Surveillance, Identification, and Characterization of Burkholderia pseudomallei Infection in Macaques and Orangutans.

Abstract

Burkholderia pseudomallei is a Gram-negative bacterium responsible for causing melioidosis in a wide range of species. It may infect susceptible hosts through cutaneous, ingestion, or inhalation routes. The disease has a moderate to high case fatality rate, and the causative microorganism is categorized as Tier 1 Select Agent by the United States CDC. Concerns regarding melioidosis were raised among nonhuman primate (NHP) researchers in Indonesia, following a fatal melioidosis case in a cynomolgus macaque (Macaca fascicularis) in a primate research center and fatal cases in orangutans. Those incidents were the driving force to conduct surveillance in primate populations. Serosurveillance was conducted on cynomolgus (Macaca fascicularis) and pig-tailed macaques (Macaca nemestrina) in several facilities breeding facilities in West Java and Banten, Indonesia: Jonggol, Darmaga, Lodaya, and Tinjil Island facilities. The serosurveillance was conducted using a panel of enzyme-linked immunosorbent assays targeting immunoglobulins G toward B. pseudomallei proteins: purified lipopolysaccharide (LPS) and recombinant proteins of alkyl hydroperoxide reductase subunit C (AhpC), hemolysin coregulated protein (Hcp1), and putative outer membrane porin protein (OmpH). Demographic factor analysis showed that species and breeding location were significant factors that were associated with seropositivity. Cynomolgus macaques and Jonggol facility have significantly association with seropositivity towards B. pseudomallei antigenic proteins. Enclosures in Jonggol are designed with soil flooring that may act as the reservoir of B. pseudomallei. Wet season and the observed grass-plucking behavior in those macaques may have contributed to the exposure to B. pseudomallei in the deeper layer of the soil. Pig-tailed macaques are predicted to be less-susceptible to the bacteria, since they showed lower immune response even though being reared in the same facility as some seropositive cynomolgus macaques. An evaluation of the immunoassay panel showed that there were strong and positive correlations of the recombinant protein assays (AhpC, Hcp1, and OmpH), whereas antibody level in LPS assay was higher but showed no correlation to the results of other assays. This may suggest that the development of antibody towards B. pseudomallei LPS is quicker and stronger, since LPS is a strong T-cell independent antigen. It may explain the reason that some animals had high level of antibody to LPS but not to other antigens. Even though the seropositivity ranges from 13-88% in several facilities, the presence of immunoglobulin G against B. pseudomallei does not necessarily mean that the macaques were currently having melioidosis, as the antibodies may reflect asymptomatic infection or a resolved infection. Efforts to culture throat, oral, wound, nasal, and rectal swabs of seropositive macaques resulted in no positive culture for B. pseudomallei. Based on previous reports by other researchers, macaques (including Macaca mulatta, Macaca fascicularis, and Macaca nemestrina) have been considered as animal species with moderate to high resistance towards melioidosis. Negative culture from seropositive animals in this study may indicate that those animals have been exposed to B. pseudomallei, but do not develop melioidosis, or the condition has been resolved. Environmental sampling from soil in several breeding facilities also resulted in no culturable B. pseudomallei, but the number of samples and area coverage was not adequate to represent the very large area of the facilities. In this study, B. pseudomallei was isolated from a fatal melioidosis case in a Bornean orangutan (Pongo pygmaeus wurmbii). Diagnosis was confirmed through culture of pulmonary abscess which was then followed by TTSS-1 real-time PCR assay of the bacterial colonies. In order to provide information regarding pathogen characteristics, morphology observation, multilocus sequence typing (MLST) and YLF/BYFC PCR was conducted on B. pseudomallei isolate obtained from pulmonary abscess. On Ashdown agar, the isolates resembled Type I and Type II morphotypes of B. pseudomallei. The isolate showed positivity to YLF gene cluster, which is almost exclusively found in isolates form Asia region. Sequence typing of seven loci of B. pseudomallei housekeeping genes (ace, gltB, gmhD, lepA, lipA, nark, and ndh) showed a combination of allele sequence type, that was further identified as B. pseudomallei sequence type (ST) 54. That sequence type was analyzed by E-BURST, a method that picture relatedness between similarity in allele types. E-BURST was performed on multiple datasets; all sequence types in Borneo or Kalimantan, all sequence types in Indonesia, all sequence types in Southeast Asia, all sequence types found in animals, all sequence types found in the environment, all sequence types found in humans, and all reported sequence types worldwide. E-BURST analysis of this ST and other STs of B. pseudomallei found in SE Asian countries showed that ST54 is clustered together with STs from Malaysia, Thailand, and Cambodia. It is predicted that ST54 shares a common ancestor with STs found in those countries. Information obtained in this study is the first milestone in discovering melioidosis in nonhuman primates in Indonesia and may serve as a stepping stone to discover the burden of the disease in other animal species, by providing the benefits of using panel immunoassays and providing information on local B. pseudomallei characteristics