Epidemiology of selected pig viral diseases in Bhutan...Eastern part of the Himalayan range between latitudes 26°45' N and 28° 10' N, and longitudes 88°45' E and 92°10' E. It is

Epidemiology of selected pig viral diseases in Bhutan

Vijay Raika Monger

2015

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Cover design: Vijay Raika Monger and Samir Pradhan (Druk Square, Bhutan)Printed by: Gildeprint drukkerijenISBN: 978-94-6233-137-2

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Epidemiology of selected pig viral diseases in Bhutan

Epidemiologie van een aantal virale varkensziekten in Bhutan.(met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor

promoties in het openbaar te verdedigen op dinsdag 9 december 2015 des middags te 12.45 uur

door

Vijay Raika Mongergeboren op 8 april 1970 te Thimphu, Bhutan


Promotor : Prof. dr. J.A. StegemanCopromotor : Dr. W.L.A. Loeffen

The research described in this thesis was financially supported by the Netherlands University Foundation for International Cooperation (NUFFIC, NFP Grant CF 7436) and Central Veterinary Institute, part of Wageningen UR (CVI, WOT01-003-010-01), Lelystad, The Netherlands.

Printing of this thesis was financially supported by the Central Veterinary Institute, part of Wageningen UR.


For Bela, Vanessa and Valin



Contents

Chapter 1 General introduction 9

Chapter 2 Serosurveillence and associated risk factors of 19 important pig viral diseases in Bhutan

Chapter 3 Longitudinal study of selected viral diseases 41 in professional pig breeding farms and village backyard farms in Bhutan

Chapter 4 Genetic characterization of porcine 55 circovirus type 2 (PCV2) in pigs in Bhutan

Chapter 5 Prevalence and phylogeny of Hepatitis 71 E virus (HEV) in pigs in Bhutan

Chapter 6 Evaluation of oral bait vaccine efficacy 85 against Classical Swine Fever Virus (CSFV) in village backyards farms in Bhutan

Chapter 7 Summarizing discussion 101 Nederlandsesamenvatting 112 Curriculum vitae 113 List of publications 114 Acknowledgements 115



Chapter 1

General introduction

9


10


Importance of livestock in Bhutan

Bhutan is a kingdom with an area of 38,394 km2 and a population of 634 982, situated in the Eastern part of the Himalayan range between latitudes 26°45' N and 28° 10' N, and longitudes 88°45' E and 92°10' E. It is a landlocked country bordered by China in the north an India in the south. It is an agrarian country with almost 70% of the population residing in the rural areas. Only 8% of its land cover is suitable for arable agriculture. Livestock are an integral part of the farming system and an important economic activity of the rural household income. The overarching policy of the Bhutanese government is to increase livestock production and productivity by implementing livestock development programs and activities through control of economically important diseases. The primary role of the Bhutanese Ministry of Agriculture and Forest (MoAF) is targeted towards poverty alleviation and enhancing food security. The Department of Livestock (DoL) is the sole government agency mandated to plan, manage and execute livestock developmental programs and national level livestock disease control programs.Bhutanese farming consists of small holder subsistence agriculture wherein farmers keep livestock and also grow food and cash crops. Livestock are integral to household livelihoods in rural communities and includes diverse domestic animal species such as yaks, cattle, horses, sheep, goats, buffaloes, poultry and pigs. These species are reared as favored the agro-ecological social conditions. In the dry-subtropical and wet subtropical zones, farmers rear multiple species including cattle, sheep, goats, poultry and pigs. In the temperate zone, farmers rear mostly cattle whereas in the alpine zone, the people live a nomadic life and rear mainly yaks.

Pig rearing system in Bhutan

Pigs are traditional components in most rural communities in the country, serving as a source of income and a means of fulfilling socio-religious functions or in exchange for other commodities [1]. The importance lies in the high consumption of pig meat, as pork is one of the most important food items in the Bhutanese daily diet if not for any ceremonies or rituals. The offering or sacrifices of pigs to local deities still prevail in some parts of the country [2]. Pig rearing is also preferred by rural communities for its ability to convert waste food and offal into meat.

Pigs in the backyard Rural backyard pig holdings are seen in the villages, raising pigs in the backyards of the house. They are fed with locally available resources, mainly from the household’s crop by-products. The number of pigs reared by an individual farmer normally varies may vary from 2-8

Introduction

11


pigs. The pigs are generally kept in confinement either in a small pigsty, constructed usually with locally available materials (stones, mud, wood, bamboo thatch), or they are tethered near the house. However, in some rural villages, farmers keep pigs in a free-range system where pigs scavenge in the villages and its surroundings [1]. Most of the time, they wander freely and eat whatever they find, sometimes the farmers also give supplementary feed like kitchen waste and farm bi-products which makes them come back home daily. The scavenging pigs are usually the local indigenous pigs, adapt to this kind of environment. The breed of pigs at the other backyard farms could be either indigenous pigs or exotic pigs supplied by the government breeding farms, though the numbers of indigenous pigs are declining due to introduction of exotic pigs.

Commercial pig farms The number of commercial pig enterprises in Bhutan is still limited. According to livestock statistics, there were a total of 27,500 pigs in 2008 (Anonymous 2008). However, the total pig population may be underestimated, especially pigs in backyard farms in the rural villages, due to the absence of central registration system of pig farms in Bhutan. Consumption of pork is quite high in Bhutanese tradition and the demand is far beyond the production capacity of the country [3], which is met through the import of pork from neighboring countries like India and Nepal. According to the Livestock statistics 2012, the import of pork was 1842 metric tons (MT) against domestic pork production of only 317 MT [4]. In view of the importance of pig farming in terms of its contribution to rural poor and possible potentials for pig rearing in the country, the Government of Bhutan initiated pig breeding farms in the early sixties with importation of exotic breeds of European origin. Currently, the National Piggery Development Centre (NPiDC) at Gelephu, situated in the southern region, is the focal agency for piggery development in the country. Under the NPiDC, there are three breeding farms which are the Nucleus Breeding Centre (NBC) at Gelephu (southern region), and two Regional Breeding Centres (RBCs) at Yusipang (western region) and Lingmethang (eastern region). The focus has been more on upgrading of the local breed using the exotic breeds like Large Black, Large White, Saddle Back and Duroc Jersey. NBC at Gelephu has 150-200 sows, RBC at Yusipang has 100-150 sows and RBC at Lingmethang has 60-100 sows. In order to move towards self-sufficiency in breeding programs and pork production, the Government has started promoting private breeding farms and establishment of commercial farms in selected regions in the form of piggery cooperatives. The selected regions are those, where the people do not have any religious disapproval of raising and slaughtering of pigs, which are mainly in the southern region of the country among Hindus [5]. Compared to rural backyard pig husbandry systems mentioned before, commercial pig farms have better housing system with concrete floor and better roofing. Adequate pen space, feed and water facilities are provided to meet the requirements of the pigs. The government assists these farmers with

Chapter 1

12


Figure 1. Map of Bhutan with veterinary infrastructures

Introduction

provision of loans to construct the pig sheds. They also avail veterinary services like vaccination and treatments free of charge. Profit is the major motivation for these kinds of commercial pig farms.

Animal Health services in Bhutan Animal health services are provided free of cost to the people by the Royal Government of Bhutan andDOL is responsible for its delivery. The delivery of animal health services like treatment, vaccination and surveillance activities to the farming communities are undertaken by the Livestock Extension Centres (LECs) or Renewable Natural Resources Extension Centres (RNRECs) which are located in all 205 sub-districts and manned by a Para-veterinarian. The LECs cater only to animal health and livestock activities, whereas, the RNRECs, also coordinate agricultural and forestry activities. There are District Veterinary Hospitals (DVHs) in 20 districts, where a veterinarian is posted to provide technical support to the LECs and RNRECs. The DVHs are further assisted by the network of Laboratory centres such as National Centre for Animal Health (NCAH) located at Serbithang in Thimphu and the Regional Livestock Development Centres (RLDCs) located in the 4 different geographical regions of the country (Figure 1).

13


The disease surveillance and early warning of any epizootic originating from the international border are being managed through these RLDCs. Any livestock imported has to be quarantined at least for 14 days under the Livestock Legislation. Within the MOAF, there is an autonomous agency called the Bhutan Agriculture and Food Regulatory Authority (BAFRA), mandated to undertake the quarantine and inspection of all livestock and livestock products that are imported or exported, including the internal movement of livestock and livestock products. The animal quarantine stations are located in the four southern border districts and the international airport at Paro district. Regulatory inspectors from BAFRA are posted in most of the police check points along the major highways. The DOL is mandated to set the standards for sanitary and zoo sanitary requirements for all activities relating to livestock and livestock products, while the BAFRA implements, monitors, and regulate these standards. The country’s passive surveillance system is essentially based on farmer-diagnosed and reported presence of diseases in their herds and villages.

Animal diseases in Bhutan

As per the Livestock Act of Bhutan and the Livestock Rules and Regulation of Bhutan, Rinderpest (RP), Foot and mouth disease (FMD), Rabies, Black quarter (BQ), Hemorrhagicsepticemia (HS), Anthrax, Classical swine fever (CSF), Highly pathogenic avian influenza (HPAI), Newcastle disease (NCD), Infectious Bursal disease (IBD), Mareks disease (MD) have been listed as notifiable livestock and poultry diseases in Bhutan. Infectious diseases are one of the limiting factors for disease control in Bhutan.

Pig diseases

With regards to pig diseases in Bhutan, there is a lack of scientific information on pig diseases. There is no good-quality data regarding viral pathogens like Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Swine Influenza Virus (SIV), Porcine Circovirus Virus type 2 (PCV2) and Ajuszky’s Disease Virus (ADV) are considered among the most relevant viral contributors to co-infections in pigs [6]. The diseases caused by these agents pose a major threat to production and for effective control, it is important to have reliable information on these diseases.

In Bhutan, except for PRRS, there was no information on the pig pathogens mentioned above. The first PRRS was diagnosed and confirmed in 2008 in a government breeding farm [7], which was caused by the highly pathogenic PRRSV genotype 2. Moreover in the early nineties, Escherichia coli was diagnosed to be one of the causes of mortality in piglets in a government pig breeding farm [8].

Chapter 1

14


CSF is a notifiable disease and thought to be endemic in Bhutan. Among repeated cases of mortality, CSF is the most common clinical diagnosis. A total of 10 outbreaks have been reported between 1998 and 2012 in the village backyard farms [9]. However, the confirmation of the disease is doubtful as most of these outbreaks were diagnosed based on clinical signs and postmortem examination only. A sero-surveillance study carried out in 1999 targeting high risk areas and vaccination free pigs indicated 5.8% seroprevalence of CSF [10]. Swill feeding, which is very common in the backyard herds serves as a risk factor for CSF transmission.

Another notifiable disease, FMD is endemic in Bhutan and is reported from almost all parts of the country, mainly in cattle, with serotype O as the most prevalent FMDV serotype involved [11]. However, there is no documentation of outbreaks of FMD in pigs although pigs were found to be seropositive [12, 13].

Disease prevention and control program

The flow of information starts from the point when a farmer reports an occurrence of disease to the LEC. In the event of a notifiable disease, the farmer can directly report to the RLDCs/NCAH, or through the heads (Gups) of sub-districts or LECs/RNRECs. By law, it is mandatory for the administration at the village and sub-district level to report any suspicions of notifiable diseases to the nearest LECs/RLDCs. The para veterinarians upon preliminary investigation are also required to report suspicions of disease to the nearest RLDCs, through the District Livestock Officers (DLOs) (DVH), or directly to RLDC/NCAH by flash report, indicating the suspected disease, date of occurrence, morbidity and mortality and the geographical location of the outbreak. The veterinarians from the RLDCs/NCAH then visits the herds/villages and undertakes a detailed epidemiological investigation and collects samples for a laboratory investigation of the outbreak. Samples are sent to the NCAH for laboratory confirmation. Based on the findings at the outbreaks, technical feedback is given to the DVH (Districts) on the measures to be applied to control the outbreaks. Immediate reporting to OIE is done for new outbreaks of diseases notifiable to the OIE and thereafter weekly reporting is done until the disease outbreak is resolved. Routinely, six-monthly and annual reports on the animal health situation are produced for the OIE.

For the animal health information system, the LECs/RNRECs are required to submit a monthly animal health report of their jurisdiction to the DLOs, in which information on clinical cases, disease outbreaks, and vaccination is documented. The DLOs then send the compiled report to all the sub-districts and to RLDCs. The veterinary laboratories are required to generate monthly reports on the samples tested, diseases diagnosed, and details of outbreak investigations performed.

Introduction

15


Vaccination is currently the main method used for disease control. Vaccines for diseases like Black Quarter (BQ), HemorrhagicSepticemia (HS), Anthrax and CSF are produced locally in the country based on conventional techniques. Other vaccines like FMD, Rabies and most poultry vaccines are procured from India or elsewhere. These vaccines are done out as per the national vaccination program. In pigs, vaccination against CSF and FMD is done only in the government breeding farms due to difficulty in restraining pigs and which are scattered herds in village back yard holdings.

Official, contingency plan is available only for Avian Influenza for implementation should there be any outbreak. Under this contingency plan, clinical and laboratory surveillance of chicken population are carried out to avoid introduction of the disease into the country. During the suspicion of the disease outbreak, activities like quarantine, declaration of the outbreak zone, protected zone, surveillance zone, culling and compensation are carried out.

Aims and outline of this thesis

Pig production in a developing country like Bhutan is characterized by traditional small scale subsistence driven production systems. As in most developing countries, very few publications on pig diseases are available.

The aim of this thesis was to generate information on selected pig viral diseases in Bhutanese pig farms through seroprevalence study, genetic characterization of virus strains and studying the dynamics of diseases in the government breeding and village backyard farms. The thesis also aimed to understand the disease dynamics between the government breeding farms and backyard holdings. This study provides baseline information for pig health and pig disease control in Bhutan that is essential to expand pig production in Bhutan.

Chapter 2 describes the sero-prevalence of important pig viral diseases in Bhutan and the risk factors associated with the occurrence and introduction of these diseases. The study focuses on Classical Swine Fever (CSF), Porcine Reproductive and Respiratory Syndrome (PRRS), Porcine Circovirus type 2 (PCV2), Aujeszky’s Disease (AD) and Swine Influenza (SI).

Chapter 3 covers a longitudinal study to elucidate the role of sows in the disease prevalence on government breeding farms and the dynamics of virus spread between the government herds and village backyard holdings in relation to virus spread within the government herds.

In chapters 4 and 5, Porcine Circovirus type 2 (PCV2) and Hepatitis E virus (HEV) in the Bhutanese pigs were characterized through sequencing of their nucleotides.

The current CSF vaccine is a lapinised, live-attenuated Chinese strain and parental vaccination village backyard pigs proved to be unsuccessful due to various practical problems. Therefore, the research described in Chapter 6, investigated the efficacy of an oral bait CSF vaccine in village backyard pig farms in Bhutan. In chapter 7, the findings in this thesis are discussed.

Chapter 1

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References

1. Timsina MP, Sherpa DL: Characterization of Bhutan’s indigenous pig genetic resources and production systems. Council of RNR Research of Bhutan, The Ministry of Agriculture 2005.2. Wangchuk R: An integral part of Bongop culture. In Kuensel, Kuensel Corporation Ltd. Thimphu, Bhutan. Accessed on 29 April 2009. 2005.3. Anonymous: Livestock population and production bulletin: data pertaining to year 2005 and earlier periods. Thimphu, Bhutan, Department of Livstock, MoA. 2007.4. Anonymous: Livestock statistics. Department of Livestock, Ministry of Agriculture and Forests, Thimphu Bhutan 2012.5. Nidup K, Tshering D, Wangdi S, Gyeltshen C, Phuntsho T, Moran C: Farming and biodiversity of pigs in Bhutan. Animal Genetic Resources / Resources génétiques animales / Recursos genéticos animales 2011, 48:47-61.6. Halbur PG: Porcine viral respiratory diseaseProceedings IPVS, 15th International Pig Veterinary Society Congress, Birmingham, England. 1998:1-10.7. Raika V, Stadejek T, Tshering C, Rinzin K, Chabros K, Parchariyanoi S, : Outbreak of PRRS in government breeding farm. Disease investigation report. National Centre for Animal Health, Serbithang. In.; 2008.8. Cork SC, Moitra ND, Gurung JB, Halliwell RW: Escherichia coli as a cause of mortality in piglets in the royal Kingdom of Bhutan. The Veterinary Record 2002, 150:313-315.9. Anonymous: Animal Health Bulletin, National Centre for Animal Health, Serbithang. 2010.10. Raika V: Study of Classical Swine Fever using Ceditest ELISA, MSc thesis in Tropical Veterinary Medicine. University of Edinburgh, U.K 1999.11. Dukpa K, Robertson ID, Ellis TM: The Epidemiological Characteristics of the 2007 Foot-and-Mouth Disease Epidemic in Sarpang and Zhemgang Districts of Bhutan. Transboundary and Emerging Diseases 2011, 58(1):53-62.12. Dukpa K, Robertson I, Edwards J, Ellis T: A retrospective study on the epidemiology of foot-and-mouth disease in Bhutan. Tropical Animal Health and Production 2011, 43(2):495-502.13. Dukpa K, Robertson ID, Ellis TM: The seroprevalence of foot-and-mouth disease in the sedentary livestock herds in four districts of Bhutan. Preventive Veterinary Medicine 2011, 100(3–4):231-236

Introduction

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18


Chapter 2

Seroprevalence and associated risk factors of important pig viral diseases in Bhutan

V. R.Mongera, c, J.A. Stegemanb, G. Koopb, K. Dukpac, T. Tenzinc, W.L.A. Loeffena

aCentral Veterinary Institute of Wageningen UR, Department of Virology,P.O. Box 65, 8200 AB Lelystad, the NetherlandsbDepartment of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CL Utrecht, The NetherlandscNational Centre for Animal Health, Department of Livestock, Ministry of Agriculture and Forests, Thimphu, Bhutan

Published in: Preventive Veterinary Medicine 2014 November1; 117(1): 222-232

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Abstract

A cross-sectional serological study was conducted in Bhutan between October 2011 and February 2012 to determine the prevalence of antibodies to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), swine influenza virus (SIV) subtype H1N1 and Aujeszky’s disease virus (ADV). Furthermore, risk factors for the seropositive status were investigated.

Antibodies to SIV, subtype H1N1 (likely pandemic H1N1 2009) were detected in 49% of the pigs in the government farms, and 8% of the village backyard pigs. For PCV2, these percentages were 73% and 37% respectively. For CSFV, the percentages were closer together, with 62% and 52% respectively. It should be taken into consideration that vaccination of piglets is routine in the government herds, and that piglets distributed to backyard farms are also vaccinated. No direct evidence of CSFV infections was found, either by clinical signs or virus isolation. Antibodies to PRRSV and Aujeszky’s disease, on the other hand, were not found at all.

Risk factors found are mainly related to practices of swill feeding and other biosecurity measures. For CSFV, these were swill feeding (OR=2.25, 95% CI: 1.01-4.99) and contact with neighbour’s pigs (OR=0.31, 95% CI: 0.13-0.75). For PCV2, this was lending of boars for local breeding purposes (OR=3.30, 95% CI: 1.43-7.59).

The results of this study showed that PCV2 and SIV infections are important in pigs in Bhutan and thus appropriate control strategies need to be designed and applied which could involve strict regulation on the import of live pigs and vaccination against these diseases.

Keywords: Bhutan; Seroprevalence, Pig Viral diseases, Classical Swine Fever virus, Swine Influenza virus, Porcine Circovirus, Porcine Reproductive and Respiratory Syndrome virus, Aujeszky’s Disease virus, Virus Neutralization Test, Haemagglutination Test, ELISA

20

Chapter 2


Seroprevalence and associated risk factors

21

Introduction

Village backyard pig farming plays an integral role in providing nutrients and household income in many low-income countries [1]. In Bhutan, located between China and India, village backyard pig farming is characterized by small numbers of pigs reared by subsistence farmers [2]. In addition, there are three government-ownedpig-breeding farms in Bhutan to supply hybridized piglets to the farmers for fattening and cross-breeding purposes. These government breeding farms have been set up to improve local breeds through cross breeding with exotic breeds, which are being imported from various countries, including India, Australia, Denmark and, most recently, from the UK. The number of cross-bred pigs in the country has increased over the past years, constituting about 38 percent of the total pig population [3]. Semi-commercial back-yard farms are being established and a decline in the numbers of indigenous pigs is seen. There is a high risk of introduction of exotic diseases in the country through the import of exotic pigs and further spreading to the village backyard farms through subsequent distribution of pigs by central government breeding farms. This has important socio-economic and possibly public health consequences, constituting a threat to livelihood of the rural livestock farmers.

There has been no scientific study on pig diseases in Bhutan except for a surveillance study of CSFV, demonstrating a seroprevalence of 6% [4]. More recently, porcine reproductive and respiratory syndrome (PRRS) cases have been confirmed for the first time in government pig breeding farms [5]. Since then PRRS is under control due to an effective control program consisting of regular monitoring, serological testing of breeding sows and culling of seropositive animals. Similarly, there is a need to have information on other important pig viruses that are of economic importance, like swine influenza virus (SIV), Aujeszky’s disease virus (ADV) and infections caused by porcine circovirus type 2 (PCV2). Bhutan is at risk for introducing pig viruses; it shares a border with India in the south, across which there are imports of fresh pig meat and sometimes illegal live pig imports. In addition, Bhutan imports live pigs for breeding purposes to improve local production, importation of these pigs contributes to the risk of introd pathogens. Subsequent spread of these pathogens may be enhanced by poor biosecurity measures at the village backyard level.

The aims of this study were to determine the prevalence of antibodies to CSFV, PRRSV, ADV, SIV and PCV2 in government breeding farms and village backyard farms in Bhutan, and to identify possible risk factors associated with seropositivity of these viruses


Materials and Methods

Study area and study populationBhutan is subdivided into 20 districts. In each district a District Veterinary Hospital (DVH)

is present, headed by a District Livestock Officer (DLO) responsible for overall coordination of animal health and livestock production in their respective district and all related administrative duties. There is a concentration of pigs towards the southern regions, due to climate, landscape and religious factors [3].

The three government breeding farms are located in the districts of Thimphu (west region), Sarpang (east-central region), and Mongar (east region) (Figure1).

These breeding farms have been established with the main objectives of improving local pigs through cross-breeding programs and distribution of piglets to farmers in the country. The size of these government farms currently ranges from 65-150 sows, with accompanying piglets and some replacement stock (Table 1). The farm in Sarpang is the central breeding farm, providing replacement stock to both other government farms. There is no central registration of pig farms

Figure 1: Map of Bhutan and its 20 districts. Selected villages where backyard farms were samples, are marked as black dots. The three government herds are marked as red (open) squares. The fourregions (west, west-central, east-central and east, from left to right) are identified with different colours

Chapter 2

22



23

in Bhutan, and data on numbers of farms and pigs depend on infrequent livestock censuses. According to livestock statistics [6], there were a total of 27,500 pigs in Bhutan in 2008.

Table 1.Seroprevalences of classical swine fever (CSF), swine influenza H1N1 (SI) and porcine circovirus type 2 (PCV-2) government breeding farms. The numbers of sows and gilts in the herds (N) were the ones present at the time of sampling. The number of piglets in the herds (N) is the average number of piglets present at any time. The number of samples from each category is depicted under “n’’. The 95% confidence interval (95% CI) is the exact binomial confidence interval, corrected for finite population.

However, these figures are likely to be an underestimation because the survey depended on farmers actively reporting these numbers to the District Livestock Centres (LECs). The only vaccine that is currently licensed for use in pigs in Bhutan is a locally produced, lapinized, live-attenuated CSF vaccine. The vaccine is for intra-muscular application only, and therefore its administration requires a veterinarian. As a result, vaccination against CSF is mostly restricted to Government pig breeding farms and few semi-commercial backyard farms. At village backyard level, the use of this vaccine is very limited.

Sampling strategyA cross-sectional study was carried out in three government pig breeding pig farms and

343 village backyard pig farms in Bhutan to determine the seroprevalence of five pig diseases. Blood samples for this serological study were collected between October 2011 and February 2012. This study was conducted following approval by the Council for Renewable Natural Resources Research of Bhutan (CORRB), an organization within the Ministry of Agriculture and Forests (MOAF), mandated to coordinate research policy and programs in the country.


Government breeding pig farmsIn all three government pig breeding farms, a total of 101 sows, 74 gilts (replacement stock),

and 201 piglets (< 90 days) were included. The numbers of pigs to be sampled in each subcategory were chosen according to an expected seroprevalence of 50%, with a desired precision of 15% and a confidence level of 95% [7]. Due to local circumstances on the individual farms, the ultimate number of samples was occasionally somewhat higher or lower (Table 1). Sows and gilts were sampled randomly from the pigs present. Piglets, however, were mainly sampled among those aged approximately 45-50 days. This was because they are sold to village backyard farms shortly after they are weaned (45-50 days) and are not available beyond that age. Some older piglets (up to 90 days of age, mainly kept as replacement stocks) were also included if present. A total of 376 pigs were sampled on the three government breeding farms.

Village backyard pig farmsA multi-stage sampling approach was used for the village backyard farms from the 20

districts. Due to the lack of a central registration of pig farms in Bhutan, data from the last livestock census [6] and information from the District Livestock Officers (DLOs) were used to select pig backyard farms. The DLOs are the animal health coordinators in the Districts who have the best information on the livestock population in their respective districts. Three districts were excluded from the study: two contained less than 20 pigs according to the last livestock census, and the third district, with less than 400 pigs, is one of the most inaccessible regions of Bhutan. Leaving out these districts was expected to have little impact on the results of the study. Having the DLOs select the villages (figure 1) may have resulted in villages with higher number of backyard holdings (four holdings or greater) more likely to be selected than those with fewer holdings. Also this was expected to have little impact on the results. Once a village was selected, a list of farmers owning backyard pig farms was generated and then 4-5 farmers were randomly selected per village. Finally, from the selected backyard farm, 1-5 pigs were selected for sampling, by simple random sampling (Table 2). In total, 465 pigs were sampled from 343 backyard farms of the 69 villages.

Herd size (Total No. of pigs) Herd size (Total No. of pigs)

1

2 to 4

5 to 10

10 to 20

> 20

1

2

3

4

5

Chapter 2

24

Table 2.Number of samples taken in village backyard farms, depending on number of pigs present.



25

Sample CollectionBlood samples were collected, using Sarstedt Monovette syringes from all selected pigs.

For each animal sampled date, location (village, sub-district. district), animal identification, age, sex, and sample number were recorded. Serum was obtained after centrifugation of the coagulated blood samples and stored at -20o C until testing.

Questionnaire

A questionnaire was used to collect data on risk factors associated with the seroprevalence of the pig diseases through face-to-face interviews. The interviews were carried out by the first author in one region (west), and one senior animal health officer in each of the other three regions, after receiving training from the first author. The data were gathered using only “close ended” questions. In general, the aspects covered in the questionnaire were herd size, presence of other livestock (mixed herd), origin of pigs, housing conditions, practice of using boars for breeding within the locality, feeding practices (swill feeding), presence of wild boar in the locality, farm bio-security (cleaning of sheds and use of disinfectant), access to veterinary services and purchase of pork. Farmers were also asked whether they had basic training in pig husbandry practices from the government. A full version of the questionnaire is available on request. A total of 243 farmers agreed to be interviewed across 69 villages.

Laboratory tests

Antibodies against classical swine fever virus (CSFV)Serum samples were pre-screened in a commercial blocking enzyme linked immunosorbent

assay (ELISA) (Herd Check CSFV antibody test kit; IDEXX Laboratories, Switzerland). The protocol was carried out according to the manufacturer’s specifications. The test was considered positive if the inhibition percentage was ≥ 30%. The diagnostic sensitivity and specificity, based on the validation done at Central Veterinary Institute (CVI), Lelystad, The Netherlands were 91.2% and 99.9% respectively. The positive sera in the ELISA test were subsequently tested in virus neutralisation test (VNT) against a cell culture adapted C-strain [8] and Alfort/187 strain [9] of CSFV as described by [10], with validated sensitivity of 93% and specificity of 100% at CVI, to confirm the ELISA results and try to differentiate between antibodies against the CSFV vaccine strain (C-strain) and field strain (Alfort/187). Although there is no published data on the CSFV field strain in Bhutan, Alfort/187 strain was selected because the Indian CSFV isolates in the region close to Bhutan also belonged to subgroup 1.1, [11, 12]. Similar strains were most likely to circulate in Bhutan. The cell-adapted C-strain was chosen as most representative for the C-strain vaccine from Bhutan. Serum samples were ultimately considered to have antibodies against CSFV if it was positive to either of the CSFV strains in the VNT (titres ≥ 10).


Antibodies against swine influenza virus (SIV)Serum samples were pre-screened in an indirect double antibody sandwich (IDAS) ELISA

to detect antibodies against the nucleoprotein (NP) of swine influenza type A virus as described by [13] with some modifications. In short, ELISA plates (Costar EIA/RIA, Cat. No. 3590, Costar, USA) coated with monoclonal antibody (MAbs) (HB65) against the NP and incubated with 100 μl of antigen (H7N1 – V371) for one hour. Subsequently, 100 μl of a 1:8 diluted serum was added and incubated for another hour. Then conjugated MAb HB65 was added, followed by TMB after one more hour of incubation. After stopping the reaction, The plates were read at 450 nm with an ELISA reader. This was an in-house ELISA kit produced at CVI with validated sensitivity and specificity of 92% and 99% respectively.

Samples that gave an inhibition percentage (PI) ≥ 40% against swine influenza type A were confirmed in a haemagglutination inhibition (HI) test. The HI test was performed according to the standard procedures (OIE, 2008) and standardized using four haemagglutinin units (HU) per well. The starting dilution for testing sera was 1:10. Sera with a titre ≥10 were considered positive.

Given the absence of data on SIV in Bhutan, prior to conducting the HI test on the ELISA positive samples, 26 ELISA positives (≥40%) and 6 ELISA negatives (< 40%) were selected and tested in the HI test to select the most relevant influenza strains to be used in the test. For this, five reference antigens of influenza were used: A/swine/Netherlands/Best/96 (European swine H1N1) [14], A/swine/Gent/7625/99 (European swine H1N2) [15], A/swine/Netherlands/St Oedenrode/96 (European swine H3N2) [16], A/Port Chalmers/1/73 (H3N2 of human origin) [17] and A/Netherlands/602/2009 (pandemic H1N1) [18].

Antibodies against porcine circovirus (PCV2)Serum samples were tested in a commercially available indirect ELISA (Porcine Circovirus

2 Antibody Test Kit; BioChek BV, The Netherlands) according to the manufacturer’s instruction. The sensitivity and specificity of the kit were previously estimated at 85% and 95.6% respectively [19]. The presence or absence of antibody to PCV2 was determined by calculating the test sera-to-positive (S/P) ratio. Samples were considered positive if the S/P ratio was ≥ 0.5.

Antibodies against porcine reproductive and respiratory syndrome Virus (PRRSV)

Serum samples were tested in an immuno peroxidase monolayer assay (IPMA) as previously described [20], The sensitivity and specificity of the test are unknown, but expected to be high [21].

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Furthermore, the test showed a high degree of accordance with an ELISA in the serodiagnosis of PRRS [22]. In short: MA-104 monolayers were infected with 1000-2000 TCID

50 of a PRRSV

isolate and incubated for 24 hours. Samples were incubated on the fixated cells for 1 hour and subsequently cells were stained with a conjugated rabbit anti-swine immunoglobulin. An intense red staining of the cells indicates the presence of PRRSV antibodies.

Antibodies against Aujeszky’s disease virus (ADV)Serum samples were tested in a commercially available ELISA kit (PrioCHECK® PRVgB;

Prionics AG, Switzerland) according to the manufacturer’s instructions. Samples were considered positive if the inhibition percentage was ≥ 50%. The kit has a diagnostic sensitivity of 96% and specificity of 100%, validated at CVI.

Statistical analysis

Seroprevalences in the government farms were calculated for each animal category (piglets, gilts and sows) separately. A 95% confidence interval was calculated using the exact binomial confidence intervals [23]. Furthermore, a correction for finite populations was applied [24]. Seroprevalences in the backyard holdings were also calculated separately for animal categories as close as possible resembling those in the government farms.

Seroprevalence in backyard village farms and governmental farms was compared for each of the diseases using a random effects logistic regression model. The outcome of a specific disease was the dependent variable (1 = positive, 0 = negative). The individual villages and government farms were transformed into a new variable “cluster” that included the ID of the villages (clusters 1-69) and the government farms (clusters 70 - 72), which served as the random effect variable to take into account the dependence of pigs within a cluster. Age of pigs and type of farm (0 = backyard; 1 = government farm) were added as explanatory variables. The Intra-class correlation coefficient (ICC) was calculated for the random effects term using the latent variable approach [25].

Risk factors analysis was done for village backyard holdings only and not for government breeding farms, because of just three government pig breeding farms. For each disease, sero-status of a herd was coded as a dichotomous variable (positive/negative) and all variables from the questionnaire were recorded as dichotomous variables. Risk factors from the questionnaire that were potentially associated with seropositivity of CSFV, SIV and PCV2 positive herds were identified using Chi-squared tests; if the expected number in one of the cells of the 2x2 tables was < 5, the Fishers exact test was used.Variables with P-values < 0.25 based on the chi-square test were tested in the subsequent multivariable logistic regression model, including a random effect for village (nested within region). A backward-elimination procedure was used by fitting the full model and reducing the model based on significance of the variables one by


one, whilst checking for collinearity and confounding at each step. Confounding was deemed to be present when a beta-estimate changed more than 25% upon exclusion of a confounder from the model. If a variable could not be fitted, because of empty cells, it was excluded from further analyses. Variables that were significant at P < 0.05 according to the likelihood ratio test were considered as risk factors for the respective disease seropositivity, the strength of association between a variable and disease prevalence was presented in terms of odds ratio (OR). Fixed effects in a random-effects logistic regression model correspond to cluster specific (CS) estimates, which in our models represented the effect of a risk factor within a village. This makes sense for risk factors that truly happen at village level, such as the variable ‘using the breeding boar from neighbour’, but is meaningless for the variable ‘region’ for instance. Therefore, OR’s were calculated based in population-averaged (PA) beta-estimates or on cluster estimates, depending on the nature of the variables. PA beta-estimates were calculated from CS estimates according to [25].

where PA is population averaged, CS is cluster specific and σ2 is the variance of the random village effect. Based on these beta-estimates, ORs were calculated by exponentiating the beta-estimate.

Logistic regression models were built in R (version 3.0.0; R Foundation for Statistical Computing, Vienna, Austria). For the random effects logistic regression models, the GLMER function from the LME4 library was used.

Results

All tested sera were serologically negative for ADV and PRRSV antibodies. There were significant differences in the age distribution over the three government breeding farms and in the village backyard farms ( = 16.67; P < 0.01), therefore age was found to be a confounder and was corrected for in subsequent analyses.

The seroprevalences of SIV (H1N1) and PCV2 antibodies in government breeding farms were significantly higher than in the village backyard farms (P < 0.001 and P = 0.001 respectively). Also for CSFV antibodies, the prevalence in government breeding farms was higher than in village backyard farms, but this difference was not statistically significant (P = 0.06). The estimated ICC (and proportion of variance at the cluster level) in the final model for antibodies against CSFV, SIV H1N1 and PCV2 at village backyard farms (regions) was 0.32, 0.27 and 0.23 respectively, indicating a clustering of seropositive animals within villages/government farms.

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Antibodies against CSFV

Government breeding farmsA total of 234 out of 376 pigs tested (62%) were seropositive (Table 1). All positive sera

had neutralizing antibody titres in the VNT against both CSFV C-strain and Alfort/187 strain that were mostly similar. No differentiation was therefore possible between vaccination and

infection titres. Seroprevalence differed between the government breeding farms (P = 0.025).

Village backyard farmsA total of 240 out of 465 pigs tested (52%) were seropositive (Table 3).Again, titres against

C-strain and Alfort/187 strain were similar and no differentiation between vaccination and infection titres was possible. After correcting for other risk factors, there was no significant effect of region on the seroprevalence (P = 0.15).

Antibodies against SIV

Selection of influenza strains to be used in HI testAll six samples that were negative in the ELISA (< 40% inhibition) were also negative in all

the HI-tests. Using a cut-off of 40% in the ELISA for confirmation testing in the HI-tests was therefore considered to be appropriate.

From the 26 positive samples in the ELISA ( ≥ 40% inhibition), 21 were positive in at least one of the HI tests. Nineteen samples had titres against influenza subtype H1N1, ranging from 20 to 10,240. Titres against European swine H1N1 and pandemic H1N1 were mostly comparable. Four samples had low titres against H1N2 (3 x 20, 1 x 80). In three of these, high titres against H1N1 were observed and cross-reaction was assumed. Three samples had low titres against H3N2 (3 x 20). These were considered likely to be false-positives.

The remaining five ELISA-positive samples were negative in all the HI-tests. Some of these were likely false-positive in the ELISA (3 samples had results of 54%, 55%, and 56% PI respectively), but especially for the other two, with PI of 75% and 84%, circulation of strains that did not match with the strains that were used in the HI-test is likely.

Based on these results it was decided to confirm all ELISA-positive samples using HI-tests against both the European swine H1N1 and pandemic H1N1 strain.


Table 3. Seroprevalences of classical swine fever (CSF), swine influenza H1N1 (SI) and porcine circo virus type 2 (PCV-2) in village backyard farms, separate for 4 main regions and total for the country. The number of samples from each category is depicted under “n”. The 95% confidence interval (95%CI) is the exact binomial confidence interval.

Government breeding farmsA total of 180 out of 376 pigs tested (47%) were seropositive against H1N1 (Table 1). From

these, 179 were positive against pandemic H1N1 and 161 against European swine H1N1. Titres against pandemic H1N1 were higher than against European swine H1N1 in 161 samples.

In the initial ELISA, 185 out of the 376 pigs had an inhibition percentage ≥ 40%. Four of those samples were not retested in the HI-test, while 3 samples with PI of 45%, 47% and 78% were negative in the HI-test. There was a significant difference in the seroprevalence of H1N1 among Government breeding farms (P < 0.001).

Village backyard farmsA total of 36 out of 465 pigs tested (8.0%) were seropositive against H1N1 (Table 3). All

36 were positive against pandemic H1N1 and 35 against European swine H1N1. Titres against pandemic H1N1 were higher than against European swine H1N1 in 34 samples.

In the initial ELISA, 55 out of the 465 pigs had an inhibition percentage ≥ 40%. Six of those samples were not retested in the HI-test, while 12 were negative in the HI-test (all in the range of PI of 40-49%). There was a significant effect of region on seroprevalence (P = 0.005), with the highest seroprevalence in the West, and the lowest in the East.

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Antibodies against PCV2

Government breeding farmsA total of 267/376 pigs tested (71%) were seropositive to PCV-2 (Table 1). Seroprevalence

varied among farms (P < 0.001).

Village backyard pig farmsOne hundred and seventy sera out of 465 (37%) pigs tested were seropositive to PCV2

antibodies (Table 3). Region was significantly associated with PCV2 seroprevalence (P = 0.021).

Risk factors for seropositivity of CSFV, SIV and PCV2

The results of the univariate analysis are shown in Table 4. The final logistic regression model for seropositivity of CSFV, SIV and PCV2 is summarized in Table 5. After performing backward elimination for each disease seropositivity (CSFV, SIV and PCV2) as dependent variables, no significant predictors, except region were found to be associated with SI. Swill feeding and contact with other pigs in the neighbourhood was associated with seropositivity of CSFV. Lending of breeding boars to neighbors for breeding purpose was associated with risk of animals being PCV2 seropositive. The factor ‘using boar from neighbor’ was excluded from the multivariable model due to collinearity with the factor ‘lending of boar to neighbour’, therefore only the latter was included in the final model, in which lending of a boar, was found to be a risk factor for seropositivity of PCV2. The variable ‘Do you buy pork from the local market’ was excluded from the SIV-modeling process because of empty cells and could not be tested further.


Table 4. Characteristics of possible risk factors for seroprevalence of classical swine fever (CSF), swine influenza H1N1 (SI) and porcine circovirus type 2 (PCV-2). Significant factors (P < 0.25) in the univariate analyses are marked with *.

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Table 5. Multivariable analysis of risk factors for seropositivity of classical swine fever (CSF), swine influenza H1N1 (SI) and porcine circovirus type 2 (PCV-2), expressed as odds ratio (OR) and 95% confidence intervals (95% CI).

OR based on population based averaged beta-estimates = 1OR based on cluster-specific beta-estimates = 2

Discussion

This is the first seroprevalence study of antibodies against CSFV, SIV, PCV2, PRRSV and ADV in the pig population ever conducted in Bhutan. The study demonstrated the presence of antibodies to CSFV, SIV (H1N1) and PCV2 but the absence of antibodies to PRRSV and ADV with a high degree of certainty (upper 95% confidence limit of seroprevalence 0.4%).

The estimated ICC of the random effects for seropositivity for CSFV, SIV H1N1 and PCV2 were 0.32, 0.27 and 0.23 indicating that a substantial proportion of the total variation was explained at village and government farm level.

The antibodies to SIV, H1N1 and PCV2 were higher in the government breeding farms than in the village backyard farms. This may be due to the low pig density in Bhutan, with scattering of pigs at the backyard level, divided by large distances and limited contact between each other. In combination with the short life span of these pigs as finishers, this likely limits disease transmission. In the government farms, the conditions for disease transmission are more favorable, with a high density of pigs of all ages, and a lot of direct and indirect contact


between the pigs. Most importantly, some of these diseases may have entered the farms with imported pigs. Once present, infectious agents will have easily spread to young piglets. While central (government) breeding farms, providing piglets to backyard farms, can have important advantages in the pig disease control country wide, these breeding farms might on the other hand also play an important role in the transmission of certain diseases to backyard farms through the distribution of piglets.

A previous surveillance study in Bhutan suggested that CSF was endemic in the country, although at a low prevalence [4]. In this study, no differentiation between the antibodies against CSFV vaccine strain (C-strain) and field strain (Alfort/187) could be made by VNT. Consequently, no conclusions could be drawn regarding the circulation of CSFV in Bhutan. Nevertheless, most of the seropositives are expected to be a result of the vaccination. The lack of difference in the seroprevalence of CSFV between the government farms and village backyard farms can be explained by the fact that most of the pigs in the backyard farms have been supplied from the government breeding farms which have been vaccinated against CSF prior to distribution to the farmers.

The detection of antibodies against SIV was carried out using ELISA and further verification done by HI test against two H1N1 strains. In a limited pilot study, only H1N1 antibodies were detected with high confidence. Almost all positives in the NP-ELISA could subsequently be explained by infections with H1N1, providing evidence that Bhutanese pigs were mainly exposed to influenza subtype H1N1. Other SIV subtypes are either absent, or present at very low levels only. Antibody titres against pandemic H1N1 were in general somewhat higher than titres against European swine H1N1. This suggests that pandemic H1N1 is the most likely cause of infection in Bhutan. No confirmed cases of pandemic H1N1 being transmitted from humans to pigs have been established in Bhutan despite reports of pandemic H1N1 in humans [26]. However, many cases of human-to-pig transmission have been reported from other countries [27], and due to the intensive contacts between pigs and humans in Bhutan, this is also likely to have happened in Bhutan. In pigs, the pandemic H1N1 virus could co-circulate with other swine influenza viruses providing opportunity for reassortment[28], posing challenges to public health importance. It is therefore important for regular monitoring, especially at the human-animal interface [29]. The higher seroprevalence of SIV H1N1 in government breeding farms might be associated with high animal densities [30]. In government breeding pig farms, pigs are kept in large groups when compared to village backyard farms, where pigs are kept in small numbers. Single introductions can therefore easily be followed by further pig-to-pig transmission, resulting in high within herd seroprevalence. Furthermore, the higher seroprevalence of SIV H1N1 in the western region as compared to the eastern region may be due to the remoteness of the eastern region.

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Antibodies against PCV2 turned out to be widespread among pigs in Bhutan in both government breeding farms and the village backyard farms with significantly higher seroprevalence in government breeding farms. Currently PCV2 is one of the most economically important viral pathogens of pigs world-wide and known to cause post-weaning multisystemic wasting syndrome (PMWS), including respiratory signs, reproductive failure and porcine dermatitis and nephropathy syndrome (PDNS), collectively known as porcine circovirus associated disease (PCVAD) [31-35]. The government pig breeding farms in Bhutan have a history of the above mentioned clinical diseases which might be attributed to infection by PCV2. Since pigs in Bhutan are not vaccinated against PCV2 virus, seropositive results in this study indicate presence of infection in pigs in Bhutan. However, it is important to carry out further investigation including virological studies to confirm the infection and associations between the presence of virus and clinical disease. If there is enough evidence of an association of the virus with clinical disease, vaccination may be initiated as a control measure.

All the pigs tested did not have antibodies against PRRSV and ADV, indicating absence of these diseases in the sampled population. There was a PRRS outbreak in one government pig breeding farm in 2008, but this was controlled by culling of all infected and seropositive pigs and no further outbreak was reported [5]. The absence of seropositive pigs in this study suggests that the virus has not spread beyond this previously infected farm. However, there should be strict import restrictions on pigs from those countries that are endemic for PRRS and AD to ensure ongoing freedom of these diseases in the Bhutanese pig population.

Our study has shown that swill feeding and government farms as the source of pigs are associated with seropositivity against CSFV, whereas contact with neighbour’s pigs was associated with lower seropositivity. The latter is surprising as it is hard to think of a causal mechanism explaining that more contact leads to less seropositiveness. A possible explanation may be that farmers who have purchased vaccinated animals are less worried about picking up infections from neighbouring farms and therefore allow more contact than farmers who have unvaccinated animals. The fact that pigs sourced from the government breeding farms have higher risk of being seropositive against CSFV is obviously the result of prior vaccination. However, the risk factor “swill feeding” might indicate that natural infection is occurring in village backyard farms. Swill feeding has been cited as a risk factor for CSFV infections [36, 37]. Pig feed in the village backyard pig farms in Bhutan mainly comprises kitchen wastes (swill) and sometimes local feed like bran and weeds. Cooking of swill before feeding to the pigs and an improved biosecurity could further reduce the chance of an introduction of CSFV. Though wild boar are known to be one of the sources of CSFV transmission to domestic pigs [38], the presence of wild boar was not found to be a risk factor for seropositivity of CSFV in the current study. Wild boars are found to be present in almost all parts of Bhutan.


Lending of domestic breeding boars in the neighborhood backyard farms for breeding purposes would increases the number of contacts between farms. This kind of contact would probably also associated with a higher chance of virus transmission per contact, therefore increasing the odds of pigs being seropositive to PCV2. Once breeding boars get infected themselves, PCV2 is shed in the semen as well [39]. Therefore, indiscriminate use of breeding boars in village backyard farms should be avoided.

Buying pork from the local market could not be included in the modeling process for association with SIV, as zero farms who did not buy pork tested Buying pork from the local market could not be included in the modeling process for association with SIV, as zero farms who did not buy pork tested.

Acknowledgements

We thank Bernie Moonen-Leusen, Sjaak Quak and Yvon Geurts, Central Veterinary Institute, Lelystad, in the Netherlands for their assistance in carrying out the laboratory works. We thank the Royal Government of Bhutan for facilitating the conduct of this research. We thank Dr.Tashi, Dorji, in Bhutan, for his technical guidance. The authors thank the National Centre for Animal Health and Regional Livestock Development Centres in Bhutan for their logistic and technical support during the field work. The authors acknowledge the veterinarians, extension agents, farm managers and laboratory technicians involved in the survey. This study was funded by the Netherlands University Foundation for International Cooperation (NUFFIC) and Central Veterinary Institute (CVI), Lelystad, The Netherlands.

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17. Schild GC, Oxford JS, Dowdle WR, Coleman M, Pereira MS, Chakraverty P: Antigenic variation in current influenza A viruses: evidence for a high frequency of antigenic 'drift' for the Hong Kong virus. Bulletin of the World Health Organization 1974, 51(1):1-11.18. Munster VJ, De Wit E, Van Den Brand JMA, Herfst S, Schrauwen EJA, Bestebroer TM, Van Vijver DD, Boucher CA, Koopmans M, Rimmelzwaan GF et al: Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets. Science 2009, 325(5939):481-483.19. Štukelj M, Toplak I, Vengušt G: Prevalence of antibodies against selected pathogens in wild boars (sus scrofa) in Slovenia. Slovenian Veterinary Research 2014, 51:21-28.20. Wensvoort G, Terpstra C, Pol JM, ter Laak EA, Bloemraad M, de Kluyver EP, Kragten C, van Buiten L, den Besten A, Wagenaar F: Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Veterinary Quarterly 1991, 13(3):121-130.21. Nodelijk G: Porcine Reproductive and Respiratory Syndrome (PRRS) with special reference to clinical aspects and diagnosis: A review. Veterinary Quarterly 2002, 24(2):95-100.22. Bøtner A: Diagnosis of PRRS. Veterinary Microbiology 1997, 55(1–4):295-301.23. Clopper CJ, Pearson ES: The Use of Confidence or Fiducial Limits Illustrated in the Case of the Binomial. Biometrika 1934, 26(4):404-413.24. Isserlis L: On the Value of a Mean as Calculated from a Sample. Journal of the Royal Statistical Society 1918, 81(1):75-81.25. Dohoo I, Martin W, Stryhn H: Mixed model for discrete data. In: Veterinary Epidemiologic Research, 2nd edn. pp. 582. 2009(2nd Edition):582.26. Wangchuk S, Thapa B, Zangmo S, Jarman RG, Bhoomiboonchoo P, Gibbons RV: Influenza surveillance from November 2008 to 2011; including pandemic influenza A (H1N1) pdm09 in Bhutan. Blackwell Publishing Ltd 2012.27. Keenliside J: Pandemic Influenza A H1N1 in Swine and Other Animals Current Topics in Microbiology and Immunology 2013, 370:259 - 271.28. Martha I, Nelson., Marie R, Gramer M, Amy L, Vincent., Holmes. EC, : Global transmission of influenza viruses from humans to swine. 2012, 93:2195-2203.29. Vijaykrishna D, Poon LLM, Zhu HC, Ma SK, Li OTW, Cheung CL, Smith GJD, Peiris JSM, Guan Y: Reassortment of Pandemic H1N1/2009 Influenza A Virus in Swine. Science 2010, 328:1529.

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30. Liu W, Wei MT, Tong Y, Tang F, Zhang L, Fang L, Yang H, Cao WC: Seroprevalence and genetic characteristics of five subtypes of influenza A viruses in the Chinese pig population: A pooled data analysis. Veterinary Journal 2011, 187(2):200-206.31. West KH, Bystrom JM, Wojnarowicz C, Shantz N, Jacobson M, Allan GM, Haines DM, Clark EG, Krakowka S, McNeilly F et al: Myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus. Journal of Veterinary Diagnostic Investigation 1999, 11(6):530-532.32. Stevenson GW, Kiupel M, Mittal SK, Choi J, Latimer KS, Kanitz CL: Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors. Journal of Veterinary Diagnostic Investigation 2001, 13(1):57-62.33. Connor B, Grauvreau H, West K, Bogdan J, Ayroud M, Clark E, G. , Konoby C, Allan G, Ellis JA: Multiple porcine circovirus 2-associated abortion and reproductive failure in a multisite swine production unit. . Canadian Veterinary Journal 2001, 42:551 - 553.34. Huang YL, Pang V, Lin CM, Tsai YC, Chia MY, Deng MC, Chang CY, Jeng CR: Porcine circovirus type 2 (PCV2) infection decreases the efficacy of an attenuated classical swine fever virus (CSFV) vaccine. Veterinary Research 2011, 42(1).35. Segalés J, Allan GM, Domingo M: Porcine circovirus diseases. Animal Health Research Reviews 2005, 6(02):119-142.36. Edwards S, Fukusho A, Lefèvre P-C, Lipowski A, Pejsak Z, Roehe P, Westergaard J: Classical swine fever: the global situation. Veterinary Microbiology 2000, 73(2–3):103-119.37. Edwards S, Sands JJ: Evidence of circovirus infection in British pigs. Veterinary Record 1994, 134(26):680-681.38. Fritzemeier J, Teuffert J, Greiser-Wilke I, Staubach C, Schlüter H, Moennig V: Epidemiology of classical swine fever in Germany in the 1990s. Veterinary Microbiology 2000, 77(1–2):29-41.39. Rose N, Larour G, Le Diguerher G, Eveno E, Jolly JP, Blanchard P, Oger A, Le Dimna M, Jestin A, Madec F: Risk factors for porcine post-weaning multisystemic wasting syndrome (PMWS) in 149 French farrow-to-finish herds. Preventive Veterinary Medicine 2003, 61(3):209-225


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Chapter 3

Longitudinal study of selected viral diseases in professional pig breeding farms and village backyard farms in Bhutan

V. R.Mongera, c, J.A. Stegemanb, W.L.A. Loeffena

aCentral Veterinary Institute of Wageningen UR, Department of Virology,P.O. Box 65, 8200 AB Lelystad, The NetherlandsbDepartment of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CL Utrecht, The NetherlandscNational Centre for Animal Health, Department of Livestock, Ministry of Agriculture and Forests, Thimphu, Bhutan

In preparation

41


Abstract

The pig production system in Bhutan is mostly in the form of traditional backyard farms. Seroprevalences of porcine circovirus type 2 (PCV2), swine influenza virus H1N1 (SIV, H1N1) and hepatitis E (HEV) were previously found to be significantly higher in government breeding pig farms than in the backyard farms. Thus, there was a concern on the risk of transmission and spread of diseases to the backyard pig farms through the distribution of piglets. The main aim of this study was to to assess the dynamics of these diseases in piglets originating from large breeding farms, with possible difference in infection dynamics between piglets being sold to backyard farms and piglets remaining in the breeding farms. A longitudinal study was also carried out in three large government breeding farms (from two of which the piglets in this study originated) over a period of more than one-and-a-half year to determine infection dynamics in sows.

A survival analysis revealed no differences in overall seroconversions between piglets raised in government farm or in backyard farms, even though seroprevalences for PCV2 and HEV where significantly higher in piglets raised in government herds. In the sows in the government herds, seroprevalences against all four viruses [CSFV, SIV (H1N1), PCV2 and HEV] were high and increasing towards the end, showing that active circulation of these viruses, except for CSFV took place.

Large breeding farms providing piglets for fattening to backyard farms may be good for controlling notifiable diseases. However, this system also promotes the spread of diseases endemic in the breeding farms. Depending on the damages these endemic diseases may cause in the backyard system, in such a system an increased level of disease control in the breeding farm may be warranted,

Keywords: Longitudinal, CSF, SIV, PCV2, HEV, Bhutan, Backyard farm, Government farm, seroprevalence, Dynamic, piglets, sows

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Chapter 3


Longitudinal study of viral diseases in pig breeding and village farms

43

Introduction

Pig farming constitutes the livelihood of rural poor to socio-economically disadvantaged people in developing countries. It is a low investment alternative to produce meat and provide economic support for the family [1]. The majority of pig farmers in developing countries raise a small number of pigs in their backyards, primarily supplementing feed with kitchen waste [2]. Also in Bhutan the pig production system is mostly in the form of traditional backyard farms usinglocally available resources including kitchen wastes.

Biosecurity measures in the backyard holdings are generally poor or do not exist. Pigs are often purchased locally, which creates uncontrolled pig movement and a contact structure that promotes the spread of diseases. Poor biosecurity, uncontrolled pig movements and limited knowledge regarding pig health and diseases can significantly enhance the spread of infectious diseases between backyard farms. An alternative to this structure of backyard farms is a pyramidal structure with large breeding farms producing piglets for backyard farms, which creates a top-down contact structure that may help not only to improve the genetics of the backyard pigs, but also to control spread of diseases in backyard farms. However, when infections are present in the breeding farms there will be the risk of disseminating infectious agents very efficiently.

In a study by Monger et al (2014), a concern was raised on the risk of transmission and spread of diseases like porcine circovirus type 2 (PCV2), and swine influenza virus H1N1 (SIV, H1N1) from breeding farms to the backyard farms through the distribution of piglets. Seroprevalences of these diseases were found to be significantly higher in government breeding farms than in the backyard farms.

The main aim of this study was to to assess the dynamics of classical swine fever virus (CSFV), SIV (H1N1), PCV2 and hepatitis E virus (HEV) infections in piglets originating from large breeding farms. Of specific interest was the possible difference in infection dynamics between piglets being sold to backyard farms and piglets remaining in the breeding farms, thus exposed to a different environment. Infection dynamics were assessed through a longitudinal serological study on several batches of piglets. During the same period, a longitudinal study was carried out in three large government breeding farms (from two of which the piglets in this study originated) over a period of more than one-and-a-half year to determine infection dynamics in sows.


Figure 1.The survival curves for each individual batch (batch 1, 2 and 3) raised in backyard vs those raised in government farms.


Study areaThe study was carried out in three government breeding farms which are located in the

districts of Thimphu (west region), Sarpang (east-central region) and Mongar (east region).

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45

These breeding farms distribute piglets to the village backyard farms to improve local pigs through cross-breeding programs for fattening purpose. The size of these government farms currently ranges from 65-150 sows, with accompanying piglets and some replacement stock. The farm in Sarpang is the central breeding farm, providing replacement stock to both other government farms. A vaccination against CSF and foot-and-mouth disease (FMD) is carried out in all government breeding farms on an annual basis.

Table1. Selection of piglets from two government breeding farms, Thimphu and Sarpang, grouped into three batches for a longitudinal study.

Batch

1

2

3

Government Farm, Group

Thimphu,BackyardThimphu,Government

Sarpang, BackyardSarpang, Government

Thimphu,BackyardThimphu,Government

Total Animal

1017

10152020

Sampling strategy

PigletsThree batches of piglets from two government breeding farms, Thimphu (batches 1 and 3)

and Sarpang (batch 2), were selected for this part of the study (Table 1). The study of batches 1 and 2 was carried out from August 2011 until January 2012. The

study of batch 3 was carried out from August 2012 until December 2012. Within each batch, individual piglets were either sold to a backyard farm at the age of approximately 70 days or remained in the government farm as replacement breeding stock. The pigs in batch 1 and 2 were sampled five times and in batch 3 four times. The mean age of these pigs on each sampling was respectively 21, 70, 84, 132, and 242 days (the latter batch 1 and 2 only)

SowsA total of 101 sows were selected in the government breeding farms and followed up to

determine the seroprevalence and incidence of CSFV, SIV, PCV2 and HEV infections for a period of one-and-a-half year. Sows were sampled four times on a six-monthly basis in each of the farms. First sampling was done in September 2011, second sampling in March 2012, third sampling in September 2012 and the final sampling in March 2013.


Laboratory tests

Antibodies against classical swine fever virus (CSFV)The serum samples were tested in a virus neutralisation test (VNT) against a cell culture

adapted Alfort/187 strain [3] of CSFV as described by [4], with validated sensitivity of 93% and specificity of 100% at CVI.

Serum samples were considered to have antibodies against CSFV if it was positive to the CSFV strain in the VNT with titres >10. Antibodies against CSFV were mainly the result of vaccination, with no possibility to discriminate between vaccination and infection.

Antibodies against swine influenza virus (SIV)Serum samples were tested in a haemagglutination inhibition (HI) test using A/

Netherlands/602/2009 (pandemic H1N1) antigen [5]. The HI test was performed according to the standard procedures (OIE, 2008) and standardized using four haemagglutinin units (HU) per well. The starting dilution for testing sera was 1:10. Sera with a titre ≥ 20 were considered positive.

Sample collectionBlood samples were collected, using Sarstedt Monovette syringes from all selected pigs.

Serum was obtained by centrifugation of the coagulated blood samples and stored at -20oC until testing.

Antibody detection against HEVAntibody against HEV (IgG) in serum samples was measured using an enzyme-linked

immunosorbent assay (ELISA) kit, as described earlier [7]. This essay has an estimated sensitivity and specificity of 84% and 89% respectively. Samples with Percent Positivity (PP) values ≥ 25 PP were considered positive.

Antibodies against porcine circovirus type 2 (PCV2)Serum samples were tested in a commercially available indirect ELISA (Porcine Circovirus 2

Antibody Test Kit; BioChek BV, The Netherlands) according to the manufacturer’s instruction. The sensitivity and specificity of the kit were previously estimated at 85% and 95.6% respectively [6]. The presence or absence of antibody to PCV2 was determined by calculating the test sera-to-positive (S/P) ratio. Samples were considered positive if the S/P ratio was ≥ 0.5.

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47

1) A pig with a positive test results in any of the samplings, showing a significant rise in titre in the subsequent sampling (for CSFV and SIV defined as a fourfold increase and for PCV2 and HEV an increase of the S/P and PP by 0.3 and 20, respectively.2) A pig with a positive test result in the first sampling, showing at least the same titre (CSF, SIV), S/P (PCV2) or PP (HEV) in the second sampling. This criterion assumes that titres of maternally derived antibodies should significantly be reduced between the first and second sampling, with a lack of such a reduction suggesting an active immune response.3) A pig with a positive test result in the first sampling, showing at least the same titre (CSF, SIV), S/P (PCV2) or PP (HEV) in the second sampling. This criterion assumes that titres of maternally derived antibodies should significantly be reduced between the first and second sampling, with a lack of such a reduction suggesting an active immune response.

A cox proportional hazard survival model was used to find out the effect of group (backyard and government) and batch (1, 2 and 3) on the seroconversion against specific viruses ((CSF, SIV, PCV2 and HEV) over time in the piglets. The dependent variable was a seroconversion against a specific virus (CSFV, SIV, PCV2 and HEV) (1 = seroconversion, 0 = no seroconversion) and the independent variables were the group (backyard = 1, government farm = 2), batch (Thimphu 1 = 1, Sarpang = 2, Thimphu 2 = 3) and time (in days).

The statistical analysis was done in R (version 3.0.0; R Foundation for Statistical Computing, Vienna, Austria)


The seroprevalence in sows and piglets was calculated for each government farm and each virus at every time point. Seroprevalences in the three government farms were compared for each of the diseases using a random effects logistic regression model. The outcome of a specific infection was the dependent variable (1 = positive, 0 = negative). The collection time (four collections) and government farms served as explanatory variables and sows as random effect variable.

For each of the piglets in the study, the approximate moment of seroconversion was determined. The observation of seroconversions was hampered by the presence of maternal antibodies. Antibodies against each of these viruses in the first sampling were therefore assumed to be maternal antibodies and not yet the result of a seroconversion. Thereafter, the pigs were considered to have seroconvert as soon as they fulfilled one the following criteria:

A pig with a negative test result in any of the samplings becoming positive in the subsequent sampling.


Results

Seroprevalence in pigletsSeroprevalences against PCV2 and HEV were significantly higher in piglets raised in the

government farm, compared to those raised in backyard farms (P<0.001) (Tables 2 and 3). An overall increase of the seroprevalence was observed for these two viruses.

The seroprevalence against SIV decreased in both groups, with no detectable antibodies at the end of the study (Table 4).

The seroprevalence against CSFV, on the other hand, remained between 85 and 100% in both groups, during the whole study period (Table 5).

Seroconversions in pigletsOnly five seroconversions against SIV were observed in batch 3. Besides that, no

seroconversions against SIV occurred in any of the piglets.For CSFV, PCV2 and HEV the cox proportional hazard survival model showed that

differences between the batches were statistically significant. In Figure 1, the survival curves for each individual batch are therefore shown separately. There were no significant differences in seroconversions between pigs raised in the government farms vs. those raised in the backyard farms.

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Table 2.Seroprevalence against PCV2 indifferent batches of piglets in a longitudinal study. The total period of sampling in batch 1 and 2 are five times and in batch 3 is four times.



49

Table 3.Seroprevalence against HEV indifferent batches of piglets in a longitudinal study.The total period of sampling in batch 1 and 2 are five times and in batch 3 is four times.

Table 4.Seroprevalence against SIV (H1N1) indifferent batches of piglets in a longitudinal study. The total period of sampling in batch 1 and 2 are five times and in batch 3 is four times.


Seroprevalence in sowsFrom the 101 sows that were initially included in the study, 74 were still present at the end.

Most loss to follow up occurred between the third and fourth sampling. Seroprevalences against all four viruses were high, up to 100% in the end (Table 6).

All seroprevalences increased during the study period, although only for SIV statistically significant (P<0.001). Statistically significant differences between the government farms were only observed for CSF during the first sampling (Thimpu lower seroprevalence than the other two herds, P<0.1) and for SIV in general (highest seroprevalence in Mongar, followed by Sarpang and then Thimpu (P<0.001).

Discussion

The main aim of this study was to to assess the dynamics of classical swine fever virus (CSFV), SIV (H1N1), PCV2 and HEV infections in piglets originating from large breeding farms, either being raised in the government farm or in backyard farms. A survival analysis revealed no differences in overall seroconversions between piglets raised in government farm or in backyard farms, even though seroprevalences for PCV2 and HEV were significantly higher in piglets raised in government herds. In the sows in the government herds, seroprevalences against all four viruses where high [CSF, SIV (H1N1), PCV2 and HEV] and increased towards the end of the study, showing that active circulation of these viruses, except for CSF took place, which will have contributed to the exposure of the piglets. The antibodies against CSFV were most likely due to the vaccination.

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Table 5.Seroprevalence against HEV in different batches of piglets in a longitudinal study.The total period of sampling in batch 1 and 2 are five times and in batck 3 is four times.



51

Table 6.Seroprevalence against CSF, SIV (H1N1), PCV2 and HEV in sows from three government farms in a longitudinal study over a period of two years.

Differences between the three batches of piglets, depending on time and herd of origin, were quite large. It’s likely that exposure of the piglets in the government herds is highly variable. In some cases the majority of the seroconversions had already taken place at the age of 70-85 days, which indicates that the piglets had been exposed to the virus in the government breeding herd. These infections do not only lead to similar serological profiles in pigs raised in government farms vs. those raised in backyard farms, but may also contribute to spread of the viruses to backyard farms. In general, our finding about time of seroconversion were comparable to previous studies, in which the majority of pigs seroconvert to PCV2 between 60-120 days of age [8, 9], and to HEV between 30 and 90 days of age [10].

Except for five seroconversions against SIV (H1N1) in one batch, no seroconversions against SIV (H1N1) were observed in any of the piglets. Virus circulation amongst the sows did take place, however, as evidenced by the increasing seroprevalence in time. Nevertheless, the batches of piglets that were involved in the study were apparently not exposed to the virus in the government herds. In a previous study [11], it was suggested that part of the infections with SIV in swine, especially the ones in the backyard farms, may have been the result of exposure to infected humans. In the current study period there were no indications that this has happened to the piglets. Exposure of these piglets to human influenza may have been limited, as the end of the study period coincided with the start of the influenza season in humans, which is generally with the start of the winter from December on [12].


Due to the practice of vaccination against CSF in the government farms, no conclusions could be drawn regarding the circulation of CSFV from this study, as most, if not all seropositives were the result of the vaccination. The CSF vaccination in the government breeding farms is usually carried out soon after the piglets are weaned at 45-50 days, before they are sold to the farmers in the backyard farms. The differences that were observed between the batches in the survival analysis are likely due to irregularities in the vaccination of these different batches.

For diseases endemic in the government farms, the government farm seems to be the main source for transmission of the virus to the backyard farms. Nevertheless, this type of system (professional breeding farms) is often promoted to control notifiable diseases. It is easier to control these notifiable diseases in large breeding herds than in the backyard farms. The latter are often scattered and under a poor management system, with an intricate contact structure. Controlling the disease in the breeding herds and providing piglets free of the disease to backyard farms should thus result in a disease-free population. This type of pyramidal system may be good for controlling notifiable diseases, which are being well controlled in the breeding farm. However, this system also promotes the spread of diseases endemic in the breeding farm. Depending on the damages these endemic diseases may cause in the backyard system, in such a system an increased level of disease control in the breeding farm may be warranted, to guarantee freedom of certain diseases in the backyards. Part of such a disease control can be surveillance systems, quarantine measures when new pigs enter the breeding herd, vaccination and other biosecurity measures.

Acknowledgements

We thank Bernie Moonen-Leusen and SjaakQuak, Central Veterinary Institute, Lelystad, in the Netherlands for their assistance in carrying out the laboratory works. We thank Jan van den Broek for his assistance in the statistical analysis of the data. The authors thank the Program Directors of the National Centre for Animal Health and Regional Livestock Development Centres in Bhutan for their logistic and technical support during the field work. The authors acknowledge the veterinarians, extension agents, farm managers and laboratory technicians for their assistance in the study. This study was funded by the Netherlands University Foundation for International Cooperation (NUFFIC, NFP Grant CF 7436) and Central Veterinary Institute (CVI, WOT01-003-010-01), Lelystad, The Netherlands.

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53

References1. Huynh TTT, Aarnink AJA, Drucker AG, Verstegen MWA: Pig production in Cambodia, Laos, Philippines and Vietnam. A review.Asian Journal of Agriculture Development 2007, 4(69-90).2. Furukawa T, Nirasawa K, Ishii K, Thuy LT, Satoh M:Comparison of production systems for efficient use of indigenous pig breeds in developing countries. Animal Science Journal 2013, 84(3):200-205.3. Dahle J, Liess B: Comparative-study with cloned classical swine fever virus-strains alfort and glentorf-clinical, pathological, virological and serological findings in weaner pigs. Wiener Tierarztliche Monatsschrift 1995, 82(8):232-238.4. Terpstra C, Bloemraad M, Gielkens ALJ: The neutralizing peroxidase-linked assay for detection of antibody against swine fever virus. Veterinary Microbiology 1984, 9(2):113-120.5. Munster VJ, De Wit E, Van Den Brand JMA, Herfst S, Schrauwen EJA, Bestebroer TM, Van Vijver DD, Boucher CA, Koopmans M, Rimmelzwaan GF et al: Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets. Science 2009, 325(5939):481-483.6. Štukelj M, Toplak I, Vengušt G: Prevalence of antibodies against selected pathogens in wild boars (sus scrofa) in Slovenia. Slovenian Veterinary Research 2014, 51:21-28.7. Van der Poel WHM, Pavio N, van der Goot J, van Es M, Martin M, Engel B: Development and validation of a genotype 3 recombinant protein-based immunoassa for hepatitis E virus serology in swine. Brazilian Journal of Medical and Biological Research 2014, 47:334-339.8. Larochelle R, Magar R, D'Allaire S: Comparative serologic and virologic study of commercial swine herds with and without postweaning multisystemic wasting syndrome. Canadian Journal of Veterinary Research 2003, 67(2):114-120.9. Sibila M, Calsamiglia M, Segalés J, Blanchard P, Badiella L, Dimna ML, Jestin A, Domingo M: Use of a polymerase chain reaction assay and an ELISA to monitor porcine circovirus type 2 infection in pigs from farms with and without postweaning multisystemic wasting syndrome. American Journal of Veterinary Research 2004:88-92.


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10. Li W, She R, Wei H, Zhao J, Wang Y, Sun Q, Zhang Y, Wang D, Li R: Prevalence of hepatitis E virus in swine under different breeding environment and abattoir in Beijing, China. Veterinary Microbiology 2009, 133(1–2):75-83.11. Monger VR, Stegeman JA, Koop G, Dukpa K, Tenzin T, Loeffen WLA: Seroprevalence and associated risk factors of important pig viral diseases in Bhutan. Preventive Veterinary Medicine 2014(0).12. Hsieh Y-H: Pandemic influenza A (H1N1) during winter influenza season in the southern hemisphere. Influenza and Other Respiratory Viruses 2010, 4(4):187-197.

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Chapter 4

Genetic characterization of porcine circovirus type 2 (PCV2) in pigs of Bhutan

V.R.Monger1, 4, W.L.A.Loeffen1, K.Kus3, J.A.Stegeman2, K.Dukpa4, K.Szymanek3, K. Podgórska3

1Central Veterinary Institute of Wageningen UR, Department of Virology,8200 AB Lelystad, The Netherlands2Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CL Utrecht, The Netherlands3National Veterinary Research Institute, Department of Swine Diseases, Partyzantow 57, 24-100 Pulawy, Poland4National Centre for Animal Health, Department of Livestock, Ministry of Agriculture and Forests, Thimphu, Bhutan

Published in: Transboundary and emerging diseases, 2015 February 23doi: 10.1111/tbed.12383.

55


Abstract

Porcine circovirus (PCV) is a small non-enveloped virus with a single-stranded circular DNA with two antigenically and genetically different species, PCV1 and PCV2. Among these two, PCV2 is responsible for multifactorial disease syndromes, the most important disease known as PCV2-systemic disease (PCV2-SD), previously known as post-weaning multi-systemic wasting syndrome (PMWS). The epidemiological situation is dynamically changing and new strains including recombinant PCV2 have emerged in Asia. In Bhutan, pigs are important livestock and play a very important role in providing meat and income for rural farmers. Although high rate of pigs seropositive against PCV2 was described in Bhutan, there was no virological evidence for PCV2 infections. This study was conducted to confirm the presence of PCV2 through detection of PCV2 DNA and molecular characterization of PCV2 strains in tissue and blood samples collected from Bhutanese pigs. PCV2 genome was detected in 16 out of 34 tissue samples pigs from the government farm. In 9 pigs, very high level of viral replication indicated that PCV2-SD was detected. Phylogenetic analysis performed with a set of GenBank sequences revealed that the Bhutanese PCV2 strains belonged to the PCV2b genotype and grouped with cluster 1C.

Keywords: Porcine circovirus type 2, Phylogenetic analysis, Sequence, Genome, pigs, Bhutan

56

Chapter 4


Introduction

Porcine circovirus (PCV) is a small non-enveloped virus with a single-stranded circular DNA and a genome of about 1.76 kb classified in the Circoviridaefamily[1]. Two antigenically and genetically different species, PCV1 and PCV2, were identified. PCV1 is considered nonpathogenic whilst the course of infection with PCV2 may be subclinical or evolve into a range of multifactorial disease syndromes collectively described as porcine circovirus associated disease (PCVD). One of the most important diseases in this group is PCV2-systemic disease (PCV2-SD), previously known as post-weaning multi-systemic wasting syndrome (PMWS). The disease causes elevated mortality in young weanling pigs and significant economic losses worldwide [1, 2]. PCV2 infection has also been linked with porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), reproductive failure, granulomatous enteritis, necrotizing lymphadenitis, exudative epidermitis and congenital tremor [3].

Four functional, partially overlapping open reading frames (ORFs) identified in a PCV2 genome encode a Rep protein involved in replication and its splicing variants (ORF1), a capsid protein (ORF2), a protein involved in pathogenesis (ORF3) and a protein involved in apoptosis suppression (ORF4) [4]. Although a relatively low level of diversity is observed within PCV2, three major genotypes were distinguished, including PCV2a (previously Group 2), PCV2b (previously Group 1) and PCV2c, based on retrospective analysis in Denmark [5-7]. Existence of two additional genotypes was also suggested [3, 8].

Phylogenetic analysis indicated that around 2003 a genetic shift from PCV2a to the currently predominating and probably more pathogenic PCV2b strains took place. Recent studies in Asia suggest that the epidemiological situation is dynamically changing and new strains including recombinant PCV2 have emerged [9-11].

In Bhutan, pigs play a very important role in providing meat and income for rural farmers. There are three government breeding farms that supply pigs to village backyard farms (Figure 1).

In a serological study conducted in 2013, 73% of the pigs sampled in Bhutan were seropositive against PCV2 [12]. Since these pigs were not vaccinated against PCV2, seropositivity indicated circulation of PCV2 in examined farms. In Bhutan, so far no studies have been carried out to find any virological evidence of PCV2, although there have been history of clinical diseases related to PCV2 like reproductive failure in sows, wasting and increased mortality amongst weaner piglets in the government breeding farm.

This study was conducted to confirm the presence of PCV2 in the Bhutanese pigs through detection of PCV2 DNA and molecular characterization of PCV2 strains circulating in the Bhutanese pigs.

57

Genetic characterization of porcine circovirus type 2 in pigs of Bhutan


Materials and methods

Sample collectionA total of 62 tissue samples (mostly spleen and lungs) were collected from 34 pigs in a

government pig breeding farm located at Yusepang, Thimphu district of Bhutan (Figure 1). All pigs had died with multi-systemic lesions and clinical signs such as respiratory distress, wasting and enteritis. Samples were collected at the time of autopsy of these pigs, submitted to the National Centre for Animal Health (NCAH). The age of pigs ranged from 1 to 12 months. Additionally, serum samples from 28 unthrifty pigs from 8 village backyard farms from Samtse district (southern region of the country) (Figure 1) were submitted by the District Veterinary Hospital to NCAH. All samples were stored at -80°C until laboratory analyses. All examined farms, including the government breeding farm, Yusipang, received pigs from the main nucleus farm in Gelephu, which is located in the south of Bhutan. Details of origin, type of samples collected are summarized in Tables 1 and 2.

Figure 1. Map of Bhutan showing the location of three government pig breeding farms (red triangle); and selected district (Samtse) highlighted in blue colour. The samples were submitted to NCAH from a Regional Pig breeding Centre, Yusepang, indicated by a red triangle and backyard farms from four villages under Samtse District, highlighted by red stars.

58

Chapter 4


Laboratory analysisSerum samples were tested with Ingezim IgM/IgG ELISA (Ingenasa, Spain) for the presence

of antibodies specific to PCV2. Approximate stage of infection (active infection, recent infection or past infection) was estimated based on relation of IgM to IgG antibodies according to the manufacturer’s recommendations. Both sera and tissue homogenate samples were submitted for extraction of total nucleic acids with MagNa Pure LC extraction station and compatible MagNA Pure LC Total Nucleid Acid Isolation Kit (Roche, Germany). PCV2 DNA was detected using previously described oligonucleotides [13] and protocol [14]. Quantitative results were obtained by extrapolation of obtained Ct values to a standard curve constructed based on duplicates

Figure 2. Phylogenetic tree constructed based on whole genome sequences (A) and ORF2 328 sequences (B). Phylogenetic trees were constructed based on N-J algorithm, 65 sequences from 329 GenBank were included into the analysis. Names include three letter country code and a year of 330 submission to GenBank. The arrows indicate Bhutanese (BTN) strains. Classification of 331 genotypes and clusters was based on Olvera et al. (2007). Rec. cluster = recombinant cluster.

59



of serial dilutions of pIC-PCV2 plasmid carrying whole PCV2 genome (Queens University, Belfast, UK). The reaction was linear within a range of 106-101 DNA copies/reaction with a correlation coefficient (R2) above 0.98. Results were expressed as copies/ml (serum samples) or copies/g (tissue samples).

All the samples with Ct values below 30 (corresponding to the limit of detection of the sequencing PCR, appr. 102 copies/reaction) were submitted for sequencing. PCV2 genome was sequenced in two overlapping fragments based on previously described primer pairs CBB1/CBB2 and CBB3/CSZ2 [15].

Phylogenetic analysis was performed using CLC DNA Workbench 6.6.1 software (CLC bio), based on an accurate alghorithm for alignment and neighbor-joining algorithm for construction of a phylogenetic tree. Sequences obtained in the present study were analysed in comparison to 65 sequences downloaded from GenBank, representing known genetic diversity of PCV2.

Due to the low positivity of samples from backyard farms, it was not possible to use the same sequencing system. Partial ORF2 sequence (493 nt; pos. 1094-1587) from sample No. 13 was obtained by sequencing a PCR product amplified with previously described primers [16].

Results

Detection of antibodies specific to PCV2Antibodies specific to PCV2 were detected in the serum samples collected from the

backyard farms. Based on comparison of IgM/IgG level, the infection status was interpreted as past infection in 14 samples, active infection in 1 sample and recent infection in 4 samples. Moderate viral loads were detected in 2 samples collected from pigs classified as recently infected (Table 1).

60

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61

Table 1. Results of serological and quantitative real-time PCR analysis of sera samples collected in backyard farms.



Chapter 4

62

Genetic characterization of PCV2 strainPCV2 genome was detected in tissues from 16 out of 34 pigs from the government farm.

Detected viral loads ranged from 104.45 to 1011.5 copies/g depending on the pig and tissue tested (Table 2). In 11 pigs, viral loads exceeding107 copies/g in at least one examined tissue sample indicated very high level of viral replication.

Overall, 18 whole genome sequences from tissues of 10 pigs were obtained, from 1 to 3 sequences per pig. Two strains, differing by 1 nucleotide substitution C or T at a position 1673, were identified in the government farm (KM975682 and KM975683 respectively). The substitution was at a 3rd codon position and did not lead to an amino acid change. Seven pigs were infected with strain KM975682 (1673C), 2 pigs with strain KM975683 (1673T) and 1 pig was infected with both detected strains (Table 2). A 493 nucleotides fragment of ORF2 sequence (pos. 1094-1586)from serum sample No. 13 collected in a backyard farm was compared to other sequences obtained in the study. Phylogenetic analysis revealed that the corresponding fragments of sequences obtained in the government and backyard farmswere identical.

Phylogenetic analysis performed with a set of GenBank sequences revealed that the Bhutanese PCV2 strains belonged to PCV2b genotype and grouped within cluster 1C according to the classification proposed by Olvera et al. [5] (Figure 2 (A)).

The length of the genome was 1 nucleotide longer compared to other PCV2b sequences (1768 nt) due to the T insertion at a position 1005 in the intergenic region between ORF1 and ORF2. Similar to other strains in cluster 1C, one amino acid lysine extension at the C-terminus of the deduced amino acid capsid protein sequence was observed. The signature motif proposed to distinguish PCV2a (positions 1479-1468, amino acid residues TNKI) and PCV2b (positions 1479-1468, amino acid residues SNPR(L) strains was identical to those previously reported for 1C strains (SNPRL) [10, 17]. Also other marker positions in the deduced amino acid sequence represented composition typical for 1C strains (Table 3) [10, 18]

BLAST search revealed multiple Chinese PCV2 sequences of high similarity to the Bhutanese strains. The highest percentage of genetic identity was observed in case of AY291317 (99.83%) and AY943819 as well as KF742543 (99.77%) strains collected in 2003, 2005 and 2007 respectively (Figure 2 A, B). The strain AY291317 of highest similarity was isolated from a pig with PCV2-systemic disease. No information on clinical status of pigs infected with two other strains was available. Considering that ORF2 carries the highest level of genetic variability in the PCV2 genome and that multiple GenBank submissions are restricted to this fragment, an additional analysis focused on ORF2 of the strains from group 1C was performed (Figure 2 (B)). Phylogenetic analysis revealed that one ORF2 sequence originating from China from 2003 (DQ231516) was identical to KM975682 (Bhutanese strain with C substitution at position 1117).



63

Table 2. Results of quantitative real-time PCR analysis of tissue samples from a government breeding farm. Two strains were detected differing with C/T substitution at pos. 1673 – strain KM975682 (C) and strain KM975683 (T).


64

Tabl

e 3.

Com

paris

on o

f put

ativ

e am

ino

acid

subs

titut

ions

in m

arke

r pos

ition

s of a

cap

sid

gene

(OR

F2) r

epor

ted

prev

ious

ly a

nd o

bser

ved

in th

is st

udy

(BTN

).

Chapter 4


65

Apart from Chinese strains, two European strains from Belgium (EU909686) and Serbia (HQ378158) were identified in the same branch of the phylogenetic tree with the genetic identity to Bhutanese strains ranging from 99.38 to 99.43%.

Comparison of sequences obtained in Bhutan to other sequences from GenBank indicated a high level of genetic similarity. However, Bhutanese sequences differed at several positions, including a T-insertion at position 1005 changing the overall length of the genome, a substitution of A to C at a genome position 587 and a substitution of A to G at a genome position 1117 (1116 in other PCV2b strains). Also, none of the highly related sequences had a T substitution at 1673 nt position that was detected in strain KM975683. Only the A1117G substitution located at the first codon position lead to a change of a putative amino acid sequence within the capsid protein. The change from Tyr to His occurred at the last position of a linear immune-reactive epitope (193-207 aa) within a predicted sulphate binding site [19]. Although the above mentioned nucleotide features were only attributed to the Bhutanese strains in this particular cluster of highly similar sequences, they previously occurred in other PCV2 sequences of different origin and variable level of nucleotide similarity to the Bhutanese strains.

Discussion

A previous seroprevalence study revealed the presence of antibodies against PCV2 in the Bhutanese pig population, especially amongst pigs in the government pig breeding farms [12]. However, no information was available on clinical PCVD and type of PCV2 strains circulating in the country, although clinical signs such as abortions, mummified fetuses, progressive weight loss and respiratory lesions were frequently reported in the government pig breeding farms.

This study is the first report on the molecular characteristics of the Bhutanese PCV2 strains. The Bhutanese PCV2 strains belong to PCV2b genotype and group within cluster 1C according to classification criteria of Olvera et al. [5]. This finding is in agreement with the recently observed evolutionary trend in this geographic area. Strains belonging to cluster 1C became predominant in Southern China during the last few years [10].

Recent experimental infection studies indicated that strains of that cluster with a lysine extension at the C-terminus of the capsid protein displayed increased virulence in vivo [20]. However, the occurrence of clinical PCVD in Bhutan was not previously investigated. According to widely accepted criteria defined by the international PCVD Consortium (www.pcvd.net), the diagnosis of PCVD is based on the presence of clinical lesions and detection of abundantvirus associated with histopathological lesions in tissues [1]. In this study, although recommended in situ analysis was not possible to perform due to the lack of formalin-fixed tissue samples, high viral loads detected in some of the examined tissue samples may indirectly indicate the occurrence of PCV2-SD. There is a correlation between high viral loads in tissues and severity of histopathological lesions as well as the intensity of in situ antigen detection [21].



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The association of high levels of PCV2 DNA with clinical PCV2-SD was reported in other studies as well [5,14]. A threshold of viral load indicating clinical PCVD with high sensitivity and specificity was estimated for 106.8-108.4copies/g depending on the type of tissue [21]. For spleen, lung and tonsils used in the present study, cut off values 107.4, 106.9 and 108.4 copies/g were proposed respectively. Based on these criteria, 9 of examined pigs could be classified as affected with PCV2-SD.

A genetic identity of 100% between the 493 nucleotide ORF2 fragments suggests that the same PCV2 strain circulates in the backyard and government farm. It is most likely that PCV2 infection was transmitted from government breeding farms which supplied pigs to the backyard farms. The government breeding farms import live pigs from other countries and supply hybridized piglets to the farmers for fattening and cross-breeding purposes [12]. The most recent import of live pigs to Bhutan was from the United Kingdom in 2003. The comparison of partial ORF2 sequences from PCV2 strains collected in UK during that period (AY325515, AY325517, AY325905, AY325516, AY325906, AY325518 and AY325519) did not reveal any epidemiological relationships with Bhutanese strains (data not shown). Most of the UK sequences belonged to cluster 1A/1B and two short 246 nt fragments (AY325518 and AY325519) clustered with archive Danish sequences classified in PCV2c genotype.

The phylogenetic study revealed that Bhutanese PCV2 strains belonged to PCV2b genotype and grouped within cluster 1C having high similarity with multiple Chinese PCV2 sequences and two European strains from Belgium (EU909686) and Serbia (HQ378158). Based on this result, it was not possible to unequivocally trace the origin of the Bhutanese PCV2 strains. Although Bhutan and China share a border, there are no official records of live pig trade between the two countries and there has been no official record on the import from Belgium and Serbia. However, there has been import of several exotic breeds before 2003 from countries like India, Thailand, Philippines and Australia [22].

In conclusion, this study confirmed the presence of PCV2 in Bhutan as evidenced from the results of quantitative real-time PCR and sequencing data. The results indirectly indicated the presence of not only subclinical infections but also clinical PCVD. The government breeding farms are likely the source of PCV2 transmission to village backyard farms. Therefore, vaccination against PCV2 may be initiated in the government pig breeding farms in order to limit the spread of the disease to backyard farms. However, it is necessary to have more insight into the occurrence of PCVD and the losses incurred before the initiation of vaccination. It is also recommended that piglet mortality, especially in weaners, should be thoroughly investigated and samples submitted for regular monitoring of the presence of PCVD in the farms. At the same time, strict quarantine regulations and testing of imported live pigs should be carried out to prevent introduction of new PCV2 strains into the country.

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Acknowledgements

The authors thank the Veterinary Research Institute, Department of Swine Diseases, Partyzantow 57, 24-100 Pulawy, Poland for carrying out the laboratory analysis. We would like to sincerely thank prof. Gordon Allan (Queens University, Belfast, UK) who kindly provided pIC-PCV2 plasmid within NMSACC-PCVD 518432 SSA (FP6) collaboration. We thank Bernie Moonen-Leusen and SjaakQuak, Central Veterinary Institute, Lelystad, in the Netherlands for their assistance in preparation of samples. The authors wish to thank the laboratory technicians in Bhutan for collecting the samples for the study.

The study was supported by Polish Ministry of Science and Higher Education, project PBS2/A8/27/2014 and the Netherlands University Foundation for International Cooperation (NUFFIC, NFP Grant CF 7436).

References1. Segales J: Porcine circovirus type 2 (PCV2) infections: clinical signs, pathology and laboratory diagnosis. Virus Res 2012, 164(1-2):10-19.2. Allan G, Krakowka S, Ellis J, Charreyre C: Discovery and evolving history of two genetically related but phenotypically different viruses, porcine circoviruses1 and 2. Virus Res 2012, 164(1-2):4-9.3. Segales J, Kekarainen T, Cortey M: The natural history of porcine circovirus type 2: from an inoffensive virus to a devastating swine disease? Vet Microbiol 2013, 165 (1-2) :13-20.4. Lv QZ, Guo KK, Zhang YM: Current understanding of genomic DNA of porcine circovirus type 2. Virus Genes 2014.5. Olvera A, Cortey M, Segales J: Molecular evolution of porcine circovirus type 2 genomes: phylogeny and clonality. Virology 2007, 357(2):175-185.6. Dupont K, Nielsen EO, Baekbo P, Larsen LE: Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case-control study supports a shift in genotypes with time. Vet Microbiol 2008, 128(1-2):56-64.7. Grau-Roma L, Crisci E, Sibila M, Lopez-Soria S, Nofrarias M, Cortey M, Fraile L, Olvera A, Segales J: A proposal on porcine circovirus type 2(PCV2) genotype definition and their relation with postweaning multisystemic wasting syndrome (PMWS) occurrence. Vet Microbiol 2008, 128(1-2):23-35.



8. Guo LJ, Lu YH, Wei YW, Huang LP, Liu CM: Porcine circovirus type 2 (PCV2): genetic variation and newly emerging genotypes in China. Virol J 2010, 7:273.9. Cai L, Ni J, Xia Y, Zi Z, Ning K, Qiu P, Li X, Wang B, Liu Q, Hu D et al: Identification of an emerging recombinant cluster in porcine circovirus type 2. Virus Res 2012, 165(1):95-102.10. Wei C, Zhang M, Chen Y, Xie J, Huang Z, Zhu W, Xu T, Cao Z, Zhou P, Su S et al: Genetic evolution and phylogenetic analysis of porcine circovirus type 2 infections in southern China from 2011 to 2012. Infect Genet Evol 2013, 17:87-92.11. Huynh TM, Nguyen BH, Nguyen VG, Dang HA, Mai TN, Tran TH, Ngo MH, Le VT, Vu TN, Ta TK et al: Phylogenetic and Phylogeographic Analyses of Porcine Circovirus Type 2 Among Pig Farms in Vietnam. Transbound Emerg Dis 2013.12. Monger VR, Stegeman JA, Koop G, Dukpa K, Tenzin T, Loeffen WL: Seroprevalence and associated risk factors of important pig viral diseases in Bhutan. Prev Vet Med 2014.13. Opriessnig T, Yu S, Gallup JM, Evans RB, Fenaux M, Pallares F, Thacker EL, Brockus CW, Ackermann MR, Thomas P et al: Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus. Vet Pathol 2003, 40(5):521-529.14. Podgorska K, Stadejek T: Porcine circovirus type 2 viremia and seroconversion in pigs from a farm affected by postweaning multisystemic wasting syndrome. Pol J Vet Sci 2011, 14(4):667-669.15. Csagola A, Kecskemeti S, Kardos G, Kiss I, Tuboly T: Genetic characterization of type 2 porcine circoviruses detected in Hungarian wild boars. Arch Virol 2006, 151(3):495-507.16. Ouardani M, Wilson L, Jette R, Montpetit C, Dea S: Multiplex PCR for detection and typing of porcine circoviruses. J Clin Microbiol 1999, 37(12):3917-3924.17. Cheung AK, Lager KM, Kohutyuk OI, Vincent AL, Henry SC, Baker RB, Rowland RR, Dunham AG: Detection of two porcine circovirus type 2 genotypic groups in United States swine herds. Arch Virol 2007, 152(5):1035-1044.

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18. Li W, Wang X, Ma T, Feng Z, Li Y, Jiang P: Genetic analysis of porcine circovirus type 2 (PCV2) strains isolated between 2001 and 2009: genotype PCV2b predominate in postweaning multisystemic wasting syndrome occurrences in eastern China. Virus Genes 2010, 40(2):244-251.19. Mahe D, Blanchard P, Truong C, Arnauld C, Le Cann P, Cariolet R, Madec F, Albina E, Jestin A: Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes. J Gen Virol 2000, 81(Pt 7):1815-1824.20. Guo L, Fu Y, Wang Y, Lu Y, Wei Y, Tang Q, Fan P, Liu J, Zhang L, Zhang F: A porcine circovirus type 2 (PCV2) mutant with 234 amino acids in capsid protein showed more virulence in vivo, compared with classical PCV2a/b strain. PLoS One 2012, 7(7):e41463.21. Harding JC, Baker CD, Tumber A, McIntosh KA, Parker SE, Middleton DM, Hill JE, Ellis JA, Krakowka S: Porcine circovirus-2 DNA concentration distinguishes wasting from nonwasting pigs and is correlated with lesion distribution, severity, and nucleocapsid staining intensity. J Vet Diagn Invest 2008, 20(3):274-282.22. Nidup K, Tshering D, Wangdi S, Gyeltshen C, Phuntsho T, Moran C: Farming and biodiversity of pigs in Bhutan. Animal Genetic Resources © Food and Agriculture Organization of the United Nations, 2011 2011, 48:47 - 61.

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Chapter 5

Prevalence and phylogeny of Hepatitis E virus (HEV) in pigs in Bhutan

V. R.Mongera,c, R. Hakze-van der Honinga, W. H. M. van der Poela, J.A. Stegemanb, W.L.A. Loeffena


In preparation


Abstract

HEV is the major cause of fecal-orally transmitted and waterborne epidemics of hepatitis in humans in the developing countries of Asia and Africa and an important public-health concern. It is caused by non-enveloped RNA virus classified in the genus Orthohepevirus in the family Hepeviridae. Currently, the situation of HEV infection in humans and pigs in Bhutan is not known. The aim of this study was to determine the HEV seroprevalence in three government pig breeding farms and village backyard farms and genetically characterize HEV strains circulating in the Bhutanese pig population. A total of 829 pig serum samples were obtained from three government breeding pig farms and village backyard farms between October 2011 and February 2012. Subsequently, 431 faecal samples were collected towards the end of 2014 from three government breeding farms and village backyard farms, representing animals of age groups from 48 days to 6 months. Enzyme-linked immunosorbent assay (ELISA) revealed that 73% [95% confidence interval (CI) 69–75%] of the pigs in the government breeding farms and 51% [95% (CI) 44–58%] of the pigs in the village backyard farms tested positive for anti-HEV IgG respectively. HEV RNA was detected in the feces of 106 out of 431 pigs (25%, 95% CI 21–29%). Through phylogenetic analysis using the ORF2 region, the Bhutanese isolates were characterized as genotype 4 with highest similarity (93.7%) with the Indian isolate IND-SW-00-01. The serological prevalence of anti-HEV IgG and genetic characterization of HEV in pigs from Bhutan was documented in this study for the first time with a high prevalence of HEV antibodies and observation of genotype 4.

Keywords: Bhutan, serosurveillance, Hepatitis E virus, ELISA, PCR, Genetic characterization, government farm, Backyard farm, Exotic pigs, Local pigs.

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Introduction

Hepatitis E virus (HEV) is a non-enveloped RNA virus classified in the genus Orthohepevirus in the family Hepeviridae[1], consisting of a genome with three open reading frames [2]. The virus is transmitted primarily by the faecal-oral route and is associated with both sporadic infections and epidemics in areas with poor sanitation and weak public-health infrastructures. HEV is the major cause of faecal-orally transmitted and waterborne epidemics of hepatitis in humans in the developing countries of Asia and Africa. As such it is an important public-health concern [3]. Lately, infections have also become a problem in industrialized countries mainly due to eating raw or undercooked pork [4] and wild boar meat [5], but also due to transfusion of blood from infected persons to immune compromised recipients.

The first swine HEV was isolated and characterized in the United States [6]. In the human population there are currently 4 genotypes of HEV recognized [3]. Genotypes 1 and 2, which cause infection only in humans, and genotypes 3 and 4, which also infect pigs and other mammals [7]. Pigs are believed to be a major animal reservoir, being involved in natural cross-species infections [8] and can be infected with strains of HEV genotypes 3 and 4 [9]. It is still unclear whether genotypes 1 and 2 HEVs exist in pigs at all. Domestic pigs have been reported to be susceptible to infection with a human HEV strain under experimental conditions [10].

High seroprevalences of HEV antibodies in pig populations have been reported around the world, with up to 95% in U.S. pigs[11], 45% among 6-month-old piglets in Canada [12] and up to 90% in Australian pigs [13]. The seroprevalence of HEV in pigs in several countries in Europe has been found to be around 80% [14]. In the US (genotype 3) and Taiwan (genotype 4), both human and swine HEV isolates belong to the same genotype [15, 16]. This suggests a zoonotic connection. In India, on the other hand, human and swine HEV isolates belong to different genotypes, 1 and 4 respectively [17], which points to independent circulation of the virus in both populations. Also in Nepal, HEV was found in domestic pigs [18]. Currently, the situation of HEV infection in humans and pigs in Bhutan is not known. In Bhutan many piglets that are raised in backyard farms are provided by a few, governmental, breeding farms only, following a highly pyramidal system. Such a system is sometimes suggested as a solution in the control of epidemic diseases, but it is not yet known what the effect would be on the spread of endemic diseases. Furthermore, the results of this study will contribute to a better understanding of the epidemiology of HEV on a multi-country level, comparing the situation in Bhutan to neighboring countries.

The aim of this study was to determine the HEV seroprevalence in three government pig breeding farms and village backyard farms and genetically characterize HEV strains circulating in the Bhutanese pig population. All collected serum samples were tested in an ELISA and faeces samples were tested by real-time RT-PCR, and subsequent characterizations were performed through sequencing of the amplified genomicfragment.

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Samples

Serum samplesCollection of serum samples has been described by Monger et al. (2014). In brief, a total

of 829 pig serum samples was obtained from three government breeding pig farms and village backyard farms between October 2011 and February 2012 [19] from different age categories (Table 1).

Blood samples were collected, using SarstedtMonovette syringes from all selected pigs. For each animal sampled date, location (village, sub-district. district), animal identification, age, sex, and sample number were recorded. Serum was obtained after centrifugation of the coagulated blood samples and stored at -20o C until testing.

RNA extractionIn the laboratory, a volume of 400 μl of the Trizol/supernatant mixture was used to extract

RNA from: 80 μl chloroform was added to the sample, and after mixing the sample was incubated for 5-10 minutes and subsequently centrifuged in a table top centrifuge at 4oC for 10 minutes at 13,000 g. For further RNA isolation the water phase was transferred to a Magna Pure (MP96), using the external lysis protocol (Roche, Mannheim, Germany). The MagNa Pure 96 DNA and viral NA small volume kit was used according to the manufacturer’s description.

Table 1. Prevalence of antibodies against Hepatitis E virus (HEV) in government pig breeding farmsand backyard farms in Bhutan.

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Laboratory tests

Antibody detection against HEVAnti-HEV IgG antibodies in serum samples were measured using an enzyme-linked

immunosorbent assay (ELISA), as described [20]. This essay has an estimated sensitivity and specificity of 84% and 89% respectively. Samples with Percent Positivity (PP) values ≥ 25PP were positive.

Molecular Detection of HEVHEV detection in faecal samples was performed by real-time RT-PCR on undiluted RNA

samples with primers JVHEVF and JVHEVR and probe JVHEVP [21]. The HEV real time RT-PCR was performed using the RNA UltraSenceTM One-Step Quantitative RT PCR System Kit (Invitogen).

Nucleotide sequencing and phylogenetic analysisFaecal samples positive for HEV RNA by real-time RT-PCR were amplified using a nested

RT-PCR format targeting an ORF2 fragment of HEV. Briefly, 2 μl of 10 μM RH-HEV-Rnested primer was added to 5 μl RNA. The RT mix contained 2 μl (5X) first strand buffer (Invitrogen, Breda, The Netherlands), 50 mM DTT, 1 mMdNTP mix (TaKaRa, City, Country), 20 U RNasin (Promega, Madison, USA) , 100 U Superscript III reverse transcriptase (Invitrogen, Breda, The Netherlands) and 2,5μM primer HEV222alt: GAR AAi GGR CGiGAi GGR GCi GG. Five μl RNA was added to 5μl RT-mix, thus the RT reaction was performed at a final volume of 10 μl.

The mixture was incubated for 10 min at 22o C followed by 60 min at 42o C, heated for 5 min at 95o C and then placed on ice. Forty μl of the PCR1 mixture was added to the RT mixture. The PCR1 mix contained 2,5μM primer HEV224alt: AAY CAR GGi TGG CGY TCiGTi GAR AC, 10 X PCR buffer (Invitrogen, Breda, The Netherlands), 1,5 mM MgCl2, 0.2 mMdNTP and 2.5 U Taq DNA polymerase. The PCR reaction was performed in a final volume of 50 μl. Cycling conditions were denaturation at 95o C for 5 minutes followed by 35 amplification cycles (95o C 30 s, 42o C 30 s and 60o C 45 s). One μl of the first PCR was used in the nested PCR. The PCR2 mix contained 10x PCR buffer (FS Taq polymerase 2161567, Roche), 2,5 U FS Taq polymerase (FS Taq polymerase 2161567, Roche), 0,2mM dNTP and 1μM of each primer HEV-AN89alt: GAG GAG GAA GCT ACC TCY GGY YTiGTi ATG CTY TGY AT and HEV-ANalt: GGA GAA GGA GTT GGT CGR TCY TGY TCR TGY TGR TT.

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The PCR reaction was performed in a final volume of 50 μl. Cycling conditions were denaturation at 95o C for 6 min followed by 40 amplification cycles (95°C 30 s, 60°C 20 s, and 72°C 15 s). With this PCR, fragments of 493 nucleotides were obtained. The PCR products were separated in a 1.5% agarose gel and visualized under UV after ethidium bromide staining. Positive RT-PCR products were excised from the gel, purified by using a Gel DNA Recovery kit (Zymo Research, CA, USA), and sequenced in both directions subsequently. For sequencing we used the primers HEV-AN232: GAG GAG GAA GCT ACC TC and HEV-AN233: GGA GAA GGA GTT GGT CG. Nucleotide sequences were aligned and clustered using Bionumerics version 5.1 (Applied Maths) using the Jukes and Cantor correction for evolutionary rate. Evolutionary trees were drawn using Neighbour-Joining clustering. The confidence values of the internal lineages were calculated by performance of 1000 bootstrap analysis.


Seroprevalences and their exact binomial 95% confidence intervals were calculated [22]. Seroprevalences in the governmental breeding farms and backyard farms were compared using a random effects logistic regression model. The response variable was the serostatus of a pig (1 = positive, 0 = negative). The cluster where the pig was housed (village or government farm) was categorised as a new variable “herd” (village 1 – 69 and the government farms (70 - 72), which served as random effect in the model to account for the dependence of pigs within a cluster. Age category (1 = <3 months, 2 = 3-9 months; 3 = >9 months) of pigs, breed (1 = Local; 2 = Exotic) and type of farm (1 = backyard; 2 = government farm) were added as fixed effect (predictor) variables. The predictor variables in the model were also assessed for potential interactions. Statistical analysis were done in SPSS software, IBM SPSS version 20 (SPSS, Chicago, IL, USA) and R (version 3.1.3; Foundation for Statistical Computing, Vienna, Austria).

Results

Detection anti-HEV IgG in serum samplesIn the government breeding farms, 273 out of 376 pigs tested (73%; 95% CI: 69-75%) were

seropositive against anti-HEV IgG. The seroprevalence increased with age, and in the oldest pigs, the seroprevalence in each of the government breeding farms was above 95% (Table 1).

In the village backyard farms, 231 out of 453 samples (51%; 95% CI: 44-58%) were seropositive against anti-HEV IgG. A significant interaction between farm type and age was

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obtained in the model, which indicates that there was only a significant effect of age on the seroprevalence of HEV in government breeding farms.

The seroprevalence of HEV in pigs of the age groups 3-9 months and greater than 9 was significantly higher (P = 0.04 and P < 0.001) than in the age group less than 3 months. This effect was not observed in the village backyard farms. The seroprevalence was significantly higher in exotic pigs compared to local pigs. The latter comparison only applies to backyard farms since there are only exotic breeds on the government breeding farms.

Detection of HEV RNA by Real-Time Rt-PCROverall, 106 out of 431 examined faecal samples (25%; 95% CI: 21-29%) tested positive

for HEV RNA. HEV-positive faeces were detected mainly in two government breeding farms, 39/39 (100%; 95% CI: 91-100%) of the pigs from Thimphu and 35/100 (35%; 95% CI: 26-45%) from Sarpang. Only one out of a hundred samples from Mongar breeding farm tested positive (Table 2).

Sequencing and phylogenetic analysisA total of 11 HEV samples, positive by real-time RT-PCR were sequenced. These were

selected to represent the main positive clusters (two government farms and the village backyard farms from two different regions). All these PCR positive samples had CT values less than 30 and were exotic pigs. Only three local pigs from the backyard farms were positive in the PCR against HEV, but with CT values greater than 30 and sequencing attempts were not successful.

Through phylogenetic analysis using the ORF2 region, all eleven isolates were characterized as genotype 4. Between the isolates from Sarpang farm and Thimphu farm, there was a difference of 4 nucleotides. Isolates from the backyard farms in the western region (Chhukha district) were identical to the isolates from the Thimphu government breeding farm, while the isolates from a backyard farm in the west-central region (Wangdue district) differed 2 nucleotides from the isolates on both the government breeding farms. Based on the ORF2 region, the similarity with the Indian isolate IND-SW-00-01 was the highest (93.7%) (Figure 1).

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Table 2. Real time RT-PCR positive positive faecal samples against Hepatitis E (HEV) from different age groups of government farms and backyard farms in Bhutan.

Figure 1. Phylogenetic analysis of HEV strains from pigs in Bhutan.

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Discussion

The aim of this study was to determine the HEV seroprevalence in Bhutanese pigs from government pig breeding farms and backyard farms and genetically characterized HEV strains circulating in the Bhutanese pig population.

The seroprevalence study showed that HEV is widespread among pigs in Bhutan in both government breeding farms and the village backyard farms. This was confirmed by detecting the virus in faeces samples from 3-9 month old pigs in the government farms and village backyard farms. The phylogenetic study revealed that the Bhutanese HEV isolates belonged to HEV genotype 4, having the highest similarity to an Indian HEV isolate (IND-SW-00-01).

Seroprevalences of more than 90% were found in the oldest pigs in the three government farms. Our study showed that, although the Bhutanese pig population is only small, HEV is widespread with 61% of the pigs tested having antibodies against HEV. The HEV is also highly prevalent in pigs in neighbouring countries like India [17, 23], Nepal [24] and Thailand [25]. Similar to what was found in previous reports [12, 26], the seroprevalence of HEV increases as animals grow older. The detection of HEV RNA in pig faeces provides further evidence of circulation of HEV in Bhutanese pigs.

Pig HEV isolates of genotype 4 has been isolated from Asian countries like India, Thailand and China [17, 25, 27]. Our results of the phylogenetic analysis indicate that the Bhutanese HEV strains are of local origin and were not imported with breeding stock purchased from Europe in recent history, suggesting that Bhutanese HEV strains could be more closer to strains from neighbouringasian countries. More so, genotype 4 has been restricted to Asia and was found exclusively in Asia [28].

In our study, within the two government farms, the isolates where identical, while there was a difference of 4 nucleotides between both farms. It is possible for isolates from different regions to be closely related [17].

This suggests a recent common ancestor, which can be expected, as the Thimphu farm obtains breeding stock from the Sarpang farm. Backyard farms in the west region (Chhukha district) usually obtain their piglets from the Thimphu farm, explaining why these isolates were also identical to each other.

All in all, the close relationship between the strains support the hypothesis of the HEV being endemic in government herds, and being introduced into the backyard farms with the piglets obtained from these government farms.

Both exotic pigs and local pigs in the backyard farms were included in the study. The local pigs are indigenous pigs which are usually purchased locally, while the exotic pigs are purchased from the government breeding farms. The seroprevalence in exotic pigs was higher than in local pigs which further supports the hypothesis that the virus is mostly introduced in


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the backyard farms through the piglets obtained from the government farms. Circulation in the backyard farms themselves seems more limited. Conditions for HEV transmission are more favorable with a high density of pigs and therefore more chances of direct and indirect contact between the pigs [29]. It has also been found that pigs in free range system which have access to outdoor areas have lower risk of exposure to HEV infection [30].

Out of 127 samples from local pigs, only 3 samples were positive in PCR for HEV RNA. Unfortunately, none of the samples selected from local pigs could be sequenced due to higher CT value (> 30), so no phylogenetic data could be obtained to find out whether the same strains circulate in both type of pigs or whether the HEV strains in both populations are circulating independent from each other.

Even though the seroprevalence in the three government farms was comparable, only 1 out of 100 samples from the Mongar farm, located in the east-central region of Bhutan, was tested PCR-positive. This farm is located far from the central veterinary laboratory, where the initial processing of the samples took place. Relatively long shipping times and exposure to high temperatures may have caused degradation of the RNA, in which case the low prevalence of PCR-positive faeces samples in the Mongar farm might be an artifact.

HEV is an important public health concern in developing countries [29]. All HEV real-time RT-PCR positive samples from Bhutan were found to belong to genotype 4, which is found to be responsible for human infection in many countries including industrialized countries [31]. There is frequent exposure of humans to pigs or pig waste, and poor sanitation in Bhutanese villages, where most pigs are reared in the backyards. This might pose a potential risk for HEV transmission from pigs to the people. However, there are currently no available data on the viral hepatitis incidence due to HEV in humans in Bhutan. This study supports the need for surveillance jointly with the public health counterpart to investigate the prevalence in humans and understand whether the strains in humans and pigs are the same.

Conclusion

In conclusion, the serological prevalence of anti-HEV IgG and genetic characterization of HEV in pigs from Bhutan was documented in this study for the first time. The study revealed that HEV is prevalent in Bhutanese pig population. The partial ORF2 sequence analysis revealed that the HEV isolates of Bhutan belonged to the genotype 4. Findings of study suggested that HEV in pigs is endemic in the government breeding farms and from there is introduced into the backyard farms of Bhutan.

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Acknowledgements

We thank Visser-Hendricse, Yolande de, Central Veterinary Institute, Lelystad (CVI) , in the Netherlands for her assistance in carrying out the laboratory works. We thank Jose Luis Gonzales, CVI for his assistance in statistical analysis of the data. The authors thank the Program Directors of the National Centre for Animal Health and Regional Livestock Development Centres in Bhutan for their logistic and technical support during the field work. The authors acknowledge the farm managers and laboratory technicians for their assistance in the study. This study was funded by the Netherlands University Foundation for International Cooperation (NUFFIC, NFP Grant CF 7436) and Central Veterinary Institute (CVI, WOT01-003-010-01), Lelystad, The Netherlands.

References1. Smith DB, Simmonds P, Group motICotToVHS, Jameel S, Emerson SU, Harrison TJ, Meng X-J, Okamoto H, Van der Poel WHM, Purdy MA: Consensus proposals for classification of the family Hepeviridae. Journal of General Virology 2014, 95(10):2223-2232.2. Vasickova P, Psikal I, Kralik P, Widen F, Hubalek Z, Pavlik I: Hepatitis E virus: a review. Veterinarni Medicina 2007, 52(9):365-384.3. Meng XJ: Swine Hepatitis E Virus: Cross-Species Infection and Risk in Xenotransplantation. In: Xeno-transplantation. Volume 278, edn. Edited by Salomon D, Wilson C: Springer Berlin Heidelberg; 2003: 185-216.4. Meng XJ: Recent advances in Hepatitis E Virus. Journal of Viral Hepatitis 2010, 17(3):153-161.5. Matsuda H, Okada K, Takahashi K, Mishiro S: Severe hepatitis E virus infection after ingestion of uncooked liver from a wild boar. Jounal of Infectious Diseases 2003,188(6):944.6. Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, Haynes JS, Thacker BJ, Emerson SU: A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of National Academy of Sciences of the United States of America 1997, 94:9860–9865.7. Song YJ: Studies of hepatitis E virus genotypes. Indian Journal of Medical Research 2010, 5:487-488.8. Meng X-J: Novel strains of hepatitis E virus identified from humans and other animal species: is hepatitis E a zoonosis? Journal of Hepatology 2000, 33(5):842-845.


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9. Nishizawa T, Takahashi M, Endo K, Fujiwara S, Sakuma N, Kawazuma F, Sakamoto H, Sato Y, Bando M, Okamoto H: Analysis of the full-length genome of hepatitis E virus isolates obtained from wild boars in Japan. Journal of General Virology 2005, 86:3321-3326.10. Balayan MS, Usmanov RK, Zamyatina NA, Djumalieva DI, Karas FR: Experimental hepatitis E infection in domestic pigs. Journal of Medical Virology 1990, 32(1):58-59.11. Meng X-J, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, Haynes JS, Thacker BJ, Emerson SU: A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences 1997, 94(18):9860-9865.12. Meng X-J, Dea S, Engle RE, Friendship R, Lyoo YS, Sirinarumitr T, Urairong K, Wang D, Wong D, Yoo D et al: Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. Journal of Medical Virology 1999, 59(3):297-302.13. Chandler JD, Riddell MA, Li F, Love RJ, Anderson DA: Serological evidence for swine hepatitis E virus infection in Australian pig herds. Veterinary Microbiology 1999, 68(1–2):95-105.14. McCreary C, Martelli F, Grierson S, Ostanello F, Nevel A, Banks M: Excretion of hepatitis E virus by pigs of different ages and its presence in slurry stores in the United Kingdom. The Veterinary Record 2008, 163(9):261-265.15. Hsieh S-Y, Meng X-J, Wu Y-H, Liu S-T, Tam AW, Lin D-Y, Liaw Y-F: Identity of a Novel Swine Hepatitis E Virus in Taiwan Forming a Monophyletic Group with Taiwan Isolates of Human Hepatitis E Virus. Journal of Clinical Microbiology 1999, 37(12):3828-3834.16. Huang FF, Haqshenas G, Guenette DK, Halbur PG, Schommer SK, Pierson FW, Toth TE, Meng XJ: Detection by Reverse Transcription-PCR and Genetic Characterization of Field Isolates of Swine Hepatitis E Virus from Pigs in Different Geographic Regions of the United States. Journal of Clical Morcobiology 2002, 40:1326-1332.

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17. Arankalle VA, Chobe LP, Joshi MV, Chadha MS, Kundu B, Walimbe AM: Human and swine hepatitis E viruses from Western India belong to different genotypes. Journal of Hepatology 2002, 36(3):417-425.18. Clayson E, Innis B, Myint K, Narupiti S, Vaughn D, Giri S, Ranabhat P, Shresha M: Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal. The American Journal of Tropical Medicine and Hygiene 1995, 53:228-232.19. Monger VR, Stegeman JA, Koop G, Dukpa K, Tenzin T, Loeffen WLA: Seroprevalence and associated risk factors of important pig viral diseases in Bhutan. Preventive Veterinary Medicine 2014(0).20. Van der Poel WHM, Pavio N, van der Goot J, van Es M, Martin M, Engel B: Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine. Brazilian Journal of Medical and Biological Research 2014, 47:334-339.21. Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR: A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. Journal of Virological Methods 2006, 131(1):65-71.22. Clopper CJ, Pearson ES: The Use of Confidence or Fiducial Limits Illustrated in the Case of the Binomial. Biometrika 1934, 26(4):404-413.23. Shukla P, Chauhan UK, Naik S, Anderson D, Aggarwal R: Hepatitis E virus infection among animals in northern India: an unlikely source of human disease. Journal of Viral Hepatitis 2007, 14(5):310-317.24. Clayson ET, Innis BL, Myint KS, Narupiti S, Vaughn DW, Giri S, Ranabhat P, Shrestha MP:Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal. The American journal of tropical medicine and hygiene 1995, 53(3):228-232.25. Hinjoy S, Nelson KE, Gibbons RV, Jarman R, G., , Chinnawirotpisan P, Fernandez S, Tablerk P, Labrique AB, Patchanee P: A Cross-Sectional Study of Hepatitis E Virus Infection in Pigs in Different-Sized Farms in Northern Thailand. Foodborne Pathogens and Disease 2013, 10:698-704.


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26. Takahashi M, Nishizawa T, Miyajima H, Gotanda Y, Iita T, Tsuda F, Okamoto H: Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus. Journal of General Virology 2003, 84(4):851-862.27. Dalton HR, Bendall R, Ijaz S, Banks M: Hepatitis E: an emerging infection in developed countries. The Lancet Infectious Diseases 2008, 8(11):698-709.28. Lu L, Li C, Hagedorn CH: Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis. Reviews in Medical Virology 2006, 16(1):5-36.29. Emerson SU, Purcell RH: Hepatitis E virus. Reviews in Medical Virology 2003, 13(3):145-154.30. Rutjes SA, Bouwknegt M, van der Giessen JW, de Roda H, A. M., , Reusken CBEM: Seroprevalence of Hepatitis E Virus in Pigs from Different Farming Systems in The Netherlands 77 2014, Journal of Food Protection:640-642.31. Nishizawa T., Takahashi M., Endo K, Fujiwara S, Sakuma N, Kawazuma F, Sakamoto H, Sato Y, Bando M, Okamoto H: Analysis of full lenth genome of hepatitis e virus isolates obtained from wild boars in Japan. Journal of General Virology 2005, 86:3321-3326.

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Chapter 6

Evaluation of oral bait vaccine efficacy against Classical Swine Fever in village backyard pig farms in Bhutan

V. R.Mongera, c, J.A. Stegemanb, K. Dukpac, R.B. Gurungc, W.L.A. Loeffena


Published in: Transboundary and Emerging Diseases, 2014 December 12 doi: 10.1111/tbed.12333.

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Abstract

Control and eradication of Classical Swine Fever (CSF) in countries with a high proportion of backyard holdings is a challenge.Conventional attenuated Chinese-C strain vaccines, though safe and effective, are difficult to use in backyard farms due to various practical reasons. The aim of this study was to evaluate the efficacy of the CSF oral bait vaccine in village backyard pig farms and to assess the farmers’ knowledge on CSF and motivation on using oral vaccines. The pigs were fed the bait by the farmers themselves; one bait was given on day 0, followed by second bait on the next day. Seventy-three percent (140 out of 193 pigs) of vaccinated pigs had either a slight (two-threefold; 60 pigs) or significant (at least fourfold; 80 pigs) increase of the antibody titre against CSFV. A significant increase of the antibody titres was mainly observed in pigs with no pre-vaccination titre (OR = 12, 95% CI = 4-40). The number of pigs with protective antibody titres (≥40) rose from 47 (24%) to 115 (60%) following vaccination. Only 30% of the farmers claimed to be familiar with CSF, although clinical signs they mentioned were rather unspecific and could relate to many other pig diseases. Most of the farmers claimed to be motivated to use oral vaccines if made available. The oral vaccine could be a substitute for the conventional attenuated CSF vaccines in areas where it is logistically difficult for veterinarians to visit. It may therefore be a useful tool to combat endemic CSF disease in regions where the disease continues to have a serious impact on the backyard farmers who depend on pig farming for their sustenance and livelihoods.

Keywords: Bhutan, Classical Swine Fever, Village backyard farms, oral bait vaccine, Exotic pigs, Local pigs, Virus Neutralization Test.

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Chapter 6


Evaluation of oral bait vaccine efficacy against classical swine fever

Introduction

Classical swine fever (CSF) is one of the most economically important pig diseases worldwide, affecting profitability in large farms and livelihood of small backyard farms [1]. In Bhutan, CSF is a notifiable pig disease with sporadic outbreaks. The disease is usually diagnosed by the veterinarian from the clinical signs or case history, but many cases are likely unreported due to remoteness of the outbreak area or failure of farmers to notify a suspicion. There is no scientific data on CSF in Bhutan except for a purposive surveillance study, demonstrating a seroprevalence of 6% [2].

Control and eradication of CSF in countries with high proportion of backyard holdings poses challenges for veterinary services since backyard holdings may act as a reservoir for CSF virus and possible source of infections for commercial farms [3]. Lack of proper control strategies coupled with inadequate veterinary services can limit control of CSF especially in the backyard production systems.

Many countries have eradicated the disease through vaccination [4] and it is still the choice in CSF-endemic countries. This is particularly relevant in developing countries, where control measures such as stamping out and zoning are difficult to apply due to socio-economic reasons. Conventional attenuated Chinese-C strain live vaccines are the most commonly used vaccines against CSF [5]. They are safe and efficiently reduce the CSFV prevalence by increasing herd as well as individual animal immunity [6]. Systematic vaccination using conventional vaccines would be a good option for control and eradication of CSF in intensive and industrialized pig production system. However, many developing countries, including Bhutan, have small-scale production systems like backyard farms and free ranging system, lacking proper registration of pigs and farms [7]. Furthermore, these farms may be scattered and in remote areas, they are difficult to reach. Vaccines are also commonly administered via the intra-muscular route and therefore require a veterinarian or trained para-veterinarian to administer the same. Therefore, parenteral vaccination in Bhutan is limited only in government breeding farms and most backyard farms in the village backyard holdings are rarely vaccinated [2]. The above mentioned factors and a lack of adequate veterinary services seriously affect the vaccination coverage in backyard holdings in general.

The use of the oral vaccine RIEMSER® Schweinepestoralvakzine, a conventional CSF live vaccine based on the “C” strain has been successfully used to control CSF in wild boars [8, 9]. The use of oral vaccines against CSF in backyard pigs as an alternative to parenteral vaccination was tested under field conditions and found to be effective [10]. However, along with successful use of oral vaccines, it is important to look at the cultural and socioeconomic structure and lack of

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adequate knowledge and awareness on CSF and its control among backyard farmers, as this will ultimately determine the potential success or failure of oral vaccination. It is therefore important to evaluate the farmer’s views on CSF and its control using vaccine and their willingness to use oral vaccine.

This study aims to evaluate the efficacy of the oral bait CSF vaccine used in village backyard pig farms in Bhutan. The study was also conducted to assess the farmers’ knowledge of CSF disease and motivation about vaccination at backyard farms.


Study area and selection of animalsThe study was conducted in Bhutan from November 2013 to February 2014. Bhutan is

subdivided into 20 districts and in each district there are several villages [7]. The village backyard farm in Bhutan is characterized by small numbers of pigs (usually 2-4 pigs) reared by the subsistence farmers, either in a small confined pigsty or free-range scavenging system [11]. There is inadequate biosecurity measure and poor management in these backyard farms and pig feeds consist of mainly kitchen wastes (leftover foods, vegetable peels) and locally available plants and wild weeds. These backyard farms consist of either local (indigenous) pigs or exotic pigs. Exotic pigs are mainly of European origin, supplied by government breeding farms. These exotic pigs are routinely vaccinated against CSF at weaning age (45-50 days) at the government farms before being sold to the farmers. Local pigs are mainly native pigs produced in the backyard holding system or purchased from elsewhere locally and are in general not vaccinated. They are generally smaller in size compared to exotic pigs.

Twenty-one villages from six districts with different ecological zones were purposively included in the study. These villages are located in the southern regions and selected for the study due to more concentration of pigs in these districts. The villages were selected based on the pig population data provided by the District Livestock Officer from the selected districts. From the selected villages, the backyard farms were selected based on the list of backyard pig farms provided by the livestock extension agent. A total of 224 pigs from 71 village backyard farms from those 21 villages were included in the study. Most of these pigs were confined, with only a few scavenging pigs. The study was conducted following approval by the Council for Renewable Natural Resources Research of Bhutan (CORRB), an organisation within the Ministry of Agriculture and Forests (MOAF), mandated to coordinate research policy and programs in Bhutan.

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The vaccine and vaccination procedureOral vaccination was carried out using oral bait vaccine commercially available and licensed

RIEMSER®Schweinepestoralvakzine according to the manufacturer's recommendations (RiemserArzneimittel AG, Greifswald-InselRiems, Germany). The vaccine is made up by 1.6 ml C-strain “Riems” vaccine with a minimum titre of 104 TCID

50 ml filled into a blister

incorporated in a corn-based bait matrix [12]. The study was conducted involving farmers. The farmers were explained about the oral bait vaccine and given specific instruction on how to feed the pigs with the bait. Based on the instruction, the farmers fed their pigs by providing two baits per pigs; one bait given on day 0, followed by the second bait subsequently on the next day. The baits were laid on the floor of the shed before feeding time in the early morning hours letting each pig feed on them. Farmers were asked to observe the bait uptake and any abnormal behaviour or disease after the intake of the bait. The vaccines were stored frozen until use. For the entire study period, required vaccines were transported in cool boxes with ice pack and handed over to the farmer who fed the bait on the same day. The second bait supposed to be fed on the next day were asked to be stored at 4o C.

Sampling procedureBlood of all vaccinated animals were collected on day 0 and 28 days post-vaccination

(dpv) for serological examination to monitor the antibodies against CSF oral vaccine. Whole blood was collected using 10-ml sterile plain vacutainer tubes and stored overnight at room temperature for serum separation. Each serum was then transferred into a sterile cryo vial and transported in an icebox to the laboratory, sorted and stored at -20o C until analysis. Names of the owner, herd size, age and sex of sampled animal was recorded.

Thirty one animals (14 %) out of the 224 animals were not available for sampling at 28 dpv because they had either been sold or slaughtered. Therefore, they were excluded from the study.

QuestionnaireFace-to-face interviews were applied to the pig farmers by the first author using a structured

questionnaire during both the first and second visits to the farm. The questionnaire contained only “close ended” questions in three parts. The first part covered general aspects such as herd size, knowledge of farmer on CSF, vaccine and vaccination program, and experiences in pig rearing. The second part of the questionnaire contained information on individual pigs such as age, breed, source of pigs, vaccination status, and number of baits eaten. The third part covered information collected at the end of the field trial about farmers’ experience on feeding the bait, their view on the use of oral bait vaccine, willingness to pay if charged and pig’s response to the bait.

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Detection of antibodiesIn order to determine the effectiveness of oral vaccination, all sera collected before and

after vaccination, were tested for antibody titres by means of the virus neutralisation test (VNT) based on CSFV strain, Alfort/187 [13] as described by [14, 15]. The tests were conducted at the Central Veterinary Laboratory, Lelystad, Netherlands.

Briefly, 100 TCID50

of CSFV strain ‘Alfort/187’ were incubated at 37o C for 1 hour with two-fold serial dilutions of the serum to be investigated. Then, to each serum-virus mixture, PK-15 cells were added and after an incubation at 37o C (5% CO

2) for 4 days, medium was

removed and cells were fixed by adding 4% formaldehyde. A mixture of two horseradish peroxidase-conjugated monoclonal antibodies against CSFV was used to label virus infected cells. Antibody titres were expressed as the reciprocal value of the neutralisation titre (ND

50).

Vaccine response investigationResponse to CSF oral bait vaccine was interpreted by comparing pre- and post-vaccination

titres of individual pigs. “No change” was defined as a post-vaccination titre that was less than twice the pre-vaccination titre. A “slight increase” was defined as a post-vaccination titre that was 2 to 3 times the pre-vaccination titre. A four-fold (or more) rise in titre was considered a “significant increase”. Furthermore, pigs were considered to have a protective titre whenever the neutralizing antibody titres were above 40 ND

50 [16]. This cut-off was also used by Milicevic et

al (2013) to determine whether pigs were protected or not against disease post oral vaccination.

Data management and analysis

Data management and analysis was carried out using SPSS software, IBM SPSS version 20 (UK). For prevalences, a 95% confidence interval was calculated using the exact binomial confidence intervals [17]. A logistic regression was performed to assess the effect of age (> 90 days or < 90 days), breed (exotic or local) and pre-vaccination titer (≥10), as independent variables) on seroconversion rate of vaccinated animals using either titer (≥40 = 1; else 0) as dependent variable.

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Results

Antibody titres at the time of vaccination (Pre-vaccination)On the day of the vaccination (prior to vaccination), 51 out of the 193 pigs (26%; 95% CI:

20-33) already had an antibody titre of at least 10 ND50

(Tables 1 and 2, with further details on type and age of pigs). Exotic pigs were significantly more likely of having pre-vaccination titres >40 than local pigs (OR = 28, 95% CI: 10-76). Fifty-six percent of exotic pigs (42/75) had pre-vaccination antibody titres greater than 40 ND

50 compared to 4% (5/118) local pigs.

Table 1. Response to oral vaccination with classical swine fever bait vaccines in exotic and local pigs, depending on pre-vaccination titre. No change is a post-vaccination reponse < 2x the pre-vaccination titre, a slight increase 2-3x the pre-vaccination titre, and a significant increase > 4x the pre-vaccination titre; n denotes the number of pigs for each category of pre-vaccination titres.

Table 2. Response to oral vaccination with classical swine fever bait vaccines in young and older pigs, depending on pre-vaccination titre. No change is a post-vaccination reponse < 2x the pre-vaccination titre, a slight increase 2-3x the pre-vaccination titre, and a significant increase > 4x the pre-vaccination titre; n denotes the number of pigs for each category of pre-vaccination titres.

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Oral vaccine responseIn 140 out of the 193 pigs (73%, 95% CI: 66-78), either a slight (60 pigs) or significant

(80 pigs) increase of the antibody titre against CSFV was observed (Tables 1 and 2). Figure 1 demonstrates detailed plots showing the relation between pre-vaccination and post-vaccination titre for individual pigs.

From the logistic regression analysis, a significant increase of the antibody titres were mainly observed in pigs with no pre-vaccination titre (OR = 12, 95% CI = 4-40). Conversely, age (OR = 1, 95% CI = 0.9-1) and breed (OR = 0.9, 95% CI = 0.4-2) did not have an influence on the significant increase in the antibody titres 28 dpv.

Protective antibody titres post-vaccinationTitres that were considered protective (≥ 40) were observed in 47 of the 51 pigs with

pre-vaccination titres (Tables 1 to 3). Following vaccination, the number of pigs with protective antibody levels (≥ 40) rose from 47 (24%) to 115 (60%) (Table 3)

Pigs that had pre-vaccination antibody titres >40 are most likely to remain protected with protective titres (>40) 28 dpv (OR = 7, 95% CI: 2.3-19; P = 0.001).

Figure 1: Relation between pre-vaccination and post-vaccination titres for young pigs (A), older pigs (B), local pigs (C) and exotic 394 pigs (D).

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Table 3. Protective antibody titres against CSF at 0 and 28 days post vaccination.

Pigs

Total

TypeExotic pigLocal pigAgeYoung pig (<90 days)Older pig (>90days)

Titre≥ 40Pre-vaccination

47 (24%)

42 (56%)5 (4%)

2 (5%)45 (29%)

n

193

75118

39154

Post-vaccination115 (60%)

59 (79%)56(47%)

18 (46%)97 (63%)

Farmer's knowledge and perception on CSF and vaccination through a questionnaire

The feedback of the pig farmers based on the questionnaire is shown in Table 4. The median herd size was 3 (with 2 and 8 at the 10 and 90 percentile). Of the farmers interviewed, 41% were familiar with CSF and clinical signs related to CSF. The main three clinical signs regarding the CSF, that the farmers answered were off-feed, fever and recumbency. Vaccination against CSF at the backyard level is not a regular practise and none of the farmers interviewed said that they vaccinate at their backyard farms. The main reasons being that the pigs were already vaccinated, the vaccine was not available, or they didn’t know how and where to obtain the vaccine.

With regards on the use of oral bait vaccine, all farmers successfully fed the vaccine during the trial period and, except for two farmers, they said the administration of vaccine as a bait was easy. These two farmers had fed the bait after feeding time. All farmers were willing to vaccinate their pigs if the oral vaccine is made available, but ten (14%) farmers still preferred a veterinarian to come and vaccinate their pigs.

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Table 4. Characteristic of farmer’s response to the questionnaire on experience in pig rearing, knowledge and perception of CSF, vacci-nation and willingness to use oral vaccine against CSF.

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Discussion

The objective of this study was to evaluate the efficacy of oral bait CSF vaccine in village backyard pig farms in a field experiment. Here we report on the oral vaccine trial in village backyard farms involving farmers to give the vaccine themselves. Furthermore, a questionnaire was used during and after the trial to know farmer’s perception about CSF and its control and their motivation to use the oral vaccine.

In our study, it was found that 73% (140/193) of pigs responded to the vaccine with an increase of the antibody titre 28 dpv. Out of these, 43% responded by developing a “slight increase” in antibody titres (two- or three-fold) and 57% by a “significant increase” (four-fold or higher) in antibody titres, showing a response to the vaccine in vaccinated pigs in a field situation.

Because this study was carried out in a field situation, 26% of pigs were found to have antibody titres against CSFV before vaccination (pre-vaccination antibodies). These pigs were mainly exotic pigs, supplied from the government breeding farms with vaccination history. The exotic pigs having pre-vaccination antibody titres greater than 40 ND

50 remained protected

(titres > 40) after 28 dpv. Pre-vaccination antibody titres against CSFV in local pigs may be attributed to past exposure to natural infection or vaccination. Vaccination in local pigs is rare and recovery from a previous infection is therefore the most likely cause for the antibody titres. This could, however, not be verified because there was no information on a history of any illness associated with CSF in these pigs.

The seroconversion rate after vaccination was influenced by the presence or absence of antibodies against CSFV at the time of vaccination (pre-vaccination titres). Pigs that were seronegative (<10 ND

50) at the time of vaccination, responded better to the vaccine by developing

a “significant rise” in antibody titres (four-fold or higher) than pigs that already had pre-vaccination antibody titres. In this study, age did not have any influence on the seroconversion. In previous studies, carried out in wild pigs and domestic pigs, the oral vaccines based on the “Chinese-strain” (C-strain) were found to provide a better immune response in older animals (> 3 months) because of a better uptake of vaccine baits than in younger pigs [8, 10, 12]. We found that bait uptake was not a problem, irrespective of the age groups. Most young pigs were between the age of 2-3 months and were able to feed on the bait. This may also be because all the pigs were fed individually by farmers and therefore had an equal chance of taking up the baits, while otherwise older pigs may dominate the younger pigs. This makes the use of oral baits ideal in backyard farms with small numbers of pigs where individual pig can easily be fed.

Four weeks after vaccination, 60% of the vaccinated pigs was considered to be protected (titre ≥40) in our study, which was less than the 68% found in a previous study by Milicevic et al. (2013). One of the reasons for this difference may be the number of baits fed to the

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pigs: 2 per pig in our study and 4 in the Milicevic study. Theoretically, the vaccine dose in one bait is sufficient to induce a full protective immunity. However, both the uptake of the bait itself, and the uptake of the vaccine from the blister inside the bait are uncertain factors in the oral vaccination. Multiple baits may therefore be needed to increase the success rate of the oral vaccination. Vaccine baits are relatively expensive compared to vaccine for parenteral application. The need to use multiple baits may therefore increase the costs of oral vaccination in backyards to an unacceptable level. What is still acceptable will depend on the overall losses due to classical swine fever and the perceived chance for an individual farmer to be confronted with CSF in his pigs. This will differ for different regions and no general rule on the maximum number of baits for a cost-effective vaccination can therefore be given. An alternative approach may be to feed each pig 2 baits, but with 2 weeks in between. Thus, the immune response may benefit from a booster response, which may result in a higher rate of protection compared to giving the two baits with only one day in between. This needs to be evaluated in a further study though. In this study, we used questionnaires to understand the farmer’s background knowledge of CSF and their motivation to use an oral vaccine. They said that feeding the pigs with oral vaccine by themselves were much easier and convenient, compared to a parenteral vaccine. For one, they didn’t have to catch the pigs individually. They were willing to vaccinate themselves if oral vaccines were made available to them. This will overcome the difficulty faced in many developing countries having predominantly backyard farms and a free ranging system. Furthermore, this will also save time and resources without having to involve a veterinarian or para-veterinarian. There will be a better vaccination coverage by covering scattered and remote areas, which otherwise would be difficult to reach by a vaccinating team. For successful control of CSF in endemic areas, alongside successful vaccination program, it is also important to have adequate knowledge and awareness on the disease. In this study, only 30% of the farmers interviewed claimed to be familiar with CSF and its clinical signs. When asked about the clinical signs, those most often mentioned were very general and could apply to many pig diseases. Except for two farmers that mentioned reddening of the skin, no other clinical signs that are more typical for CSF were mentioned. It is therefore important that the farmers are sensitized through effective awareness on CSF and its clinical symptoms. Many countries rely on vaccination for the control of CSF.

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However, vaccination using conventional parenteral vaccine is difficult in backyard farms due to various practical difficulties, one reason being to be applied via the intra-muscular route requiring the participation of a veterinarian or trained para-veterinarian. Based on previous studies and results of this study, we can conclude that the oral bait vaccine using the “Chinese-strain” of CSFV can successfully induced a protective immunity in the vaccinated pigs reared under backyard village conditions. The oral vaccine may be a substitute for parenteral vaccines in areas where it is logistically difficult for veterinarians to visit, owing to remote and scattered location of the pig farms. Such easy-to-administer oral bait vaccines could be cost-effective, as there is no need to involve a veterinarian, but this also depends on the number of baits needed for a sufficient success rate of the vaccination. It has the potential to be a useful tool to combat endemic CSF disease in regions where it continues to have a serious impact on the backyard farmers who depend on pig farming for their sustenance and livelihoods, although an optimal oral vaccination scheme with the right balance between efficacy and cost-effectiveness will require some more research.

Conflict of interests

Authors do not have conflict of interest in regard to the findings from this study

Acknowledgements

The authors wish to thank all laboratory technicians and farm attendant of the National Centre for Animal Health (NCAH), Regional Livestock Development Centres (RLDCs) and Regional Pig Breeding Centres (RPBCs) in Bhutan for their assistance in sampling of pigs throughout the field work. The authors thank the Department of Virological Diagnostics, Central Veterinary Institute, Lelystad, in the Netherlands for their assistance in carrying out the laboratory analysis. The authors acknowledge the Program Directors, NCAH and RLDCs for their support to carry out the field works. The authors thank Dr. Tenzin, Deputy Chief Veterinary Officer, NCAH for review of the manuscript. This study was funded by the Netherlands University Foundation for International Cooperation (NUFFIC, NFP Grant CF 7436) and by the Dutch Government under project WOT-01-003-010-01.

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12. Brauer A, Lange E, Kaden V: Oral immunisation of wild boar against classical swine fever: Uptake studies of new baits and investigations on the stability of lyophilised C-strain vaccine. European Journal of Wildlife Research 2006, 52(4):271-276.13. Dahle J, Liess B: Comparative-study with cloned classical swine fever virus-strains alfort and glentorf-clinical, pathological, virological and serological findings in weaner pigs. Wiener Tierarztliche Monatsschrift 1995, 82(8):232-238.14. Terpstra C, Bloemraad M, Gielkens ALJ: The neutralizing peroxidase-linked assay for detection of antibody against swine fever virus. Veterinary Microbiology 1984, 9(2):113-120.15. Kaden V, Lange E, Riebe R, Lange B: Classical swine fever virus strain ‘‘C’’. How long is it detectable after oral vaccination? Journal of Veterinary Medicine 2004 b, 51:260-262.16. Terpstra C, Wensvoort G: The protective value of vaccine-induced neutralising antibody titres in swine fever. Veterinary Microbiology 1988, 16(2):123-128.17. Clopper CJ, Pearson ES: The Use of Confidence or Fiducial Limits Illustrated in the Case of the Binomial. Biometrika 1934, 26(4):404-413.

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Chapter 7

Summarizing Discussion

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Summarizing discussion

In many developing countries, pigs play an important role in the livelihood of rural communities with economic and socio-cultural functions, and with small-scale pig rearing in the backyard as the most predominant practice [1-4].

In Bhutan also, pigs are regarded as important livestock, which serve as a source of animal proteins, income, and a means of fulfilling socio-religious functions in the life of rural people who rear pigs in a backyard setting [5]. Productivity of pigs in developing countries is, however, generally low. Many reasons may account for this, including lack of proper housing and adequate feed, limited genetic potential, and poor disease control [6-9]. The overarching policy of the Bhutanese government is to increase livestock production and productivity by implementing livestock development programs and activities to control economically important diseases. However, for effective disease control, it is essential to have reliable information on the important diseases. Prior to the research presented in this thesis, information on pig diseases in Bhutan was either limited or absent.

The aim of the research described in this thesis was to obtain epidemiological information on a number of important pig viral diseases in Bhutan to have baseline data as a first step for developing effective disease control programs in the country. To meet the aims of this study, first, a serological study on selected pig viral diseases in Bhutan was conducted (Chapter 2). While Aujeszky’s disease (AD) and porcine reproductive and respiratory syndrome (PRRS) were not detected, swine influenza (H1N1) and porcine circovirus type 2 (PCV2) were detected on both the government farms and backyard farms, with a higher seroprevalence in the government breeding farms.

In chapter 3, a longitudinal study is described to assess the dynamics of infections by swine influenza virus (SIV) (H1N1), PCV2 and hepatitis E virus (HEV) in piglets. Piglets born on government breeding farms were either raised on backyard farms, or remained on the government farms, and were tested 4 to 5 times. No significant differences were observed between the two groups of piglets. The government farms seemed to be the main source for spread of the viruses to the backyard farms.

For PCV2 and HEV additional studies were carried out to determine the possible origin of the viruses and to gain insight in the epidemiological situation (Chapter 4 and 5). The Bhutanese PCV2 strains belong to the PCV2b genotype, grouped with cluster 1C. Its closest matches had been found in China. The Bhutanese HEV strains belong to group 4 with a high similarity to an Indian strain, IND-SW-00-01. Results suggest that both viruses have been introduced from neighbouring countries and not through import of the original breeding stock, which amongst others originated from Europe. Results also suggest that the origin of the two viruses was not similar.

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The efficacy of the oral bait classical swine fever (CSF) vaccine was assessed in backyard pig farms in Bhutan and farmers’ knowledge of CSF and motivation to use oral vaccine was evaluated using a questionnaire (Chapter 6). The results showed that oral bait vaccine can successfully induce a protective immunity in vaccinated pigs reared under backyard village conditions.

CSF in Bhutan

Due to the vaccination against CSF in Bhutanese pigs, serological testing cannot provide evidence for infections with field virus. During the time the studies were carried out, no direct evidence of CSFV infections was found, either by clinical signs or virus isolation. Nevertheless, an association between seropositivity against CSFV and swill feeding was found (Chapter 2).

Swill (kitchen waste) feeding is known as a potential source of introduction and spread of CSFV [10]. The outbreak of CSF in the United Kingdom in 2000 is believed to be caused by swill feeding [11]. The positive association between swill feeding and CSF seroprevalence therefore suggests circulation of CSFV in Bhutan. Also the detection of antibodies in pigs that were according to the owners not vaccinated [Chapters 2 and 6;[12]] suggests infections with field virus. On the other hand, it may not always be clear to the owner whether a pig has been vaccinated or not. This applies especially to the backyard farmers that receive pigs that have already been vaccinated before purchase.

Most developed countries have strict regulation not to feed swill to pigs [13]. However, in a country like Bhutan, where most pigs are raised at the backyard level by the rural poor, local farmers are unable to buy specialised pig feed and have to rely on locally available feed. The feeding of swill as pig feed is therefore a common practise. There is a poor awareness among pig owners on CSF and the risk of swill feeding. Better awareness and proper heating of swill before feeding to the pigs could contribute to the control of CSF.

In Bhutan, most of the mortalities in pigs that are reported from the field are thought to be due to CSF. However, this diagnosis is mainly based on clinical signs and post-mortem examination. Between 2011 and 2014, 18 cases of CSF were recorded at the National Centre for Animal Health (TADinfo database), but all based on the clinical signs and post-mortem examination and none of them confirmed by virus detection. Without virological evidence (virus isolation, PCR, antigen-ELISA) it is actually very difficult to estimate the true impact of CSF in Bhutan. Neither the current study, nor a previous study by Raika (1999), could demonstrate the extend and severity of CSF in Bhutan. There is an acute need for diagnostic tools that can be used to either confirm or rule out suspicions of CSF in Bhutan, and that can be used in surveillance programs. The laboratory at the National Centre for Animal Health (NCAH) has been equipped with PCR recently for diagnosing diseases like Avian Influenza (H5N1) and Foot and Mouth Disease (FMD). PCR testing could be extended to the detection of CSF. Also,

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an antigen-ELISA kit for CSFV could be procured for detection of the virus in blood or tissue samples from pigs suspected of CSF.

Vaccination is one of the important tools to prevent and control CSF in countries where control measures such as stamping out and zoning are difficult to apply due to socio-economic reasons [14, 15]. However, in less accessible production systems, like backyard or free ranging systems, effective vaccination campaigns proved to be difficult and demanded alternative approaches and delivery schemes [16]. Based on previous studies [16, 17] and results of the study presented in chapter 6, the oral bait vaccine using the “Chinese-strain” of CSFV may successfully induce a protective immunity in vaccinated pigs reared under backyard village conditions. In our study, the pigs were orally fed vaccine by farmers themselves, which they found quite convenient and most of them were willing to vaccinate using this method. Therefore oral vaccine could be useful in places where parenteral vaccination cannot be used. However, there are still issues to be resolved, like storage conditions of oral vaccines, which currently need a temperature of -20o C, and distribution of vaccines to remote areas. Moreover, the cost of the vaccine is relatively high compared to that of the vaccine administered parenterally.

Alongside vaccination, there is a need to create a general awareness on biosecurity and pig management with the Bhutanese farmers for prevention and control of pig diseases at the backyard level. Awareness on biosecurity through training can play a significant role in reducing the disease spread [18].

Molecular findings of PCV2 and HEV

High seroprevalences were observed against PCV2 and HEV (Chapters 2, 3 and 5). In general, seroprevalences were higher in the government farms than in the backyard farms. The longitudinal study (chapter 3) in sows and piglets suggests an ongoing exposure through endemic infection in the government breeding farms and these farms being the source for infections in backyards.

To gain more insight in the epidemiology of PCV2 and HEV, an attempt was undertaken to detect and sequence viruses from both the government farms and backyard farms. Detection of viruses proves active circulation or recent introduction. Moreover, sequencing allowed the comparison of Bhutanese strains to foreign strains, in an attempt to determine the most likely origin. Both PCV2 and HEV were detected (Chapters 4 and 5) and the sequencing results suggest that they originated from neighbouring countries and had not been introduced with the import of the original breeding stock. The results also suggest that the sources of the two viruses are different and the result of independent introductions in the country. PCV2 [19-21] and HEV [22-24] are endemic in the countries surrounding Bhutan and the virus may have been introduced through import of live pigs, meat or meat products.

For HEV, sequences were obtained from two government farms and from backyard farms in

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two different regions (Chapter 5). The results provided additional evidence for the government farms being the source of the infections in the backyard farms, which corroborated the results of serological study presented in chapter 3. Although sequences from PCV2 could not be obtained from backyard farms, the epidemiological connection between government farms and backyard farms is most likely similar for this virus.

Current pig husbandry system in Bhutan

As mentioned above, in Bhutan pig production is mostly in the form of traditional backyard farms usinglocally available resources including kitchen wastes. Backyard farm sizes typically range from 2-10 pigs. Biosecurity measures in those holdings are generally poor or do not exist. Pigs are often purchased locally, sometimes illegally from across the border, which creates uncontrolled pig movement and a contact structure that promotes the spread of diseases. This was one of the reasons to distribute piglets from government breeding farms, not only to improve the genetics for pig production, but also to improve disease control by putting piglets from known origin on the market. There are three government-ownedpig-breeding farms in Bhutan to supply hybridized piglets to the farmers for fattening and cross-breeding purposes. Many backyard farms are getting upgraded to semi-commercial back-yard farms, especially farms that are near to the towns. The results presented in this thesis indicate that the disease control goal has only been partly successful.

The serological and longitudinal study (repeated sampling) on government breeding farms revealed freedom from two important pig diseases, Aujeszky’s Disease and PRRS. Both diseases are highly prevalent in other Asian countries [25-31]. Both diseases have also not been observed in the backyard farms, demonstrating successful disease prevention in these farms in a situation where the supplier farms are free from the infection. In contrast, a high proportion of sows on the government breeding farms had antibodies against CSF, PCV2 and HEV. For CSF it is likely that the vaccination on the government farms has a positive effect on the disease control in the country as a whole, backyard farms included.

Immunized piglets are supplied to the backyard farms, which will most likely reduce transmission of the virus in the backyard system. However, the observations with PCV2 and HEV also demonstrate the weak points of the supply system. The high seroprevalence of these two pathogens on the government breeding farms indicates their endemic presence on these farms, and according to the results presented in this thesis it is likely that they serve as a source of infection for the backyard farms through piglet distribution. A comparable observation has been made in Vietnam where it was found that PRRS was introduced from state farms to the backyard farms [26]. Consequently, the current system where piglets are supplied from government breeding farms can only succeed in its goal for disease control at the national level

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if infections with major pig pathogens within the government breeding farms are prevented and managed effectively. This requires a good evaluation of the external and internal biosecurity of these farms. Important weaknesses found in the system need to be adjusted. Moreover, the possibility of vaccination against pig diseases other than CSF (e.g. PCV2) can be considered in such an evaluation, not only to help disease control within the government breeding farms, but also to reduce exposure to infection in the backyard farms and possibly supply backyard farms with piglets immune against more pig diseases than CSF alone.

Improving pig production in Bhutan

Pork consumption in Bhutan relies heavily on the import of pork from neighbouring countries. The import of pork was 1842 metric tons (MT) against domestic pork production of only 317 MT, where the demand is far beyond the production capacity of the country [32]. However, the government of Bhutan is aiming to gear towards greater self-sufficiency. It is obvious that the gap cannot be filled with backyard farms only (the country would need 5 times the number of backyard farms currently present). More professional fattening farms or farrow to finish farms are needed. In addition it is crucial to increase the piglet production capacity of breeding farms in the country.

In order to realize the goal of self-sufficiency, the Bhutanese government should stimulate the increase of the pig breeding and fattening capacity. For example, government breeding farms could take more a role to produce gilts as nucleus farms for the replacement of sows on (commercial) breeding and farrow to finish farms. Development of the latter farms would need to be stimulated. As mentioned above, from a disease management control point of view, this would require better health programs in the government breeding farms and the multiplier farms in order to prevent infections to follow the pig flow. Whether or not new breeding farms should be government farms or privately owned commercial farms, is not a point of discussion for this thesis, but will be an important decision to be made.

Independent of the decision, who owns the new breeding farms, is the need for a good disease management system in these farms and the nucleus farms. Especially during the time of growth of the pig husbandry sector, this will be challenging. However, it is crucial to pay attention to this now, before disease management is complicated because of an uncontrolled growth of the sector.

Disease management will highly depend on the origin of the pigs for the new breeding farms. One option would be to have the current government breeding farms grow larger and become a source for gilts to stock new breeding farms. Currently several important pig diseases are absent from the government farms (Aujeszky’s disease, PRRS), while CSF is being controlled by vaccination and other diseases are endemic and are actively circulating (PCV2,

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HEV, possibly SIV), (Chapters 2, 3 and 5). Having the government farms grow larger will take time and diseases present will not so easily be eradicated. As a consequence, stocking new breeding farms from these government farms will result in a similar health status, with the same diseases likely being endemic.

Another option for stocking new breeding farms is purchasing breeding pigs from abroad. In principle, pigs with a desired health status can be purchased and imported to populate new farms. Monitoring of such farms for a certain period may be necessary to make sure no unwanted pathogens were brought in from abroad with the pigs, or are introduced in the farm after it has been established.

A final option is to establish SPF herds in Bhutan, including a nucleus breeding herd which operates at the top of the production pyramid. Such a herd could be populated by Caesarean section derived piglets to guarantee the highest health level. This is an expensive and rather time consuming option, which needs to be planned thoroughly. It also requires a thorough risk assessment to estimate the risk of infections being introduced from the environment, and possible intervention tools (e.g. zoo sanitary measures, vaccination, etc.) to reduce either the chance of introduction or the consequences of an introduction of an unwanted infectious agent in the farm. The knowledge on the current disease status of the government and backyard farms could be valuable in carrying out such a risk assessment.

Next to a more professional (governmental or commercial) pig husbandry system, backyard farming is expected to play an important role in Bhutan society for many years to come. The current system, where backyard farms are provided with piglets from large (government) breeding farms is a good option for the future as well. For exotic diseases, for which there is a desire to completely eradicate them from the country, this seems to be the safest option. It follows a pyramidal structure, with limited movements and other contacts between backyard farms, thus reducing the spread of these diseases between backyard farms. It also allows for easier control, for instance through vaccination of piglets before they are being sold to backyard farms. In this thesis, evidence was shown for the spread of endemic diseases from breeding farms to backyard farms. In relation to other factors that have an effect on productivity in backyard farms, this may or may not be an important factor to take into consideration. Better diagnostic tools and a proper surveillance system need to be implemented in Bhutan to get more insight in the damages these infections may cause in backyard farms and the need for specific intervention measures. It may well be that for certain infections, accepting the current situation is the most balanced outcome, though.

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Concluding remarks

This thesis provides the information on the disease prevalence of selected viral pathogens in pigs in Bhutan and dynamics of SIV (H1N1), PCV2 and HEV infections in piglets through epidemiological studies. For PCV2 and HEV, molecular studies provided additional information on the epidemiology (origin, circulation) of these viruses. Given the objective of the government of Bhutan to increase the countries’ production of pork towards more self-sufficiency, a significant growth of the pig sector is foreseen. This will be a challenge from a logistic and operational point of view, but also from a disease management point of view. Large farms and an overall higher (regional) density of pigs require more elaborate disease management measures. The research in this thesis provides background information on choices to make in the desired growth of the pig husbandry sector in Bhutan, the need for laboratory capacity and surveillance systems for relevant pig diseases.

References

1. Deka RP, Grace D, Lapar ML, Lindahl J: Sharing Lessons of Smallholders’ Pig System in South Asia and Southeast Asia: a Review. National Conference on Opportunities and strategies for Sustainable Pig Production organized by the ICAR-National Research Centre on Pig, Rani, Guwahati, India 2014.2. Lemke U, Valle Zárate A: Dynamics and developmental trends of smallholder pig production systems in North Vietnam. Agricultural Systems 2008, 96(1–3):207-223.3. Kunavongkrit A, Heard TW: Pig reproduction in South East Asia. Animal Reproduction Science 2000, 60–61:527-533.4. Rahman S: Adoption of improved technologies by the pig farmers of Aizawl district of Mizoram, India. Livestock Research for Rural Development 2007, 19.5. Timsina MP, Sherpa DL: Characterization of Bhutan’s indigenous pig genetic resources and production systems. Council of RNR Research of Bhutan, The Ministry of Agriculture 2005.6. Shrestha NP, Edward SA, Robertson JF: Factors affecting reproductive performance in the Nepalese Pakhribas pig: effect of nutrition and housing during gilt rearing. Asian-Australian Journal of Animal Sciences 2002, 15:72-78.7. Rekwot P, YU Abubakar Y, Jegede J: Swine production characteristics and management systems of smallholder piggeries in Kaduna and Benue States of north central Nigeria. Nigerian Veterinary Journal 2003, 24:34-40.8. Lekule FP, Kyvsgaard NC: Improving pig husbandry in tropical resource-poor communities and its potential to reduce risk of porcine cysticercosis. Acta Tropica 2003, 87(1):111-117.

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9. Peltoniemi OAT, Tast A, Love RJ: Factors effecting reproduction in the pig: seasonal effects and restricted feeding of the pregnant gilt and sow. Animal Reproduction Science 2000, 60–61:173-184.10. Edwards S, Fukusho A, Lefèvre P-C, Lipowski A, Pejsak Z, Roehe P, Westergaard J:Classical swine fever: the global situation. Veterinary Microbiology 2000, 73(2–3):103-119.11. Sharpe K, Gibbens J, Morris H, Drew T: Epidemiology of the 2000 CSF outbreak in East Anglia: preliminary findings. The Veterinary record 2001, 148(3):91.12. Raika V: Study of Classical Swine Fever using Ceditest ELISA, MSc thesis in Tropical Veterinary Medicine. University of Edinburgh, U.K 1999.13. Cole CGRR, Henley CN, Dale LO, J. M: Council directive 1774/2002/EC of October 3, 2002 of the European parliament and of the council of October 3 laying down health rules concerning animal by-products not intended for human consumption. Official Journal of the European Communities, CEC (Commission of the European Communities) 200214. Moennig V: Introduction to classical swine fever: virus, disease and control policy. Veterinary Microbiology 2000, 73(2–3):93-102.15. Paton DJ, Greiser-Wilke I: Classical swine fever – an update. Research in Veterinary Science 2003, 75(3):169-178.16. Milicevic V, Dietze K, Plavsic B, Tikvicki M, Pinto J, Depner K: Oral vaccination of backyard pigs against classical swine fever. Veterinary Microbiology 2013, 163(1-2):167-171.17. Brauer A, Lange E, Kaden V: Oral immunisation of wild boar against classical swine fever: Uptake studies of new baits and investigations on the stability of lyophilised C-strain vaccine. European Journal of Wildlife Research 2006, 52(4):271-276.18. Nampanya S, Suon S, Rast L, Windsor PA: Improvement in small-holder farmer knowledge of cattle production, health and biosecurityin southern Cambodia between 2008 and 2010. Transbundary and Emerging Diseases 2011, 59:117-127.19. Anoopraj R, Rajkhowa TK, Cherian S, Arya RS, Tomar N, Gupta A, Ray PK, Somvanshi R, Saikumar G: Genetic characterisation and phylogenetic analysis of PCV2 isolates from India: Indications for emergence of natural inter-genotypic recombinants. Infection, Genetics and Evolution 2015, 31:25-32.20. Jantafong T, Boonsoongnern A, Poolperm P, Urairong K, Lekcharoensuk C, Lekcharoensuk P: Genetic characterization of porcine circovirus type 2 in piglets from PMWS-affected and -negative farms in Thailand Virology Journal 2011, 8:1-6.

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21. Wen L, Guo X, Yang H: Genotyping of porcine circovirus type 2 from a variety of clinical conditions in China. Veterinary Microbiology 2005, 110(1–2):141-146.22. Arankalle VA, Chobe LP, Joshi MV, Chadha MS, Kundu B, Walimbe AM: Human and swine hepatitis E viruses from Western India belong to different genotypes. Journal of Hepatology 2002, 36(3):417-425.23. Shukla P, Chauhan UK, Naik S, Anderson D, Aggarwal R: Hepatitis E virus infection among animals in northern India: an unlikely source of human disease. Journal of Viral Hepatitis 2007, 14(5):310-317.24. Sharma S, Parida M, Shukla J, Rao PVL: Molecular epidemiology of novel swine origin influenza virus (S-OIV) from Gwalior, India, 2009. Virology Journal 2011, 8.25. Tummaruk P, Tantilertcharoen R: Seroprevalence of porcine reproductive and respiratory syndrome, Aujeszky’s disease, and porcine parvovirus in replacement gilts in Thailand. Tropical Animal Health and Production ` 2012, 44(5):983-989.26. Kamakawa A, Thu HTV, Yamada S: Epidemiological survey of viral diseases of pigs in the Mekong delta of Vietnam between 1999 and 2003. Veterinary Microbiology 2006, 118(1-2):47-56.27. Mahesh K.C., Joshi. BR, Shrestha. SP, Prajapati. M, Kathayat. D, Dhakal. S: Sero- Prevalence of Porcine Reproductive and Respiratory Syndrome (PRRS) in Pigs of Different Developmental Regions of Nepal. International Journal of Applied Sciences and Biotechnology 2015, 3:218-222.28. Feng Y, Zhao T, Nguyen T, Inui K, Ma Y, Nguyen TH, Nguyen VC, Liu D, Bui QA, To LT et al: Porcine Respiratory and Reproductive Syndrome Virus Variants, Vietnam and China, 2007. Emerging Infectious Diseases 2008, 14(11):1774-1776.29. Yu X, Zhou Z, Hu D, Zhang Q, Han T, Li X, Gu X, Yuan L, Zhang S, Wang B et al:Pathogenic Pseudorabies Virus, China, 2012. Emerging Infectious Diseases 2014, 20(1):102-104.30. Sharma R, Saikumar G: Porcine parvovirus- and porcine circovirus 2-associated reproductive failure and neonatal mortality in crossbred Indian pigs. Tropical Animal Health and Production 2010, 42(3):515-522.31. Thanawongnuwech R, Morilla A, Yoon K-J, Zimmerman JJ: Aujeszky's Disease in Asia. In: Trends in Emerging Viral Infections of Swine. edn.: Iowa State Press; 2008: 221-224.32. Anonymous: Livestock population and production bulletin: data pertaining to year 2005 and earlier periods. Thimphu, Bhutan, Department of Livstock, MoA. 2007

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Nederlandse Samenvatting

Curriculum vitae

List of publications

Acknowledgements

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Nederlandse Samenvatting

Varkenshouderij in ontwikkelingslanden als Bhutan wordt meestal gekarakteriseerd door kleinschalige productiesystemen voor eigen levensonderhoud. Er is vaak weinig informatie over het vóórkomen van varkensziektes in deze landen. Het doel van dit proefschrift was dan ook om informatie te verzamelen over het vóórkomen van een aantal virusziekten bij varkens in Bhutan.

Er werden seroprevalentiestudies uitgevoerd naar een aantal belangrijke virusziekten bij varkens. Antistoffen tegen porcine circovirus type 2, hepatitis E en varkensinfluenza werden bij varkens door het hele land verspreid gevonden. Antistoffen tegen porcine reproductive and respiratory syndrome en de ziekte van Aujeszky werden daarentegen niet gevonden. Tegen klassieke varkenspest wordt gevaccineerd, en aanwijzingen voor veldinfecties waren er niet.

In een longitudinale studie, waarbij biggen van overheidsbedrijven werden gevolgd, bleek dat virussen als porcine circovirus type 2 en hepatitis E virus vermoedelijk vooral vanuit de overheidsbedrijven in de kleinschalige varkenshouderij terechtkomen. Door het volgen van een aantal zeugen in de drie overheidsbedrijven werd aangetoond dat beide virussen, en ook varkensinfluenzavirus, actief circuleren in deze bedrijven.

Porcine circovirus type 2 op overheidsbedrijven werd gekarakteriseerd door sequencing van het genoom. De gedetecteerde virussen behoorden tot het PCV2b genotype en waren het meest verwant aan stammen die in India circuleren. Hepatitis E virus werd eveneens middels sequencing verder gekarakteriseerd. Het behoort tot genotype 4 en virussen afkomstig van overheidsbedrijven en uit de kleinschalige varkenshouderij bleken vrijwel identiek.

Tenslotte werd een studie naar de effectiviteit van orale vaccinatie tegen klassieke varkenspest uitgevoerd. In 60% van de gevallen leidde dit tot een voldoende hoge, beschermende titer.

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Curriculum vitae

Vijay R. Monger was born on April 8th, 1970 in Thimphu, Bhutan. He graduated with a degree of Veterinary Science and Animal Husbandry from Bombay Veterinary College in the year 1994. He joined the civil service as a veterinary officer under the Department of Livestock, Ministry of Agriculture and Forests in 1994 and worked in various field under the Department of Livestock. He obtained a scholarship under the EU project in Bhutan to pursue his Master’s degree at the University of Edinburgh, Scotland, UK, where he obtained his degree of Master of Science in Tropical Veterinary Medicine in the year 2000. Since then he was working at National Centre for Animal Health as Principal Livestock Health Officer. In 2011, he started his PhD at the Veterinary Faculty of Utrecht University, the Netherlands. The study was conducted as a sandwich research project; six months in the Netherlands and six months in Bhutan for a period of four years and three months. In the Netherlands, he performed his research work at the Central Veterinary Institute part of WUR (CVI-WUR) in Lelystad, the Netherlands. Since July 2015, he works at National Centre for Animal Health as Principal Livestock Health Officer

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List of publications

Monger, V. R., Loeffen, W. L. A., Kus, K., Stegeman, J. A., Dukpa, K., Szymanek, K and Podgorska, K. 2015.Genetic Characterization of Porcine Circovirus Type 2 (PCV2) in Pigs of Bhutan.Transboundary and Emerging Diseases.Doi: 10.1111/tbed.12383.

Monger, V. R., Stegeman, J. A., Dukpa, K., Gurung, R.B and Loeffen, W. L. A. 2014. Evaluation of Oral Bait Vaccine Efficacy Against Classical Swine Fever in Village Backyard Pig Farms in Bhutan.Transboundary and Emerging Diseases.Doi: 10.1111/tbed.12333.

Monger, V. R., Stegeman, J. A., Koop, G., Dukpa, K., Tenzin, T and Loeffen, W. L. A. 2014.Seroprevalence and associated risk factors of important pigviral diseases in Bhutan.Preventive Veterinary Medicine, 117(1):222-32.

Tenzin.,Wacharapluesadee, S., Denduangboripant, J., Dhand, N.K., Dorji, R., Tshering, D., Rinzin, K., Raika, V., Dahal, N and Ward, M.P. 2010. Rabies virus strains circulating in Bhutan: implications for control. Epidemiology and Infection. Doi:10.1017/S0950268810002682

Conference contributions

Tomasz, S., Raika, V., Chabros, K., Rinzin, K., Parchariyanon, S. 2008. Highly pathogenic variant of porcine reproductive and respiratory syndrome (PRRS) virus in Bhutan. An abstract submitted in EPIZONE annual meeting, Turkey 2009 and World Association of Veterinary Laboratory Diagnosticians (WAVLD) meeting, 2009, Madrid Spain.

Monger, V.R., C.Tshering, C., Stadejek, T., Rinzin, K., Chabros, K and Parchariyanoi, S. First Detection of Porcine Reproductive and Respiratory Syndrome Virus from Pigs of Bhutan. Field Epidemiology Conference, 2010, Luang Prabang Lao PDR.

Monger, V. R., Stegeman, J. A., Koop, G., Van der Poel, R.W.H., Hakze-van der Honing, R and Loeffen, W. L. A. Serological study of pig associated viral zoonotic pathogens in Bhutan. Third International Congress on Pathogen at Human-Animal Interphase. One Health for Sustainable development. August 6-8 2015, Chanmai, Thailand.

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Acknowledgements

Firstly, I wish to thank Professor Arjan Stegeman, my principal supervisor, for his constant guidance, encouragement, and timely feedback. My sincere appreciation to Dr. Willie Loeffen, co-supervisor, for his words of wisdom and constant support and guidance at all stages of my project and for facilitating my enrolment at Utrecht university and providing me this opportunity to carry out my PhD. Professor Arjan Stegeman and Dr. Willie Loeffen were critical and inspiring and constantly kept me on my toes to complete this study. And I sincerely acknowledge them for their support and guidance to complete my thesis in time.

I thank the Netherlands University Foundation for International Cooperation and Central Veterinary Institute, Lelystad, The Netherlands for the generous financial support.

My sincere appreciation to Bernie Moonen-Leusen, Sjaak Quak, Yvon Geurts, Klaas and Froukje, Central Veterinary Institute, Lelystad, in the Netherlands for their assistance in carrying out the laboratory works. I wish to express my sincere appreciation to all the people at CVI. I would like to list them all, but the list would be too large and I might miss some names.

I would like to sincerely thank Katarzyna Podgorska, Veterinary Research Institute, Department of Swine Diseases, Partyzantow 57, 24-100 Pulawy, Poland for carrying out the laboratory analysis against Porcine circovirus (PCV2).

My stay in the Netherlands wouldn’t have been so smooth without the support of my colleagues, Jose, Amos, Tasfelem, Bram, Betty and Runa. Thanks to Jaan, my house renter for lending me a room and making me feel at home during my stay in Lelystad.

I would like to thank the Royal Government of Bhutan for granting the study leave. I thank the Secretary, Dasho Tenzin Dhendup, Ministry of Agriculture and Forests and Director General, Dr. Tashi Samdup, Department of Livestock for supporting my study. I appreciate the support of my colleagues: Drs. Kinzang Dukpa, Pasang Tshering, Karma Rinzin, Narapati Dahal, Nirmal Thapa, Ratna Bdr. Gurung, Basant Sharma, Birdoz Rai, Tenzin, Tshering and all veterinarians for facilitating my field and laboratory works in Bhutan. I thank the District Livestock Officers, Field Extension Officers, Laboratory Technicians, and other support staff who were involved in my project.

I am thankful to my wife Bela, children Vanessa and Valin for their continued support, patience and bearing my absence, while I was in the Netherlands. I acknowledge the selfless effort made by my wife in taking care of our children during my absence.

Acknowledgements

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Epidemiology of selected pig viral diseases in Bhutan...Eastern part of the Himalayan range between latitudes 26°45' N and 28° 10' N, and longitudes 88°45' E and 92°10' E. It is