Medycyna Wet. 2007, 63 (1) 62
Praca oryginalna Original paper
Enteric infections along with severe diarrhoea are very important for the neonates of many animal spe-cies and humans because of their widespread occur-rence all over the world and the mortality they cause (10, 14, 22). Bovine rotaviruses (BRV) are the major reason for the neonatal diarrhoeas of the various ani-mal species and humans (22). The agent multiplies in the intestine and spreads with the infected animals faeces (5). Bovine coronaviruses (BCV) is the cause of severe diarrhoea, especially in neonatal calves, and is associated with winter dysentery (WD) in adult cows, along with severe diarrhoea (10, 16, 22). Infection by BRV and BCV is via the oral route, with food and water contaminated with infected faeces. Adult animals are generally subclinically infected with BRV and BCV and therefore play an important role in shedding of the disease within the herd (5, 11).
Clinically, BCV causes respiratory and gastroin-testinal symptoms in calves (10). In previous studies, BRV isolation from fecal specimens and BCV isola-tion in nasal swabs and fecal specimens of healthy--looking calves showed that these calves were shed-ding the virus in low titers and played an important role in creating clinical infections within the herd (20).
Several tests are used routinely in diagnostic laborato-ries for the detection of BRV and BCV in fecal sam-ples. These include enzyme-linked immunosorbent assay (ELISA) electron microscopy, passive hemagglu-tination, latex agglutination assays and polymerase chain reaction (PCR) (2, 10, 13).
The aim of the study was, clinical and subclinical BRV and BCV infections rates in calves and serologic prevalence were examined in cows in northern Turkey.
Material and methods
Fecal samples. Samples were collected from 100, 0-3 months old calves (49 healthy and 51 with acute diarrhaeal symptoms) around three different province in Samsun. Collected fecal samples werekept at 20°C until analysed. Sera samples. Blood specimens collected from 256 unvaccinated healthy-looking cows older than 3 months and from five different districts of the Middle Black Sea region Amasya (I), Bafra (II), Çatalçam (III), Tokat (IV-a, IV-b; two different barn), Vezirköprü (V) were placed in tubes with kaolin. Sera were separated from the collected blood specimens, inactivated at 56°C for 30 minutes and stored at 20°C until used for test procedures.
Direct ELISA test for determination of bovine rota-virus antigens and bovine coronarota-virus antigens from fecal samples. Direct ELISA commercial test kits were used to detect rotavirus and coronavirus antigens in fecal
sam-Rotavirus and coronavirus prevalence
in healthy calves and calves with diarrhoea*
)
SEMRA OKUR GUMUSOVA, ZAFER YAZÝCÝ, HARUN ALBAYRAK*, YUCEL MERAL**
Department of Virology, **Department of Internal Medicine Faculty of Veterinary Medicine, Ondokuz Mayis University, 55139 Samsun, Turkey
*Virology Laboratory, Samsun Veterinary Control and Research Institute, Ministry of Agriculture, 55139 Samsun, Turkey Okur Gumusova S., Yazýcý Z., Albayrak H., Meral Y.
Rotavirus and coronavirus prevalances in healthy calves and calves with diarrhoea
Summary
The aim of the study was to examine clinical and subclinical BRV and BCV infection rates in calves and serologic prevalance of cows in northern Turkey.
In this study, 100 fecal specimens collected from healthy calves (0-3-months-old) and calves with diarrhoea were examined for bovine rotavirus (BRV) and bovine coronavirus (BCV) antigens, with 23% and 1% positivity detected respectively. BRV antigen positivity was 4.08% in healthy calves but BCV antigen was not detected. BRV and BCV antigen positivity was 41.17% and 1.96% in calves with diarrhoea. Antibody titers against BRV and BCV in 256 adult cows sampled in the same region were 71.48% and 98.43%, respectively.
As the result, it is concluded that BRV-sourced infections in calves with diarrhoea are frequently encountered (41.17%), but BCV infections are rarely detected (4.08%). In contrast, BCV infection in cows are more frequently encountered than BRV infections in Northern Turkey. Data from this study reflects the widespread distribution of BRV infections in 0-3-month-old animals and BCV infections in animals older than 3 months.
Keywords: antibody, antigen, calves, coronavirus
*) This research was supported by Research Fund of Ondokuz Mayis
Medycyna Wet. 2007, 63 (1) 63
ples (Rotavirus Elisa Kit Cat.Nr.Bio K 067 and Corona-virus Elisa Kit, Cat Nr.Bio K 068, Bio-X Diagnostics, Jemelle, Belgium). Tests were performed according to the manufacturers recommendations. After the incubation of microplates at room temperature for 10 minutes, the pro-cess was terminated by adding 50 µl of stop solution (1 M phosphoric acid) to all wells. Optical densities were measured with an Elisa reader (Daf, Italy) at 450 nm filter absorbance, according to the recommendations in the test procedure. Samples giving values equal to or greater than 0.150 were accepted as positives and values less than 0.150 as negatives.
Competitive ELISA test for determination of antibo-dies against bovinerotavirus and bovine coronavirusin sera samples. Competitive-ELISA commercial test kits were used for bovine rotavirus and coronavirus sero-diagnosis (Rotavirus Elisa Kit Cat.Nr.Bio K 126and Coro-navirus Elisa Kit , Cat Nr.Bio K 127, Bio-X Diagnostics, Jemelle, Belgium). Tests were performed according to the manufacturers recommendations. ELISA microplates coated with specific monoclonal antibody for Rotavirus and coronavirus were used and BRV and BCV cultures were added later. At the end of the test, optical densities were measured with an Elisa reader (Daf, Italy) at 450 nm filter absorbance, according to the recommendations of the test procedure. The mean optical densities for the positive and negative sera (OD pos and OD neg) and those of all the samples (OD samples) were calculated. For each tested sample and the positive serum, the percentage inhibition was calculated (inhib %) by means of the following for-mulas:
% inhibition of sample = [(ODneg ODsample)/ODneg]*100 % inhibition of positive = [(ODneg ODpos)/ODneg]*100
Results and discussion
ELISA test results for 51 calves with diarrhoea symp-toms showed rotavirus antigens in 21 calves (41.17%) andcoronavirus antigen in one calf (1.96%). From the 49 fecal samples of healthy calves, rotavirus antigens were detected in only two calves (4.08%) and corona-virus antigene was detected in none of the animals. In total, rotavirus antigen was detected in only 23% (23/100) and coronavirus antigen was detected in only 1% of the total of 100 fecal specimens examined. No infections caused by both of the agents together were detected (tab. 1). According to the ELISA test results of the 256 cow sera samples studied, 183 (71.48%) carried rotavirus and 252 (98.43%) carried corona-virus antibodies (tab. 2).
Coronavirus and rotavirus infections are mostly ob-served in young animals. Bovine coronavirus (BCV) causes diarrhoea and respiratory system disease in calves and winter dysentery in adult cows, while rotaviruses frequently cause digestive system diseases characterised by diarrhoea in calves. In different countries around the world, 16.7-67% positivity against BRV and 4-38.9% positivity against BCV has been detected by ELISA tests performed on calves with diahorrea (1, 6, 17, 18). Widespread distribution of
BRV and BCV infection in Turkey has also been demonstrated by many other researchers. From ELISA tests of fecal samples from calves with diarrhoea, 17-71.1% positivity against BRV and 15.4% positivi-ty against BCV was determined (3, 8, 15, 24).
In this study, ELISA test results of the 51 calves with diarrhoea symptoms reflected rotavirus antigenin 21 calves (41.17%) andcoronavirus antigen in one calf (1.96%). This rate is similar to the studies performed in our country and in various countries around the world. Myers et al. (20) identified rotavirus antigen in 3 of 136 healthy calf faeces while Crouch and Acress (11) detected 5% coronavirus antigen, but no rota-virus antigen. In the present study, rotarota-virus antigen was diagnosed in only two (4.08%) of the 49 healthy calves and coronavirus antigen was not identified in any of them.
In antibody surveys for BRV and BCV infections in cows by various researchers, 44-98.1% positivity for rotavirus and 62.4-95% positivity for coronavirus has been described (10, 19, 21, 23). Rotavirus and corona-virus seroprevalence studies performed in Turkey de-termined positivity rates of 28.3-54.1% and 4.4-100%, respectively (4, 5, 7-9, 12). In our study, seroprevalen-ces determined in cows against BRV and BCV infec-tions were 71.48% and 98.43%, respectively. This rate is similar to the studies performed in our country and in various countries around the world.
As the result, it is concluded that BRV-sourced infections in calves with diarrhoea are frequently en-countered (41.17%), but BCV infections are detected (4.08%) rarely. In contrast, BCV infection in cows are Tab. 1. The Elisa results for BRV and BCV antigens in the fecal samples collected from healthy calves and calves with diarrhoea
Tab. 2. Seroprevalance of BRV and BCV antibodies in 256 cattle sera collected from six different farms
s e v l a C Notoefsftaeedces BPRoVsiAitvntiyitgefonrs ) % ( r o f y ti v it i s o P s n e g it n A V C B ) % ( a e o h rr a i d h ti W 151 21(41.17) 1(1.96) y h tl a e H 149 2(4.08) l a t o T 100 23(23) 1(1) d n a n o it a c o L n r a b f o s e d o c s m r a f f o d e t s e t f o o N a r e s SoefroBpRoVsi(it%v)tiy SoefroBpCoVsi(it%v)tiy I 50 43(86) 50(100) II 28 ..18(64.2) 28(100) II I 20 15(75) 20(100) a -V I 50 32(64) 50(100) b -V I 58 ..40(68.9) .57(98.2) V 50 35(70) 47(94). l a t o T 256 ..183(71.48) .252(98.43)
Medycyna Wet. 2007, 63 (1) 64
more frequently encountered than BRV infections in Northern, Turkey. Data from this study reflects the widespread distribution of BRV infections in 0-3 months old animals and BCV infections in animals older than 3 months. These results may be reflected to winter dysentery, which caused by BCV. As a result, the protection of young animals against BRV and pro-tection of adult cows against BCV by vaccine programs designed would seem very necessary.
References
1.Abraham G., Roeder P. L., Zewdu R.: Agents associated with neonatal diar-rhoeain Ethiopian dairy calves. Tropic. Anim Health Prod. 1992, 24, 74-80. 2.Al-Yousif Y., Anderson J., Chard-Bergstrom C., Bustamante A., Muenzen-berger M., Austin K., Kapil S.: Evaluation of a Latex Agglutination Kit (Virogen Rotatest) for Detection of Bovine Rotavirus in Fecal Samples. Clin. Diagn. Laborat. Immunol. 2001, 8, 496-498.
3.Alkan F.: Buzaðý ishallerinde rotavirus ve coronaviruslarýn rolü. Ankara Üni-versitesi. Vet. Fak. Derg. 1998a, 45, 29-37.
4.Alkan F., Bilge-Dagalp S., Oguzoglu Ç., Yeþilbag K.: Rotavirus enfeksiyo-nunun epidemiyolojisinde eriþkin sýðýrlarýn rolü. Ankara Üniversitesi. Vet. Fak. Derg. 1999b, 46, 85-92.
5.Alkan F., Bilge-Dagalp S., Can-Sahna K., Ozgunluk I.: Sýðýrlarda Coronavi-rus Enfeksiyonunun Epidemiyolojisi. Ankara Üniversitesi. Vet. Fak. Derg. 2003c, 50, 59-64.
6.Bendali F., Bichet H., Schelcher F., Sana M.: Pattern of diarrhoea in new-born beef calves in South-west France Vet. Res. 1999, 30, 61-74.
7.Burgu I., Akça Y.: Sýðýrlarda rotavirus daðýlýmý üzerine serolojik araþtýrmalar. Ankara Üniversitesi. Vet. Fak. Derg. 1983, 30, 35-44.
8.Burgu I., Akça Y., Alkan F., Ozkul A., Karaoglu T.: Yenidoðan ishalli buzaðýlarda rotaviruslarýn elektron mikroskopi (EM), enzyme linked immu-nosorbent assay (ELISA) ve polyacrylamide gel elektrophoresis (PAGE) tek-nikleri ile çabuk teþhisi ve antijenik karakterizasyonu. Ankara Üniversitesi. Vet. Fak. Derg. 1995, 42, 491-498.
9.Can Sahna K., Alkan F.: Sýðýrlarda rotavirus enfeksiyonunun epidemiyoloji-sinde gebeliðin ve maternel antikorlarýn rolü. Fýrat Üniversitesi Sað. Bil. Derg. 2003, 17, 203-209.
10.Cho K. O., Hoet A. E., Loerch S. C., Wittum T. E., Saif L. J.: Evaluation of concurrent shedding of bovine coronavirus via the respiratory tract and ente-ric route in feedlot cattle. Am. J. Vet. Res. 2001, 62, 1436-1441.
11.Crouch C. F., Acress S. D.: Prevalance of rotavirus and coronavirus antigens in faeces of normal cows. Canad. J. Comp. Med. 1984, 48, 340-342.
12.Çabalar M.: Van yöresindeki bazý süt sýðýrcýlýðý iþletmelerinde Rotavirus Enfeksiyonunun Serolojik Araþtýrýlmasý. Yüzüncü Yýl Üniversitesi Vet. Fak. Derg. 2004, 15, 33-35.
13.De Beer M., Peenze I., De Costa V. M., Steele D.: Comparison of electron microscopy, enzyme-linked immunosorbent assay and latex agglutination for detection of bovine rotavirus in faeces. J. South Afr. Vet. Ass. 1997, 68, 93-96.
14.Fukutomi T., Tsunemitsu H., Akashi H.: Detection of bovine coronaviruses from adult cows with epizootic diarrhea and their antigenic and biological diversities. Arch. Virology 1999, 144, 997-1006.
15.Hasoksuz M., Kayar A., Dodurga T., Ilgaz A.: Detection of respiratory and enteric sheedding of bovine coronaviruses in cattle in Northwestern Turkey. Acta Vet. Hung. 2005, 53, 137-146.
16.Jeong J. H., Kim G. Y., Yoon S. S., Park S. J., Kim Y. J., Sung C. M., Jang O. J., Shin S. S., Koh H. B., Lee B. J., Lee C. Y., Kang M., Kim H. J., Park N. Y., Cho K. O.: Detection and isolation of winter dysentery bovine coronavirus circulated in Korea during 2002-2004. J. Vet. Med. Sci. 2005, 67, 187-189. 17.Kaminjolo J. S., Lathrop S. L., Wittum T. E., Loerch S. C., Perino L. J., Saif L. J.: Antibody titers against bovine coronavirus and shedding of the virus via the respiratory tract in feedlot cattle. Am. J. Vet. Res. 2000, 61, 1057-1061.
18.Lucchelli A., Lance S. E., Bartlett P. B., Miller G. Y., Saif L. J.: Prevalance of bovine group A rotavirus sheding among dairy calves in Ohio. Am. J. Vet. Res. 1992, 53, 169-174.
19.Mahin L., Pastoret P. P., Dewulf M., Mazouz A., Maenhoudt M., Michaux C.: Prevalence of antibody to rotavirus in Moroccan cattle. Comp. Immunol. Microbiol. Infect. Dis. 1984, 7, 69-73.
20.Myers L. L., Firehammer B. D., Border M. M., Hoop D. S.: Prevalence of enteric pathogens in the faeces of healthy beef calves. Am. J. Vet. Res. 1984, 45, 1544-1548.
21.OConnour A., Martin S. W., Nagy E., Menzies P., Harland R.: The relation-ship between the occurence of undifferentiated bovine respiratory disease and titer changes to bovine coronavirus and bovine viral diarrheavirus in 3 Ontario feedlots. Canad. J. Vet. Res. 2001, 65, 137-142
22.Parreno V., Costantini V., Cheetham S., Vierra J. B., Saif L. J., Fernandez F., Leoni L., Schudel A.: First isolation of Rotavirus associated with neonatal Diarrhoea in Guanacos (Lama guanicoe) in the Argentian Patagonia Region. J. Vet. Med. B 2001, 48, 713-720.
23.Schlafers A., Scott F. W.: Prevalence of neutralizing antibody to the calf rota-virus in New York cattle. Cornell Vet. 1979, 69, 262-271.
24.Yazici Z., Akca Y.: Yeni doðmuþ buzaðýlarda rotavirus enfeksiyonlarýnýn sero-epidemiyolojisi ve ELISA testi ile rotavirus antijenlerinin identifikasyonu. Ankara Universitesi Vet. Fak. Derg. 1993, 40, 231-240.
Authors address: Dr. Okur Gumusova Semra DVM, PhD, Department of Virology, Faculty of Veterinary Medicine, University of Ondokuz Mayis, 55139, Samsun, Turkey; e-mail: semragumusova@hotmail.com
