This is the first study to report the seroprevalence of four viral zoonoses in the pig population of Laos. Previous published studies on JEV in Laos have focused on human populations,46,47
and our study represents the first assessment of the role pigs might play in transmission to humans in Laos. Previous swine HEV studies have been reported from Laos,15,48
and our survey supports and adds weight to the argument that pigs have a potential role in the natural history of human HEV disease. No published studies have reported seroprevalence of NiV and SIV in the pig population in Laos, and we provide strong evidence that pigs pose little risk for human NiV and SIV disease, at least at the time the survey was conducted. Antibodies against NiV were not detected, and only a limited number of animals had serologic evidence of a previous SIV infection with a non-pathogenic subtype.
This study demonstrates unequivocally that JEV was widespread in the pig population of northern Laos, and a seroprevalence of 74.7% was indicative of a hyper-epizootic state. Pigs are not vaccinated against JEV in northern Laos, and these results represent natural transmission. Furthermore, maternal antibodies wane after two months,49
and the youngest pigs in our survey population were four months of age, indicating that detected antibodies were raised against active JEV infections rather than via passive immunity.
Antibodies to JEV were detected in all provinces, and significant differences in prevalence were observed between provinces for both temporal sampling frames. Because HI can detect antibodies against JEV in pigs up to three years post-infection50
and the median age at slaughter was 12 months, it was unlikely that the observed seroprevalence in the two temporal sampling frames was influenced by the timing of sample collection. The differences we encountered might have been caused by factors such as pig density, rice paddy production, and Culex
mosquito abundance. This hypothesis was further supported by the finding that pigs purchased for slaughter from free-range production systems had lower seroprevalences than penned pigs, and free-range production systems were encountered predominantly in upland rice-growing areas with limited paddies. However, the observed prevalence in all four provinces was high.
Prevalence of IgM against JEV peaked in June and July, corresponding to the start of the wet season, and water filling of rice paddies providing suitable breeding conditions for Culex
mosquitoes. In pigs, IgM is detected within 2–3 days post-infection and can be detected in serum for up to 3 weeks,35
which indicated that IgM-positive pigs we detected were recently infected and that peak transmission and greatest risk for human infection corresponds with the first half of the wet season. This peak in pigs in Laos was consistent with peak transmission to humans in Thailand in June and July 1983.51
Because we did not present a single sampling frame over a complete year, caution should be exercised in interpreting seasonal transmission patterns. However, although we would not expect highly active transmission in the dry season months because of a lack of mosquito breeding sites, the impact of irrigated rice production on Culex
mosquito abundance in the dry season of northern Laos remains to be determined.
The IgM ELISA results for pigs provide limited evidence that JEV is not maintained in the pig population throughout the year, which is consistent with an epizootic pattern of transmission. This finding could be caused by a combination of relatively low animal densities,52
a short duration of viremia, ranging from 1 to 3 days,53
and a decrease in mosquito vector abundance in the dry season winter months. The migration patterns of ardeid birds could therefore have a strong influence on JEV transmission patterns, and several ardeid bird species breed in Laos during the wet season months and other species overwinter during the dry season months.29,54
The role of these migratory birds in maintaining JEV in an epizootic state in Laos warrants further investigation.
In pigs, the most clinically significant manifestation of JEV infection is reproductive failure in sows because of abortion and abnormal farrowing.6
The high seroprevalence of JEV in young pigs ≤ 6 months of age indicates that JEV would have little or no impact on the reproductive potential of local indigenous breed sows. Indigenous breed sows in Southeast Asia sexually mature at 6–8 months of age,55
and most sows in Laos would have protective immunity by the age of first estrus. However, the impact on the reproductive potential of indigenous breed boars may be more significant. Indigenous breed boars in Southeast Asia can reach sexual maturity at 2–3 months of age,55
and infection of sexually mature boars can cause infertility.6
Because the smallholder pig sector in Laos has low productivity,56,57
we believe that the effect of JEV on this pig producing sector warrants greater scrutiny, with particular reference to boar infertility.
Two recent swine HEV studies in Laos15,48
and the present study demonstrate the relative importance of pigs as a reservoir of human HEV disease. Blacksell and others15
observed a high seroprevalence of HEV in pigs sampled at provincial slaughterhouses in northern Laos; 85.7%, 47.1%, 60.0%, and 72.1% for Huaphan, Luangprabang, Oudomxay, and Xiengkhuang Provinces, respectively. These data are substantially higher than those observed during this current study; 29–30% was observed in Xiengkhuang and Oudomxay Provinces in the wet season and 12–15% in Huaphan and Luangprabang in the dry season. The difference may have been caused by seasonal variation because Blacksell and others15
observed high seroprevalence in Huaphan and Luangprabang Provinces when sampling was conducted in the wet season months. Further work will be required to confirm seasonal peaks of transmission. However, data for Oudomxay and Xiengkhuang Provinces provides evidence that the peak seroprevalence was at the start of the wet season.
Age-related seroprevalence in the current study peaked in 4–6-month-old pigs sampled in the wet season and in 7–12-month-old pigs sampled in the dry season. The combined temporal and age prevalence data indicates that young animals are an important reservoir of HEV at the beginning of the wet season. We can speculate that management of young animals differs from that of older animals and predisposes them to infection during the wet season. However, further research will be required to understand the specific production practices associated with increased risk of HEV transmission. The importance of young animals in the epidemiology of HEV in Laos was further demonstrated by Conlan and others,48
who observed that 11.6% of pigs ≤ 6 months of age were shedding virus during the dry season months (January–March). Only genotype 4 HEV has been recognized in northern Laos,48
and this same genotype has been identified as the most common cause of human HEV disease in southern and eastern China.16,17
The seroprevalence of human HEV in Laos has been estimated to be 16–18%, and 2–4% of acute hepatitis hospital admissions were caused by HEV.13,14
To date, no data exist that describe the genotypes causing human HEV disease in Laos, and work should now be undertaken to establish the source of human infections.
Nipah virus has been detected in Pteropid fruit bats in Malaysia, Cambodia, and Thailand by serologic analysis, virus isolation, or RNA amplification,24,26,58
and NiV antibodies have also been detected in fruit bats in Indonesia,25
providing evidence that NiV is endemic to Southeast Asia. The most human cases and deaths of any outbreak to date occurred during the outbreaks in Malaysia and Singapore in 1999, and pigs were the source of human infections, demonstrating the importance of pigs as an intermediary amplification host. We found no serologic evidence of NiV infection of pigs in Laos, but this finding does not confirm the absence of NiV in this country. Pteropid fruit bats are present in Laos and to better understand the epidemiology and risks of NiV, surveys of these competent reservoir host species will be required.
Serologic data for SIV indicated low levels of virus circulation in the pig survey population during May 2008–January 2009. We found no evidence of H1NI (Ratchaburi or pandemic 2009 strain) infection; only the H3N2 subtype was detected. We could only confirm HI positivity for 2 of 23 ELISA-reactive serum samples, both of which were inconclusive in the ELISA, indicating that the ELISA has not been fully optimized for pig serum or that different subtypes are circulating in pigs in Laos. The influenza ELISA is well defined for testing avian and equine serum but less well defined for testing pig serum. This finding was further evident in our finding that two ELISA-negative samples were positive for H3N2 by HI. Influenza HI is affected by the degree of homology between the assay virus and the virus to which animals have been exposed. It was possible that many of the 14 serum samples that were ELISA positive, but not HI positive, could represent animals with exposure to a sufficiently different but homologous subtype, or to an untested subtype such as H9 or H5. In southern China, near the border region with northern Laos, the H9 subtype has been shown to have a prevalence of 10.5%30
and could explain some of the discrepancies we observed between ELISA and HI results. However, the 2 of 14 ELISA-negative HI (H3N2)-positive serum samples suggests that additional work is required to define ELISA sensitivity with pig serum in Southeast Asia. The low frequency of antibody to SIV may also result from a low-density pig population acting as a natural barrier to maintenance of virus endemicity and therefore to timing of the test cohort relative to the opportunity for periodic spread of the virus.
We provide evidence that pigs are an important reservoir of the viral zoonoses JEV and HEV in Laos, and limited evidence suggests the early months of the monsoonal wet season (May–July) coincide with peak transmission in pigs and greatest risk for human disease. Our study supports the need for continued surveillance of pig-associated viral zoonoses and the integration of human and veterinary public health authorities to control these important diseases.