Genetic Characterization of Porcine Circovirus Type 2 (PCV2) in Pigs of Bhutan.

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 multisystemic 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. Porcine circovirus type 2 genome was detected in 16 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.

Evaluation of Oral Bait Vaccine Efficacy Against Classical Swine Fever in Village Backyard Pig Farms in Bhutan.

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 per cent (140 of 193 pigs) of vaccinated pigs had either a slight (2-fold-3-fold; 60 pigs) or significant (at least 4-fold; 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.

Emergency surveillance for novel influenza A(H7N9) virus in domestic poultry, feral pigeons and other wild birds in Bhutan.

Following the March 2013 outbreak of novel avian influenza A(H7N9) virus in humans and the subsequent isolation of the virus from chickens, ducks and pigeons in the People's Republic of China, concerns were raised that the H7N9 virus would spread beyond China through the poultry value chain linking to a number of bordering countries. For this reason, a rapid emergency surveillance exercise took place in Bhutan between May and July 2013 with the objective of determining whether influenza A(H7N9) virus was silently circulating in domestic poultryandwild birds in Bhutan.Atotal of 1716 oropharyngeal,tracheal and cloacal swabs together with faecal droppings were collected from poultry, wild birds and feral pigeons throughout the country; these samples included 150 that had been previously collected for surveillance of influenza A(H5N1) virus. Overall, 733 of the samples were tested. A QIAamp Viral RNA Mini K it was used to extract viral RNA from a mix of oropharyngeal, tracheal and cloacal swabs and faecal droppings. The matrix gene of avian influenza type A virus was detected using a specific real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay, and positive samples were further tested in RT-PCR for simultaneous detection of the H7 and N9 genes. Among the 733 samples tested, 46 (26 prospective, 20 retrospective) were confirmed positive for influenza A, a prevalence of 6.3% (95% CI: 4.6 to 8.3). The influenza A-positive samples were from areas in the south of Bhutan that had experienced previous outbreaks of highly pathogenic influenza A(H5N1). None of the samples tested positive for H7N9 strains, providing evidence that influenza A(H7N9) virus was not present in the sampled population. A risk-based approach for surveillance of influenza A(H7N9) and H5N1 is recommended in Bhutan, based on the epidemiology of the disease in China and other countries in South and Southeast Asia.

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

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.

Risk factors for foot-and-mouth disease in sedentary livestock herds in selected villages in four regions of Bhutan.

AIMS: To identify livestock husbandry practices important for transmission of foot-and-mouth disease (FMD) in the herds and villages of four regions in Bhutan. To consider using this information to enhance the current prevention and control programme, a consideration arising from the failure to control FMD in spite of a control programme in place. METHODS: Between March and May 2009, 383 livestock farmers originating from 80 villages in four districts of Bhutan were interviewed, using a structured questionnaire, about the livestock management practices and incidence of FMD in their herds. Multivariable logistic regression was used to quantify the risk factors that predicted the outcome variable 'farmer-diagnosed FMD in Bhutan'. RESULTS: Sixty-two percent (49/79) of the villages and 87/355 (24%) of herds surveyed had at least one outbreak of FMD within the 5 years preceding the survey. The odds of having FMD in a herd increased substantially (OR=39.2; p<0.0001) when cattle mixed with herds from other nearby villages compared with those where mixing did not occur. Those cattle herds mixing with six or more other herds within the same village were 5.3 times (p<0.0001) more likely to have had FMD than those mixed with fewer than six herds. Farmers who fed kitchen waste to cattle were 14.1 times (p<0.0001), and those who sent their animals for grazing in the forest were 3.1 times (p=0.014), more likely to report FMD in their herds than those who did not. Farmers who kept their cattle always housed in a shed during the day (OR=0.033) or at night (OR=0.29) were less likely to report FMD than those who did not (p<0.04). CONCLUSIONS: Mixing of cattle at grazing areas was identified as a risk factor for FMD. This indicates that spread from infected herds and villages, through close contact, could be an important source of disease for non-infected herds in Bhutan. Therefore, quarantining of early cases in affected herds or villages could reduce the spread of disease within and between villages. This study also highlights the potential role of feeding kitchen waste to cattle as a risk factor for FMD. The findings from this study could be considered for strengthening of the FMD control programme in Bhutan.

The epidemiological characteristics of the 2007 foot-and-mouth disease epidemic in Sarpang and Zhemgang districts of Bhutan.

This study was undertaken to compare the epidemiological characteristics of the 2007 foot-and-mouth disease outbreak in two districts of Sarpang and Zhemgang in Bhutan. Zhemgang district recorded a significantly higher cumulative incidence in all species (26.9%) as well as for cattle (29.3%) compared to Sarpang (6.5% and 7.4%, respectively). The case fatality for cattle in Zhemgang (14.1%) was significantly higher than in Sarpang (3.3%). A total of 404 cattle and 73 pigs died of FMD in Zhemgang, whereas only 21 cattle died in Sarpang. Although all four species were affected in Sarpang, no sheep or goats were affected in Zhemgang. Spatiotemporal analyses showed the existence of four significant clusters, a primary one in Sarpang and three secondary clusters in Zhemgang. The virus belonged to the PanAsia strain of the Middle-East South-Asia topotype (O serotype), and the strain was closely related to the PanAsia strain that circulated in Bhutan during the 2003/2004 outbreaks. The severity of FMD infection in Zhemgang district could be attributed to low vaccination coverage (36.5% in 2006 when compared to 87.6% in Sarpang), inadequate biosecurity, poor nursing care of the sick animals and delayed reporting to the livestock centre. This study highlights the ability of the PanAsia strain of the O serotype to cause unprecedented morbidity and mortality, especially in a naïve population. The study also highlights the benefits of maintaining good herd immunity in the susceptible population, through adequate vaccination coverage, to minimize the severity of infection and limit the spread of disease from infected to non-infected herds.