doi:10.5455/vetworld.2013.343-345 Prevalence of Bovine Herpesvirus-1 in cattle and buffaloes in Punjab

Aim: The aim of the present study was to identify the prevalence of Bovine Herpesvirus-1 (BHV-1) in cattle and buffaloes in the Punjab using PCR as diagnostic tool. Materials and Methods: A total of 63 samples (Semen- 57, placental cotyledons-1, vaginal secretions-1, foetal stomach contents-1 and tracheal swabs-3) from cattle and buffaloes were processed for identification of BHV-1 using PCR Results: From January 2007 to December 2010 (Semen- 57, placental cotyledons-1, vaginal secretions-1, foetal stomach contents-1 and tracheal swabs-3) from cattle and buffaloes were collected. The DNA was extracted from a total of 63 samples and subjected to PCR revealed that none of the sample positive for the BHV-1 infection. Conclusion: From the study it was concluded that the farms screened were free from BHV-1 infection.


Introduction
method for the detection of BHV-1 in the suspected clinical samples.However, this method is time consuming and Bovine herpesvirus type-1 (BHV-1) is a member of the laborious.The other conventional methods for detection of family Herpesviridae, subfamily Alphaherpesvirinae.It is BHV-1, such as the fluorescent antibody test, ELISA and an important pathogen of cattle worldwide [1] and is serological testing, lack sensitivity, particularly when classified as BHV-1.1 (respiratory form), BHV-1.2 (genital applied to semen donor bulls [8].Molecular technique such form) and BHV-1.3 (encephalitic form) [2].It is responsible as PCR has been standardized and is commonly used to for great economic losses to livestock industry in terms of detect the presence of BHV-1 in various samples effectively loss of milk production and abortion.The virus causes because of its enhanced sensitivity, specificity and rapidity numerous clinical manifestations like rhinotracheitis, [9].Thus, in the present study clinical samples were vulvovaginitis, balanoposthitis, abortion and encephalitis.
examined with the help of PCR for the detection of BHV-1 in Besides this infection, it can cause acute gastroenteritis, these animals.conjunctivititis, mastitis and repeat breeding in cattle [3].

Materials and Methods
BHV-1 generally infects cattle greater than 6 months of age once maternal immunity has waned [4].The virus is excreted Samples: A total of 63 samples including semen (57), through nasal, ocular secretion, placenta of aborted animals placental cotyledons (1), vaginal secretions (1), foetal and through semen.In the bovine semen, BHV-1 is the most stomach contents (1) and tracheal swabs (3) from various farms, common viral pathogen and it poses a great threat to cattle submitted in the Department of Veterinary Microbiology, industry as the virus can spread by artificial insemination College of Veterinary Science, GADVASU from January causing variety of genital disorders [5].Following an acute 2007 to December 2010 were processed for the BHV-1.infection, the latent virus is established mainly in the sensory Sample Processesing: The frozen semen straws were ganglia, for the rest of the life of the animal [6].This latent o thawed at 37 C and diluted to 1:10 in sterile normal saline virus can be reactivated and consequently excreted, with the solution and was clarified by centrifugation at 8000 rpm for risk of infecting herd mates which are free from BHV-1.
10 minutes in a refrigerated centrifuge.The supernatant was Thus, vaccination can control the clinical disease but latent o collected and kept at -20 C till further use.infection cannot be prevented, therefore, once cattle that The placental cotyledons and vaginal secretions were have been infected must be regarded as lifelong potential collected in phosphate buffer glycerol (PBG) and were then shedders of BHV-1 [7].Bulls infected with BHV-1 are triturated in mortar and pestle using sterilized sand and lifelong carriers and potentially shed virus intermittently in normal saline solution.It was then clarified by centrifugation their semen.
at 8000 rpm for 10 minutes in refrigerated centrifuge.The Laboratory examinations are required for the BHV-1 particularly from clinical samples [12,13,14].The of the each of the above processesed sample.In brief, twenty greatest problem with the infections caused by herpes virus microlitres of proteinaseK (20mg/ml) and of 10% SDS was o is the induction of carrier state in the animals, due to which, added, mixed by vortex for 15 seconds and incubated at 56 C the presence of antibodies in an animal does not indicate the for 2 hours.Subsequently, equal volume of phenol: true picture of active infection.Considering this major chloroform: isoamyl alcohol (25:24:1) was added to the drawback in serum based tests, detection of virus becomes above solution.It was vortexed for 15 seconds and then mandatory to designate an animal as positive for BHV-1 centrifuged at 13000 rpm for 15 minutes.To the supernatant [15].Out of a total of 63 samples tested in the present study, equal volume of chloroform: isoamyl alcohol (24:1) was none of the samples was positive when tested with PCR added, mixed gently and centrifuged at 13000 rpm for 15 (Table -1).The reference DNA used in the study showed minutes.To the supernatant, 1/10 volume of 3M sodium positive amplification of 468bp in PCR suggests that the acetate solution (pH 5.2) and equal volume of isopropanol o assay used was specific and sensitive for BHV-1 and could was added and stored at -20 C overnight to allow be used to detect BHV-1 in different samples from infected precipitation of DNA.DNA was pelleted by centrifugation at o animals (Fig. 1).In an earlier study conducted in 1998 in the 12000 rpm for 10 minutes at 4 C.The pellet was then washed Lusaka province of Zambia, the prevalence of BHV-1 with 70% ethanol twice, air dried and resuspended in 50µl of o antibodies in bovine sera was reported to be in between 0% Tris-EDTA buffer (pH 8.0) and stored at -20 C till further to 80% [16].In another study, two polymerase chain reaction use.
assays specific for glycoprotein B (gB) and glycoprotein C Polymerase chain reaction (PCR): For the PCR, Forward (gC) gene detection were used for the detection of bovine primer 5'CACGGACCTGGTGGACAAGAAG3' and herpesvirus-1 in naturally infected cattle and reported that Reverse primer 5'CTACCGTCACGTGAGTGGTACG3' as out of 12 bovine tissue samples tested for gB and gC only one reported by Vilcek [10] was used in the present study.The sample was found to be positive for presence of viral reaction mixture for the PCR included 10µl of template genome, producing an amplified PCR product of size 173 bp DNA, 1.0µl of forward and reverse primers (10pm/µl) each, [17].5.0µl of PCR buffer (10x), 2.0µl of MgCl (25mM), 1.0µl of PCR was used in another study for the detection of 2 dNTPs (10mM each), 0.2µl of Taq Polymerase (5units/µl) BHV-1 directly from semen samples and it was reported that and 29.8µl of nuclease free water to make the final reaction PCR failed to amplify in some of the semen samples which volume to 50µl.
was attributed due to the inhibitory effect of the bovine The PCR conditions included an initial denaturation at semen [18].However, it was also reported that by using a o o purification protocol that eliminated the interfering 95 C for 3 minutes and then 35 cycles of denaturation at 95 C o components, the sensitivity of PCR increased markedly [19].for 60 seconds, annealing at 55 C for 60 seconds and o Out of a total of 523 semen samples collected and screened extension at 75 C for 60 seconds and then a final extension at o by isolation of virus for BHV-1 in bovine turbinate (BT) and 72 C for 10 minutes.The PCR products were analyzed using Mardin Darby Bovine Kidney (MDBK) cell lines from the 1% agarose gel with 0.05ug/µl ethidium bromide.It was four southern states of India revealed that, only four semen electrophoresed at 5-10 volts/cm for 1 hour.Gene Ruler samples showed cytopathic changes indicating that the ladder plus 100 bp (MBI, Fermentas) was run for calculating prevalence of BHV-1 is scarce [20] similar to our findings in the relative molecular weight.
which we too failed in detecting BHV-1.Similarly in another In the PCR BHV-1 isolate (IBR 216 II batch No. 9/98), study, the prevalence of BHV-1 was found to be 0.8% obtained from IVRI, Izatnagar available in the Department (2/250) [21].However, in a study on 574 semen samples of Veterinary Microbiology, Ludhiana was used as positive from seropositive cattle and buffaloes tested by real time control in the study.
PCR indicated that 1.97% cattle and 3.36% buffalo semen assumes significance as far as farm management is o supernatant was also collected and stored at -20 C till further diagnosis of BHV-1.Isolation of virus is the most frequent use.The foetal stomach content was diluted 1:10 in phosphate buffer saline and then clarified by centrifugation at 8000 rpm for 10 minutes in refrigerated centrifuge.The o Results and Discussion supernatant was collected and stored at -20 C till further use.The tracheal swab was collected in a test-tube having Infectious bovine rhinotracheitis (IBR) is a highly 2ml of sterilized tryptose phosphate broth.It was centrifuged contagious disease caused by BHV-1 affecting cattle and at 8000 rpm for 10 minutes.The supernatant was collected buffaloes [11] and it is present worldwide.BHV-1 infects o and stored at -20 C till further use.respiratory and genital tracts of cattle and buffaloes causing various diseases.PCR is an invaluable tool for detection of Isolation of DNA: The total DNA was extracted from 600µl

Table - 1
. Total number of samples tested via PCR for the detection of BHV-1 PCR exhibiting a product in a known positive sample (M: Marker; PC: Positive Control) bovine herpesvirus type 1 is an efficacious and safe vaccine, batches were positive for BHV-1 indicating that real time Vaccine, 12(5): 439-444.PCR in adjunct could be used in diagnostic laboratories for 8. Zhou J., Lyaku J., Fredrickson R.A. and Kibenge F.S.B. identifying BHV-1[22].(1999)Improved detection of bovine herpesvirus 1 in Conclusion artificially infected bovine semen by protein amplification, J. Virol.Meth., 79 (2): 181-189.It could be concluded that none of the animals screened 9. Mahmoud M. A. and Ahmed S. A. (2009) Prevalence of using PCR, yielded positive results indicating that the farms bovine herpesvirus-1 (BHV-1) in sheep and goats in Egypt.screened were free from BHV-1 infection.The finding Figure-1.