The seroprevalence of Bovine Herpes Virus Type 4 (BHV4) infection in dairy herds in Turkey and possible interaction with reproductive disorders. S. BLGE DAG ALP *, A.B. DEMİR., E. GÜNGÖR and F. ALKAN Department of Virology, Faculty of Veterinary Medicine, University of Ankara, 06110, Dı kap/ankara-turkey *Corresponding author: E-mail: bilge@veterinary.ankara.edu.tr SUMMARY n this study, serum samples of 877 cattle from 6 dairy herds located in three different regions of Turkey were tested for presence of antibodies against BHV4 using commercial ELSA. Out of herds, three herds (group ) included cattle with reproductive disorders as postpartum metritis, abortus, repeat breeders, whereas the 3 other herds without reported reproductive pathologies constituted the group. All the selected herds had seropositive animals and BHV4 seroprevalences were ranged between 33.1% (herd ) to 69.5% (herd ) in the 6 dairy herds and the overall BHV4 seropositivity was 54.3%. Additionally, the BHV4 seroprevalence was significantly higher in the group (56.8%) than in the group (44.9%) (p < 0.05) and a significant interaction between reproductive pathologies and BHV4 seropositivity was evidenced in the group (OR: 1.615, p < 0.05). Moreover, BHV4 infection was significantly correlated with the age of animals in the overall population (p < 0.01), as well in the group (p < 0.01) and in the group (p < 0.05), the older animals being more frequently infected than the youngest animals (< 2 year old). This is the first report showing the presence of BHV4 infection in cattle in Turkey, and these results suggest a possible role for BHV4 alone or in association with other viral or bacterial agents in the aetiology of reproductive disorders in cattle. Keywords : BHV4, cattle, seroprevalence, Turkey, reproductive disorders. RESUME Séroprévalence du virus BHV4 chez les vaches laitières en Turquie et possible association avec des troubles de la reproduction. Dans cette étude, la présence d anticorps dirigés contre le virus BHV4 a été recherchée sur 877 sérums de vaches laitières issues de 6 troupeaux de 3 régions distinctes de Turquie à l aide d une technique ELSA. Des troubles de la reproduction (métrites post-partum, avortements, infertilité) ont été rapportés dans 3 de ces troupeaux (groupe ), alors que les 3 autres élevages semblaient exempts de pathologies de la reproduction (groupe ). Des animaus séropositifs ont été identifiés dans tous les troupeaux et les séroprévalences de l infection par BHV4 ont varié au sein des différents élevages entre 33.1% (élevage ) et 69.6% (élevage ). La séropositivité globale a été de 54.3%. De plus, la séroprévalence a été significativement plus élevée dans le groupe (56.8%) que dans le groupe (44.9%) (p < 0.05) et une interaction significative entre la présence de troubles de la reproduction et l infection par BHV4 a été mise en évidence dans le groupe (OR: 1.615, p < 0.05). La séroprévalence pour BHV4 s est révélée significativement corrélée à l âge des animaux au sein de l ensemble de la population (p < 0.01) ainsi que dans le groupe (p < 0.01) et dans le groupe (p < 0.05), les animaux âgés étant plus fréquemment infectés que les animaux les plus jeunes (de moins de 2 ans). C est la première étude réalisée qui montre la présence de BHV4 au sein du cheptel turc et ces résultats suggèrent un rôle possible du virus BHV4 seul ou associé à d autres agents viraux ou bactériens dans l étiologie des troubles de la reproduction chez la vache. Mots-clés : BHV4, vache, séroprévalence, Turquie, troubles de la reproduction ntroduction Bovine herpesvirus type 4 (BHV4) is a member of the subfamily Gamma-herpesvirinae within the family of the Herpesviridae [16]. The virus was first isolated in Hungary from calves with respiratory infection and keratoconjunctivitis [1]. t has also been isolated from cattle showing a variety of clinical signs, such as conjunctivitis, pneumonia, metritis, enteritis, mastitis, skin lesions, and tumours of the rumen and urinary bladder [9] but also in apparently healthy animals [2]. BHV4 infections have been detected in several countries such as Swiss [13], Belgium [18], Holland [21], Germany [17], and Spain [15]. However, there is no report on BHV4 infections in Turkish cattle. The role of BHV4 in bovine respiratory and reproductive tract infections have been studied [15, 17, 18]. Whether BHV4 was the causative agent of the sporadically reported postpartum metritis cases still not clear, but metritis symptoms were recorded in BHV4 infected cows after parturition under experimental conditions [19]. MONGE et al. [15] reported BHV4-associated acute post-partum metritis as an important problem in Spanish herds. CZAPLCHE and THRY [3] reported that the BHV4 seropositivity were 17.2% and 10% in aborted cows and healthy controls, respectively. The aim of the present study was firstly to evaluate BHV4 seroprevalence in dairy cows and secondly to look for a potential association between this virus and the occurrence of reproductive disorders.
202 BLGE DAG ALP (S.) AND COLLABORATORS Materials and methods SAMPLED ANMALS The study was carried out in 6 dairy herds (i.e. a total of 877 animals) belonging to the Ministry of Agriculture and Rural Affairs located in 3 different regions of Turkey (Figure 1). Many reproductive disorders (post-partum and chronic metritis, repeat breeder, abortus) occurred in 3 of them (Group : herd n o, and ) and 692 cows stemming from these herds were sampled (Table 1). Among them, 48% presented clinical signs of reproductive pathologies (postpartum and chronic metritis, repeat breeder and abortus) and 52% were apparently healthy. The Group (185 cows) was constituted by the 3 last herds (herds n o V, V and V) in which no reproductive problem was reported. Commercial combined vaccine (Virashield Somnus, Novartis/USA) including Bovine Herpes Virus type 1 (BHV1), Bovine Viral Diarrhoea Virus (BVDV), Bovine Respiratory Syncitial Virus (BRSV), Parainfluenza Virus 3 (PV-3) and Haemophilus somnus has been used in the herd n o, whereas animals were only vaccinated against BHV1 (Pfizer/USA) in the other herds. Cows were 1 to 9 years old (average 3.67 ± 2.14 years) and the repartition of ages was similar in the 2 groups. Animals in all sampled herds are breeding intensively at similar management conditions. Blood samples were taken from cows by puncture of the jugular vein directly into kaolin coated polystyrene tubes and centrifuged at low speed (1 500 g at 4 C for 10 minutes) for separating the serum and stored at -20 C until use. Figure 1: The localisation of the 6 sampled dairy herds throughout 3 different regions in Turkey. Marmara region: herds, and Aegean region: herd V Central Anatolia region: herds V and V. THE DETECTON OF BHV4 ANTBODES A commercial indirect ELSA (BioX, Belgium, Bio K066) was used to detect antibodies for BHV4, according to the manufacturer s instructions. Briefly, 1/100 diluted serum samples were put into duplicate wells of the plate, one of them coated with the BHV4 viral antigen and other coated with cell lysates. The plate was incubated at 37 o C for 1h. After three washings with washing solution, the conjugate, a peroxidase-labelled anti bovine gg1 monoclonal antibody, was added to the wells and incubated at room temperature for one hour. Plates were washed as before, and then the chromogenic marker, tetramethyl-benzidine, and hydrogen peroxide as substrate were added. The optical density values were recorded at 450 nm using a platereader (Titertek Multiskan Plus). STATSTCAL ANALYSS Differences among seroprevalence values of the 2 groups (Groups and ) and of animals with healthy appearance and reproductive disorders in Group were analysed by the Pearson s chi-square test (2). n addition, the association between the BHV4 seroprevalence and reproductive problems was estimated by the calculated odds ratio (OR) [8]. Differences were considered as significant with p values less than 0.05. Results The seroprevalence determined on the overall sampled dairy cows (877) was 54.3%, and this parameter ranged from 33.1% (herd n o ) to 69.6% (herd n o ) according to the herds (Table 2). The seroprevalence observed in the Group (herds with reproductive disorders) was higher than in the Group (herds without reproductive pathology). Out of the 692 cows from the Group, 393 (56.8%) were found to be positive for BHV4 while 83 (44.9%) of the 185 cows from the Group were positive (Table 2). Although the seroprevalences obtained in cows with reproductive disorders and cows with healthy appearance from the Group were found to be similar (57.2% and 56.4% respectively), a significant interaction between the presence of BHV4 infection and reproductive disorders was evidenced ( 2 value was 8.367; df = 1; P = 0.004) and the estimated OR value was 1.615 with a 95% confidence interval [1.165-2.239]. Seropositivity rates were estimated according to the age of animals (Table 3). The BHV4 seroprevalences observed in the overall population as well in cows of the Group or of the Group significantly differed according to the age of cows (respective 2 values were: 12.45, p < 0.01; 11.82, p < 0.01 and 7.83, p < 0.05): adult and older cows were more often infected then younger animals (< 2 year old) (Table 3). Discussion BHV4 is a widespread viral agent in the cattle population throughout the world [5, 9, 13, 15, 17, 18]. n this study, the presence and seroprevalence of BHV4 infection was detected in dairy cows from 6 dairy herds in Turkey. This is the first report giving information about BHV4 infection prevalence in Turkey. The BHV4 seroprevalences in cattle determined in some countries are comparable with those obtained in Turkish cattle. Serological evidence of BHV4 infection in cattle has been reported in Taiwan [11], Belgium [18], taly [17], and in others [13, 15]. The herd prevalences of BHV4 infection vary from country to country and from herd to herd in these studies. WELLENBERG et al. [21]
BHV4 SEROPREVALENCE AND REPRODUCTVE DSORDERS N DARY HERDS. 203 Groups Herds Region Herd size Sampled cows (number) Number of cows (or %) with RD Balikesir / Marmara Bursa / Marmara Kirklareli / Marmara - 704 1872 605 1060.3 176 368 148 692 89 (50.6) (M, RB, A) 169 (45.9) (M) 74 (50.0) (M) 332 (48.0) V V V Mugla / Aegean Kirsehir / Central Anatolia Eskisehir / Central Anatolia - 1250 710 706 888.7 51 40 94 185 TOTAL 877 332 (37.9) RD: reproductive disorders; M: Metritis; A: Abortus; RB: Repeat breeder Group : dairy herds with reproductive disorders; Group : dairy herds without reproductive disorders TABLE 1: Repartition of the 877 sampled cows according to their geographic origin and the dairy herd. Groups Herds and animals Seroprevalence in % (BHV4 seropositive / tested animals) 53.9 (48 / 89) 46.0 (40 / 87) 50.0 (88 / 176) 69.8 (118 / 169) 69.0 (138 / 199) 69.6 (256 / 368) 32.4 (24 / 74) 33.8 (25 / 74) 33.1 (49 / 148) 57.2 (190 / 332) 56.4 (203 / 360) 56.8 (393 / 692) V 60.8 (31 / 51) V 45.0 (18 / 40) V 36.2 (34 / 94) 44.9 (83 / 185) TOTAL 54.3 (476 / 877) TABLE 2: The BHV4 seroprevalence in the 2 groups of dairy herds with (Group ) or without (Group ) reproductive disorders (RD). reported that 16 to 18% of the 750 tested sera samples reacted positively for BHV4, based on the results of three BHV4 tests. VAN MALDEREN et al. [18] detected that the BHV4 seropositivities were 15% and 29% in the northern part of Belgium and Ballony [Belgium], respectively. n the present study, all of the sampled herds were positive and the detected prevalences were extremely high in the different herds (above 30%). The reason for this accumulation could not be elucidated by the present work. These herds are state farms in which the management of animals is similar. An extensive investigation on the virus source is required for analyzing this situation. t is known that BHV4 has been isolated from various disease conditions including ocular, respiratory diseases, genital diseases, enteric diseases and skin lesions [3, 4, 14, 15, 20]. n the present study, prevalences detected in the 2 groups were found to be significantly different. Moreover, a significant association between reproductive disorders occurrence and BHV4 infection was evidenced in the Group even if prevalences in cows with or without reproductive pathologies seemed to be closed. t may be concluded from these data that BHV4 might be a causative agent (or together with other agents as BHV1, BVDV...) for reproductive problems in cows of the Group. Similarly, CZAPLCHE and
204 BLGE DAG ALP (S.) AND COLLABORATORS Groups Herds Seroprevalence in % (BHV4 seropositive / tested animals) according to age classes (in year) [1 3[ [3 6[ [6 and more] 50.0 (88 / 176) 69.6 (256 / 368) 33.1 (49 / 148) 56.8 (393 / 692) 24.5 (13 / 53) 59.6 (98 / 164) 27.5 (19 / 69) 45.5 (130 / 286) 55.2 (32 / 58) 78.5 (135 / 172) 42.3 (22 / 52) 67.0 (189 / 282) 66.2 (43 / 65) 71.9 (23 / 32) 29.6 (8 / 27) 59.7 (74 / 124) V V V 60.8 (31 / 51) 45.0 (18 / 40) 36.2 (34 / 94) 44.9 (83 / 185) 6.7 (1 / 15) 22.2 (2 / 9) 00.0 (0 / 2) 11.5 (3 / 26) 80.0 (24 / 30) 44.4 (8 / 18) 31.5 (17 / 54) 48.0 (49 / 102) 100.0 (6 / 6) 61.5 (8 / 13) 44.7 (17 / 38) 54.4 (31 / 57) TOTAL 54.3 (476 / 877) 42.6 (133 / 312) 62.0 (238 / 384) 58.0 (105 / 181) TABLE 3: The BHV4 seropositivity rates according to the age of cows from the Group (herds with reported reproductive disorders) and from the Group (herds without reported reproductive disorders). THRY [3] reported that the BHV4 seropositivities were 17.2% and 10% in aborted cows and in healthy controls respectively. Also, MONGE et al. [15] reported that BHV4 infection was detected by virus isolation and BHV4gB PCR in 83% of cows with clinical signs of acute postpartum metritis. Despite the antibiotic treatment for a long period, postpartum metritis and chronic metritis have continued to be an important problem in the herds of the Group. Additionally, based on the high level of reproductive problems despite vaccinations [especially against BVDV and BHV1] in some herds [Herd and ], the interaction between reproductive problems and the presence of BHV4 was supported. t is obviously that the decreasing of the fertility is an important cause of economical losses, together with additional treatment costs (veterinary service, medication cost...). The level of economical losses were not estimated in the herds sampled in this study, but detected rates of reproductive disorders and of seropositivity in herds of the Group were pointed high level economical losses. t is interesting that the seroprevalences obtained in cows with reproductive disorders and in healthy cows of the Group were found to be similar. The reason of these results is not also clear for us. t is possible that BHV4 causes sub clinical infections with a high level, leading to an underestimation of the number cows with reproductive disorders. Similarly, BHV4 seroprevalence was also detected with a high level in herds from the Group in which no reproductive disorders have been diagnosed. An extensive investigation on the interaction between the BHV4 seroprevalence and sub clinical and clinical infections is required to clarify this situation. t is known that BHV-4 can persist in a latent form in cells of the mammary gland or in peripheral blood leukocytes [7, 12]. Additionally, replication of latent BHV4 in various tissues has been reported after dexamethasone treatment [6]. Similarly, WELLENBERG et al. [21] reported that S. uberis infections may probably have triggered BHV4 replication. Because of the viral latency period, seroprevalences observed in the Groups and did not directly evaluate and would underestimate the BHV4 role in the aetiology of different clinical and sub clinical reproductive pathologies. Studies investigated the association between animal ages and BHV4 status are scarce [10]. When the prevalence rates of BHV4 antibodies among animals in different age groups were compared, it was seen that seropositivity rates were higher in the animals over 2 years than in the younger cows. Similarly, GUO et al. [10] reported that there is a significant interaction between the age and the BHV4 infection. As a result, farm managers would be informed about the importance of sanitary measures, which could allow to prevent the introduction of the infection into herds and to control the BHV4 infection. Actually, BHV4 vaccine has not yet been produced because of some unresolved questions as diverse tropism and level of the protection offered by a monovalent vaccine. The isolation of the viral agent from various tissues during different clinical conditions and their molecular analysis would help to the resolution of these questions. For this aim, we planned a study investigating the influence of the BHV4 infection on reproductive disorders by virus isolation and its detection using PCR technique in vaginal swap samples. References 1. BARTHA A., JUHASZ M., LEBERMANN H.: solation of a bovine herpesvirus from calves with respiratory disease and keratoconjunctivitis. Acta Vet. Acad. Scient. Hung., 1966, 16, 357-358. 2. BELAK S., PALF V.: Characterization of a herpesvirus isolated from spontaneously degenerated bovine kidney cell culture. Acta Vet. Acad. Scient. Hung., 1974, 24, 249-253. 3. CZAPLCH G., THRY E.: An association exists between bovine herpesvirus seropositivity and abortion in cows. Prev. Vet. Med., 1998, 33, 235-240. 4. DONOFRO G., VAN SANTEN V.L.: A bovine macrophage cell line supports bovine herpesvirus 4 persistent infection. J. Gen. Virol., 2001, 82, 1181-1185. 5. DONOFRO G., CAVRAN S., VAN SANTEN V., FLAMMN C.F.: Potential secondary pathogenic role for bovine Herpesvirus 4. J. Clin. Microbiol., 2005, 43 (7), 421-3426. 6. DUBUSSON J., THRY E., BUBLOT M., THOMAS., VAN BRESSEM M.F., COGNOUL F., PASTORET P.P.: Experimental infection of bulls with a genital isolate of bovine herpesvirus-4 and reactivation of latent virus with dexamethasone. Vet Microbiol., 1989, 21, 97-114.
BHV4 SEROPREVALENCE AND REPRODUCTVE DSORDERS N DARY HERDS. 205 7. EGYED L., BARTHA A.: PCR studies on the potential sites for latency of BHV-4 in calves. Vet. Res. Commun., 1998, 22, 209-216. 8. FLESS J.L.: Statistic Methods for rates and proportions. Second Edition, John Wiley and Sons, New York, USA, 1981. 9. GOYAL S.M., NAEEM K.: Bovine herpesvirus-4: a review. Vet. Bull., 1992, 62, 181-201. 10. GUO W.Z., SHEN D.T., EVERMANN J.F., GORHAM J.R.: Comparison of an enzyme-linked immunosorbent assay and a complement-fixation test for the detection of g G to bovine herpes virus type 4 (bovine cytomegalovirus). Am. J. Vet. Res., 49, 667-670. 11. KWANG M.J.: Virus isolation and serological investigation of bovine herpesvirus type 4 in Taiwan. J. Chin. Soc. Vet. Sci., 1999, 25, 50-54. 12. LOPEZ O.J., GALEOTA J.A., OSORO F.A.: Bovine herpesvirus type-4 (BHV4) persistently infects cells of the marginal zone of spleen in cattle. Microbiol. Pathogenesis, 1996, 21, 47-58. 13. METZLER A.E., WYLER R.: Pravelenz des bovinen herpesvirus 4 in der schweizerischen Rinderpopulation und mögliche serologische Kreuzreaktion mit dem Bovinen Herpesvirus 1 (BR-PV virus) (Prevalence of bovine herpesvirus 4 in the Swiss cattle population and possible serological cross-reaction with bovine herpesvirus 1 (BR-PV virus). Schweiz. Archiv. Tierheilkunde, 1986, 128, 459-467. 14. MOHANTY S.B., HAMMOND R.C., LLLE M.G.: A new bovine herpesvirus and its effect on experimentally infected calves. Archiv. Virusforschung, 1971, 33, 394-395. 15. MONGE A., ELVRA L., GONZALEZ JV., ASTZ S., WELLEN- BERG G.J.: Bovine herpesvirus 4 associated postpartum metritis in a Spanish dairy herd. Res. Vet. Sci., 2006, 80 (1), 120-125. 16. ROZMAN B., DESROSERS R.C., FLECKENSTEN B., LOPEZ C., MNSON A.C., STUDDERT M.J.: The family herpesviridae: an update. Arch. Virol., 1992, 123, 425-449. 17. TRUMAN D., LUDWG H., STORZ J.: Bovine Herpesvirus Type 4: Untersuchungen zur Biologie und verbreitung in Rinderbestanden (Biology and distribution of bovine herpesvirus type 4 in cattle herds and insemination bulls.), J. Vet. Med. B, 1986, 33, 485-501. 18. VAN MALDEREN G., VANOPDENBOSCH E., WELLEMANS G.: Bovine herpesvirus 4: a seroepidemiological survey of the Belgian cattle population. V. Diergeneesk. Tijdschr., 1987, 56, 364-371. 19. WELLEMANS G., VAN OPDENBOSCH E., MAMMERCKX M.: Experimental inoculation of Bovine Herpes Virus 4 (Strain LVR 140) in pregnant and non pregnant cows. Ann. Rech. Vet., 1986, 17(1), 89-94. 20. WELLENBERG G.J., BRUSCHKE C.J.M., WSSELNK H.J., BARKEMA H.W., VAN ORSCHOT J.T.: Simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpes virus 4 induce sub clinical mastitis. Vet. Microbiol., 2002, 86, 115-129. 21. WELLENBERG G.J., VAN ROOJ E.M.A., MASSAN J., VAN ORSCHOT J.T.: The evaluation of newly developed immunoperoxidase monolayer assays to detect antibodies against bovine herpesvirus 4. Clin. Diag. Lab. mmunol., 1999, 6, 447-451.