REGULAR ARTICLES

First report on serological evidence of bovine viral diarrhea virus(BVDV) infection in farmed and free ranging mithuns(Bos frontalis)

Vidya Singh1& Niranjan Mishra2 & S. Kalaiyarasu2

& R. K. Khetan2& D. Hemadri3 &

R. K. Singh1& K. Rajukumar2 & J. Chamuah1

& K. P. Suresh3& S. S. Patil3 & V. P. Singh2

Received: 30 January 2017 /Accepted: 4 May 2017# Springer Science+Business Media Dordrecht 2017

Abstract Despite reports of BVDV infection in several do-mestic and wild ruminants, no information exists for mithun(Bos frontalis) species. Hence, this study was undertaken todetermine prevalence of BVDV infection in mithuns, whichcontribute significantly to local economy in the North Easternregion of India. Blood and serum samples were collected be-tween 2013 and 2016 frommithuns (n = 466) belonging to thestates of Nagaland, Mizoram, and Arunachal Pradesh. Serumsamples were tested for BVDV antibodies by a commercialELISA and leukocytes were tested for BVDV by real-timeRT-PCR. The overall true seroprevalence rate was 13.1%(95% confidence interval, CI: 6.9–17.8%) with higher preva-lence in mithuns reared under semi-intensive system (27.5%)than in free-ranging mithuns (7.6%). Among the three states,seroprevalence (16.2%) was highest in Nagaland, while prev-alence rates varied markedly among geographical locations.Age-wise data showed highest seroprevalence rate in >6-year-old animals (20.6%) than 2–6 years old (16.9%), 6 months–2 years old (8.5%), and <6-month-old animals (11.3%). Theseroprevalence was higher in males (20.9%) than in females(12.1%). Among the four mithun strains, higher prevalencewas evident in Manipur (30.3%) than Arunachal (21.3%),Nagaland (11.7%), and Mizoram strain (10.2%). However,no BVDV genomic RNA could be detected. The results

provide first serological evidence of BVDV infection inmithun species and extend the knowledge on BVDV hostrange. The baseline data will help further investigations onepidemiology of BVD in mithun and its impact on mithunproduction.

Keywords Bovine viral diarrhea virus . Bos frontalis .

Mithun . Seroprevalence . India

Introduction

Pestiviruses cause significant economic losses to the livestockindustry worldwide. The genus Pestivirus belongs to the fam-ily Flaviviridae and comprises of four accepted species: bo-vine viral diarrhea virus 1 (BVDV-1), bovine viral diarrheavirus 2 (BVDV-2), border disease virus (BDV), classical swinefever virus (CSFV), and a tentative species, Giraffe (King et al.2011). Several emerging pestiviruses including recently pro-posed BVDV-3 (HoBi-like) viruses found in cattle await for-mal taxonomic approval. BVDV in cattle causes variety ofclinical syndromes including reproductive failure, abortion,still birth, respiratory disease, poor growth rate, diarrhea, ner-vous signs, and the fatal mucosal disease. Acute infection inimmunocompetent animals usually causes transient mild dis-ease followed by seroconversion, whereas infection of fetusbefore development of immune system leads to birth of per-sistently infected (PI) animals which remain the main sourcesof transmission throughout their life. However, in case of se-vere acute BVDV infection, severe clinical diseases with highmortalities have been reported (Corapi et al. 1989; Schirrmeier2013). The BVDV genome is about 12.3 kb long singlestranded positive sense RNA containing a single ORFencoding about 4000 amino acids flanked by untranslatedregions (UTR) at 5′ and 3′ ends (Meyers and Thiel 1996).

* Niranjan Mishramishranir@rediffmail.com

1 ICAR-National Research Centre on Mithun, Medziphema,Dimapur, Nagaland 797106, India

2 ICAR-National Institute of High Security Animal Diseases, AnandNagar, Bhopal, Madhya Pradesh 462022, India

3 ICAR-National Institute of Veterinary Epidemiology and DiseaseInformatics, Yelahanka, Bengaluru, Karnataka 560064, India

Trop Anim Health ProdDOI 10.1007/s11250-017-1310-z

Although cattle are the main sources of BVDV infection,BVDV infects a wide range of ruminants, both domesticatedand wild suggesting interspecies transmission (Becher et al.1997). Serological surveys in free-ranging and captive rumi-nants have detected antibodies against BVDV in more than 40species and mostly species within Bovidae and Cervidae(Hamblin and Hedger 1979; Van Campen et al. 2008; Wolffet al. 2016). Of bovids, cattle and buffaloes (Bubalus bubalis)are commonly infected with BVDV-1, BVDV-2, or BVDV-3whereas yaks (Bos grunniens) and canadian bison (Bison bi-son bison) have been found infected with BVDV-1 (Mishraet al. 2008a, b; Deregt et al. 2005). In addition, BVDV-1 hasrecently been detected in free-ranging mountain goat and big-horn sheep (Wolff et al. 2016).

The genus Bos consists of several species of wild and do-mestic cattle including mithun (Bos frontalis), gaur (Bosgaurus), and yak (Bos grunniens). The mithun is a uniquebovine species that lives in the North Eastern hilly region(NER) of India and also in adjoining areas of China,Myanmar, Bhutan, and Bangladesh. As per the latest livestockpopulation census data (2012), the mithun population in Indiais estimated at about 0.29 millions. Mithuns are reared mainlyfor meat and hide under free-ranging conditions in the forest atan altitude of 1000 to 3000 m above mean sea level in thestates of Arunachal Pradesh, Nagaland, Manipur, andMizoram and play an important role in the socioeconomicand cultural life of the local tribal population (Mondal et al.2010). Mithuns are kept mostly in community herds in fencedforest areas and thrive on natural green vegetation. AlthoughBVDVinfection has been reported from a variety of ruminantsother than cattle, there is a complete lack of information on theprevalence of BVDV infection in mithun species.

In India, BVDV is prevalent in cattle, buffaloes, sheep andgoats and all the three species of BVDV, BVDV-1, BVDV-2,and BVDV-3 have been detected (Mishra et al. 2004, 2008a,b, 2014). Moreover, yaks have been found infected withBVDV in Arunachal Pradesh state, in the NER (Mishra et al.2008a). Since mithuns play an important role for the livestocksector in the NER, and the ecosystem is favorable for inter-species transmission of BVDV, the aim of this study was todetermine the extent of BVDV infection in mithuns in theNER, India.

Materials and methods

Study area and sampling

The study was carried out in three locations (Champhai,Khuangleng, and Samthang villages) in Champhai district ofMizoram state, which is on the Indo-Myanmar border, fivelocations (NRCM Porba, Pholami, Mesulumi, Thipuzu, andPorba) within Phek district, Jotsoma and Khonoma villages in

Kohima district, NRCM Medziphema and Chumukedima inDimapur district and Xuvi village in Zunheboto district ofNagaland state, and Papumpare in Papumpere district ofArunachal Pradesh state in North Eastern hilly region (NER)of India. The geographical locations of the study sites arebetween 350 and 1852 m amsl and are shown in Table 1 andFig. 1. The mithuns at ICAR-National Research Centre onMithun (NRCM), Medziphema, and Porba in Nagaland statewere farm-reared under semi-intensive system of manage-ment, while at other sites, they were free-ranging in forestareas.

Blood and serum samples from mithuns (n = 466) wereaseptically collected from the jugular vein in sterilevacutainers (with and without K2 EDTA) using separateneedles and were shipped in ice to the ICAR-NationalInstitute of High Security Animal Diseases (NIHSAD),Bhopal within 3–4 days. Some of the samples were processedat ICAR-NRCM, Medziphema, and separated sera or buffycoats were stored at −70 °C before being sent in dry ice toNIHSAD, Bhopal. The samples were collected duringOctober 2013 to March 2016 from the free-ranging animalsrandomly, based on total number of animals available forbleeding, whereas from the farms, majority of the animalswere sampled. The animals were of 2 months to >6 years ofage and were apparently healthy or had a history of reproduc-tive problems, respiratory problems (coughing, nasal dis-charge, pneumonia), or diarrhea. These animals have neverbeen vaccinated against BVD. The sampled mithuns belongedto all the four strains found in India, Arunachal, Manipur,Mizoram, and Nagaland.

ELISA for detection of BVDVantibodies

All the sera samples from mithun (n = 466) were tested forpresence of BVDVantibodies using IDEXX BVDV total anti-body test kit (IDEXX, Liebefeld-Berne, Switzerland) accord-ing to the manufacturer’s protocol. The kit has a sensitivity of96.3% and specificity of 99.5% in detection of antibodiesagainst BVDV-1 and BVDV-2. The test was performed using1:5 serum dilution and 90 min incubation time of serum withthe coated antigen. The results were expressed as S/P values(sample to positive ratio) by calculating optical density (OD)values of test samples and corrected OD value of positive con-trol. Samples with S/P values >0.3 were considered positive forBVDVantibodies, while samples with S/P values of 0.20–0.29were considered as doubtful. The doubtful samples were testedagain. The samples which remained doubtful following repeattesting were not included in the seropositive data.

BVDV real-time RT-PCR

For detection of BVDV RNA, a TaqMan real-time RT-PCRassay (Hoffmann et al. 2006) with minor modifications (in

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thermal profile) was used. Extraction of viral RNAwas carriedout from leukocytes using RNeasy mini kit (Qiagen,Germany) following manufacturer’s protocols, and the RNAwas subjected to the pestivirus generic TaqMan assay using

LightCycler 480 (Roche, USA). The assay targeting the high-ly conserved 5′-UTR of pestivirus genome was conducted in25 μl reaction volume using the primers BVD190-F(Hoffmann et al. 2006), V326 (Vilcek et al. 1994), probe

Table 1 Geographic source of the samples collected form mithuns in the North Eastern Region, India and prevalence of BVDVantibodies by ELISA

S. no. State District Place/village Type of rearing No. tested No. Positive TP FN True prevalence (95% CI)

1 Nagaland Kohima Jotsoma Free range 24 1 1 0 6.9 (0.7–17.3)

Khonoma Free range 30 1 1 0 5.5 (0.5–14.1)

Phek Porba Free range 51 8 8 0 14.9 (5.4–25.4)

NRCM-Porba Semi-intensive 38 7 7 0 18.0 (6.4–30.5)

Pholami Free range 20 0 0 0 0 (0.3–14.4)

Mesulumi Free range 9 0 0 0 0 (0.6–27.3)

Thipuzu Free range 3 0 0 0 0 (1.5–53.8)

Dimapur NRCM, Medziphema Semi-intensive 140 42 42 2 29.1 (20.6–37.1)

Chumukedima Free range 32 5 5 0 15.6 (4.5–28.6)

Zunheboto Xuvi Free range 4 0 0 0 0 (1.2–46.3)

Sub Total 351 64 64 2 16.2 (9.3–21.5)

2 Mizoram Champhai Champhai Free range 6 0 0 0 0 (0.9–36.2)

Khuangleng Free range 43 1 1 0 3.9 (0.3–10)

Samthang Free range 30 4 4 0 13.6 (3.4–26.2)

Sub total 79 5 5 0 5.6 (0.9–11.6)

3 Arunachal Pradesh Papum pare Papumpare Free range 36 2 2 0 6.6 (0.9–15.4)

Sub total 36 2 2 0 6.6 (0.9–15.4)

Grand total 466 71 71 2 13.1 (6.9–17.8)

TP true positive, FN false negative

Fig. 1 Sampling locations of the study area

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TQ-Pesti (Gaede et al. 2005), 2 μl of RNA and SuperScript IIIPlatinum one-step real-time RT-PCR reagent set (Invitrogen,USA).

Statistical analysis

The true prevalence was estimated using Bayesian estimationof true prevalence from survey testing with one test imple-mented in epitools (Sergeant 2016). For estimating the trueprevalence prior estimate of 15% prevalence and a test sensi-tivity of 96.3% and test specificity of 99.5% was used keepingdefault number of iterations (25000) and discards.Significance was calculated by using online rxc contingencytable.

Results

The overall, state-wise and rearingmethod-wise true seroprev-alence of BVDVinfection inmithuns in NER, India are shownin Table 1. BVDV antibodies were detected in 71 of the 466mithun sera tested by ELISAwith a prevalence rate of 13.1%(95% confidence interval, CI: 6.9–17.8). Symptoms compat-ible with BVDV infection such as pyrexia, nasal discharge,pneumonia, abortion, or diarrhea were evident in 21 (29.5%)of the BVDV antibody positive animals. When compared atstate level, the prevalence rates varied from 5.6 to 16.2%,highest in Nagaland state and lowest in Mizoram state(Table 1). When looked at the rearing method prevailing, theoverall prevalence rate in farmed mithuns was 27.5%, while itwas 7.6% in free-ranging mithuns. The highest rate of preva-lence (29.1%) was found in farmed (semi-intensive) mithunsat NRCM, Medziphema, Dimapur district followed by 18.0%prevalence at NRCM, Porba, Phek district. Both of thesemithun farms belong to Nagaland state and they accountedfor approximately two thirds of the seropositive samples(69%) out of total 71 seropositive samples detected. Therewas wide variation in prevalence rates in free-rangingmithunsin various locations within three states, which ranged from 0to 15.6%. Although BVDV antibodies were detected in free-ranging mithuns in all the three states, highest prevalence(15.6%) was found at Chumukedima, Dimapur district,Nagaland, followed by 14.9% at Porba, Phek district,Nagaland, and 13.6% at Samthang, Champhai district,Mizoram (Table 1).

Investigations on prevalence of BVDV antibodies amongvarious age groups of both farmed and free-ranging mithunsshowed that prevalence rates increased with the age of animals(Table 2). Although not statistically significant, in ArunachalPradesh state, seroprevalence (12.2%) was found only in an-imals of 2–6 years age, whereas in Mizoram, although notrend is evident, the seroprevalence was significantly high in<6 months age group (18.6%) and >6 years age group (20%). T

able2

Age-w

iseBVDVseroprevalence

inmith

uns

ArunachalPradesh

Mizoram

Nagaland

<6months6months–

2years

2–6years

>6yearsTo

tal

<6months

6months–

2years

2–6years

>6years

Total

<6months

6months–

2years

2–6

years

>6years

Total

No.tested

110

205

3613

2133

1279

3074

177

70351

No.positive

00

20

22

01

25

39

3616

64Truepositive

00

20

22

01

25

39

3616

64Falsenegative0

00

00

00

00

00

01

12

Apparent

prevalence

00

100

5.56

15.38

03.03

16.67

6.33

1012.16

20.34

22.86

18.23

True prevalence

0(2.7–76.1)

0(0.6–25.4)

12.2(1.9–26.7)

0(1–40.5)

6.6(0.8–15.4)

18.6(3.7–38.4)

4.8(0.3–13.7)

5.1(0.5–12.9)

20(4.3–40.8)

5.6(0.9–22.8)

10.7(2.0–22.3)

10.8(3.0–18.9)

18.7(11.0–25.4)21.7(11.4–32.0)16.1(9.1–21.4)

P=0.683

P=0.072+

P=0.193

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Moreover, a positive association was found between age andseroprevalence and increasing prevalence with increasing agein Nagaland. The combined data showed that the prevalencewas higher in animals of >6 years of age group (20.6%) com-pared to the animals of 2–6 years age group (16.9%),6 months–2 years age group (8.5%), and <6months age group(11.3%).

The results (Table 3) showed that the overall BVDV sero-prevalence was higher in males (20.9%; 95% CI: 15.43–27.89) than in females (12.1%; 95% CI: 8.96–16.33). Butthe prevalence was higher in females than males in the statesof Arunachal Pradesh (8.3 and 0%) and Mizoram (6.1 and5.7%). When comparison was made between the four differ-ent of strains of mithun investigated (Table 4), the highestBVDV seroprevalence was evident in Manipur strain ofmithun (30.3%) followed by Arunachal (21.3%) andNagaland (11.7%), while the lowest prevalence was recordedin Mizoram strain (10.2%). However, testing of all the bloodleukocyte samples from mithuns (n = 466) by pestivirus ge-neric TaqMan real-time RT-PCR yielded negative results forBVDV genomic RNA.

Discussion

Mithun is a unique bovine species and the precious semi-wildspecies lives at high altitudes in the NER, India besidesBhutan, Bangladesh,Myanmar, and China. Although reportedin several species of bovine and non-bovid domestic and wild

animals, there is no information on BVDV infection inmithuns. This is the first report of serological evidence ofBVDV infection in mithun species which provides importantadvancement in extending the pestivirus host range.

While the role of cattle in BVDV epidemiology is wellunderstood (Houe 1999), the susceptibility of semi-wildmithuns to BVDV infection is not known. In this study, wedetermined BVDV seroprevalence in the NER, India, and theresults suggest that both the farmed and free-ranging mithunsare susceptible to BVDV infection. Since vaccination againstBVD has never been carried out in India, prevalence ofBVDV antibodies in mithuns is due to natural BVDV infec-tion. The IDEXX BVDV Total Ab ELISA used in this studyhas been used for BVDV seroprevalence studies in a variety ofdomestic and wild animals (Lanyon et al. 2013; Rodríguez-Prieto et al. 2016). Moreover, as claimed by the manufacturer,compared to the VNT, this kit has high sensitivity (96.3%) andspecificity (99.5%) for detecting BVDVantibodies in cattle.

The overall BVDV seroprevalence in mithuns in this studywas 13.1%, which approximated well with the previous re-ports on prevalence rates varying from 0 to 90% in cattle(Houe 1999), 0–50% in sheep (Nettleton et al. 1998) and 0–25% in goats (Loken 1995). The seroprevalence was lowerthan that reported in wild ruminants (60%) in Canada(Elazhary et al. 1981) but was higher than that reported infree-ranging wild ruminants (1.7%) in Switzerland(Casaubon et al. 2012). BVDVantibodies have been detectedin sera from several species of free-ranging bovids in Africa,North America, and Europe (Hamblin and Hedger 1979;

Table 3 Sex-wise BVDV seroprevalence in mithuns from NER, India

Arunachal Pradesh Mizoram Nagaland

Male Female Male Female Male Female

No. tested 7 29 16 63 139 212

No. tested positive 0 2 1 4 33 31

True positive 0 2 1 4 33 31

False negative 0 0 0 0 1 1

Apparent prevalence 0 6.9 6.25 6.35 23.74 14.62

True prevalence (95% CI) 0 (0.08–32.5) 8.3 (1.1–18.8) 5.7 (1.1–24.9) 6.0 (0.9–12.8) 22.3 (13.6–30.0) 12.6 (5.6–18.4)

Table 4 Prevalence of BVDVantibodies among four Indian strains of mithun (Bos frontalis)

Mithun strain No. tested No. tested positive True positive False negative Apparent prevalence True prevalence (95% CI)

Arunachal 63 14 14 1 22.22 21.3 (11.1–31.8)

Mizoram 109 13 13 0 11.93 10.2 (3.1–17.2)

Nagaland 277 39 39 1 14.08 11.7 (4.6–17.1)

Manipur 17 5 5 0 29.41 30.3 (12.4–50.2)

Total 466 71 71 3 15.24 13.1 (6.9–17.8)

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Depner et al. 1991; Marco et al. 2011). Although no compar-ison could be made on the BVDV serorevalence in mithunsfrom other countries due to lack of published data, when com-pared with bovids other than cattle, the prevalence rate inmithuns was lower than the rates of 29.5% reported in free-ranging European bison in Poland (Salwa et al. 2007), 31% infree-ranging American bison in Wyoming, USA (Taylor et al.1997), and 37.5% in yaks in North West China (Casaubonet al. 2012).

In addition, the seroprevalence rate in mithuns in thisstudy was also lower than that reported earlier in cattle(37.6%), buffaloes (30.76%), sheep (23.4%), and goats(16.9%) from India (Bhatia et al. 2008; Mishra et al.2009). This observation is however not unusual, sincethe mithuns are mostly reared in wild and existence of alow population density of cattle in the NER. The sero-prevalence rates varied widely among the geographicallocations within the three states with higher seropreva-lence found in farmed than in free-ranging mithuns.However, a moderate rate of seroprevalence was foundin free-ranging mithuns in a few locations in Nagalandand Mizoram states. The influence of farm managementpractices and regional variation in BVDV seroprevalencehas been reported in cattle (Graham et al. 2001).Seroprevalence against BVDV in unvaccinated cattle ishighly variable and is based on location, type of produc-tion, management practices, source of animals, age, sex,breed, contact with wild animals, and other environmentalfactors (Houe 1999). Although more detailed investiga-tions need to be done in future, the data obtained in thisstudy suggest that some of these factors might also havean important role in epidemiology of BVDV in mithunspecies.

In case of cattle, it has been shown that BVDV sero-prevalence status in various age groups indicates the in-fection status and serological testing of young animals ispreferable to identify infected herds (Houe 1999). We de-tected seroprevalence in mithuns of <6 months to >6 yearsof age and highest seroprevalence rates in mithuns of>6 years of age followed by 2–6 years age group andother two younger age groups which provide evidenceof infection at an early age besides indicating their expo-sure with BVDV for long. This is in agreement with theearlier reports in cattle (Lanyon et al. 2013), sheep andgoats, (Mishra et al. 2009) and red deer (Rodríguez-Prietoet al. 2016). When comparison was made among the threestates, a trend of increasing BVDV seroprevalence inmithuns with increasing age was seen in ArunachalPradesh, whereas seroprevalence was significantly highin both lower (<6 months) and higher age group (>6 years)in Mizoram and a positive association between age andseroprevalence, and increasing prevalence with increasingage was found in Nagaland. Nonetheless, further studies

are required to substantiate correlation between age andseroprevalence since the sampling size for mithuns in<6 months age group was limited. However, it is a factthat it is very difficult to collect large number of bloodsamples from mithuns in the wild due to inaccessibility oftheir natural habitats in the deep forests and the difficul-ties in restraining these heavily built animals.

In cattle, maternal antibodies against BVDVare detectablein unvaccinated calves up to about 6 months of age. Butwhether a similar situation exists in mithun is not known atpresent. It was unclear whether the prevalence of BVDV an-tibodies in <6-month-old mithuns found in this study was dueto the presence of maternal antibodies or was due to directexposure with BVDV. In any case, the results provide evi-dence of BVDV infection in mithun species both in youngand adults.

Sex-wise comparison of mithuns showed no statisticallysignificant difference between seroprevalence rates in malesand females but seroprevalence was higher in males than fe-males. However, further studies are required to ascertain it.This is in contrast to earlier studies in cattle where higherseroprevalence was found in cows than bulls (Daves et al.2016). One of the contributing factors may be that most ofthe farmers involved in dairy cattle production usually sell themale calves, while in case of mithun production, animals ofboth sexes are reared mostly for beef and hide. Another con-tributing factor may be differences in number of samples be-ing tested.

Of the four strains of mithuns found in India and studiedhere, BVDV seroprevalence was highest in Manipur strain(30.3%) than Arunachal (21.3%), Nagaland (11.7%), andMizoram (10.2%) strains. A significant association betweencattle breeds and BVDV seroprevalence has been reportedwith higher BVDV seroprevalence in Jersey and Holstein thanthe local breeds (Daves et al. 2016). Although the reasons forstrain dependent differences and responses to BVDVinfectioncan be ascertained in future, this study provides importantpreliminary insights on mithun strain susceptibility toBVDV infection.

A moderate number of farmed and wild mithuns werefound positive for BVDVantibodies by ELISA but no geno-mic RNA could be detected by real-time RT-PCR. This indi-cates absence of viraemia during the study period or exposureof animals with BVDV long before the study period, sinceBVDV antibodies are known to persist for long in cattle(Van Campen et al. 2008). However, concerted efforts shouldbe made in the future in detection and genetic typing ofBVDV strains circulating in mithun.

The serological test (IDEXX BVDV total antibodyELISA) used in this study for detection of BVDV anti-bodies has a high specificity to BVDV-1 and BVDV-2.Recently, in a field validation study involving Australiancattle, when compared with VNT, high specificity

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(97.1%) of this kit has been demonstrated (Lanyon et al.2013). However, cross-reactions with other pestivirusessuch as BVDV-3 (HoBi–like pestiviruses) and border dis-ease virus (BDV) can occur. But cross reactions with oth-er pestiviruses occur also with the VNT, the gold standardtest for serological diagnosis of BVD, where a range ofprevailing pestiviruses need to be used to determine thehomologous and heterologous neutralizing titer. SinceBDV rarely infects bovids and natural BVDV-3 infectionhas not been documented in bovids other than cattle andbuffaloes, cross-reactivity with other pestiviruses inmithun species is highly unlikely. However, the possibil-ity of cross-reactivity cannot be excluded completely.

Seroprevalence against cattle diseases such as infec-tious bovine rhinotracheitis (Rajkhowa et al. 2004) andbluetongue (Rajkhowa et al. 2008) has earlier been report-ed in mithuns. Although the sample size may not be truerepresentative of the target population, this is the firstepidemiological study in determining the prevalence ofBVDV antibodies in mithun species. There is a growingconcern all over the world with regard to transmission ofpathogens between wild and domestic animals and inter-species transmission is well documented for pestiviruses.But several studies suggest that BVDV can also be main-tained in wild animals without transmission from cattle(Elazhary et al. 1981; Frölich 1995). In India, BVDV isprevalent in cattle, buffaloes, sheep, and goats (Mishraet al. 2004, 2008b, 2014) besides yaks in the NorthEastern region (Mishra et al. 2008a). Since mithuns aremostly free ranging in their habitats and frequently comein contact with domestic and other wild ruminants duringgrazing and browsing, they may play a role in epidemiol-ogy of BVDV infection. The most probable mechanism ofBVDV infection in mithun is through contact with cattleduring grazing and browsing. Hence, to have a clear pic-ture on this more extensive serological and virologicalsurveillance studies are warranted in future.

In conclusion, the results of this study provide sero-logical evidence of BVDV infection in mithun speciesfor the first time and extend the knowledge on BVDVhost range. In addition, highest seroprevalence rate wasobserved in mithuns of >6 years of age, in males, inManipur strain and those found in semi-intensive rearingconditions in North Eastern Region, India. Further workis required to verify and investigate the role of mithunsin epidemiology of BVD and impact of BVDV onmithun production.

Acknowledgements N. Mishra, Vidya Singh, and D. Hemadri weresupported by a project grant (BT/392/NE/TBP/2012) from theDepartment of Biotechnology, Government of India. The authors thankthe Director, NIHSAD, Bhopal, and Director, NRCM, Medziphema,Nagaland for providing the infrastructure facilities and the persons in-volved during sampling.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict ofinterest.

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