A novel Rickettsia, Candidatus Rickettsia takensis, and the first record of Candidatus Rickettsia laoensis in Dermacentor from Northwestern Thailand.

Three hundred and forty-four tick samples were collected from vegetation at Taksin Maharat National Park, Tak province, northwestern Thailand. They were morphologically identified and molecularly confirmed by 16S rRNA and COI genes as Dermacentor laothaiensis (n = 105), D. steini (n = 139), and D. auratus (n = 100). These ticks were examined for the spotted fever group rickettsiae (SFGRs) using PCR and DNA sequencing of six genes; 17-kDa, gltA, 16S rRNA, ompA, ompB, and sca4. Of these ticks, 6.10% (21/344) gave positive results for the presence of SFGRs. Phylogenetic analyses of the SFGRs clearly indicated that a novel genotype assigned as Candidatus Rickettsia takensis was detected in D. laothaiensis (19/105) and at lesser frequency in D. steini (1/139). Furthermore, Candidatus Rickettsia laoensis was also found at a low frequency in D. auratus (1/100), the first record in Thailand. Although, the pathogenicities of these SFGRs remain unknown, our findings suggest potential risks of SFGRs being transmitted via ticks near the border between Thailand and Myanmar, a gateway of daily migrations of local people and visitors both legal and illegal.

Spotted fever group Rickettsia, Anaplasma and Coxiella-like endosymbiont in Haemaphysalis ticks from mammals in Thailand.

Ticks are ectoparasites of vertebrates and vectors of various pathogenic microorganisms. In this study, the presence of bacteria and protozoa was evaluated by PCR and DNA sequencing in 233 mammal ticks collected from 8 provinces in Thailand. Sequence and phylogenetic analyses of partial rickettsial ompA, ompB, sca4 and partial Coxiella 16S rRNA, GroEL, rpoB genes clearly revealed, for the first time, a co-infection of SFG Rickettsia belonging to R. massiliae subgroup and Coxiella-like endosymbiont (CLE), Cox-hein, in a male of Haemaphysalis heinrichi tick infesting Burmese ferret-badger in Loei province. Moreover, a male of H. hystricis tick infesting the same host was infected with another CLE, Cox-hys. Based on the 16S rRNA gene sequence, Anaplasma sp., closely related to Anaplasma bovis was also detected in a male of H. heinrichi infesting the same Burmese ferret-badger. In addition, the third CLE, Cox-asia, found in H. asiatica collected from Asian palm civet in Chiang Rai province, was different from both Cox-hein and Cox-hys. This study provided important data and broadened our knowledge on tick-borne pathogens and endosymbionts in Thailand and Southeast Asia.

Horizontal transmission of Rickettsia felis between cat fleas, Ctenocephalides felis.

Rickettsia felis is a rickettsial pathogen primarily associated with the cat flea, Ctenocephalides felis. Although laboratory studies have confirmed that R. felis is maintained by transstadial and transovarial transmission in C. felis, distinct mechanisms of horizontal transmission of R. felis among cat fleas are undefined. Based on the inefficient vertical transmission of R. felis by cat fleas and the detection of R. felis in a variety of haematophagous arthropods, we hypothesize that R. felis is horizontally transmitted between cat fleas. Towards testing this hypothesis, flea transmission of R. felis via a bloodmeal was assessed weekly for 4 weeks. Rhodamine B was used to distinguish uninfected recipient and R. felis-infected donor fleas in a rickettsial horizontal transmission bioassay, and quantitative real-time PCR assay was used to measure transmission frequency; immunofluorescence assay also confirmed transmission. Female fleas acquired R. felis infection more readily than male fleas after feeding on a R. felis-infected bloodmeal for 24 h (69.3% and 43.3%, respectively) and both Rickettsia-uninfected recipient male and female fleas became infected with R. felis after cofeeding with R. felis-infected donor fleas (3.3-40.0%). Distinct bioassays were developed to further determine that R. felis was transmitted from R. felis-infected to uninfected fleas during cofeeding and copulation. Vertical transmission of R. felis by infected fleas was not demonstrated in this study. The demonstration of horizontal transmission of R. felis between cat fleas has broad implications for the ecology of R. felis rickettsiosis.

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.

Borrelia sp. phylogenetically different from Lyme disease- and relapsing fever-related Borrelia spp. in Amblyomma varanense from Python reticulatus.

BACKGROUND: Species of the genus Borrelia are causative agents of Lyme disease and relapsing fever. Lyme disease is the most commonly reported vector-borne disease in the northern hemisphere. However, in some parts of the world Lyme borreliosis and relapsing fever may be caused by novel Borrelia genotypes. Herein, we report the presence of a Borrelia sp. in an Amblyomma varanense collected from Python reticulatus. METHODS: Ticks were collected from snakes, identified to species level and examined by PCR for the presence of Borrelia spp. flaB and 16S rRNA genes. Phylogenetic trees were constructed using the neighbour-joining method. RESULTS: Three A. varanense ticks collected from P. reticulatus were positive for a unique Borrelia sp., which was phylogenetically divergent from both Lyme disease- and relapsing fever-associated Borrelia spp. CONCLUSION: The results of this study suggest for the first time that there is a Borrelia sp. in A. varanense tick in the snake P. reticulatus that might be novel.

Detection of Rickettsia and Anaplasma from hard ticks in Thailand.

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.

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.

Molecular evidence for novel tick-associated spotted fever group rickettsiae from Thailand.

Ticks are of considerable medical and veterinary importance because they directly harm the host through their feeding action and indirectly through vectoring many bacterial pathogens. Despite many ticks being known from Thailand, very little is known about the bacteria they may harbor. We report here the results of a survey of tick-associated bacteria in Thailand. A total of 334 individuals representing 14 species of ticks in five genera were collected from 10 locations in Thailand and were examined for the human pathogens, Borrelia, Francisella, Rickettsia, and the common arthropod endosymbionts, Wolbachia, by polymerase chain reaction (PCR) assay using specific primers. Rickettsial DNA was detected in 30% (9/30) of Amblyomma testudinarium (Koch, 1844) collected from Khao Yai National Park, Nakhon Nayok Province and 16.84% (16/95) of Hemaphysalis ornithophila (Hoogstraal and Kohls, 1959) collected from Khao Yai National Park, Nakhon Nayok Province and Khao Ang Rue Nai Wildlife Sanctuary, Chachoengsao Province. Rickettsial DNA was not detected in any of the other tick species and no DNA of Borrelia, Francisella, or Wolbachia was detected in any of 14 tick species. Phylogenetic relationships among the rickettsiae detected in this study and those of other rickettsiae were inferred from comparison of sequences of the 17-kDa antigen gene, the citrate synthase gene (gltA), and the 190-kDa outer membrane protein gene (ompA). Results indicated that the three Thai rickettsiae detected in this study represent new rickettsial genotypes and form a separate cluster among the spotted fever group rickettsiae.

Phylogenetic relationships of Francisella-like endosymbionts detected in two species of Amblyomma from snakes in Thailand.

Ticks are important vectors of several bacterial pathogens, including Francisella. In this study, a total of 24 adult ticks (Amblyomma varanense and Amblyomma helvolum) collected from 4 species of snake from 3 provinces of Thailand was screened for the presence of Francisella-like endosymbionts (FLEs) by PCR. FLEs were detected in 46% (11/24) of all ticks examined. A phylogenetic analysis of the 16S rDNA sequence indicated that the 11 distinct genotypes of FLEs amplified from A. varanense and A. helvolum (from Khon Kaen and Pichit provinces, respectively) are in the same group, along with other FLEs amplified from the other tick genera. Interestingly, these FLEs are closely related to, but distinct (different clade) from, the FLEs isolated from different tick species previously reported. This work represents the first report of Francisella spp. in snake ticks from 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.

Dissemination of bloodmeal acquired Rickettsia felis in cat fleas, Ctenocephalides felis.

BACKGROUND: Cat fleas, Ctenocephalides felis, are known biological vectors for Rickettsia felis. Rickettsial transmission can be vertical via transovarial transmission within a flea population, as well as horizontal between fleas through a bloodmeal. The previously undescribed infection kinetics of bloodmeal-acquired R. felis in cat fleas provides insight into the R. felis-flea interaction. FINDINGS: In the present study, dissemination of R. felis in previously uninfected cat fleas fed an R. felis-infected bloodmeal was investigated. At weekly intervals for 28 days, rickettsial propagation, accumulation, and dissemination in gut epithelial cells, specifically in the hindgut and the specialized cells in the neck region of midgut, were observed on paraffin sections of infected cat fleas by immunofluorescence assay (IFA) and confirmed by PCR detection of R. felis 17-kDa antigen gene. IFA results demonstrate ingested rickettsiae in vacuoles during early infection of the gut; lysosomal activity, indicated by lysosome marker staining of freshly-dissected gut, suggests the presence of phagolysosome-associated vacuoles. Subsequent to infection in the gut, rickettsiae spread to the hemocoel and other tissues including reproductive organs. Densely-packed rickettsiae forming mycetome-like structures were observed in the abdomen of infected male cat fleas during late infection. Ultrastructural analysis by transmission electron microscopy (TEM) confirmed the presence and infection characteristics of Rickettsia including rickettsial destruction in the phagolysosome, rickettsial division, and accumulation in the flea gut. CONCLUSIONS: This study intimately profiles R. felis dissemination in cat fleas and further illuminates the mechanisms of rickettsial transmission in nature.

Evaluation of mosquito densoviruses for controlling Aedes aegypti (Diptera: Culicidae): variation in efficiency due to virus strain and geographic origin of mosquitoes.

Four mosquito densovirus strains were assayed for mortality and infectivity against Aedes aegypti larvae from different geographic regions. The viral titers were quantified by real-time PCR using TaqMan technology. Firstinstar larvae were exposed to the same titer of each densovirus strain for 48 hours. All strains of densoviruses exhibited larvicidal activity and caused more than 80% mortality and infectivity in the three mosquito strains. AalDNV-exposed larvae had the highest mortality rate. The mean time to death of AalDNV-exposed larvae was shorter than other DNVs-exposed larvae. We can conclude that different densovirus strains exhibit some variations in their pathogenicity to different populations of Ae. aegypti mosquitoes. A few mosquitoes from Chachoengsao and Bangkok exposed to AeDNV and AThDNV survived to the adult stage to lay eggs and showed 22% to 50% vertical transmission in the F1 generation. Phylogenetic analysis of four densovirus strains indicated that mosquito densoviruses are separated into two distinct clades.

Mosquito densonucleosis virus non-structural protein NS2 is necessary for a productive infection.

Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins. Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny.