Seroprevalence of Bovine Herpes Virus Type 1 in the Industrial Dairy Cattle Herds in Suburb of Shiraz-iran

Australian Journal of Basic and Applied Sciences, 4(10): 4650-4654, 2010 ISSN 1991-8178 Seroprevalence of Bovine Herpes Virus Type 1 in the Industrial Dairy Cattle Herds in Suburb of Shiraz-iran 1 Badiei, K., 1 Ghane, M., 1 Mostaghni K. 1 Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Abstract: A cross-sectional study was made to investigate the prevalence of infectious bovine rhinotracheitis (IBR) virus using an indirect enzyme-linked immunosorbent assay (ELISA) test in a large number of industrial dairy cattle in suburb of Shiraz (Iran). Blood samples were collected from 952 dairy cows of different parities in 43 herds. None of the herds were vaccinated against IBR. The cows were divided into different groups according to herd geographical location (North, West, East and South of Shiraz city), herd size (small, medium and large), parity and production level (low, average and high yielding cows). Two hundred and thirty seven (27.68%) of cows were ELISA seropositive. However, the true BHV-1 seroprevalence was 27.93%. All of the herds were antibody positive against BHV-1. The prevalence ranged from 16.66 to 86.19% within the herds. No relationship was observed between the presence of antibodies to IBRV and the herd size. The proportion of seropositive cows increased with their parity (P<0.05), but there were no differences among the seroprevalences of BHV-1 in cows of different production groups and four geographical regions. According to the results obtained, it was concluded that the presence of latent infection in animals within the herds in suburb of Shiraz-Iran, is responsible for the presence antibody. Key words: Infectious bovine rhinotracheitis, Prevalence, Industrial dairy cattle, ELISA, Shiraz, Iran INTRODUCTION Bovine herpesvirus type 1 (BHV-1) is a member of the family Herpesviridae, subfamily Alphaherpesvirinae and is an important pathogen of cattle (Ackermann and Engels, 2006). By restriction enzyme analysis, two subtypes of BHV-1 have been described. BHV-1 subtype 1 represents strains that cause respiratory disease, infectious bovine rhinotracheitis (IBR), whereas subtype 2 includes strains that cause genital diseases, e.g. infectious pustular vulvovaginitis (IPV) and infectious balanoposthitis (IBP) (Engels et al., 1981; Radostits et al., 2007). Metzler et al. (1985) used DNA restriction enzyme analysis to further classify subtype 2 into 2a and 2b. Subtypes 1 and 2a have been isolated from aborted fetuses, whereas the less virulent subtype 2b has not been reported to be associated with abortion (Miller et al., 1991). By conventional serological assays there is however only one antigenic serotype of BHV-1. The virus is most commonly transmitted by close contact between infected and susceptible animals by means of trade (Hage et al., 2003). Indirect transmission may occur via contaminated equipment and semen (van Oirschot et al., 1993; Hage, et al., 2003). Infectious bovine rhinotracheitis (IBR) usually occurs as a subclinical respiratory infection but also can be a cause of a severe disease. Morbidity and mortality may reach 100% and 10%, respectively, particularly if complication occurs (Radostits, et al., 2007). Unilateral or bilateral conjunctivitis, often with profuse lacrimation, is a common clinical sign of the infection although bronchitis and bronchiolitis have occasionally been observed. In most cases pulmonary pathology has not been found unless secondary bacterial bronchopneumonia occurs (Radostits, et al., 2007). Secondary bacterial infections with, for example, Pasteurella spp., can give rise to more severe clinical signs due to deeper airways being affected. Abortions may occur in the course of IBR during the third trimester of pregnancy (Murphy et al., 1999). Where natural mating is practiced, genital infection can lead to pustular vulvovaginitis or balanoposthitis. These are characterized by mild to severe necrotic lesions in the vaginal or preputial mucosae. After artificial insemination with infected semen, endometritis can arise (Kendrick and McEntress, 1987). Uncomplicated cases of respiratory or genital disease caused by BHV-1 are usually last 5-10 days. As a member of the herpesvirus family, BHV-1 can cause latency. The sciatic and trigeminal ganglia and the tonsils are the sites of latency after genital and respiratory disease, respectively (Winkler et al., 2000). Latency allows the virus to persist, so that the introduction of a Corresponding Author: Dr. K.Badiei, Department of Clinical Studies, School of Veterinary Medicine, Shiraz University, Shiraz, Iran E-mail: badiei@shirazu.ac.ir Fax: +98-711-2286950 Tel: +98-711-2286940 4650

latent infected carrier into a non infected herd is the principal way for the virus to spread. The persisting BHV- 1 can be reactivated and re-excreted by several stimuli, including transport, parturition, and treatment with glucocorticoids (Thiry et al., 1987). When BHV-1 is reactivated, the virus establishes a new life cycle of replication and can be transmitted to susceptible animals (van Oirschot, et al., 1993). The BHV-1 infection can be identified by detection of a specific antibody response and detection of the virus or viral antigens. Detection of immune response against BHV-1 specific antibodies to BHV-1 can be first detected at 7 to10 days postinfection. After the acute phase and during latency, BHV-1-infected cattle are mainly detected by the presence of specific antibodies to BHV-1. Virus neutralization tests and various ELISAs (Enzyme-linked Immunosorbent Assay) are usually used for detecting antibodies against BHV1 in serum. ELISA tests appear to gradually be replacing VN tests. Several types of BHV-1 ELISA tests are commercially available and most of them are also suitable for detecting antibodies in milk. The test on bulk milk, used with an appropriate cut-off value, has been reported to estimate the prevalence of BHV-1 in a herd (Pritchard, 2001). However, data from individual or pooled serum from non-milking groups will more precisely provide the herd s status (Pritchard, 2001). In addition, some ELISAs can be used in conjunction with marker vaccines to detect infected cattle in a vaccinated population (van Oirschot et al., 1997). Considering this fact that there is a lack of information on the seroprevalence of bovine herpes virus type 1 in suburb of Shiraz, the study was carried out to clarify different aspects of the disease in a large number of the industrial dairy cattle of the region. MATERIALS AND METHODS The cross-sectional study was conducted on the industrial dairy herds in the Shiraz Suburb of Fars province, southern Iran. Two dairy cattle production systems are described in this area. One is a system of small independent farms which make up about 38% of all the cows in suburb of Shiraz. The herd density is about 5 50 animals per farm with a low technology level and milk production (average milk yield about 3575 Kg/cow/year). The second system is the commercial industrial dairy herds with an approximate herd size of more than 50 cows, with more advanced technology and average milk production of about 7193 Kg/cow/year. In this study, we included only commercial industrial dairy farms. The population of the tested industrial dairy cattle herd complexes was between 50 and 1700. The total dairy cow population under the study was 18643. All of the cows were Holstein breed. Cows had never been vaccinated against IBR. Most of the farmers used artificial insemination in their herds. The herds were stratified to small (50 100 cows), medium (101 200 cows) and large size (>200 cows). The study was carried out with a random cluster sampling design, from July 2008 to May 2009. Herd selection was based on geographical location and density of cattle in the region. Samples were collected based on 5 percent of herd population in 4 geographical regions: North, West, East and South of Shiraz City. Totally, 856 blood samples were tested from 39 industrial dairy cattle herds. In order to study the effect of cows age on distribution of IBRV antibodies, cows were divided into different age groups (under 2 years old, 1 parity, 2 parity, 3 parity, 4 parity and over cows). The effect of level of milk production on distribution of IBRV antibodies was determined via grouping the cattle into low (<27 kg, based on daily milk production at day 50 postpartum), average (27-42 Kg) and high (>42 kg). The blood samples were taken from the tail vein into a plain vacutainer tube. The samples were allowed to clot at room temperature for 40 minutes and then centrifuged at 3000 g for 10 minutes and serum was collected and stored at -20 C until testing. Undiluted serum samples were tested for antibodies to IBRV using a commercially available indirect ELISA (SVANOVIR IBR-Ab ELISA; Svanova Biotech) according to the manufacturer s instructions. Relative to the serum neutralisation test, as it was described in the manual, this assay has a sensitivity of 97.4 percent and a specificity of 92.4 percent. The plates were read in an automatic plate reader (Immunoskan Plus) at 450 nm, and the results were expressed as optical density (OD). Samples with a corrected OD value below 0.20 were considered negative. The Rogan and Gladen s (1978) correction of apparent prevalence was used for estimation of the true prevalence of seropositive samples. It was equated the true prevalence= (apparent prevalence +Sp-1)/ (Se+Sp- 1). Differences in prevalence among the herds and groups were tested using Chi-square statistical method. P value less than 0.05 considered statistically significant. Results: The results showed that 237 (apparent prevalence = 27.68%) out of 856 samples were IBR-antibody positive (Table 1). However, 619 (72.31%) cows had corrected OD value below 0.20 and were IBR-antibody negative. However, the true prevalence of IBRV antibody-positive was 28.19%. All of the herds had antibody 4651

against IBR virus. The prevalence ranged from 16.66 to 86.19% within the herds. The results of our study also indicated that the number of seropositive animals increases with the age. The infection rate in animals in different age and parity was shown in Table 2. The number of seropositive animals in groups 1 and <2 years old cattle was significantly lower than the number of animals in other parity groups (P<0.05). The numbers of seropositive animals in parity groups of 2, 3 and 4 were not significantly different from each other (Table 2). There was a significant difference between the number of seropositive animals in groups of lower and more than 2 years old (P<0.05). However, the difference in the number of seropositive animals among different parities (older than 2 years old) was not significant (Table 2). No Significant differences were observed among IBRV prevalence in four different geographical regions (Table 3). The herd size had no effect on the IBRV seroprevalence (Table 4). There were no significant differences among the prevalences of IBRV in different milk producing groups (Table 5). Table 1: Prevalence of IBRV-seropositive and seronegative cows in industrial dairy cattle herds in suburb of Shiraz-Iran Number of Herds Number of Samples Number (%) Seropositive Number (%)Seronegative Total 39 856 237 (27.68) 619 (72.31) Table 2: Number and percentage of IBRV-seropositive and seronegative cows of different parities Parity and Age Number of samples Number of seropositive Number of seronegative 1 & <2 years old 219 (25.58%) 18 (7.59%) a 201 (32.47%) 2 181 (21.14%) 33 (13.92%) b 148 (23.90%) 3 197 (23.01%) 71 (29.95%) c 126 (20.35%) 4 259 (30.25%) 115 (48.52%) d 144 (23.26%) In each column, different letters show significant difference (P<0.05). Table 3: Number and percentage of IBRV-seropositive and seronegative cows in different geographical regions in industrial dairy cattle herds in suburb of Shiraz-Iran Location Number of Samples Numbe of Seropositive Number of Seronegative North 187 (22.16%) 57 (24.05%) 130 (21.00%) West 251 (32.14%) 61 (25.73%) 200 (32.31%) East 252 (28.99%) 53 (23.36%) 199 (32.14%) South 156 (16.70%) 66 (27.84%) 90 (14.54%) Table 4: The seroprevalence of IBRV in small, medium and large herds in industrial dairy cattle herds in suburb of Shiraz-Iran Herd size Number of tested cows within the herds --------------------------------------------------------------------------------------------------------------------------------------------------------------------------- No. of herds examined Positive Negative Positive Negative Small 14 14 - Small 294(34.34%) 81(34.18%) 213(34.41%) Medium 15 15 - Medium 278(32.47%) 77(32.49%) 201(32.47%) Large 10 10 - Large 284(33.17%) 79(33.33%) 205(33.11%) Total 39 - Total 856(100%) 237(27.68%) 619(72.31%) Table 5: Number and percentage of IBRV-seropositive and seronegative cows in low, average and high yielding dairy cattle Production Levels Number of Samples Numbe of Seropositive Number of Seronegative Low 316(36.91%) 32(34.60%) 234(37.80%) Average 331(38.66%) 77(32.49%) 254(41.03%) High 209(24.41%) 78(32.91%) 131(21.16%) Discussion: Prevalence of BHV-1 antibody-positive cows (27.68%) in suburb of Shiraz-Iran didn't differ greatly from those reported from Iran and some parts of the world. Serological studies on BHV-1 in different parts of Iran indicated a seroprevalence of 31.48% in Ahvaz (Haji Hajikolaei and Seyfiabad Shapouri, 2006), 30.39% in Kerman (Sakhaee et al., 2009), 46.68% in Chaharmahal and Bakhtiari province (Hemmat Zade et al., 2002) and 48.9% in Urmia (Mahmodian et al., 2002). Paton et al. (1998) reported 69% seropositive animals in England and Wales and a similar percentage were reported from the State of Portuguesa, Venezuela (Obando et al., 1983). Investigations on dairy herds have established IBR prevalence in the range 17.3-54.0% (Eiras et al., 2009). Our investigation showed that the IBR prevalence in the region was in the lower limit of this range. The IBR status of countries in the European Economic Area (EEA) is highly variable. Studies established herd prevalence for Europe of 10-80% (Borchers, 2006). After several decades of eradication programmes, Finland, Norway, Sweden, Austria, Denmark, Switzerland and the Bolzano region of Italy, have been declared IBR-free by the OIE since 2003 (Borchers, 2006). 4652

The prevalence obtained in this study was lower to those observed in the European countries that have not tried to control IBR. These prevalences include 47.2% of beef cattle in Portugal (Soares et al., 2006), 61.0% of the unvaccinated dairy herds of Italy (Cavinari, 2006), and 84.0% of the dairy herds, 53.0% of the beef herds, 89.0% of the mixed herds and 35.0%, 31.0% and 43.0% of the dairy cows, beef cows and mixed dairy/beef animals of Belgium (Boelaert et al., 2000). The same fact was true about the prevalences recorded at the start of eradication programmes such as the 50.0% for Germany (Teuffert, 2006) or 80% for Hungary (Pálfi and Foldi, 2006). The prevalence of BHV-1 seropositive cows may reflect the proportion of BHV-1 carriers because after a primary infection, the virus remains latent and it may be reactivated and disseminated (resulting in spread to susceptible animals) (Hage et al., 1996). Since vaccination against BHV-1 was not practiced in the herds in suburb of Shiraz, serological response reflected natural infection. Findings also indicated an increased proportion of seropositive animals with increasing age. The older the animal, it would be more likely to be exposed to a natural infection source (Nardelli et al., 1999; Solis-Calderón et al., 2003). Antibodies against natural infection start to appear 7-10 days postinfection and persist throughout the animal s life (Muylkens et al., 2007). Eiras et al. (2009) reported that as the age of the animals increased, a significant increase in the percentage of seropositive animals for BHV-1 (p < 0.05) was recorded. Difference in the number of seropositive animals between the younger and older animals in other studies may be due to differences in herd size, cow rearing systems and animal keeping conditions. In the present study BHV-1 antibody prevalence was not significantly different among four different geographical regions. This might be explained by the fact that the animals were kept in intensive similar systems and minimum interchange of animals between herds occurred. Another factor could be the preponderance of artificial insemination rather than natural mating in the area. Our results also showed that the herd size did not affect the number of seropositive animals. It is probably due to the similar intensive management system in industrial dairy cattle herds around Shiraz- Iran. The results of our study also showed that the BHV-1 virus affected different classes of milk producing dairy cows at the same level. It is probable that the housing conditions of these different classes were similar and no action was taken to control the disease in herds. Based on our results it was concluded that BHV-1 seroinfection was present noticeably in industrial dairy cattle herds in suburb of Shiraz-Iran. Furthermore, the situation may show a high number of BHV-1 carriers in this area. ACKNOWLEDGMENT This Study was supported by the center of excellence for studying on high producing dairy cows. REFERNCES Ackermann, M. and M., Engels, 2006. Pro and contra IBR-eradication. Veterinary Microbiology, 113: 293-302. Boelaert, F., P. Biront, B. Soumare, M. Dispas, E. Vanopdenbosch, J.P. Vermeersch, A. Raskin, J. Dufey, D. Berkvens, and P. Kerkhofs, 2000. Prevalence of bovine herpesvirus-1 in the Belgian cattle population. Preventive Veterinary Medicine, 45: 285-295. Borchers, K., 2006. BoHV-1 eradication: opening lecture. In the Proceedings of BHV-1 eradication Symposium, Berlin, Germany, P: 4. Cavinari, S. 2006. Epidemiological data on IBR in Italy and experience of control in the field. In the Proceedings of BHV-1 eradication Symposium, Berlin, Germany, PP: 8-9. Eiras, C., F.J. Dièguez, M.L. Sanjuán, E. Yus, and I. Arnaiz, 2009. Prevalence of serum antibodies to bovine herpesvirus-1 in cattle in Galicia (NW Spain). Spanish Journal of Agricultural Research, 7: 801-806. Engels, M., F. Steck and R. Wyler, 1981. Comparison of the genomes of infectious bovine rhinotracheitis and infectious pustular vulvovaginitis virus strains by restriction endonuclease analysis. Archives of Virology, 67: 169-74. Hage, J.J., Y.H. Schukken, H.W. Barkema, G. Benedictus, F.A.M. Rijsewijk and G.H. Wentink, 1996. Population dynamics of bovine herpesvirus 1 infection in a dairy herd. Veterinary Microbiology, 53: 169-180. Hage, J.J., Y.H. Schukken, H. Schols, M.A. Maris-Veldhuis, F.A. Rijsewijk and C.H. Klaassen, 2003. Transmission of bovine herpesvirus 1 within and between herds on an island with a BHV1 control programme. Epidemiology and Infection, 130: 541-52. Haji Hajikolaei, M.R. and M.R. Seyfiabad Shapouri, 2006. Seroepidemiological study of bovine hrpesvirus 1 (BHV-1) infection in cattle in Ahvaz. Iran Veterinary Journal, 2: 23-31(In persian). 4653

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