DISTRIBUTION OF BOVINE HERPESVIRUS 1 IN CATTLE POPULATION AND BULLS FROM CENTERS FOR ARTIFICIAL INSEMINATION AND PRIVATE FARMS IN BULGARIA

Th e aim of the study is determination of the BHV 1 incidence among the bovine population in Bulgaria by using various ELISA tests for antibody detection and diff erent methods for proving viral antigen. Commercial diagnostic ELISA kits were used for screening as well as for confi rmation of the antibodies. Serological examination encompassed 2973 serum samples from bovine population in Bulgaria (cattle, calves and bulls) originating from 21 country regions. Total 408 cattle and 150 bulls’ samples originating from artifi cial insemination centers (AIC), commune and private farms from 21 country regions were subjected to virology testing. Identifi cation of isolated viruses was performed using conventional and nested PCRs for BHV 1 gB gene detection. Th e percentage of positively reacting cattle sera were signifi cantly (p < 0.001) higher (38.3%) then that of bulls (29.3%) aft er ELISA testing of 2240 cattle serum samples and 733 bulls’ sera. Seven strains with BHV 1 characteristics were isolated from Dobrich, Lovech – 2 strains, Plovdiv, Targovishte, Pazardjik and Svishtov regions aft er cultivation 408 samples from cattle and calves (buff y coats, nasal, eyes, vaginal swabs, tissues and organs) on cell cultures. Th ree BHV 1 strains were isolated from Sliven, Shumen and Blagoevgrad regions aft er examining 150 bulls’ samples (nasal, eyes, preputial, semen samples and buff y coats). Confi rmation of virus isolation was accomplished using PCR gB gene based primers (amplifi ed 478 bp) Th e amplicons from 3 bulls isolated strains with the same size and location as the referent Oxford strain were observed. Aft er performance of nested PCR for gB gen, products with size of 385 bp were obtained in all samples as well as in the referent Oxford strain.

has a narrow specter of hosts in comparison with other representatives of this subfamily having broad range of hosts. A range of clinical signs are observed, depending on the virus introduction and BHV 1 infection route -infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV), infectious pustular balanopostitis in bulls (IPB) (Gibbs and Rweyemamu, 1977). BHV 1 infection is initially manifested as a venereal form aff ecting genital tract in cattle (IPV) and bulls (IPB). IPV infection is characterized by small pustules on the vulva mucosa and caudal parts of the vagina. In IPB, the clinical symptoms are restricted to the mucosa of the preputium, penis and distal parts of urethra.
Th e virus can cross the placenta to infect the fetus and potentially cause the abortion. It can infect the male genital tract inducing balanopostitis or penopostitit. Aft er initial or reactivated infection of bulls, the virus is shed through the preputium and semen infection occurs in 2 to 7 days (Straub, 1990). Th e virus spreading can occur through female animals (so-called vertical transmission) (Straub, 1990). Th e initial period of virus dissemination can prolong for several weeks and during the initial stage of infection, the virus cannot be isolated (Huck et al., 1971). Latently infected bulls can re-excrete the virus in stressful situations (transportation or dexamethasone treatment). Th e animals usually do not manifest clinical symptoms and can disseminate the virus over a long time period, even for years (Bitch, 1978;Pastoret, 1982). Aft er natural or artifi cial insemination with the infected semen material for in vitro fertilization, the virus can infect female animals and the risks for national and international spread of the virus is increasing (Kendrick and Mc Entee, 1967).
Identifi cation of latent virus carriers among the bulls requires adequately sensitive and specifi c serological tests. Usually, the serum neutralization tests (SNT) is used as the gold standard for detecting BHV 1 specifi c antibodies. Diff erent laboratories apply diverse SNT variants. Positive correlation between duration of serum-virus mixture incubation period and reaction sensitivity is well established. For example, the serum titers are 16 times higher aft er 24 h incubation of the serum-virus mixture as compared with titers aft er 1 h incubation (Bitch 1978, OIE, 2004. Despite the fact that SNT is highly sensitive and specifi c; however, it requires cell cultures and sterile conditions. Moreover it is time consuming, laborious and cannot be automated. Th us, variants of ELISA are preferred techniques for detection of BHV 1 antibodies (Van Oirschot, 2000). Commercially available ELISAs are developed for accomplishing the control and eradication programs and their sensitivity is comparatively studied (Kramps et al., 1993).
Detection, isolation and identifi cation of BHV1 is accomplished by the use of cell culture and various methods (Wyler et al., 1989): ELISAs with monoclo-nal antibodies for antigen detection (Collins et al., 1988) as well as a range of polymerase chain reactions (PCR) proving gB, gC, gD genes and thymidine kinase (TK) (Wiedmann et al., 1993;Kibenge et al., 1994 andVilcek et al., 1994). Highly sensitive PCR determining three to fi ve molecules of BHV-1 in bovine semen (Engelenburg, 1993) and nested PCR detecting 0.25-2.5 TCID50 of BHV 1 in bovine semen (Masri et al., 1996) was developed.
Th e aim of this study is determination of BHV 1 incidence among the bovine population in Bulgaria by using various ELISAs for antibody detection and diff erent methods for detection of virus antigens

Serological analysis
Serological examination of 2973 serum samples from bovine population in Bulgaria (cattle, calf and bulls) originating from 21 country regions was performed. Blood samples were coagulated, harvested, inactivated at 56 o C for 30 min and subsequently, antibiotics were added. Th e samples were frozen at -20 o C.
Commercial diagnostic ELISA kits were used for screening as well as for the confi rmation of the antibodies (Institute Pourquier, France). Th e readings were performed at 450 nm optical density (OD) for both reactions.
Th e screening reaction is based on specifi c bounding of the antibodies from the examined sample to the BHV1 antigen immobilized on the ELISA microtiter plate. Th e bound antibodies were detected using the anti bovine immunglobulin G (IgG) monoclonal antibodies marked by horse-radish peroxidase (HRPO). Aft er addition of chromogen substrate, the presence of BHV1antibodies in the sample results in specifi c color reaction indicating the seropositive result Minimal mean OD value of positive control 0.350 and proportion between mean values for positive and negative controls equal or more than 3.5 was adoptedas the criteria for reaction validity.
Percentage of positive (S/P) samples was determined by the formula: Mean OD 450 nm positive control -OD 450 nm negative control Th e sera with S/P ≤ 45% were considered negative and with S/P ≥ 55% as positive to BHV 1 antibodies. Sera with S/P between 45 and 55% were considered doubtful and were retested aft er 3-4 weeks.
Verifi cation of the results obtained in screening test was performed in a subsequent confi rmation test. Wells with BHV 1 antigen and corresponding wells loaded with control antigen (uninfected cell culture) were used. Th e sera samples were fi lled into the both wells. Th e results were considered valid if a minimal mean OD 450 value for positive control was 0.350 (uncorrected) and the ratio between mean corrected OD 450 value for the positive control and the negative control was equal or greater than 3.5.
Th e result was obtained aft er OD correction using the following equation: where OD BHV 1 wells coated with the BHV 1 antigen, OD control uncoated wells Th e results were obtained by calculation of sample positive control (S/P) by the ratio: Positive control (mean OD corrected ) All samples with S/P ≤ 45% were considered negative, with S/P between 45 and 55% doubtful and with S/P >55% were considered BHV1 positive .

Virus analysis
Total 408 cattle and 150 bulls' samples originating from artifi cial insemination center (AIC), community and private farms from 21 country regions were analyzed. Buff y coats, nasal, eye, preputial and vaginal swabs, as well as samples of the semen, tissues and organs from infected animals were treated by the method of Dilovski et. a.,l (1982) and inoculated onto cell cultures. Primary rabbit and continuous cell lines from calf kidney -Madin-Darby bovine kidney (MDBK) were used for isolation and culturing. Eagle minimal essential medium (E MEM) with Hank's salts were used as growing and maintenance medium. Antibiotics penicillin 100 UI/mL, Streptomycin 100 γ/mL and 10% fetal calf serum (FCS) and 2% FCS were added as a growing and maintenance medium, respectively. Semi-confl uent or confl uent cell culture monolayers were inoculated with 0.2 mL of treated specimens and adsorbed for 2 h at 37 o C with exception of buff y coats and semen samples (adsorbed for 1 h at 37 o C).
Aft er absorption, the cell monolayers were washed and maintenance medium was added. Th e cytopathic eff ect was determined microscopically 120 h aft er inoculation. Th ree consecutive passages of cell cultures were performed. Uninfected cell cultures were used as the negative control. Th e virus titers and serum neutralization titers were determined by the method described by Dilovski et al. (1982).
Conventional PCR was developed for BHV 1 gB gene for the identifi cation of isolated viruses (Fucsh et al., 1999). Primers gB 1 multiplying part from gB gen at position 883 -902 and gB 2 at position 1341 -1360 amplifying product with 478 bp size were used. Nested PCR multiplying BHV 1 gB gen was performed with nested primers gB N 1 at position 899 -918 and gB N 2 at position 1264 -1283 creating the product with 385 bp size. Th e strains DNA isolated from the infected cell cultures and DNA of referent BHV 1 were subjected to electrophoresis in 2% agarose-gel containing (1mg/mL) ethidium bromide. Th e electrophoresis was carried out at 120 V for 45 min in 0.5 TBE buff er (0.045 M Tris borate, 0.001 M EDTA pH 8.6). As positive and negative controls, the referent BHV 1 Oxford strain and uninfected MDBK cells were used, respectively. To control the contamination, sterile distilled deionized water was added instead of DNA.

RESULTS
Th e percentage of positive sera aft er using the confi rmatory test was insignifi cantly (p>0.05) higher than that in the screening test.
Analysis using ELISA revealed that out of 2240 serum samples from bovine population and 733 from bulls the rate of positive cattle serums (38.3%) was signifi cantly higher(p < 0.001) then that of the bulls (29.3%) (fi g.1). Th e mean optical density of sera samples for cows and calves from 16 regions varied between (26.4% and 44.3%). For bulls' samples from the same regions the mean optical density ranged between (87.1% and 147.6%). Th e signifi cantly highest (p<0.01) OD rate was established in bulls from Smolian, Lovech, Pernik, Varna and Pazardjik regions. In the other investigated regions, the percent was between 65% and 96% (table1). Proportional distribution of positively reacting cattle and calf serums was signifi cantly (p<0.01) higher than that of the bulls in all years of investigation, except for 2009 and 2011 (fi g.2). Aft er cultivating 408 samples originating from cattle and calves (buff y coats, nasal, eye, vaginal swabs, tissues and organs) on cell culture , seven strains manifesting BHV 1 properties were isolated from Dobrich, Lovech -2 strains, Plovdiv, Targovishte, Pazardjik and Svishtov regions.
Th e morphological characteristics were typical for BHV 1. In primary and permanent cell cultures, the cytopathic eff ect was visible 12 h aft er inoculation. Rounding and detachment of cell cultures were advanced 18-20 h later, and the number and space of clusters with damaged cells increased and vacuolation and granulation of cells were evident. A lot of spherical cells undergoing ballooning degeneration linked with cytoplasmic bridges and progressive detachment of cells aft er 72 h were visible. Single degenerating cells and full cell detachment 96-120 h aft er inoculation were observed.
Confi rmation of virus isolation was accomplished using PCR gB gene based primers (amplifi ed 478 bp) Th e amplicons from 3 bulls isolated strains with the same size and location as the referent Oxford strain were observed. Aft er performing nested PCR for gB gen, products 385 bp in size were obtained in all samples as well as in the referent Oxford strain. Virus amplifi cation was established neither in uninfected MDBK cells nor in sterile distilled water, which were used as negative controls in conventional and nested PCRs (fi g. 3).

DISCUSSION
In the last years, ruminants' population in Bulgaria has been decreasing dramatically and the number of animals on small private farms ranges between 2 and 5, while the number of animals on big farms is 100-200. Changes in the breeding system resulted in changes of the epizootological situation in Bulgaria. Results on seroprevalence in some regions indicate that BHV 1 infection is widespread in bovine population. In our previous investigation, we established the prevalence of infection in diff erent country regions being 16-33% (Peshev et al., 2009) whereas in the current investigation revealed a higher incidence rate (38.3%). Most probably, the reason for that is application of more sensitive ELISAs in current research than in the previous one.
Newly designed indirect BHV 1 ELISA method that we have applied for this screening is based on monoclonal antibodies with higher specifi city and sensitivity, and the results can be compared with the data obtained by gB blocking ELISA or serum neutralization test (Beer et al., 2003;Isa et al., 2003). Most of the blocking and competitive reactions proving BHV 1 antibodies in infected or vaccinated animals are based on monoclonal antibodies created against epitopes of BHV 1 gB glycoprotein. Th is protein is essential for BHV 1 and is highly immunogenic for infected animals. We obtained higher percentage of positive serums (cattle, calf and bulls) than in the screening test because of the using highly sensitive confi rmative ELISA . Higher sensitivity of this ELISA is due to the using of corrected OD (subtraction of control uninfected cell culture OD values). It can be concluded that the use of more sensitive confi rming ELISA is mandatory for such epidemiological studies.
Th e BHV 1 infection is easily spread from animal to animal via the respiratory or reproductive transmission route, since aft er native or artifi cial insemination semen samples are potential source of BHV 1 infection (Guerin et al. 1993). Contaminated semen samples are potential threat to bovine population because BHV 1 can cause infectious pustular vulvovaginitis, endometritis, short estrus cycles, repeat breeding, abortion in cows and balanopostitis in susceptible bulls (Schultz and Sheff y, 1980). Th e virus in semen participates in seminal fl uids, non-sperm cells and is adsorbed on spermatozoa (Engelenburg et al. 1993). Th e cryoprotected semen in AIC is harvested at -196 o C and distributed to other country regions and farms. Th e distribution rate of IBR infection determined in this research is lower in regions where the number of positive bulls is also lower. Th e bulls can spread the virus during clinical and subclinical infection. In that respect, regular testing and control of such animals is of crucial importance.
Th e specifi c humoral immune response to BHV 1 is the result of viral infection and the specifi c antibody titers can persist for several years (Kashooek et al., 1996). In our research, we established high percentage of serologically positive cows in seven regions, with an average rate of OD positivity varying between 26 and 44%, which can be as attributed to a recent BHV 1 infection. Aft er examining the bulls originating from the same regions, we established three to four times higher average percentage of OD positive results, which strongly indicates the circulation of active BHV 1 infection among bulls' population.
Th e virus isolation test on diff erent cell culture is frequently used and is adopted as a gold standard for sensitive and specifi c BHV 1 detection (Edwards et al., 1983;Brunner et al., 1998). Analytical sensitivity of virus isolation varied between <1 TCID 50 to 5 TCID 50 /mL. BHV1 antigen can be determined by direct or indirect immunofl uorescent tests or by enzyme immune test for antigen detection. Both immunofl uorescence reactions exhibit lower sensitivity as compared to the enzyme immune methods (Collins et al., 1988). We used cell cultures for BHV 1 isolation and the highly sensitive ELISA, classical and nested PCRs for the confi rmation of isolated BHV 1. Seven newly isolated strains from cows and three from bulls are an evidence for broad spreading of BHV 1 in bovine population in Bulgaria Health status of animals depends on the raising system and the fi nancial capacity of owners to implement the control and immunoprophylactic measures. Th e situation with BHV 1 distribution in diff erent European countries is variable. Th e herd prevalence ranges from 10 to 80%. In Portugal, the prevalence is 47.2%, in dairy herds in Italy from 61 to 84%, for beef herds 59% and for mixed herds 89%. In Spain, the prevalence is between 38.4% and 50.4% Eiras et al. (2009). In Belgium, the positivity rate for dairy herds is 35%, for beef cows 31% and for mixed herds 43% (Boelaert et al. 2000). In Lithuania, using the Pourquier (ELISA IBR-IPV gB glycoprotein antibodies) the seroprevalence rate was established to be 33.86% for cows, 8.77% for heifers and 1.69% for bulls, whereas the average percentage is 14.01%.
Diff erent European countries apply diverse strategies of BHV 1 control and eradication. In some countries, the disease is eradicated by testing and removing of positive animals. Th is is an expensive method and it is economically reasonable only at low level of virus infection in premises. In other countries, the protection against disease is carried out by intensive immunization programs using conventional inactivated, attenuated and gene-deleted vaccines. By the fact that the percent of infected animals in Bulgaria is high, the method for testing and removing is economically unprofi table. In Netherlands and Germany, disease control is accomplished using so-called DIVA strategy (diff erentiating infected from vaccinated animals) applying gene deleted vaccines and specially created tests discriminating infected from vaccinated animals. In conditions of broad distribution of infection, as in our country, this strategy can be used, and when the percentage of positive animals decreases to minimum, other positive animals can be removed from the farms. Animals imported from countries eradicating BHV 1 have to be vaccinated against BHV 1 on their farms of origin or during obligatory quarantine in the accepting country.