION examined animals’ sera using SNT and ELISA, respectively. SNT proved to be Bovine viral diarrhea virus (BVDV), sensitive and specific technique as well as which is the prototype of the Pestivirus cIEA for detection of anti-BVD antibodies, genus of the Flaviviridae family, causes but cIEA technique was more specific test early embryonic death, abortion, for detecting anti-BVDV antibodies teratogenesis, respiratory problems, against non- structural proteins from chronic wasting syndrome, and immune persistently infected animals.

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Accurate diagnosis of BVDV infection also with those of Saliki et al. [19] who depends upon isolating the virus from proved that IPMA had a relative sensitivity nasal swabs or blood or tissue samples of 85% and a relatively specificity of from affected animals in a diagnostic 100% to identify PI cattle. laboratory [20]. Buffy coat samples from It was concluded that diagnosis of BVD suspected cattle and their apparently BVD can be studied via detection of healthy contacts from different specific anti-BVD-antibodies in sera by governorates in Egypt were subjected to SNT and ELISA, However, serological antigen capture ELISA for BVDV diagnosis of BVDV infections in cattle is diagnosis. From a total number of 151 only complicated by the widespread use of 22 (14.56%) samples were positive for BVDV vaccines, as indicated by the high DVDV. The highest detection rate was SN titers found in cattle that had received found in cattle at Kaluobia 10/54 (18.5%) multiple BVDV vaccinations and in cattle and Gharbia 6/44 (13.63%) governorates exposed to Pl animals. Although cIEA has followed by 3/24 Menofeia (12.5%) and many advantages over SNT being rapid, Giza 3/29 (10.34%) governorates using simple, cheap and does not need ELISA ELISA technique. These results agreed reader for detection of specific anti-BVD- with those obtained by previous studies [3, antibodies, diagnosis must be confirmed 10, 17] who used ELISA kits with specific with isolation, biotyping and identification monoclonal antibodies directed to NSP2-3 of BVDV using suitable sensitive and for detection of BVDV strains which specific methods as ELISA, IFAT and offered sensitivity equivalent to virus IPMA. isolation. All of the 22 BVDV positive buffy coat 5. REFERENCES samples using ELISA were inoculated in MDBK cell line. It was found that 19 1. Allam, N. 2000. Growth behavior of samples (8 from Kaluobia, 3 from Giza, 3 BVDV. Iman strain on the level of NA from Menofeia and 5 from Gharbia) synthesis and genotyping of its viral induced cytopathic effect by 3 blind serial RNA. M.V.Sc. Thesis, Fac. Vet. Med., passages in MDBK cell culture suggesting Cairo University. 2. Aly, N.M., Shehab, G.G., Abd el-Rahim, a cytopathogenic biotype of BVDV. This I.H. 2003. Bovine viral diarrhoea, agreed with that found in earlier reports [1, bovine herpesvirus and parainfluenza-3 10], which found that MDBK cell line is virus infection in three cattle herds in considered the most common cell culture Egypt in 2000. Rev Sci Tech. 22: 879- suitable for BVDV virus isolation. Only 3 892. samples gave no CPE suggesting a non- 3. Aymen, H.A. 2000. Virological studies cytopathogenic biotype of BVDV. These on Bovine virus diarrhea in camels, results agreed with Aly et al. [2] who used M.V.Sc. Thesis. Fac. Vet. Med., Zagazig the cell culture method for biotyping and Univ. then identification of both cpBVDVand 4. Bezek, D.M., Baker, J.C., Kaneene, J.B. ncpBVDV. Diagnosis of 3 isolates of 1988. Immuno-fluorescence of bovine virus diarrhea viral antigen in white noncytopathic BVDV was identified using blood cells from experimentally infected IFAT and IPMA. These results agreed immunocompetent calves. Can J Vet Res. with previous studies [2, 10] that used 52: 288–290. immunofluorescent technique for 5. Daliri, M., Ghorashi, S.A., Morshedi, D., identification of both cpBVDVand Hajian, T., Afshar, K. 2007. Detection of ncpBVDV biotypes after using the cell bovine viral diarrhea virus in bovine culture method and characterised by semen using nested-PCR. Iranian J. specific intracytoplasmic fluorescence as Biotechnol. 5: 48-81. positive samples. These results also agreed

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6. Deregt, D., Masri, S.A., Cho, H.J. and 14. Radostits, O.M. and Littlejohns, I.R. Bielefeldt Ohmann H. 1991. Bovine 1988. New Concepts in the Pathogenesis, viral diarrhea virus proteins: relatedness Diagnosis and Control of Diseases of p175 with p80 and p125 and evidence Caused by the Bovine Viral Diarrhea of glycoprotein processing. Can J Virus. Can. Vet. J. 29: 513-528. Microbiol. 37: 815-822. 15. Radwan, G.S. 2002. Molecular detection 7. Ghazi, Y.A., El-Sherif, A.M., Azzam, of bovine viral diarrhea virus in clinical R.A. and Hussein, H.A. 2008. samples by polymerase chain reaction- Diagnostic studies on Bovine Viral chemiluminscent DNA hybridization Diarrhea Infection in Cattle and assay. Arab J. Biotech. 5: 287-298. Buffaloes with Emphasis on Gene 16. Radwan, G.S., Brock, K.V., Hogan, J.S. Markers. Global Veterinaria 2: 92- 98. and Larry Smith, K. 1995. Development 8. Givens, M.D., Heath, A.M., Carson, of a PCR amplification assay as a R.L., Brock, K.V., Edens, M.S., Wenzel, screening test using bulk milk samples J.G. and Stringfellow, D.A. 2003. for identifying dairy herds infected Analytical sensitivity of assays used with bovine viral diarrhea virus. Vet. for detection of bovine viral diarrhea Microbiol. 44: 77-92. virus in semen samples from the 17. Refaat, M., Hala A. Salem, Jehan A. Southeastern United States. Vet. Gafer and Dardiri, M.A. 2010. Microbiol. 96: 145-155. Molecular, Virological and Pathological 9. Gogorza, L.M., Morán, P.E., Larghi, J.L., diagnosis of Bovine Viral Diarrhea Seguí, R., Lissarrague, C., Saracco, M., (BVD) virus infection in calves. Egypt. J. Braun, M., Esteban, E.N. 2005. Comp. Path. & Clinic. Path. 23: 32-50. Detection of bovine viral diarrhea virus 18. Ridpath, J.F. 2005. Classification and (BVDV) in seropositive cattle. Prev. Vet. molecular biology. In: Bovine Viral Med. 72: 49-54. Diarrhea Virus: Diagnosis, 10. Hanaa, A. Ghoneim, Naglaa, I. Hassan, Management and Control. Goyal, S.M. Hanaa A. Elhalawany and Nabih, A.M. and Ridpath, J.F. Ames, I.A., Blackwell 2010. Mixed Infection of Bovine Viral Publishing. Pp. 65-89. Diarrhea Virus, Mycoplasma Species 19. Saliki, J.T., Fulton, R.W., Hull, S.R. and and Mannheimia Haemolytica in Calves Dubovi, E.J. 1997. Microtiter Virus Showed Chronic Pneumonia with Isolation and Enzyme Immunoassays for Reference to the Histopathological Detection of Bovine Viral Diarrhea Findings of the Affected Lungs. J. Virus in Cattle Serum. J. Clin. Microbiol. American Sci. 6: 538 – 555. 35: 803–807. 11. Handel, I.G., Willoughby, K., Land, F., 20. Vickers H.L. 1992. BVD: getting a Koterwas, B., Morgan, K.L., Tanya, positive diagnosis. In Proc. XVII World V.N., Bronsvoort, B.M. 2011. Buiatrics Congress and XXV American Seroepidemiology of bovine viral Association of Bovine Practitioners diarrhoea virus (BVDV) in the Conference (Vol. 2), 31 August-4 Adamawa Region of Cameroon and use September, St. Paul, Minnesota, USA. of the SPOT test to identify herds with Pp. 158-161. PI calves. PLoS One 6: e21620: 1-11. 21. Vilcek, S., Paton, D.J., Durkovic, B., 12. O.I.E. 2004. Manual of diagnostic tests Strojny, L., Ibata, G., Moussa, A., and vaccines for terrestrial animals. Loitsch, A., Rossmanith, W., Vega, S., Chapter 2.10.6: Bovine viral diarrhoea. Scicluna, M.T. and Palfi, V. 2001. (http://www.oie.int/eng/normes/mmanua Bovine viral diarrhoea virus genotype 1 l/A_00132.htm). can be separated into at least eleven 13. OIE Terrestrial Manual 2008. Bovine genetic groups. Arch. Virol. 146: 99-115. Viral Diarrhea. Chapter 2.4.8. Pp. 698- 711.

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El-Bagoury et al. (2012) مجلة بنها للعلوم الطبية البيطرية عدد 32 )1(، يونيو 3113: 130-132

مجلة بنها للعلوم الطبية البيطرية BENHA UNIVERSITY FACULTY OF VETERINARY MEDICINE

مدى انتشار فيروس االسهال البقرى الفيروسى فى االبقار من بعض المحافظات فى مصر. 3 2 1 1 جبر فكرى الباجورى ، أيمن سعيد الهباء ،نوال محمد عمى ، خالد أحمد عبد الواحد 1 قسم الفيرولوجيا- كمية الطب البيطرى-جامعة بنها، 2 معهد بحوث صحة الحيوان–الدقى–الجيزة، 3 الهيئة العامة لمخدمات البيطرية–الدقى - الجيزة

الممخص العربى تم تشخيص عدوى فيروس االسهال البقرى الفيروسى بين أبقار مصابة وأخرى مجاورة فى محافظات القميوبية والجيزة والمنوفية والغربية بدراسةمدى انتشار االجسام المضادة لمفيروس. كان العدد الكمى لألمصال المختبرة لالجسام المضادة لمفيروس ايجابيا فى 100% من الحيوانات بواسطة اختبار المصل التعادلى )151/151( و 62.25% من الحيوانات )97/ (151 بواسطة اختبار االنزيم المناعى المتسابق. يكشف اختبار االنزيم المناعى المتسابق عن االجسام المضادة ضد البروتينات 80/125 غير الهيكمية لمفيروس فى االمصال تحت الفحص. أظهر التعرف عمى فيروس االسهال البقرى الفيروسى فى عينات buffy coat ان 14.56% من العينات )22/151( كان ايجابيا باستخدام اختبار االليزاصائد االنتيجين. أظهر العزل والتعرف عمى النوع الحيوى لفيروس االسهال البقرى الفيروسى من عينات buffy coat االيجابية عمى خط خاليا MDBK ان )19/22( من العينات كان ايجابيا لفيروس االسهال البقرى الفيروسى من النوع الحيوى الممرض لخاليا الزرع النسيجى بينما )3/22( من العينات كان ايجابيا لفيروس االسهال البقرى الفيروسى وكان من النوع الحيوى غير الممرض لخاليا الزرع النسيجى والذى تم التعرف عميه من خالل اختبارات الوميض الفموروسنتى غير المباشر و IPMA. مما سبق نستنتج أن اختبار االنزيم المناعى المتسابق الكاشف عن االجسام المضادة ضد البروتينات 80/125 غير الهيكمية لمفيروس له مميزات عديدة عن اختبار المصل التعادلى فى التشخيص السريع لعدوى فيروس االسهال البقرى الفيروسى اال أنه يجب تأكيد التشخيص بوسائل العزل واالستعراف والتصنيف الحيوى لنوع الفيروس باستخدام طرق مناسبة حساسة ومتخصصة مثل اختبارات االليزا و الوميض الفموروسنتى وIPMA . )مجمة بنها لمعموم الطبية البيطرية: عدد 23 )1(، يونيو 2102: 130-021(

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