Domestic Pigs and Japanese Encephalitis Virus Infection, Australia

To determine whether relocating domestic pigs, the amplifying host of Japanese encephalitis virus (JEV), decreased the risk for JEV transmission to humans in northern Australia, we collected mosquitoes for virus detection. Detection of JEV in mosquitoes after pig relocation indicates that pig relocation did not eliminate JEV risk.

subgroup, from which Cx. annulirostris is considered to be the most important species, are the primary Australian JEV vectors (5). During an outbreak in 1998, the virus was shown to be widespread on Badu Island; isolates were obtained from mosquitoes collected throughout the community (6). A vaccination program initiated on the outer Torres Strait islands in 1995, including Badu Island, appears to have limited the number of human clinical cases (5).
To further reduce JEV risk for Badu Island residents, after the 1998 outbreak domestic pigs were removed from the Badu Island community to a piggery located ≈2.5 km away ( Figure 1, panel B). This relocation led to a significant reduction in the proportion of Cx. annulirostris feeding on pigs, and speculation was that this might reduce the number of JEV-infected mosquitoes (7). We report on the effects of pig relocation away from human habitation on virus infection rate in Cx. sitiens subgroup mosquitoes.

The Study
In response to JEV activity, as evidenced by human clinical cases or the seroconversion of sentinel pigs, adult mosquitoes were collected on Badu Island during 1995, 1998, and 2003 (6,8, and this article, respectively). Badu Island is located at 10º07′S and 142º09′E in the central western region of the Torres Strait and is a granite island of ≈101 km 2 ; its ecology has been described (6)(7)(8)  W. Hock Co., Gainesville, FL, USA) baited with either CO 2 alone or in combination with 1-octen-3-ol. Mosquitoes were killed on dry ice and placed in liquid nitrogen dry shippers or on dry ice in insulated containers for transport to Cairns for storage at -70 o C. Mosquitoes were placed on a refrigerated table for species or taxonomic group identifi cation before being placed in pools of <200 mosquitoes and sent to Queensland Health Forensic and Scientifi c Services or the University of Queensland, Brisbane, for JEV detection.
The virus isolation protocols used in 1995 and 1998 have been described (6,8). In 2003, virus RNA was detected by using a real-time TaqMan reverse transcription-PCR (9).
To facilitate the comparison of virus distribution on Badu Island, trap locations were grouped into 3 general areas: within 1.2 km of the piggery, within the area of human habitation (the community), and at a rubbish dump located ≈1.5 km from the community and 4.0 km from the piggery ( Figure 2). Dumps are a potential focus of JEV activity because mosquitoes, feral pigs, and wading birds congregate at them (10). Maximum-likelihood estimation of mosquito infection rates with 95% confi dence intervals were calculated for each of these general areas by using the PooledIn-fRate statistical software package (11).
Because of the diffi culty in morphologically separating the members of the Cx. sitiens subgroup, data for the 3 members of the group found in Australia-Cx. annulirostris, Cx. palpalis, and Cx. sitiens-were pooled for analysis. However, PCR restriction fragment length polymorphism analysis of a subsample of 135 Cx. sitiens subgroup mosquitoes collected in 2003 showed that Cx. annulirostris was the dominant member of this group on Badu Island and comprised 94.1% of polymorphic specimens processed; the other 4.4% and 1.5% were identifi ed as Cx. palpalis and Cx. sitiens, respectively.
A total of 44,328 Cx. sitiens subgroup mosquitoes were processed for detection of JEV; 2,871, 24,592, and 16,865 were processed from 1995, 1998, and 2003, respectively (Table). JEV was detected in 66 pools of Cx. sitiens subgroup mosquitoes; the highest maximum-likelihood estimation of mosquito infection rate was obtained from mosquitoes collected at the dump in 1998. Despite removal of the domestic pigs, JEV was still detected in 5 pools of Cx. sitiens subgroup mosquitoes collected within the community in 2003. However, the point estimates of infection rates were lower than those obtained in 1995 and 1998, when domestic pigs were present within the community, although the slight overlap in 95% confi dence intervals indicates that this difference in infection rate was not signifi cant.

Conclusions
We demonstrated that although removing domestic pigs from areas of human habitation may reduce contact between amplifying hosts and vectors (7), it does not elimi-nate the presence of JEV-infected mosquitoes. Thus, pig removal does not negate JEV risk for humans. Indeed, evidence for low-level virus transmission to humans is provided by Hanna et al., who found a low level (32%) of natural boosting immunity in Badu residents who had received an inactivated mouse brain-derived JEV vaccine 3 years earlier (12).
As has been observed elsewhere in the absence of pigs (13), mosquitoes may have become infected by feeding on viremic herons and egrets, populations of which are found on Badu Island (14). Analysis of host feeding patterns demonstrated that birds accounted for 23% of blood meals of Cx. annulirostris identifi ed from the dump in 2003 (S. Hall-Mendelin and A.F. van den Hurk, unpub. data). Alternately, feral pigs in the community and at the dump are a potential source of virus for mosquitoes, although <1% of mosquito blood meals were from swine at these locations. Mosquitoes could become infected by feeding on viremic pigs at the piggery and then disperse to other areas on the island. The mean fl ight distance of Cx. annulirostris is 4.4 km; some females traverse up to 12 km (15), which is considerably farther than the 2.5 km between the piggery and the community on Badu Island. Solomon recommends that domestic pigs be moved >5 km from human habitation to limit JEV transmission to humans (3). Indeed, if infected mosquitoes were fl ying from the piggery to the community, then the data from our study support this recommendation. However, we suggest that domestic pigs be removed far enough away from human habitation to encompass the fl ight range of the local Culex vectors.