Molecular Surveillance for Vector-Borne Bacteria in Rodents and Tree Shrews of Peninsular Malaysia Oil Palm Plantations.

Many human clinical cases attributed to vector-borne pathogens are underreported in Malaysia, especially in rural localities where healthcare infrastructures are lacking. Here, 217 small mammals, consisting of rodents and tree shrews, were trapped in oil palm plantations in the Peninsular Malaysia states of Johor and Perak. Species identification was performed using morphological and DNA barcoding analyses, and 203 small mammals were included in the detection of selected vector-borne bacteria. The DNA extracted from the spleens was examined for Orientia tsutsugamushi, Borrelia spp., Bartonella spp. and Rickettsia spp. using established PCR assays. The small mammals collected in this study included Rattus tanezumi R3 mitotype (n = 113), Rattus argentiventer (n = 24), Rattus tiomanicus (n = 22), Rattus exulans (n = 17), Rattus tanezumi sensu stricto (n = 1) and Tupaia glis (n = 40). Orientia tsutsugamushi, Borrelia spp. and Bartonella phoceensis were detected in the small mammals with the respective detection rates of 12.3%, 5.9% and 4.9%. Rickettsia spp., however, was not detected. This study encountered the presence of both Lyme disease and relapsing fever-related borreliae in small mammals collected from the oil palm plantation study sites. All three microorganisms (Orientia tsutsugamushi, Borrelia spp. and Bartonella phoceensis) were detected in the R. tanezumi R3 mitotype, suggesting that the species is a competent host for multiple microorganisms. Further investigations are warranted to elucidate the relationships between the ectoparasites, the small mammals and the respective pathogens.

Habitat and Season Drive Chigger Mite Diversity and Abundance on Small Mammals in Peninsular Malaysia.

Chigger mites are vectors of the bacterial disease scrub typhus, caused by Orientia spp. The bacterium is vertically transmitted in the vector and horizontally transmitted to terrestrial vertebrates (primarily wild small mammals), with humans as incidental hosts. Previous studies have shown that the size of the chigger populations is correlated with the density of small mammals in scrub typhus-endemic regions. Here, we explore interactions between the small mammals and chiggers in two oil palm plantations located in the Perak and Johor states of Peninsular Malaysia. The location in Perak also contained an aboriginal (Orang Asli) settlement. A ~5% sub-sample from 40,736 chigger specimens was identified from five species of small mammals (n = 217), revealing 14 chigger species, including two new records for Malaysia. The abundance and species richness of chiggers were significantly affected by habitat type (highest in forest border), state (highest in Perak), and season (highest in dry). The overall prevalence of Orientia tsutsugamushi DNA in small-mammal tissues was 11.7% and was not significantly affected by host or habitat characteristics, but in Johor, was positively associated with infestation by Leptotrombidium arenicola. These findings highlight the risk of contracting scrub typhus in oil palm plantations and associated human settlements.

Molecular evidence of rat bocavirus among rodents in Peninsular Malaysia.

Rat bocavirus (RBoV) and rodent bocavirus (RoBoV) have previously been detected in Rattus norvegicus; however, these viruses have not been reported in rodent populations in Malaysia. We investigated the presence of RBoV and RoBoV in archived rodent specimens. DNA barcoding of the rodent cytochrome c oxidase gene identified five different species: Rattus tanezumi R3 mitotype, Rattus tiomanicus, Rattus exulans, Rattus argentiventer, and Rattus tanezumi sensu stricto. Three spleens were positive for RBoV (1.84%; 3/163), but no RoBoV was detected. Phylogenetic analyzes of the partial non-structural protein 1 gene grouped Malaysian RBoV strains with RBoV strains from China. Further studies among rats from different geographical locations are warranted for this relatively new virus.

Replication Kinetics of Rickettsia raoultii in Tick Cell Lines.

Rickettsia raoultii is one of the causative agents of tick-borne lymphadenopathy in humans. This bacterium was previously isolated and propagated in tick cell lines; however, the growth characteristics have not been investigated. Here, we present the replication kinetics of R. raoultii in cell lines derived from different tick genera (BME/CTVM23, RSE/PILS35, and IDE8). Tick cell cultures were infected in duplicate with cryopreserved R. raoultii prepared from homologous cell lines. By 12-14 days post infection, 100% of the cells were infected, as visualized in Giemsa-stained cytocentrifuge smears. R. raoultii growth curves, determined by rickettsiae-specific gltA qPCR, exhibited lag, exponential, stationary and death phases. Exponential phases of 4-12 days and generation times of 0.9-2.6 days were observed. R. raoultii in BME/CTVM23 and RSE/PILS35 cultures showed, respectively, 39.5- and 37.1-fold increases compared to the inoculum. In contrast, multiplication of R. raoultii in the IDE8 cultures was 110.1-fold greater than the inoculum with a 7-day stationary phase. These findings suggest variation in the growth kinetics of R. raoultii in the different tick cell lines tested, amongst which IDE8 cells could tolerate the highest levels of R. raoultii replication. Further studies of R. raoultii are needed for a better understanding of its persistence within tick populations.

Current tools for the diagnosis and detection of spotted fever group Rickettsia.

Spotted fever group (SFG) rickettsiae causes a number of diseases in humans worldwide, which can range from mild to highly lethal. Since the clinical presentations of rickettsioses caused by SFG rickettsiae are variable and may be similar to the diseases caused by other rickettsiae, such as Orientia tsutsugamushi (agent for scrub typhus), Coxiella burnetii (agent for Q fever) and the typhus group rickettsiae (agents for epidemic and murine typhus), the accurate diagnosis of infections caused by SFG Rickettsia remains challenging especially in resource-poor settings in developing countries. This review summarizes the various diagnostic and detection tools that are currently available for the confirmation of infections by SFG rickettsiae. The advantages and challenges pertaining to the different serological and molecular detections methods, as well as new assays in development, are discussed. The utility of the detection tools contributing to the surveillance of SFG rickettsiae in arthropods and animals are reviewed.

Molecular detection of pathogens from ectoparasites recovered from peri-domestic animals, and the first description of a Candidatus Midichloria sp. from Haemaphysalis wellingtoni from rural communities in Malaysia.

Rural communities in Malaysia have been shown to be exposed to Coxiella, Borrelia and rickettsial infections in previous seroprevalence studies. Further research is necessary to identify the actual causative agents and the potential vectors of these infections. The arthropods parasitizing peri-domestic animals in these communities may serve as the vector in transmitting arthropod-borne and zoonotic agents to the humans. Molecular screening of bacterial and zoonotic pathogens from ticks and fleas collected from dogs, cats and chickens from six rural communities in Malaysia was undertaken. These communities were made up of mainly the indigenous people of Malaysia, known as the Orang Asli, as well as settlers in oil palm plantations. The presence of Coxiella burnetii, Borrelia, and rickettsial agents, including Rickettsia and Anaplasma, was investigated by performing polymerase chain reaction (PCR) and DNA sequencing. Candidatus Rickettsia senegalensis was detected in one out of eight pools of Ctenocephalides felis fleas. A relapsing fever group Borrelia sp. was identified from one of seven Haemaphysalis hystricis ticks tested. The results from the PCR screening for Anaplasma unexpectedly revealed the presence of Candidatus Midichloria sp., a potential tick endosymbiont, in two out of fourteen Haemaphysalis wellingtoni ticks tested. C. burnetii was not detected in any of the samples tested. The findings here provide evidence for the presence of potentially novel strains of rickettsial and borrelial agents in which their impact on public health risks among the rural communities in Malaysia merit further investigation. The detection of a potential endosymbiont of ticks also suggest that the presence of tick endosymbionts in the region is not fully explored.

Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines.

Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2-5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the "pandemic" A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.