Seroprevalence of Bluetongue virus in small and large ruminants in Punjab province, Pakistan

Accepted Manuscript Title: Seroprevalence of Bluetongue Virus in small and large ruminants in Punjab province, Pakistan Authors: Tayyebah Sohail, Tahir Yaqub, Tariq Abbas, Masood Rabbani, Jawad Nazir, Syeda Marriam Maqbool, Saima Yaqub, Momena Habib, Aziz-ul-Rahman, Nadia Mukhtar, Muhammad Shahbaz, Muhammad Yasir Zahoor, Muhammad Zubair Shabbir PII: DOI: Reference:

S0001-706X(18)30839-8 https://doi.org/10.1016/j.actatropica.2018.09.020 ACTROP 4793

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

3-7-2018 22-9-2018 22-9-2018

Please cite this article as: Sohail T, Yaqub T, Abbas T, Rabbani M, Nazir J, Maqbool SM, Yaqub S, Habib M, Aziz-ul-Rahman, Mukhtar N, Shahbaz M, Zahoor MY, Shabbir MZ, Seroprevalence of Bluetongue Virus in small and large ruminants in Punjab province, Pakistan, Acta Tropica (2018), https://doi.org/10.1016/j.actatropica.2018.09.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Seroprevalence of Bluetongue Virus in small and large ruminants in Punjab province, Pakistan Tayyebah Sohail1, Tahir Yaqub1, Tariq Abbas2, Masood Rabbani1, Jawad Nazir1, Syeda Marriam Maqbool1, Saima Yaqub1, Momena Habib1, Aziz-ul-Rahman1, Nadia Mukhtar3, Muhammad

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Shahbaz4, Muhammad Yasir Zahoor1, Muhammad Zubair Shabbir1#

Affiliation:1University of Veterinary and Animal Sciences, Lahore 54600, Pakistan; 2Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan; 3Institute of Public Health, Lahore 54600, Pakistan, 4The Women University of Azad Jammu and Kashmir, Bagh 12600, Pakistan. Corresponding author:

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Muhammad Zubair Shabbir

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E. mail: [email protected]

Abstract

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Bluetongue (BT) is a vector-borne disease of immense economic importance for small and large

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ruminants. Despite frequent disease reports from neighboring countries, a little is known about current disease status and prevalent serotypes in Pakistan. We screened a total of 1,312 healthy

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animals for group-specific antibodies and serotype-specific genome for BT virus through

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competitive ELISA and real-time PCR, respectively. An overall prevalence of group-specific VP7 antibodies [28.81% (n= 378/1312, 95% CI=26.4 – 31.4)] was observed. The prevalence was higher

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in goats [40.75% (n=194/476, 95% CI=36.4 – 45.3)] followed by buffalo [29.34% (n=81/276, 95% CI=24.3 – 34.9)], sheep [18.40% (n= 60/326, 95% CI=14.5 – 22.9)] and cattle [17.94% (n= 42/234, 95% CI= 13.56 – 23.4)]. The odds of seropositivity were more in buffalo of Nili breed (OR= 2.06, 95% CI= 1.19-3.58) as well as those found with a presence of vector (OR= 2.04, 95% CI= 1.16-3.59). Buffalo and cattle with history of abortion [(OR= 3.95, 95% CI= 1.33-11.69) and 1

(OR= 5.89, 95% CI= 1.80-19.27) respectively] were much likely to be infected with the disease. Serotype 8 was detected in all animal species while, serotypes 4 and 6 were detected in sheep, 2, 6 and 11 in goat, and 2 and 16 in buffalo. The study concludes a much frequent exposure of

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different serotypes of Bluetongue virus (BTV) in small and large ruminants and indicates its expansion to enzootic range worldwide.

Keywords: Bluetongue; Bluetongue virus; Punjab; Serotype; Small ruminants; large ruminants; Pakistan 1. Introduction

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Bluetongue (BT), caused by Bluetongue virus (BTV), is a vector-borne disease of small and large

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ruminants. Numerous species of Culicoides biting midges serve as a vector for transmission of

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virus from one susceptible host to another (Legisa et al., 2013). Sheep, goat, cattle, camels, llamas,

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deer and antelopes are major susceptible hosts. Clinical symptoms include erosions of oral mucosa, fever, salivation, oedema of face and lips, erosions in/around nostrils, apathy and

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tiredness, lameness, and dysphagia, conjunctivitis, coronitis, muscle necrosis, and stiffness in

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limbs. A total of 27 BTV serotypes are reported worldwide to-date (Maan et al., 2012; Jenckel et al., 2015). Clinical manifestation, however, varies among hosts and has been found to be much

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severe in sheep and some species of deer (Elbers et al., 2008). The disease is considered a

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“notifiable” due to direct (i.e. mortality, reduced milk yield, infertility and abortion) and indirect losses (i.e. international trade restrictions, costs of surveillance and mass vaccination, vector

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control and treatment) (Wilson and Mellor, 2009; Coetzee et al., 2012; OIE 2014). An annual loss of US$ 125 million has been estimated in US alone (Nicoletti et al., 2014). A varying prevalence of BTV has already been reported in neighboring countries of Pakistan. A seroprevalence of 26.66%, 31.25% and 52.27% has been evidenced in sheep, goat and cattle population originating from Orissa, India (Joardar et al., 2014). In a most recent survey, prevalence 2

of antibodies in sheep (20.3%) and yaks (13.3%) has been reported from Tibetan Plateau, China (Ma et al., 2017). Similarly, seroprevalance of 68.6 % in sheep, 71.8 % in goats and 48.6 % in cattle has been documented from Afghanistan (Ali et al., 2014). Seroconversion for BTV was

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evidenced in goat (67.7 %), sheep (6.57%) and cattle (2.69 %) originating from Iran (Mozaffari and Khalili, 2012; Noaman et al., 2013; Mozaffari et al., 2014). Transmission of BTV across disease endemic countries including Pakistan is possible either through active or passive flight of carrier vector (Culicoides spp.) (Saegerman et al., 2008) and cross-border movement of animals and animal’s products via Afghanistan, Iran (Raziq et al., 2010; Zahur et al., 2011) adjacent to

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Baluchistan and Khyber Pakhtun Khwa (KPK) province, and via India adjacent to Punjab

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province.

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Only a few studies have been done in Pakistan to assess an overall status of BTV in Pakistan

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(Akhtar et al., 1995; Akhtar et., 1997). We recently have reported serum based surveillance exploring BTV amongst sheep (44.21%) and goat (50.87%) in Balochistan (Sohail et al., 2018).

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These studies were limited to sheep and goats in Khyber Pakhtun Khwa (KPK) and Balochistan

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only while the socio-economically major area of Pakistan remained exclusively unexplored. Here,

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we assessed prevalence of VP7 group-specific antibodies against BTV and distribution of different serotypes among small and large ruminants originating from diverse agro-climatic region of the

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Punjab province, Pakistan.

2. Materials and methods

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2.1. Study area

The study was carried-out in select districts of Punjab province Pakistan. The province, located at 31.1704°N and 72.7097°E, is considered the homeland of five rivers that covers an area of 205,344 square kilometers. The province is bordered by the Indian states of Punjab and Rajasthan to the

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East, Kashmir to the North-East, the province of Sindh to the South, the province of Balochistan to the Southwest, the province of Khyber Pakhtunkhwa to the West, and the Islamabad Capital Territory to the North. Agriculture, livestock and their products are main source of livelihood for

livestock

sector

and

contributes

60%

of

total

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people particularly those that are living in rural settings. Indeed, the province dominates the population

in

the

country

(http://www.livestockpunjab.gov.pk/LiveStockAdmin/uploads/editor_files/livestock_census_pun jab_2018_nacel). The climate is predominantly sub-tropical with mild and relatively rainy winter followed by hot summer and short rainy period of monsoon. The average temperature varies from

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28-31ºC (northern to middle Punjab) to 31-33ºC (southern Punjab) (Khattak & Ali, 2015). Major

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parts of the province receive an annual rainfall less than 250 mm (Salma, Shah, & Rehman, 2012).

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Together, the agriculture system along with the climate conditions provides a favorable

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environment for the growth of midges and other mechanical vectors like ticks and mosquitoes. 2.2. Sample collection

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The archived sera (n = 1312) and data were collected from select districts of Punjab in 2013-2015

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under an international collaborative research project related to epidemiology of soil-borne zoonotic pathogens in Pakistan (Project ID: HDTRA1-10-1-0080). A multi-stage random

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sampling approach was used to select 1312 animals (476 goats, 326 sheep, 234 cattle and 276

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buffaloes) from small select villages where animal numbers in livestock barns ranged from 8-41. At the first stage, nine districts of the province were selected through purposive sampling

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approach. The select districts named Faisalabad, Chakwal, Attock, Gujranwala, Lahore, Dera Ghazi Khan, Sahiwal, Sargodha and Sheikhupura are main livestock rearing areas in the province, represent traditional agro-livestock mixed production system in the country, and had an increased annual incidence of human and animal diseases (Directorate of Animal and Human Health, Punjab). In the second stage, assuming 50% prevalence, 95% confidence interval, 0.4 intra-class coefficient, and 5% margin of error, a sample size of 357 villages was estimated from a total 4

number of villages (n=4883) in selected districts of Punjab province. Nevertheless, for more valid results, we randomly selected ten percent villages (n = 485) representing each of the study district (i.e., probability proportional to size sampling). The third stage involved convenient blood sampling of animals according to prior history of soil-borne pathogen detection, farmer’s consent,

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presence of adult animals at livestock barn, available resources and logistics. Given the frequent contact among animals while sharing water-canal, grazing fields and nature of disease transmission being vector-borne, a village was considered as a single large herd. In case, the owner was unavailable or unwilling to cooperate, we approached the nearest neighbor for sampling in an ethical manner. In order to ensure a better response from the farmers and its inhabitants, the

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sampling was carried out in close liaison with Livestock and Dairy Development Department of

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Punjab province and private veterinary practitioners, whichever resource was accessible in that

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particular region. A summary of the samples and herds from each district is given in Table 1. In

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addition, the different sampling locations are presented in Fig. 1. A brief history including age, gender, breed, parity, presence or absence of vector, history of abortion and herd size was collected

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and used for risk factor analysis.

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2.3. Serum analysis through cELISA

Competitive-ELISA (cELISA) was used to detect BTV group-specific IgG against VP7-protein as

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per manufacturer’s instruction (ID Screen® Bluetongue Competition, Grables, France). The kit’s reagents were equally suitable for detection of antibodies originating from small and large

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ruminants. Sample sera was added and incubated with antigen coated ELISA plate. After washing

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and with the addition of conjugate, conjugated antibodies showed color in the absence of anti-VP7 antibodies in test sera when incubated with respective substrate. The optical density (OD) values were measured at 450nm by iMark™ Microplate Absorbance Reader (BIO-RAD, California, USA). The signal-to-noise ratio (S/N % = ODSample /ODNC × 100) was calculated. A sample having ratio greater than or equal to 40% was considered negative, while less than 40% was considered 5

positive for any of the serotype of BTV. 2.4. Real-time PCR based genotyping A pool of 6-8 sera of each animal species from seropositive and seronegative herds was processed

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in equal numbers for real-time PCR based genotyping. The pooled samples (n = 30) included sheep (n = 9), goat (n = 11), cattle (n = 4) and buffalo (n = 6). The sample was subjected for RNA extraction (QIAamp® DSP Virus Spin Kit, QIAGEN, Hilden, Germany) followed by Real-time based screening for the prevalent serotypes. We used LSI VetMAXTM European BTV Typing Real Time PCR kit (Thermo-Fisher Scientific-LSI, Lissieu, France) which can individually detect eight

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serotypes (1, 2, 4, 6, 8, 9, 11 and 16) simultaneously from one sample aliquot in eight different

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reaction tubes. The kit has been reported to be highly sensitive and has high efficiency (95-100%).

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Determined by a series of dilution (usually 10-fold), the efficiency of an assay corresponds to

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constant increase in target amplicon per cycle during exponential phase. An ideal assay should have an efficiency value ranging from 90-100%, where one can have confidence on primer and

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probe specificity, reaction condition and pipetting error etc. The assay was performed in CFX96TM

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Real-time PCR Detection System (BioRad, California, USA) and Ct-values were recorded for

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interpretation as per manufacturer’s instruction. 2.5. Statistical analysis

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The data was compiled and a univariate analysis was carried out in IBM SPSS statistics for Windows version 21.0. (IBM SPSS Inc, Armonk, NY). Association between seropositivity and

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categorical predictor variables was determined by Pearson’s chi-square (χ2) test. Fisher’s Exact test was used as an alternate method when any assumption of the chi-square test was violated. All variables with p-value ≤ 0.2 (two-sided) in a univariate analysis were included in a mixed-effects logistic regression model with villages as random effect. The mixed model was used to account

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for clustering effect as BTV is a vector borne disease and frequent contact amongst animals within village is not unexpected. The regression model included serological status of animals (positive versus negative) as dependent variable. The independent fixed effects variables in the model were

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age, sex, breed, parity, presence of vector, history of abortion, and herd size depending on outcome of univariate analysis. For each explanatory variable, category with lowest prevalence was used as a reference. Any variable showing an association of ≤ 0.05 in regression analysis carried out in Rsoftware version 3.4.4 (lme4 package) was considered as statistically significant for the disease. The sampling sites and the study outcomes in terms of seropositive or seronegative herd and the

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distribution of BTV serotypes were mapped with Q GIS version 2.18.14.

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3. Results

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A total of 1,312 samples were processed for detection of group-specific (VP7 protein) antibodies

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against BTV from nine districts of Punjab province. Out of 117 total herds sampled, 71 were found positive for group-specific antibodies against BTV giving an overall herd prevalence of 60.68%

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(95% CI= 51.20-69.45) in the select geographical area of the province. A maximum herd-wise

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seroprevalence was found in DG Khan, Sahiwal, Chakwal, Attock i.e, 100% while least

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seroprevalence was detected in district Sheikhupura i.e, 30.0% (Table 1). An overall prevalence of 28.81% (95% CI= 26.39-31.36) was observed in both small and large ruminants. The level of

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seroconversion was more in goats [40.76% (n= 194/476, 95% CI= 36.33-45.34)] followed by buffalo [29.34% (n= 81/276, 95% CI= 24.12-35.16)], sheep [18.40% (n= 60/326, 95% CI= 14.43-

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23.13)], and cattle [17.94% (n= 42/234, 95% CI= 13.38-23.60)]. The seroprevalence varied with respect to species and districts. A district-wise summary of seroprevalence in each animal species is given in Table 2.

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Outcome of association between seropositivity and risk factors were determined by univariate analysis for small (Table 3) and large ruminants (Table 4), whereas the outcome of risk factors as definite disease determinants is given in Table 5. Following univariate analysis for small

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ruminants, though an initial association was observed for presence of vector in sheep, and herd size and age in goat (Table 3); the odds of seroconversion were found insignificant when further analyzed in multivariate logistic regression (Table 5). For cattle, the odds of seroconversion were five times more in animals with a history of abortion (OR= 5.89, 95% CI= 1.80-19.27, p=0.003). Age, breed and presence of vector, that initially showed an association with disease occurrence

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(Table 4), were found to be insignificant as disease determinants (Table 5). On the other hand, for

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buffaloes, the odds of seroconversion were more for Nili breed (OR= 2.06, 95% CI= 1.19-3.58, p

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= 0.010), animals with history of abortion (OR= 3.95, 95% CI= 1.33-11.69, p = 0.013) and those

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that had an exposure to vectors (OR= 2.04, 95% CI=1.16-3.59, p = 0.013) (Table 5). A village to village clustering effect was estimated by ICC value through mixed-effects logistic regression

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model (Table 5). Such a congregation effect within a cluster is used to group study farms/herds

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with similar production system and thereby, excluding any discriminatory effect of a particular

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geographical area or farm conditions comparable to any other rearing and production system in a country. A zero intra-class correlation coefficient (ICC) was observed for buffalo that showed a

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lack of clustering effect between villages or an absolute lack of correlation between the responses within the cluster. On the contrary, a highest clustering effect or ICC value was observed for sheep

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(0.547), followed by goat (0.452) and cattle (0.248) and, hence a higher reliability of responses within the cluster could be expected. Of the total samples processed for BTV genome detection, only 43.33% were found positive. The prevalence of BTV serotype specific genome was detected in 6 of 9 districts only (Table 1).

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Serotypes 2, 4, 6, 8 and 11 in small ruminants, while 2, 8 and 16 were found in large ruminants. Interestingly, serotype 8 was found exclusively in all positive districts and animal species indicating it to be the main serotype of BTV prevailing in the province. In addition, serotype 4 was

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found exclusively in sheep, serotype 11 was only found in goat while serotype 16 was found in cattle alone. The serotype distribution in different districts of Punjab along with the sampling sites is presented in Fig. 1. 4. Discussion

This is the first large-scale study on prevalence status of BTV group-specific antibodies and its

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circulating serotypes in the Punjab province of Pakistan. Different assays such as agar gel

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immunodiffusion (AGID), haemagglutination-inhibition (HI), complement fixation test (CFT),

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and serotype-specific serum neutralization (SN) test have been employed for serological evidence

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of BTV previously (Boulanger et al., 1967; Van der Walt, 1980; Herniman et al., 1983; Koumbati et al., 1999). However, these are not considered as methods of choice largely due to potential cross-

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reactivity with other arboviruses (Khezri and Azimi, 2013). We used cELISA that is highly

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sensitive, specific and has previously been validated by others (Vandenbussche et al., 2008). Since

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there is no vaccine being used for the control and management of BTV infection in the country (Pakistan), one can speculate presence of antibodies either as representative of current or previous

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exposure to virus. The presence of BTV was further confirmed by highly efficient, specific and sensitive real-time PCR for detection of genome corresponding to eight different serotype of BTV.

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Although varying, we observed seroconversion in all studied animal species. A greater proportion of small ruminants (31.79%) were seropositive than large ruminants (24.12%). The rate of seroconversion was higher in goats (40.76%) followed by buffalo (29.34%), sheep (18.40%), and cattle (17.94%). This is not surprising because we reported both an increased and varying rate of

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prevalence from another province, Balochistan indicating higher percentage of seropositive goats (50.87%) than sheep (44.21%) (Sohail et al., 2018). A seropositivity of 48.4% in sheep flocks has previously been documented from KPK province (Akhtar et al., 1997). Similar observations for

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BTV have also been reported from the neighboring countries. For instance, a prevalence of 85.3% in goat and 74.4% in sheep has been reported from Iran (Oryan et al., 2014). Another study from the same country evidenced a seroprevalence of 74.2% and 72.9% in goat and sheep, respectively (Mohammadi et. al., 2012). Joardar et al. (2013) reported a higher rate of seropositivity in sheep (58.82 %) than goat (31.79 %) from North-Eastern state of India. A study originating from West-

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Bengal (India) demonstrated a higher occurrence of seropositivity in goat (66.95%), followed by

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sheep (57.66%) and cattle (52%) (Panda et al., 2011). Another study from India (northern Kerala)

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revealed a prevalence of 16%, 7.5% and 6.9% in sheep, goat and cattle, respectively (Arun et al.,

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2014). Gaire et al. (2016) reported a seropositivity of 29.3% in dairy cattle in Nepal. Taken together, one can speculate that all the animals are equally susceptible to BTV. There may be

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differences in clinical form of disease where severity could be more often observed in one specie

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than others. For example, sheep is considered to be more vulnerable to infection and exhibit more

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severe form of infection even at same level of viremia in goat (Koumbati et al., 1999). Occurrence of BTV is considered to be subclinical in cattle (Arun et al., 2014), however it could serve as a

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source of infection through virus transmission to and from vectors to susceptible animals (Schwartz-Cornil et al., 2008). The outcomes revealed a higher ICC value for sheep followed by

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goat, cattle and buffalo. Considering cluster sampling and subsequent analysis, therefore, one can speculate a higher consistency for disease occurrence in small ruminants than large ruminants. Hence, to better elucidate clustering effect, a smaller sampling size for small ruminants than large ruminants may be considered enough to predict a reliable disease status in the region.

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The Nili breed of buffalo was found at a greater risk of BTV infection than any other study breed of small and large ruminants. A little or no impact of breed susceptibility to infection has previously been reported for small ruminants (Koumbati et al., 1999; Caporale et al., 2014). Both

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indigenous and exotic cattle breeds are found to be equally susceptible in a previous study (Adam et al., 2014). Although a significant difference in seroconversion was observed for type of animal species (Tibetan sheep vs yak), rate of difference in seroconversion within breeds of sheep and yak was found negligible (Ma et al., 2017). History of abortion was associated with an increased seropositivity in cattle and buffalo. A similar relationship between abortion and seropositvity has

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previously been documented (Akhtar et al., 1997), whereas Gaire et al. (2016) and Sabaghan et al.

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(2014) documented a lack of any association between abortion and BTV seropositivity. The

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presence of vector was also found to be a significant factor for BTV infection in buffalo. It is quite

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obvious for being a vector-borne disease of small and large ruminants where a remarkable association with presence of disease vector in animal vicinity has been reported (Bouwknegt et al.,

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2010; Khair et al., 2014). Though we did not find an association between animal age and disease

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occurrence, adult animals are reported to be more seropositive than the younger ones (Radostits et

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al., 2007; Adam et al., 2014; Khair et al., 2014) simply due to fact that chances of exposure increase with age. Numerous factors such as warm temperature, humid environment, transhumant animal

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movement, introduction of animal in a herd, rainfall and insect spray are reported to have a positive and negative association for animal seropositivity (Akhtar et al., 1997; Becker et al., 2003; Adam

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et al., 2014; Khair et al., 2014; Gao et al., 2017). Since collection of information pertaining to these factors require periodic farm visits or access to farmers, environmental variables, nationwide disease surveillance records, implementation of disease control strategy in terms of use of acaricide, we were not able to correlate study outcome with these factors and, therefore owing to

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these limitations, study outcomes and its subjective interpretation should be considered accordingly. Because determining the prevalent serotypes in a particular geographical setting provides an

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important insight towards disease epidemiology, its subsequent diagnostics and control strategies, we evidenced real-time PCR based prevalence of BTV serotype for the first time in Punjab province, Pakistan. More than one serotype was detected from different animal species where serotype 8 was the most dominant in all of the studied animals across the select districts of Punjab province. The occurrence of more than one serotype has been documented worldwide. For

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example, serotype 8 has been documented from different parts of the world such as Belgium and

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France; it caused a mortality of 5-10% of national livestock in Belgium (Saegerman et al., 2011),

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while a negligible clinical impact was reported from France (Sailleau et al., 2017). A total of 23

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serotypes have been reported from India (Sairaju et al., 2013). Despite non-virulent nature of serotype 21 worldwide, clinical cases were observed in goats (Susmitha et al., 2012; Rao et al.,

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2016). Taken together, an apparent variation in clinical nature of disease induced either by same

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and/or different serotypes within same geographical area could be ascribed to segmented BTV

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genome where reassortment among different serotypes could yield emergence of novel serotypes with varying clinical outcome (Shafiq et al., 2013; Caporale et al., 2014). Although we observed

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an increased prevalence of disease, clinical cases of BT are not much frequently reported unlike other diseases with similar clinical symptoms such as peste des petits ruminants virus. There could

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be many reasons that includes a relatively less-virulent nature of prevailing serotypes such as observed in Australia where BTV serotypes are either non-virulent or less virulent in nature leading to absence of clinical disease in the country (Kirkland, 2004), lack of routine diagnostics

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and disease surveillance, lack of differential diagnosis with other closely related diseases like vesicular stomatitis, goat pox, sheep pox, foot and mouth disease and pesti des petits. There were several study limitations. We used archived sera collected previously in another

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project. Therefore, sample number was not uniform and/or representative of animal population across select districts and a very little information was available to correlate with respect to BTV infection. Also we were not able to discriminate titers suggestive of either current or past infection because of lack of proper animal tracking system in the country where resampling from the same animal is not possible in another time. Sensitivity and specificity of cELISA has previously been

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evaluated for cattle and sheep sera only (Vandenbussche et al., 2008); nonetheless, we also used

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buffalo and goat sera. Test validity has neither been assessed for buffalo and goat sera previously

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anywhere nor available validity results are pertaining to Pakistan in particular; above reference

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study was conducted in Belgium. This is important because disease outbreaks have been reported from Europe and Belgium with BTV serotype 8 whereas, despite abundant prevalence in all animal

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species studied, there has been no such evidence of clinical disease in Pakistan so far. Though

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needs further investigation in subject matter, together it indicates potential variability in disease

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susceptibility to indigenous breed and thus, variation in prevalence rate may be expected despite using previously published sensitivity and specificity of cELISA assay. Considering real-time

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PCR, we were able to process only a limited number of pooled sera within available budget using a commercially available kit. Because the kit offers detection of 8 different serotypes in a sample

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when same genome is processed separately in 8 different reaction tubes, pooling of sera originating from a herd may not affect presence of more than one prevalent serotype in that particular sample. 5. Conclusion

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Bluetongue is prevalent in Punjab province. Though rate of prevalence is varying, both small and large ruminants are susceptible to infection. With an overall dominance of serotype 8 in study districts, more than one serotype (2, 4, 6, 8, 11 and 16) is circulating in the province. Further studies

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should be conducted to ascertain an overall disease status in the country to better elucidate disease situation and, later implement vector and disease control strategies in Pakistan. Conflict of interest No conflict of interest was declared by any author. Acknowledgement

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We thank team members involved in collection of samples under Defense Threat Reduction

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Agency (DTRA), USA funded project (Project ID: HDTRA1-10-1-0080). We also acknowledge

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Higher Education Commission of Pakistan in providing funds for kits and consumables under the

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project (ID: 20-3307/NRPU/R&D/HEC/13/674) funded to the University of Veterinary and Animal Sciences Pakistan. Furthermore, we are thankful to an English speaker “Unsa Memon”

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who did necessary edits wherever required.Ethical statement

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Blood sampling from sheep and goats was performed agreeing to the guidelines of the International

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Animal Care and Use Committee (IACUC). In addition, farmers consent was also obtained prior to sampling and collection of animal’s history. Sample collection and processing was also

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approved by Ethical Research Board at the University of Veterinary and Animal Sciences, Lahore, Pakistan (vide letter No. DR/236 dated May 16, 2013).

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References Adam, I.A., Abdalla, M.A., Mohamed, M.E., Aradaib, I.E., 2014. Prevalence of bluetongue virus infection and associated risk factors among cattle in North Kordufan State, Western Sudan. BMC Vet Res. 10, 94. Akhtar, S., Djallem, N., Shad, G., Thieme, O., 1997. Bluetongue virus seropositivity in sheep flocks in North West Frontier Province, Pakistan. Prev Vet Med. 29, 293-298. Akhtar, S., Howe, R. R., Jadoon, J. K., & Naqvi, M. A. (1995). Prevalence of five serotypes of bluetongue virus in a Rambouillet sheep flock in Pakistan. Vet Rec. 136(19), 495-495.

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17

Table. 1: Herd-wise seroprevalence and serotype distribution of BTV among different districts of Punjab. Seroprevalence

Serotype distribution

N o.

Districts

Herd s

Sam ples

% Positi prevalenc 95% CI ve e

1

Faisalaba d

25

252

15

60.00

2

Sahiwal

9

65

9

3

Chakwal

4

20

4

Sargodha

8

5

Attock

6

Goa t

Cat tle

Buffal o

38.89 78.19

6

11

8

8

100.0

62.88 100.0

8

-

-

-

4

100.0

0.000 04.61

56

3

37.50

10.24 74.11

-

-

4

29

4

100.0

29.88 98.95

4

8

Gujranwa la

15

157

11

7

Sheikhup ura

30

524

8

Lahore

20

9

Dera Ghazi Khan

2

Total

117

U

N

A

2, 8, 16

-

-

-

-

-

2, 6, 8

8

44.83 91.08

D

M

8

73.33 30.00

15.41 49.56

14

70.00

45.67 87.16

10

2

100.0

19.79 100.0

-

-

-

-

1312

71

60.68

51.20 69.45

--

--

--

--

TE

9

EP

199

A

CC

SC RI PT

She ep

18

8 -

Table. 2: Animal species-wise distribution and seroprevalence of sheep, goat, cattle and buffalo in Punjab province. Number of Type of Number of Animal level District animals that tested 95% CI animal animals tested Prevalence % positive 3

3

100.0

31.00 - 100.0

Sheep

5

5

100.0

46.29 - 100.0

Goat

21

21

100.0

80.76 - 100.0

Cattle

5

5

100.0

46.29 - 100.0

Buffalo

2

2

100.0

19.79 - 100.0

Sheep

4

2

50.00

09.19 - 90.81

Goat

9

7

77.78

40.19 - 96.05

Sheep

1

0

00.00

00.00

Goat

9

8

88.89

50.67 - 99.42

Cattle

54

10

18.52

09.70 - 31.87

Buffalo

59

28

47.46

34.48 - 60.77

Sheep

64

21

32.81

21.90 - 45.80

Goat

75

41

54.67

42.81 - 66.05

Cattle

18

1

5.56

00.29 - 29.38

Buffalo

56

19

33.93

22.16 - 47.91

Sheep

34

7

20.59

09.34 - 38.41

Goat

49

16

32.65

20.36 - 47.65

Cattle

48

6

12.50

05.19 - 25.94

Buffalo

59

12

20.34

11.39 - 33.20

Sheep

17

5

29.41

11.38 - 55.95

Goat

75

22

29.33

19.67 - 41.13

Sheep

32

8

25.00

12.13 - 43.75

Goat

33

32

96.97

82.49 - 99.84

Cattle

12

7

58.33

28.60 - 83.50

Buffalo

3

0

00.00

00.00

SC RI PT

Attock*

Cattle

N

A

Dera Ghazi Khan*

D

TE

EP CC

M

Faisalabad

Gujaranwala

U

Chakwal

A

Lahore

Sahiwal*

Sargodha

19

Sheep

6

1

16.67

00.88 - 63.52

Goat

35

10

28.57

15.24 - 46.52

Cattle

94

10

10.64

05.50 - 19.12

Buffalo

97

20

20.62

13.34 - 30.28

Sheep

163

11

06.75

03.59 - 12.06

Goat

170

37

SC RI PT

Sheikhupura 21.76

A

CC

EP

TE

D

M

A

N

U

* The districts from which no sample was available for one or two species.

20

15.96 - 28.87

Table. 3. Univariate analysis of anti-VP7 antibodies in small ruminants and their potential association to categorical variables. Animal

Variable

Levels

ELISA positive

Total tested

Seropositivit y (%)

Young

33

195

16.92

Adult

27

131

20.61

Female

45

260

17.31

Male

15

66

22.73

Buchi

19

76

25.00

Kajli

15

97

15.46

Lohi

26

153

16.99

Nulliparou s

47

259

18.15

Breed

Sheep

U

Sex

Parity 13

Presence of vector

No

18

Yes

42

249

16.87

History of abortion

No

56

309

18.12

4

17

23.53

Herd/flock size

<20

46

256

17.97

>20

14

70

20.00

Young

99

278

35.60

Adult

95

198

48.00

Female

160

397

40.30

Male

34

79

43.00

Beetal

68

141

48.20

Kajli

90

233

38.60

Nachi

25

71

35.20

Teddy

0

1

00.00

Teddy

11

30

36.70

EP

A 77

M

D

TE

Yes

67

21

CC

A

1

0.400

1.030

1

0.310

2.963

2

0.227

0.056

1

0.813

1.659

1

0.198

0.314

1

0.575

0.151

1

0.698

7.326

1

0.007

0.204

1

0.651

5.497

4

0.240

23.38

Sex

Breeds

0.710

19.40

Age

Goats

pvalue

N

Multiparou s

df

SC RI PT

Age

χ2

Nulliparou s

149

378

39.40

Multiparou s

45

98

45.90

Presence of vector

No

136

340

40.00

Yes

58

136

42.60

History of abortion

No

188

463

40.60

Yes

6

13

46.20

Herd/flock size

<20

108

291

37.10

>20

86

185

46.50

1.362

1

0.243

0.282

1

0.595

SC RI PT

Parity

0.161

1

0.688

4.115

1

0.043

* Presence of vector in sheep while, herd size and age in goat were found to be significant in goat in univariate analysis.

Levels

ELISA positive

Young

22

Adult

59

Female

67

32.35

208

28.37

214

31.31

14

62

22.58

38

97

39.18

Nili-Ravi

43

179

24.02

Nulliparou s

34

128

26.56

Multiparou s

47

148

31.76

Presence of vector

No

26

123

21.14

Yes

55

153

35.95

History of abortion

No

71

260

27.31

Yes

10

16

62.50

Herd/floc k size

<20

63

207

30.43

>20

18

69

26.09

Male

EP

TE

Nili

Breed

M

D

Sex

Parity

CC A

Seropositivit y (%)

68

Age

Buffalo

Total tested

N

Variable

A

Animal

U

Table. 4. Univariate analysis of anti-VP7 antibodies in large ruminants and their potential association to categorical variables.

22

χ2

df

pvalue

0.393

1

0.531

1.766

1

0.184

6.966

1

0.008

0.893

1

0.345

7.212

1

0.007

9.003

1

0.003

0.472

1

0.492

Young

18

131

13.74

Adult

24

103

23.30

Female

32

184

17.39

Male

10

50

20.00

Cholistani

16

65

24.62

Dajal

6

31

19.35

Rajan

6

33

18.18

Red Sindhi

7

31

22.58

Sahiwal

7

74

09.46

Nulliparou s

30

171

17.54

Age

Breed

Cattle

25

116

Yes

17

No

33

N

No

118

215

0.182

1

0.670

6.077

4

0.193

0.071

1

0.790

2.028

1

0.154

12.153

1

0.000

19.05

A

63

D

History of abortion

12

M

Presence of vector

Multiparou s

0.059

U

Parity

1

SC RI PT

Sex

3.579

21.55 14.41

15.35

A

CC

EP

TE

* Breed, history of abortion, presence of vector and gender in buffalo while breed, history of abortion, presence of vector and age were found to be significant in univariate analysis.

23

Table. 5. Multivariable mixed effects logistic regression model (p < 0.05) for BTV serological status amongst ruminants originating from traditional farms/villages in Punjab province (2013-15) Animal

Variable

OR

95% CI

Std. Error

p-value

00.16

00.08 - 00.33

00.06

<0.001

Breed (Nili)

--

02.06

01.19 - 03.58

00.58

0.010

History of abortion(Yes)

--

03.95

01.33 -11.69

02.19

0.013

Presence of vector (Yes)

--

02.04

01.16 - 03.59

00.59

0.013

Sex (Female)

--

01.24

00.62 - 02.48

00.44

0.537

Variance

00.00

--

--

--

ICC

00.00

--

U

--

--

--

--

(Intercept)

--

00.06

00.02 - 0.18

00.03

<0.001

Breed (Cholistani)

--

03.43

00.89 - 13.25

02.37

0.073

--

02.27

00.45 - 11.45

01.88

0.319

--

02.20

00.45 - 10.92

01.80

0.333

Breed (Red Sindhi)

--

03.92

00.62 - 24.95

03.70

0.148

History of abortion (Yes)

--

05.89

01.80 - 19.27

03.56

0.003

Presence of vector (No)

--

01.29

00.46 - 03.65

00.68

0.629

Age group (Adult)

--

00.90

00.29 - 02.81

00.52

0.858

Variance

1.086

--

--

--

--

ICC

0.248

--

--

--

--

SC RI PT

--

M

Buffalo

(Intercept)

EP

Fixed Parts

TE

Breed (Rajan)

D

Breed (Dajal)

A

CC

Cattle

Sheep

A

Fixed Parts

N

Random Parts

Random Parts

Fixed Parts

24

(Intercept)

--

00.12

00.04 - 00.38

00.07

<0.001

Presence of vector(Yes)

--

01.26

00.42 - 03.75

00.70

0.681

Variance

3.977

ICC

0.547

SC RI PT

Random Parts

Goat

(Intercept)

--

00.54

00.30 - 00.98

00.16

0.043

Flock size (>20)

--

01.11

00.51 - 02.40

00.44

0.790

Age group(Adult)

--

01.12

00.54 - 02.30

00.41

0.759

Variance

2.712

--

U

Fixed Parts

--

--

--

ICC

0.452

--

--

--

--

A

N

Random Parts

M

Figure legend:

A

CC

EP

TE

D

Fig. 1: Geographical distribution pattern of sites for blood sampling and identified serotypes across selected districts of Punjab province, Pakistan

25

D

TE

EP

CC

A

SC RI PT

U

N

A

M

Figr-1

26