Buffalo Bulletin Vol.27 No.1 (March 2008) p. 165-169






Jain Lata, A.N. Kanani, T.J. Patel, J. H. Purohit, M.K. Jhala,

H.C. Chuahan and B.S. Chandel


Department of Veterinary Microbiology,

College of Veterinary Science and Animal Husbandry, Anand Agriculture University,

Anand, Gujarat 388001, India.





The present study was undertaken to screen the sera of cattle and buffalo bulls belonging to five semen collection centres of Gujarat state for the presence of IBRV antibodies employing monoclonal antibody based blocking ELISA (M-ELISA). The indirect fluorescence test was performed to correlate the results obtained by M-ELISA. Out of 89 sera tested by M-ELISA, 26 (29.28%) were positive. A variable rate of seroprevalence was recorded from all the centres included in the study. Further studies, mainly isolation of virus from semen, are warranted. Results indicated the prevalence of IBR infection among cattle and buffalo bulls of semen collection centres of Gujarat state. However, it is emphasized that the cattle and buffalo population of organized farms as well as rural areas of Gujarat State should be screened for IBR to learn the exact picture. Thus, future steps can be taken to control this emerging disease.


Keywords: bovine herpesvirus, seroprevalence





Infectious bovine rhinotracheitis (IBR) caused by BHV-1 of family Herpesviridae is amongst the important emerging diseases of cattle and buffaloes in India. BHV-1 is responsible for a variety of clinical conditions in cattle and buffaloes, including infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV), infectious pustular balanoposthitis (IPB), and conjunctivitis and generalized disease in neonates and thus causes great economic losses to the livestock industry (Gibbs and Rweyemamu, 1977).

The infection has serious economic implications for India, which is emerging as the worlds biggest milk producer and has the worlds largest cattle and buffalo population.

IBR was first recognized in the United States during 1950s and it has since then recognized in many countries throughout the world. In India, the disease was first reported in Uttar Pradesh among cross bred calves in 1976 (Mehrotra et al., 1976). Various workers have since reported widespread seroprevalence and have isolated the virus from different parts of the country, including Gujarat (Renukaradhya et al., 1996; Khan, 2004).

Considering the infectious nature of the disease and its economic implications, a systemic study was undertaken to determine the evidence of IBR in breeding bulls, maintained at semen collection centres employing monoclonal antibody based blocking ELISA and the indirect immunofluorescence test.





I. Serum samples: A total of 89 serum samples were collected from cattle (38) and buffalo bulls (51) stationed at  five different semen collection centres of Gujarat state. The separated serum was collected in screw-capped plastic vials and heat inactivated at 56oC for 30 minutes, and Merthiolate (1:10,000) was added in all vials as a preservative. The sera were held at -20ºC temperature until use.


II. Reference reagents:

a) For M-ELISA: An IBR monoclonal antibody based blocking ELISA kit (Catalog no. B1004-AB01) was made available by courtesy of BV European Veterinary Laboratory, The Netherlands.

b) For indirect fluorescence: An indirect fluorescence kit was made available by VMRD, Inc. Pullman, USA.


Protocol of M-ELISA

All 89 sera were subjected to M-ELISA as per the protocol of the IBR monoclonal antibody based blocking ELISA kit. The protocol is as follow:

1. To all the wells of the microtiter strip, 50 µl of ELISA buffer was added, and then 50 µl of each positive control serum and negative control serum (supplied in kit) was added to positive and negative control marked wells,  respectively. Then, 50 µl of serum sample was added to an individual marked sample well of the strip and then incubated for 3 h at 37ºC.

2. Microtiter strips were washed with washing solution for atleast 4 times.

3. Then, 100 µl of anti-BHV-1 conjugate was dispensed to all the wells and incubated for 30 min at room temperature. Then, the washing step was repeated.

4. Equal parts of substrate A and substrate B were mixed with gentle shaking immediately before use. Then, 100 µl of substrate solution was dispensed to each well and incubated for 15 minutes at room temperature.

5. Then, 50 µl of stop solution was added to each well and mixed by tapping. The absorbence values (OD) were read immediately (within 10 minutes) at 450 nm.


Interpretating the result of M-ELISA

 Calculation:    Ratio = Sample OD/Negative control OD

The samples were considered to be positive for IBR antibodies when the ratio was equal to or less than 0.6. The sample with ratio equal to or more than 0.7 were considered negative. The samples having ratios between 0.6-0.7 were considered doubtful and retested.


Protocol of Indirect FAT: Out of 89 sera, only 8 sera (6 M-ELISA positive and 2 M-ELISA negative) were tested for presence of BHV-1 antibodies by indirect FAT as per the protocol of kit supplied by VMRD. The protocol is as follow:

1. To an individual marked well of FA Substrate Slide (Catalog no.: 210-88-10-IBR, VMRD) (i.e. well for positive control, negative control and field serum) 50 µl serum was added and then the slide was incubated in a humid chamber at 37ºC for 30 minutes.

2. The slide was gently rinsed in FA Rinse Buffer (Na2CO3 : 11.4 gm; NaHCO3 : 33.6 gm; NaCl :8.5 gm; DI/dH2O up to 1 liter; pH 9.0) and then soaked for 10 minutes in FA Rinse Buffer.

3. To each well, 50µl labelled anti-IgG FITC conjugate (Catalog no.: 020-1, VMRD) was placed and incubated in a humid chamber at 37ºC for 30 minutes.

4. The slide was rinsed in FA Rinse Buffer and then soaked for 10 minutes in FA Rinse Buffer.

5. Slide was mounted with FA Mounting Fluid [glycerol/FA rinse buffer, pH 9.0, (1:1)] and viewed with a fluorescent microscope at 100X-250X and confirmation was done at 400X.


Interpretating the result of Indirect FAT:

            In positive control wells, 2-3 positive reactions on positive cells and no reactions on negative cells were seen. While in negative control wells, no reaction was seen on positive as well as on negative cells. In wells containing field serum samples, 2-4 positive reactions on positive cells and no reactions on negative cells were seen.





            A total of 89 serum samples from breeding bulls were screened by IBR monoclonal antibody based blocking ELISA and the overall rate of seroprevalance recorded was  29.21 percent . Table 1 represents location-wise, species-wise and breed-wise seroprevalence of IBR in cattle and buffalo bull population.

            Similarly, Dhand et al., 2002 reported seroprevalence of 28.76 per cent in cattle and buffaloes in Punjab state. However, contrary to the present findings, Khan (2004) reported the slightly lower rate of seroprevalence of 21.30 percent in the cattle and buffalo population of Gujarat state. While a higher rate of seroprevalence 49.97 percent was reported by Pandita and Srivastava (1993). The variation in this may be due to the sample size, location of the samples collected, inclusion of samples from the sexes, seasons, etc.

Species-wise prevalence was found to be 34.21 and 25.49 percent in cattle and buffalo bulls, respectively. Seroprevalence recorded in cattle was in accordance with the results obtained by Dhand et al. (2002). However, contrary to the present findings, Suri Babu et al. (1984) reported a higher rate of seroprevalence, 65.78 percent, in cattle from Andhra Pradesh. While Rajesh et al. (2003) reported the very low rate of seroprevalence of 14.88 percent in cattle from Kerala state.

During the present investigation, the rate of seroprevalence recorded in buffalo bulls was 25.49 percent, which corroborates the finding of Aruna and Suri Babu (1992) who reported 21.05 percent seroprevalence of IBRV antibodies in buffaloes of Andhra Pradesh. However, Mannickam and Mohan (1987) failed to detect IBRV antibodies in buffaloes in Tamil Nadu.

Of the total 89 serum samples, eight (six M-ELISA positive and two M-ELISA negative) were tested for the presence of IBR antibodies by indirect FAT. One positive and one negative control sera were also placed for the validation of the test result. All the six M-ELISA positive sera were found to be positive by this method and both the two M-ELISA negative sera were negative. Figures 1 and 2 show the immunofluorescent reaction of positive and negative serum samples, respectively. Thus, 100% correlation was observed between these two methods. However, it is emphasized that a greater number of samples should be tested to draw a meaningful conclusion.

            Onisk et al. (1989) developed a technique of immunofluorescent assay for the detection of IBR/IPV antibodies and claimed that the technique can be performed to screen a large number of sera samples. Bratanich et al. (1990) compared SN, indirect FAT and ELISA for their sensitivity and specificity to detect antibodies to BHV-1 and found high correlation coefficients among all three techniques with a higher sensitivity for the ELISA.

Though the present study is based on limited number of samples only from breeding bulls maintained at various semen collection stations of Gujarat state, it is difficult to reach a meaningful conclusion with respect to breeds, species, locations and regions. However, it is indicative of prevalence of IBRV antibodies in Gujarat state. The prevalence of BHV-1 infection in cattle and buffalo bulls and its serious impact on the livestock industry make it one of the most important infectious agents for livestock. The serological test can be inadequate in the low antibody titer or seronegative latent infection; thus, it is essential that in addition to serological diagnosis, identification of etiological agent should be established or the existence of the antigen before isolation should be revealed by various tests (Duman et al., 2007). Moreover, further systematic seroepidemiological and isolation studies are warranted to find out the actual status of IBR in Gujarat state and elsewhere in India. Thus, further steps can be taken to control this emerging disease.



Table1. Seroprevalence of IBRV/BHV-1 in breeding bulls by M-ELISA.



Numbers tested

Number positive

Percent positive

[A] Location:

























[B] Species-wise:













[C] Breed-wise (Cattle) :

Cross bred
















[D] Breed-wise (Buffalo):





















Figure 1. Immunofluorescent staining reaction with field serum sample antibody-antigen

  complex of BHV-1 infected MDBK cells monolayer on well and FITC antibovine IgG    

  conjugate. Note the bright fluorescing specific reaction on infected cells as compared to   

  normal cells (400X)





Figure-2: Immunofluorescent staining reaction with negative control antibody-antigen complex of

  BHV-1 infected MDBK cells monolayer on well and FITC antibovine IgG conjugate. 

               Note the absence of fluorescence (400X).





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