Wachareeporn Trinachartvanit

Hard ticks and their bacterial endosymbionts (or would be pathogens)

The symbiotic microorganisms of arthropod vectors are highly significant from several points of view, partly due to their possible roles in the transmission of pathogenic causative agents by blood-sucking vectors. Although ticks are well studied because of their significance to human health, novel microbial associations remain to be described. This review summarises several endosymbiotic bacterial species in hard ticks from various parts of the world, including Coxiella-, Francisella-, Rickettsia- and Arsenophonus-like symbionts as well as Candidatus Midichloria mitochondrii and Wolbachia. New methodologies for the isolation and characterization of tick-associated bacteria will, in turn, encourage new strategies of tick control by studying their endosymbionts.

Relative Wolbachia density of field-collected Aedes albopictus mosquitoes in Thailand

ABSTRACT Female Aedes albopictus mosquitoes from natural populations of different geographical regions of Thailand were collected and allowed to oviposit to determine relative Wolbachia A and Wolbachia B densities of their offspring (F1) by using real-time quantitative PCR (RTQ-PCR). An important aspect of this work is that all Aedes albopictus mosquitoes were collected from the field. Twenty-seven offspring were from diverse areas of Thailand (Songkhla, Konkaen, Chantaburi, and Kanchanaburi). The range of relative Wolbabhia A density in F1 mosquitoes was from 0.007 to 1250.78 (bacteria-to-host ratio), whereas relative Wolbachia B densities ranged from 0 to 348.2 (bacteria-to-host ratio). These data are in contrast to those from a previous study that showed a very low amount (less than 0.10) of both relative Wolbachia density types for laboratory strains. The percent transmission of Wolbachia density from mother to each individual offspring cannot be predicted and was not related to the sex of the F1. Obtaining confirmation for variations and unpredictable Wolbachia transmission load raises some concerns about using Wolbachia as a gene-driving system in nature for population replacement if Wolbachia density is involved in cytoplasmic incompatibility in this mosquito.

Gene expression and physiological role of Pseudomonas aeruginosa methionine sulfoxide reductases during oxidative stress

Pseudomonas aeruginosa PAO1 has two differentially expressed methionine sulfoxide reductase genes: msrA (PA5018) and msrB (PA2827). The msrA gene is expressed constitutively at a high level throughout all growth phases, whereas msrB expression is highly induced by oxidative stress, such as sodium hypochlorite (NaOCl) treatment. Inactivation of either msrA or msrB or both genes (msrA msrB mutant) rendered the mutants less resistant than the parental PAO1 strain to oxidants such as NaOCl and H2O2. Unexpectedly, msr mutants have disparate resistance patterns when exposed to paraquat, a superoxide generator. The msrA mutant had a higher paraquat resistance level than the msrB mutant, which had a lower paraquat resistance level than the PAO1 strain. The expression levels of msrA showed an inverse correlation with the paraquat resistance level, and this atypical paraquat resistance pattern was not observed with msrB. Virulence testing using a Drosophila melanogaster model revealed that the msrA, msrB, and, to a greater extent, msrA msrB double mutants had an attenuated virulence phenotype. The data indicate that msrA and msrB are essential genes for oxidative stress protection and bacterial virulence. The pattern of expression and mutant phenotypes of P. aeruginosa msrA and msrB differ from previously characterized msr genes from other bacteria. Thus, as highly conserved genes, the msrA and msrB have diverse expression patterns and physiological roles that depend on the environmental niche where the bacteria thrive.

Detection of Rickettsia and a Novel Haemaphysalis shimoga Symbiont Bacterium in Ticks in Thailand

In this study, we identified two Haemaphysalis species present at the Khao Yai National Park in Thailand and investigated the presence of rickettsia in these ticks. A total of 166 Haemaphysalis specimens were collected randomly under leaves along visitor paths at five locations in the park. Male and female adults of two different Haemaphysalis species, H. shimoga and H. lagrangei, were identified. Polymerase chain reaction (PCR) analysis revealed Rickettsia bacteria in these two Haemaphysalis species; this study represents the first time such presence has been reported in Thailand. The infection rates of Rickettsia were in both H. shimoga (7.41%) and H. lagrangei (10.17%) at these locations in addition to two pools of Haemahysalis nymphs (28.57%). Furthermore, 25.93% of H. shimoga showed positive results that matched Haemaphysalis longicornis symbionts (92% sequence identity) and the Coxeilla burnetti 16S ribosomal RNA gene (90% sequence identity). We propose that this is a novel H. shimoga symbiont bacterium in Thailand and might be a novel Coxeilla-like agent or Coxeilla sp. found in H. shimoga. In contrast, we did not observe any Wolbachia bacteria, which also belong to the order Rickettsiales, in the same group of Haemaphysalis ticks. Furthermore, PCR was used to detect three other genera of bacteria, Anaplasma, Ehrlichia and Borrelia, none of which were identified in the Haemaphysalis ticks studied.

Bioaccumulation of heavy metals in water, sediments, aquatic plant and histopathological effects on the golden apple snail in Beung Boraphet reservoir, Thailand

Changes in the seasonal concentrations of heavy metals (Cu, Mn, Fe, Zn, Pb and Cd) were determined in water, sediments, snails (Pomacea canaliculata) and aquatic plants (Ipomoea aquatica) in three selected tributaries of the Beung Boraphet reservoir, Nakhon Sawan Province, central Thailand. Only Fe, Cu, Mn and Zn were detected by FAAS in all samples collected. The water quality of Beung Boraphet was medium clean with Fe, Mn, Cu and Zn concentrations well below internationally accepted limits. According to the criteria proposed for sediments by the EPA Region V, Zn and Mn concentrations were within the non-polluted range while Fe and Cu (wet season) concentrations fell into the class of severely polluted sediment. Both P. canaliculata and I. aquatica bioconcentrated more Mn in their tissues than were found in sediments, especially in the wet season. The results of Pearson correlation study and BCF values also indicated similar findings. Only Mn showed the importance of sediment-to-snail concentration and high BCF values in both snails and plants. P. canaliculata exposed to contaminated sediment for two months, showed higher accumulation of metals (Fe, Mn, Cu and Zn) in the digestive tracts and digestive glands than in the foot muscles. Histopathological changes included alterations in the epithelial lining of the digestive tracts, digestive glands and the gills. Loss of cilia and increase in mucous cells were observed in the digestive tracts and gills, while the digestive glands exhibited an increase of dark granules and basophilic cells, and dilation of digestive cells. The results indicated that both P. canaliculata and I. aquatica could be used as biomonitors of sedimentary metal contamination for the Beung Boraphet reservoir.

Detection of Coxiella-like endosymbiont in Haemaphysalis tick in Thailand

In this study, we focused on the molecular detection of Coxiella-like bacteria using a PCR technique to identify Coxiella 16S rRNA sequences in Haemaphysalis tick samples (105 adults, 8 nymph pools and 19 larval pools). Seven Haemaphysalis species obtained from 5 locations in Thailand were evaluated in this work. Coxiella endosymbionts could be detected in samples representing all 3 growth stages examined. The results also revealed that only 4 of 7 tick species were positive for Coxiella-like endosymbiont: Haemaphysalis hystricis, Haemaphysalis lagrangei, Haemaphysalis obesa, and Haemaphysalis shimoga. Haemaphysalis shimoga demonstrated the highest percentage of Coxiella-like positive samples (58.33% with n=24), while Haemaphysalis hystricis had the lowest percentage; only 1 female tick was positive for Coxiella-like bacteria (n=6). Interestingly, the results indicated that female Haemaphysalis ticks tended to harbour Coxiella symbionts more frequently than male ticks (59.32% of females and 21.27% of males of all species studied). Phylogenetic analyses based on 16S rRNA sequences illustrated that Coxiella-like spp. from the same tick species always grouped in same clade, regardless of the location from which they were isolated. Moreover, a phylogenetic tree also showed that Coxiella-like endosymbionts from other genera (for example, the tick genus Rhipicephalus) formed a separate group compared to Coxiella-like symbionts in the genus Haemaphysalis. This suggests that a high amount of DNA sequence variation is present in Coxiella-like bacteria harboured by ticks.

Hepatozoon and Theileria species detected in ticks collected from mammals and snakes in Thailand.

We report the detection of Hepatozoon and Theileria in 103 ticks from mammals and snakes in Thailand. By using a genus-specific 18S rRNA PCR, Hepatozoon and Theileria spp. were detected in 8% and 18%, respectively, of ticks (n=79) removed from mammals. Of the ticks removed from snakes (n=24), 96% were infected with Hepatozoon spp., but none were infected with Theileria. Phylogenetic analysis revealed that Hepatozoon spp. detected from Dermacentor astrosignatus and Dermacentor auratus ticks from Wild boar (Sus scrofa) formed a phylogenetic group with many isolates of Hepatozoon felis that were distantly related to a species group containing Hepatozoon canis and Hepatozoon americanum. In contrast, a phylogenetic analysis of the Hepatozoon sequences of snake ticks revealed that Hepatozoon spp. from Amblyomma varanense from King cobra (Ophiophagus hannah) and Amblyomma helvolum ticks from Indochinese rat snake (Ptyas korros), and Asiatic water snake (Xenochrophis piscator) are grouped with Hepatozoon spp. recently isolated from Monocellate cobras, Reticulated pythons and Burmese pythons, all of Thai origin, and with Hepatozoon sp. 774c that has been detected from a tick species obtained from Argus monitors in Australia. A phylogenetic analysis demonstrated that Theileria spp. from Rhipicephalus (Boophilus) microplus, Haemaphysalis obesa, and Haemaphysalis lagrangei ticks from Sambar deer (Cervus unicolor) cluster with the Theileria cervi isolates WU11 and 239, and Theileria sp. Iwate 141. We report for the first time a Hepatozoon species that shares genetic similarity with Hepatozoon felis found in Dermacentor astrosignatus and Dermacentor auratus ticks collected from Wild boars in Thailand. In addition, we found the presence of a Theileria cervi-like sp. which suggests the potential role of Haemaphysalis lagrangei as a Theileria vector in Thailand.

Molecular detection of Rickettsia species in Amblyomma ticks collected from snakes in Thailand

Some reptile ticks are potential vectors of pathogens such as spotted fever group (SFG) rickettsiae. Here, we report for the first time in detail the molecular evidence, DNA sequences and phylogenetic studies, for the presence of Rickettsia spp. in Amblyomma ticks (Amblyomma helvolum and Amblyomma varanense) from snakes in Thailand. A total of 24 tick samples was collected from 4 snake species and identified. A phylogenetic analysis inferred from the partial sequences of the gltA gene indicated that the Rickettsia spp. from 2 Amblyomma helvolum and 1 Amblyomma varanense belong to the same group as the SFG rickettsiae, which are closely related to Rickettsia raoultii strains. In contrast, there was 1 Rickettsia sp. from Amblyomma helvolum grouped into the same clade with other SFG rickettsiae (Rickettsia tamurae, Rickettsia monacensis, and a Rickettsia endosymbiont of Amblyomma dubitatum from Brazil). However, another Rickettsia sp. from Amblyomma varanense was closely related to Rickettsia bellii and Rickettsia sp. strain RDa420 from Thailand. In addition, from phylogenetic results based on the 16S rRNA gene and a concatenated tree of the 3 genes (gltA, ompA, and ompB), we found what may be a novel SFG rickettsia species closely related to Rickettsia raoultii (from both Amblyomma varanense and Amblyomma helvolum). In conclusion, our findings are the first report on the presence of novel SFG rickettsiae in 2 snake tick species, Amblyomma varanense and Amblyomma helvolum in Thailand and in south-eastern Asia.

Detection of Rickettsia and Anaplasma from Hard Ticks in Thailand

ABSTRACT: We collected a total of 169 adult hard ticks and 120 nymphs from under the leaves of plants located along tourist nature trails in ten localities. The results present data examining the vector competence of ticks of different genera and the presence of Rickettsia and Anaplasma species. The ticks belonged to three genera, Amblyomma, Dermacentor, and Haemaphysalis, comprising 11 species. Rickettsia bacteria were detected at three collection sites, while Anaplasma bacteria were detected at only one site. Phylogenetic analysis revealed new rickettsia genotypes from Thailand that were closely related to Rickettsia tamurae, Rickettsia monacensis, and Rickettsia montana. This study was also the first to show that Anaplasma bacteria are found in Haemaphysalis shimoga ticks and are closely related evolutionarily to Anaplasma bovis. These results provide additional information for the geographical distribution of tick species and tick-borne bacteria in Thailand and can therefore be applied for ecotourism management.

Rickettsia sp. Closely Related to Rickettsia raoultii (Rickettsiales: Rickettsiaceae) in an Amblyomma helvolum (Acarina: Ixodidae) Tick from a Varanus salvator (Squamata: Varanidae) in Thailand

ABSTRACT An engorged female Amblyomma helvolum Koch tick was removed from an adult Varanus salvator Laurenti lizard during field collection in Thailand. After using polymerase chain reaction to amplify three genes (16S rDNA, gltA, and OmpA), we discovered the presence of a Rickettsia sp. of the Spotted Fever Group. Phylogenetic analysis revealed that this Rickettsia sp. is closely related to Rickettsia raoultii Mediannikov. Therefore, we report herein for the first time the detection of a novel Spotted Fever Group Rickettsia in an Amblyomma helvolum from a Varanus salvator in Thailand.