Sero-prevalence and Risk Factor Analysis of BoHV-1 in Bovines in Haryana State of India

Bovine herpesvirus-1 (BoHV-1) belongs to the genus Varicellovirus in the subfamily Alphaherpesvirinae under the family Herpesviridae. BoHV-1 is known to cause several diseases including infectious bovine rhinotracheitis (IBR), vaginitis, balanoposthitis, abortion, conjunctivitis and enteritis. It is transmitted horizontally through sexual contact, artificial inseminationand aerosol transmission and may also be transmitted vertically across the placenta. Morbidity and mortality vary considerably and reported lower in dairyherds than in beef cattle (Majumder et al., 2015). Even though mortality is low, the disease shows its severe impact on growth, milk production, loss of new born calves, temporary failure of conception, insufficient feed conversion, secondary bacterial pneumonia, cost of treatment and international livestock trade. IBR has been known to exist in India since, 1976 (Mehrotra et al., 1976) and sporadic studies on sero-prevalence have been conducted in different parts of India (Nandi et al., 2011; Kollannur et al., 2014). Mallick (1986) reported the sero-prevalence of IBR disease in seven states of India and observed that 65.3% exotic, 73% crossbred and 62% indigenous cattle were seropositive. Several sero-prevalence studies have been carried out by different researchers and observed that the disease is prevalent in almost all the states of India. (Renukaradhya et al., 1996, reported the sero-prevalence of 50.9% and 52.5% in cattle and buffaloes respectively. To effectively control this disease, screening, surveillance and monitoring along with vaccination is important. But, no real-time data is available for its sero-prevalence status in the state of Haryana. Hence, to know the status of seroprevalence, a cross-sectional serological study was conducted with the objective ofestimating the apparent present prevalence of BoHV-1 and potential risk factor among unorganized cattle and buffalo herds.

Study area
 
Haryana is one of the northern states of India situated between 27° 37' to 30° 35' latitude, 74° 28' to 77° 36' longitude and 700 and 3600 ft altitude above sea level. Haryana falls into the agro-climatic zone-vi, which is further divided into two sub agro-climatic zones based on the geographical feature of the state i.e., Western zone comprising of semi-desert sandy plain and Eastern zone comprising of Shivalik hills and Ghaggar Yamuna plain (Fig 1 and Table 1).
 

 

 
Sample
 
A total of 490 blood samples were collected from cattle and buffaloes from all twenty two districts of Haryana from unorganised herd randomly. The sampling was performed during April to December 2018. Out of 490 samples, 270 samples were from cattle and 220 from buffaloes were collected. In brief, blood samples were collected via jugular venipuncture into sterile vacutainer tubes without anticoagulant. Samples were centrifuged at 1000xg for 15 min. to separate the serum and further stored at -20°C. The complete experiment/study was conducted in the Department of Animal Biotechnology, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar.
 
ELISA
 
The collected serum samples were tested for antibodies against BoHV-1 using an indirect enzyme-linked immunosorbent assay (ELISA). The assay was performed using Monoscreen Ab-ELISA kit and test was carried out according to manufacturer’s instructions. Finally, the plate (Fig 2) was read in the ELISA plate reader at 450 nm. Results of test sera were expressed as:
 



 
Statistical analysis
 
The data recorded were compiled and further categorized in to various factors such as species (cattle and buffalo), age (<2.5, 2.5-4.5, 4.5-6.5 and ≥6.5 years), sex of animal (male and female), districts and agroclimatic zone of Haryana (eastern and western). The sero-prevalence of each sample was coded yes (positive) and no (negative). The sero-prevalence between various factors viz., Species, age, sex, districts and zone was compared using Chi-square test. The association of sero-prevalence with various factors was undertaken by using logistic regression analysis. The significant differences were considered if p<0.05. All statistical analyses were done in SAS 9.3 Version software.
 
Ethical statement
 
Samples were collected with consent from animal owners. The protocol of this study does not require the approval of the Institutional Animal Ethics Committee.

The present cross sectional study revealed that, out of the 490 serum samples, 239 were positive for the presence of antibodies against BoHV-1 with an overall sero-prevalence of 48.78% (239/490) among cattle and buffalo population of the state. However, the sero-prevalence of BoHV1 in cattle was 37.77% (102/270) and in buffalo was 62.27% (137/220) as mentioned in Table 2. Statistically, overall sero-prevalence was significantly (p<0.01) higher in buffaloes (62.27%) as compared to cattle (37.77%). Out of all 22 districts of the state, highest sero-prevalence among cattle population was seen in district Faridabad (70%) and in buffalo it was Kaithal (90%). Moreover, the lowest sero-prevalence was observed in Rewari district i.e., 5% and 20% for cattle and buffalo population respectively as mentioned in the Fig 3.
 

 

               
The chi-square analysis of percent sero-prevalence with various factors was done and the results are given in Table 3. Also, the overall sero-prevalence was significantly (p<0.05) higher in eastern zone (52.83%) of the state as compared to western zone (41.78%). Significantly, higher antibodies titre was observed in older animals (³6.5 years) followed by age group 2.5-4.5 years, 4.5-6.5 years than young animals (<2.5 years). There was no significant difference observed in overall sero-prevalence between male and female animals. In case of species wise chi-square analysis, only age was found significant factor associated with sero-prevalence for buffaloes, as the highest sero-prevalence (75.61%) was observed in older buffaloes (≥6.5 years) followed by age 2.5-4.5 years (69.64%), 4.5-6.5 years (58.51%) than young animals with age of <2.5 years(41.38%). Factors like breed, sex etc. were not significantly (p>0.05) associated with BoHV1 sero-prevalence in cattle or buffaloes.
 

       
The Binary logistic regression was used to determine the likelihood of BoHV-1 due to various factors used in this study. The results of overall logistic regression analysis revealed that species, zone and age were significantly associated with sero-prevalence of BoHV-1. The likelihood of BoHV-1 was significantly 0.37 times lower (OR=0.37, 95% CI: 0.25; 0.54) in cattle than the reference category i.e. buffaloes. In case of zone, the higher (1.52 times) likelihood was observed in eastern zone (OR=1.52, 95% CI: 1.03; 2.26) than western zone (OR=1.00). As compared to older animals (Age ³6.5 years), the likelihood of BoHV-1 was lower in other age groups with minimum in animals with age <2.5 years (0.34 times), followed by 4.5-6.5 years (0.57 times) and 2.5-4.5 years (0.82 times) as mentioned in Table 4. The comparison between percent sero-positivity in cattle and buffaloes of different districts of Haryana is mentioned in Table 5.
 

 

       
Sero-prevalence represents cumulative exposure to infection. The accuracy of serology must be addressed on both the individual level and at the level of a population. Positively identifying the occurrence of disease is usually based upon the presence of antibodies for that particular disease.
 
Sero-prevalence of BoHV-1 in bovines of Haryana state
 
Among the 490 samples collected from bovines, 239 were found positive and 251 were found negative accounting to an overall positive percentage of 48.78. Almost similar sero-prevalence of 49.86% was reported by (Sulochana et al., 1981) in Kerala, 45.01% was reported by (Deka et al., 2005) in Indian breeding bulls. Low sero-prevalence of 28.76% was reported by (Dhand et al., 2002) in bovines of Punjab as compared with the present study. More or less similar result were obtained using ELISA on serum samples of cattle and buffaloes from different states like Uttar Pradesh by (Singh and Yadav, 2010), who reported  an overall sero-prevalence of 32.31% in bovines. The higher sero-positivity of 64.47% and 56.5% was reported by Vaid et al., (1991) and Samal et al., (1981) in Himachal Pradesh and Uttar Pradesh respectively. Babu et al., 1984 also reported a higher rate of sero-prevalence of 65.78 percent in cattle from Andhra Pradesh. The sero-prevalence of BoHV-1 among dairy cattle of Assam was found to be 20.34% (Chettri et al., 2015). Similar to this observations, 37.44% seropositivity was observed in cattle of Chattisgarh (Samrath et al., 2016). The overall sero-positivity of 40.70% and 23.22% were reported at organized and un-organized farm respectively in cattle of Bidar (Satbige et al., 2018).
 
Species-wise Sero-prevalence of BoHV-1
 
The higher sero-prevalence was seen in buffalo (62.27%) population as compared to cattle population (36.825%) in this study. Thakur et al., (2017) reported higher sero-prevalence (38.14%) in buffaloes than cattle (26.78%) in Uttarakhand. Trangadia et al., 2010 also reported high sero-prevalence in buffaloes (62.39%) than cattle (60.46%) in India. While in contrast to present study, Khan (2004) reported an overall sero-prevalence of 21.30% for IBR by i-ELISA test and species wise incidence was found higher in cattle (23.40%) than in buffaloes (18.97%). Gill et al., (1987) also reported a higher sero-prevalence of 47% in cattle than 14% in buffaloes.
 
Sex-wise Sero-prevalence of BoHV-1
 
An overall sero-positivity was seen to be 49.25% in female animal, while 38.10% in male. However, 38.04% female cattle and 62.62% female buffaloes were positive. Among males, 33.33% (5/15) cattle 50% (3/6) buffaloes were seen to be positive. Similarly, Samrath (2009) found an overall (34.98%) sero-positivity in female animals and (32.78%) in males respectively. Similar observation were also reported by Nandi et al., (2007) who observed 66% positivity in female, and 38% in male  in Jhansi. Sharma et al., (2006) reported higher sero-prevalence of IBR in female cattle as compared to male cattle i.e. 56.92% and 20%, respectively in Himachal Pradesh. In general, higher sero-prevalence was observed in female as compared to male and same was also observed in this study. Since, none of the animal included in the study had been vaccinated against BoHV-1, the sero-prevalence obtained indicate that the animal had been exposed to the virus, assuming that the presence of antibodies can only be caused by exposure to the pathogen (Kampa et al., 2004).

The present study found an overall higher percent sero-prevalence (48.77%) of BoHV-1among cattle and buffalo population of the state of Haryana. Most importantly the sero-prevalence was comparatively higher in buffalo as compared to cattle. The higher sero-prevalence revealing the fact that the BoHV-1 is naturally and latently existing in dairy animal of the state.

The authors acknowledge the DBT, GOI, for financial support under HRD scheme. We are equally grateful to the university administration and technical staff of the department for their cooperation throughout the study.

There is no conflict of interest among authors.