Research Article

Seroprevalence of Bluetongue Disease among Domestic Ruminants Raised in International Border Areas of Nepal

Bikash Puri1, Anil K. Tiwary2, Bharata Regmi3,4, Dinesh K. Singh1, Doj R. Khanal5 and Manoj K. Shah3*

1Department of Veterinary Pathology, Institute of Agriculture and Animal Science, Tribhuvan University, Rupandehi, Bhairahawa, Nepal; 2Department of Anatomy, Physiology and Biochemistry, Faculty of Animal Science, Veterinary Science and Fisheries, Agriculture and Forestry University, Rampur, Chitwan, Nepal; 3Department of Surgery and Pharmacology, Faculty of Animal Science, Veterinary Science and Fisheries, Agriculture and Forestry University, Rampur, Chitwan, Nepal; 4Government of Nepal, Department of Livestock Services, Veterinary Laboratory, Pokhara; 5National Animal Health Research Centre (formerly AHRD), Nepal Agricultural Research Council, Lalitpur, Nepal.

Received | April 20, 2021; Accepted | October 13, 2021; Published | February 25, 2022

*Correspondence | Manoj K. Shah, Department of Surgery and Pharmacology, Faculty of Animal Science, Veterinary Science and Fisheries, Agriculture and Forestry University, Rampur, Chitwan, Nepal; Email: mkshah@afu.edu.np

Citation | Puri, B., A.K. Tiwary, B. Regmi, D.K. Singh, D.R. Khanal and M.K. Shah. 2022. Seroprevalence of bluetongue disease among domestic ruminants raised in International Border Areas of Nepal. Sarhad Journal of Agriculture, 38(2): 555-562.

DOI | https://dx.doi.org/10.17582/journal.sja/2022/38.2.555.562

Keywords | Antibodies, Bluetongue, Disease, Ruminants, Seroprevalence

Introduction

Bluetongue disease is an emerging, economically important vector-borne viral disease of domestic and wild ruminants. It is caused by the Bluetongue virus (BTV) belonging to the Orbivirus Genus of the Reoviridae family (Mozaffari and Khalili, 2012). So far, 26 distinct serotypes of BTV have been identified (Khair et al., 2014). BTV infects animals throughout the world, lying between latitude 40oN and 35oS. However, the virus has spread far beyond the aforementioned range (Sperlova and Zendulkova, 2011). The prevalence of bluetongue disease correlates with the distribution of competent culicoides vectors and appropriate climatic conditions (Raut et al., 2013). Bluetongue disease has been included in “List A” disease of Office of International Epizootic (OIE) due to its enormous potential for rapid spread and substantial economic losses arising from poor weight gain, production loss, increase in reproductive problems and subsequent death of the infected animals. Moreover, the international trade of infected animals and its product is banned (Najarnezhad and Rajae, 2013).

Nepal is sustaining around 0.8 million sheep and 9 million goats, which together contribute to 25% of total meat production and 582 metric ton wool productions, whereas, 30% milk comes from 7.2 million cattle and 70% of milk comes from 5 million buffaloes (MoAC, 2011). Almost every household raises livestock and its products are valuable sources of protein and the main source of cash income for livestock producers in Nepal. Thus, the outbreak of bluetongue disease may deteriorate the human health and results in serious socio-economic effect.

The bluetongue is considered as an important Transboundary Animal Diseases (TADs) in Nepal but there is an unrestricted movement of livestock from the neighboring countries, i.e., India and China, where bluetongue disease is endemic. So, the disease may enter Nepal along with imported animals. Because of the high density of the vector, the disease spreads rapidly. Moreover, Nepal being a member country of WTO, it has to follow the sanitary and phytosanitary (SPS) measures and regulations on technical barriers of trade. Therefore, the control of this BTV infection is a necessity in Nepal, given a large number of animals and their importance in the national economy. Thus, to unravel the prevalence of bluetongue disease in ruminants, random serosurveillance is an utmost in the particular region. The present study was mainly aimed to detect antibodies against BTV in ruminants raised in international border areas of Nepal.

Materials and Methods

Site selection

The study was conducted in the Darchula district, the remote mountainous district of Far Western Nepal, expanding between latitude 29o01’ to 30o15’ N and Longitude 80o03’ to 810o09’ E. This district has a diverse geographical variation with altitude varying from 357 to 7132 meters high from the sea level and has subtropical to temperate climatic conditions. It shares a national border with Bhajangonon the east, Baitadi on the south, and international border with Uttarakhand (India) and Taklakot (China) on the west and north, respectively. The study was conducted in September, 2013 in 6 villages (Shankarpur, Mallikarjun, Gokuleshwor, Sikhar, Latinath and Bulgar) of Darchula District (Figure 1).

Sample design

A two-stage sampling survey was performed in the present study. The villages, taken as the primary sampling unit, were selected on a random basis. At the second stage, a fixed proportion of animals were selected from the population using simple random sampling. The sample size was calculated using a computer program which uses two-stage prevalence surveys as described earlier (Cameron, 1999). The questionnaire was prepared and a field visit was made to the research site. Information related to age, gender, breed, presence or absence of vector and history of abortion were collected and recorded from the owner in an interactive manner. Before visiting the site, consent was obtained from the Department of Livestock, District livestock service office, Darchula regarding the intention to conduct study in the area.

Sample collection and testing

Approximately, 5 ml of blood samples were collected aseptically by jugular vein puncture. The serum was separated after 3-4 hours of clotting in slanting position at sites and preserved in the icebox. Afterwards, it was dispatched to the laboratory, where further refineries of sera were carried out by centrifugation at 2000 rpm for 15 minutes. The separated serum samples were stored properly at -20˚C until testing. Competitive ELISA test kit (ID-Vet, Montpellier, France) was used for detecting the antibody to BTV in sera. The serological test was carried out according to the manufacturer’s guidelines and results were reported positive or negative based on optical density reading (450 nm) when compared with the positive and negative control.

Statistical analysis

The data related to age, gender, breed, presence or absence of vectors and history of abortion were entered into Excel. Firstly, associations between the BTV infection and its potential risk factors were calculated using Pearson’s Chi-square test (PH STAT 2) and Chi-squared with Yates’ correction was used when at least 20% of expected frequencies are less than five. Fisher’s exact test was used for the estimation of the level of significance and association of that factor was expressed as highly significant, significant and non-significant based on the p-value being less than 0.01, between 0.05 and 0.01 and greater than 0.05 respectively.

 

Results and Discussion

Seroprevalence of BTV antibody among domestic ruminants

Overall, the seroprevalence of bluetongue was 41.8% among ruminants. Out of 220 serra samples tested for antibody to BTV (Table 1), 92 samples were found positive for BTV comprising 37.5% (39) cattles, 58.3% (21) buffaloes, 40% (12) goats and 40% (20) sheep. Furthermore, the seropositivity was highest in buffaloes (58.3%) but no significant association (p>0.05) was noted among species. Such a high seroprevalence might be due to the huge vector population following monsoon. However, an earlier study of Gaire (2013) reported 28.16% seroprevalence of BTV in sheep, goat and cattle in two bio-diverse landscapes of Nepal. Similarly, Jha et al. (2008) examined 401 sera samples of sheep from 11 districts using c-ELISA and reported 28.4% seropositivity for BTV antibodies. As per our knowledge, this was the first seroprevalence survey conducted in buffalo from Nepal to detect the antibody against the BTV. The result showed 58.9% seropositivity against BTV antibodies in the buffalo population, which was in agreement with earlier researchers from India who reported 58.33% (Chauhan et al., 2004) and 60.12% (Patel et al., 2007), seropositivity of BTV. This higher seroprevalence in buffalo might be due to greater exposure to the vector and their larger body surface area as compared to sheep and goat. Moreover, the skin of buffalo is quite softer while cattle have tough skin making culicoides difficult to suck blood. Further, previous exposure to the virus may also produce a more marked antibody response (Uhaa et al., 1990). 

Similarly, 40% (12) goats were seropositive to BTV antibodies (Table 1) which were in agreement with the result of Sreenivasulu et al. (2004) who reported 43.6% seropositivity in goat in Andhra Pradesh and Shlash et al. (2012) observed 39.47% positivity in goats in Iraq. The greater prevalence of bluetongue in goats, i.e., 58.01% and 54.5% has also been reported from Goa (Barbuddhe et al., 2005) and Uttar Pradesh (Bitew et al., 2013), respectively of India. This higher seroprevalence in small ruminants may reflect their involvement in basic ecology of the virus (Sreenivasulu et al., 2004). Further, the goat was assumed to be infected with serotypes of the virus which causes disease without overt clinical signs (Chand et al., 2009).

 

Table 1: Seroprevalence of BTV antibody in domestic ruminants.

Category	Risk factors	Total samples	Positive (%)	df	χ2	p- value
Species	Cattle	104	39(37.5%)	3	4.941	0.1761
	Buffalo	36	21(58.3%)
	Goat	30	12(40%)
	Sheep	50	20(40%)
VDCs	Shankarpur	40	7(17.5%)	5	19.291a	**p= 0.0017
	Malikarjun	31	16(51.6%)
	Gokuleshwor	21	14(66.6%)
	Shikhar	36	18(50%)
	Latinath	32	16(50%)
	Guljar	60	21(35%)

a,b Indicate significant differences (p<0.05) between the means of the groups. * and **= significance level at p<0.05 and p<0.01 respectively, NS= not significant. Figures in parenthesis are percentages.

 

In this study, the c-ELISA test exhibited BTV seropositivity in 40% of sheep. However, Jha et al. (2008) reported the prevalence rate of 28.4% in a large population of sheep in Nepal. Similarly, Yadav (2012) and Gaire (2013) reported 19.3 % and 25%, respectively in sheep. The seropositivity rate seems quite higher in the present study than those reported previously in Nepal. However, the higher seroprevalence of BTV was reported in sheep as 58.82% in Assam (Joardar et al., 2013) and 57.66% in West Bengal (Panda et al., 2001) of India. The both higher as well as lower prevalence rate of BTV antibodies was detected in sheep populations of several countries. Specifically, it was reported as 54.10% in Saudi Arabia (Yousef et al., 2012), 48.8% in Pakistan (Akhtar et al., 1997), 45.7% in India (Sreenivasulu et al., 2004), 29.59% in Southeast Turkey (Gur, 2008) and 21.40% in Kazakhstan (Lundervold et al., 2003).

The present study showed lower seropositivity in cattle (37.5%) than those of sheep (40%) and goats (40%). Similar findings have been reported in earlier study, i.e., in cattle (33.4%) and sheep (45.71%) conducted in Indian subcontinents (Sreenivasulu et al., 2004). The prevalence rate of blue tongue disease observed in the present study was remarkably higher than that observed as 29.32% in Chitwan and Lamjung (Gaire, 2013). These discrepancies in rates might be due to spatial and temporal variation. On the contrary, the findings of the present study were notably lower than the earlier prevalence rates in cattle, i.e., 70% in Assam (Joardar et al., 2013) and 58.82% in Punjab (Oberoi et al., 1988) state of India. Similarly, earlier researchers reported 93.5%, 88% and 84.57% of cattle sera positive for BTV antibodies in Iran (Jafari-Shoorijeh et al., 2010), Turkey (Gur, 2008) and Khartoum and Gezira state of Sudan (Elhassan et al., 2014), respectively. Cattle are considered as the main amplifying host and thereby play a vital role in the maintenance of viruses in nature (Khezri and Bakhshes, 2014).

However, the sera samples from different location of the Darchula showed seropositivity of Shankarpur (17.5%), Mallikarjun (51.61%), Gokuleshwor (66.67%), Shikhar (50%), Latinath (50%) and Guljar (35%). Overall, samples from Gokuleshwor areas showed a higher seroprevalence for BTV (Table 1). The result showed a remarkable difference (p<0.01) in the seroprevalence of BTV antibodies at different locations of the study areas. This might be due to diverse climatic condition of Darchula including hot and humid temperature following monsoon that favours for the vector growth and activity (Kumar et al., 2018). Moreover, the district share open boarder with India and China, where several outbreaks of bluetongue disease have been reported (Joardar et al., 2013; Bitew et al., 2013; Zhang et al., 2004). Hence, the outbreak of disease in one country increases probability of disease transmission into another country. Similarly, poor husbandry practice and transhumance system of rearing in high hills might also contribute for higher prevalence of circulating BTV antibodies among ruminants in Darchula District.

Age wise seroprevalence of BTV antibodies

The age-wise seroprevalence of BTV antibodies has been listed in Table 2. The seroprevalence of antibodies to BTV among different age groups of ruminants revealed higher seropositivity in older (49.12%) age (greater than 6 months) as compared to younger (16.32%) ages (less than 6 months). Interestingly, age-wise differences were observed only in cattles and goats. Higher prevalence of BTV antibodies was

 

Table 2: Age-wise seroprevalence of BTV antibody.

Species	Age	Animal tested	Positive (%)	df	χ2	p-value
Cattle	Younger	24	4 (16.66%)	1	Fisher’s Exact test	* p=0.02
	Older	80	35 (43.75%)
Buffalo	Younger	7	2 (28.57%)	1	Fisher’s Exact test	NS p=0.10
	Older	29	19 (31.03%)
Goat	Younger	7	0	1	Fisher’s Exact test	* p=0.02
	Older	23	12 (52.17%)
Sheep	Younger	11	2 (18.18%)	1	Fisher’s Exact test	NS p=0.16
	Older	39	18 (37%)
Total	Younger	49	8 (16.32%)	1	16.837a	*** P=0.000030
	Older	171	84 (49.12%)

a,b Indicate significant differences (p<0.05) between the means of the groups. * and **= significance level at p<0.05, p<0.01 and p<0.001 respectively, NS= not significant. Figures in parenthesis are percentages.

 

Table 3: Sex wise seroprevalence of BTV antibody in cattle, buffalo, goat and sheep.

Species	Sex	Animal tested	Positive (%)	df	χ2	p-value
Cattle	Male	30	9 (30%)	1	Fisher’s Exact test	NS P=0.37
	Female	74	30 (40.5%)
Buffalo	Male	14	3 (21%)	1	Fisher’s Exact test	** p=0.0005
	Female	22	18 (81.8%)
Goat	Male	9	2 (22.2%)	1	Fisher’s Exact test	NS p=0.24
	Female	21	10 (47.6%)
Sheep	Male	14	4 (28.6%)	1	Fisher’s Exact test	NS p=0.35
	Female	36	16 (44.4%)
Total	Male	67	18 (26.86%)	1	8.853a	** P=0.0030
	Female	153	74 (48.36%)

a,b Indicate significant differences (p<0.05) between the means of the groups. * and **= significance level at p<0.05 and p<0.01 respectively, NS= not significant. Figures in parenthesis are percentages.

 

found in older ruminants (49.12%). This may correlate with increased duration of exposure, rather than increased age susceptibility to infection per species (Ward et al., 1994). The older animals are more exposed to the midges because of their larger total body surface area (Uhaa et al., 1990). Besides, some culicoides have more affinity towards ruminant derived carbon dioxide production which greatly fluctuates during their life (Kline et al., 1994). Normally, young animals are kept indoors and are well-taken care by the owner until 6 months of age. Afterwards, they are released into pasture for grazing, where vector exposure and subsequent BTV infection is high. The findings of the present study are consistent with the previous findings (Mohammadi et al., 2012; Uhaa et al., 1990; Ward et al., 1994; Lundervold et al., 2003; Gaire, 2013; Khair et al., 2014).

Sex wise seroprevalence of BTV antibodies

The seroprevalence of BTV antibody varied (p<0.01) among various sex groups (Table 3). It showed higher seropositivity in females (48.36%) than males (26.86%) The sex-wise difference was observed only in buffaloes, with higher seroprevalence in females (81.8%) than males (21%). This study also revealed the sex wise differences in prevalence rate of BTV. Sex was highly significant (p=0.002) risk factor and females were more prone to BTV infection compared to males. The reason might be due to bias in the sample size as more female ruminants were included in present study as similar to earlier study (Elhassan et al., 2014). In agreement with the present finding, Yadav (2012) also found higher seroprevalence in females (15%) than males (9.8%) in Lalitpur district. Likewise, Patel et al. (2007) reported 65.14%

 

Table 4: Relationship with abortion history and BTV antibody in ruminants.

Species	Abortion	Animal tested	Positive (%)	df	χ2	p-value
Cattle	Yes	2	0	1	Fisher’s Exact test	NS p=0.52
	No	102	39 (38.23%)
Buffalo	Yes	1	0	1	Fisher’s Exact test	NS p=0.41
	No	35	21 (60%)
Goat	Yes	7	3 (42.85%)	1	Fisher’s Exact test	NS p=1
	No	23	9 (31.13%)
Sheep	Yes	15	9 (60%)	1	Fisher’s Exact test	NS P=0.11
	No	35	11 (31.42%)
Overall	Yes	25	12 (48%)	1	0.443a	NS P=0.524
	No	195	80 (41.02%)

a,b Indicate significant differences (p<0.05) between the means of the groups. * and **= significance level at p<0.05 and p<0.01 respectively, NS= not significant. Figures in parenthesis are percentages.

 

and 51.56% seropositivity in females and males, respectively by c-ELISA in Gujrat. Also, Elhassan et al. (2014) recorded higher seroprevalence in females (74%) than males (9.8%). However, Gaire (2013) reported higher seropositivity in males (31.31%) than females (27.27%), indicating both males and females are equally susceptible to BTV infection. 

Association of BTV seroprevalence with abortion history

The seropositivity of BTV with and without abortion history was 48% and 41.02% respectively. However, no significant differences (p>0.05) in BTV antibodies were found among ruminants with or without a history of abortion (Table 4). Regarding abortion history, 48% and 41.02% seropositivity was found in ruminants with and without abortion history, respectively. But, no significant association (p>0.05) was observed between prevalence rate and abortion because some serotypes cause subclinical infection or a prolonged period of past endemic infection (Mohammadi et al., 2012). However, the previous study conducted in Chitwan and Lamjung district of Nepal showed higher (65.75%) seropositivity of BTV in animals with abortion history (Gaire, 2013). The other factors may be involved for abortion in animals. Elhassan et al. (2014) described that different viral and protozoan diseases causes abortion among cattle in Sudan. 

Conclusions and Recommendations

Vaccination against bluetongue was not in practice hence antibody detected in ruminants of international border areas of Nepal might be from natural infection. Interestingly, no clinical case of bluetongue has been reported from the study area till date indicates there no absence of disease but necessitates the intensive surveillance with improved diagnostic technique. Further study is also required to characterize the serotypes of specific viruses and their vector present in the study area.

Acknowledgements

This study was financially supported by the fund from the Zoonoses Control Project/Nepal Agricultural Research Council (NARC) and the National Animal Science Research Institute, Kumaltar, Lalitpur provided the cELISA kit to carry out this research. The livestock farmers, technical personnel from DLSO, Darchula and Animal Health Research Division, Khumaltar are highly acknowledged for their cooperation and support in this study.

Novelty Statement

The present study detected antibodies against BTV for the first time in ruminants raised in international bordered areas of Nepal. The findings may be useful to plan the prevention strategies of bluetongue diseases and likely to be of great interest to the related researchers, clinicians and epidemiologists who read this popular journal.

Authors’ Contribution

Bikash Puri: Designed the study, collected and processed samples, analysed data and wrote the manuscript.

Anil Kumar Tiwary: Analysed the data and r eviewed the manuscript.

Bharata Regmi: Processed the samples.

Dinesh Kumar Singh: Designed and supervised the research.

Doj Raj Khanal: Designed and supervised the laboratory work.

Manoj Kumar Shah: Revised the manuscript.

Conflict of interest

The authors declare that there is no conflict of interest with this research.

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