Arunee Ahantarig

Morphological changes in human neural cells following tick-borne encephalitis virus infection.

Tick-borne encephalitis (TBE) is one of the leading and most dangerous human viral neuroinfections in Europe and north-eastern Asia. The clinical manifestations include asymptomatic infections, fevers and debilitating encephalitis that might progress into chronic disease or fatal infection. To understand TBE pathology further in host nervous systems, three human neural cell lines, neuroblastoma, medulloblastoma and glioblastoma, were infected with TBE virus (TBEV). The susceptibility and virus-mediated cytopathic effect, including ultrastructural and apoptotic changes of the cells, were examined. All the neural cell lines tested were susceptible to TBEV infection. Interestingly, the neural cells produced about 100- to 10,000-fold higher virus titres than the conventional cell lines of extraneural origin, indicating the highly susceptible nature of neural cells to TBEV infection. The infection of medulloblastoma and glioblastoma cells was associated with a number of major morphological changes, including proliferation of membranes of the rough endoplasmic reticulum and extensive rearrangement of cytoskeletal structures. The TBEV-infected cells exhibited either necrotic or apoptotic morphological features. We observed ultrastructural apoptotic signs (condensation, margination and fragmentation of chromatin) and other alterations, such as vacuolation of the cytoplasm, dilatation of the endoplasmic reticulum cisternae and shrinkage of cells, accompanied by a high density of the cytoplasm. On the other hand, infected neuroblastoma cells did not exhibit proliferation of membranous structures. The virions were present in both the endoplasmic reticulum and the cytoplasm. Cells were dying preferentially by necrotic mechanisms rather than apoptosis. The neuropathological significance of these observations is discussed.

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.

PirAB Toxin from Photorhabdus asymbiotica as a Larvicide against Dengue Vectors

ABSTRACT We have evaluated Photorhabdus insect-related protein (Pir) from Photorhabdus asymbiotica against dengue vectors. PirAB shows larvicidal activity against both Aedes aegypti and Aedes albopictus larvae but did not affect the Mesocyclops thermocyclopoides predator. PirAB expressed the strongest toxicity compared to PirA, PirB, or the mixture of PirA plus PirB. Whether the presence of an enterobacterial repetitive intergenic consensus sequence in PirAB, but not in PirA, PirB, or the mixture of PirA plus PirB, has any impact on biological control efficacy needs further investigation.

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.

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.

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.

Tick-borne encephalitis virus infection of cultured mouse macrophages.

The interactions of tick-borne encephalitis virus (TBEV) with mouse macrophages were studied at the electron microscopic level. The cultured mouse macrophages were sensitive to infection with TBEV strain Hypr (a highly neuroinvasive and neurovirulent strain for laboratory mice) and produced relatively high virus titers. However, these macrophage cells remained morphologically inactivated. Viral particles were located mainly in the ER but were also present in other exocytic compartments. No virus production was observed in cells infected with the attenuated, non-neuroinvasive TBEV strain 263. In this case, the infection led to a clear morphological activation of the macrophages. In conclusion, the virus replication process in mouse macrophage cells might be different from that in other mammalian cell lines since the smooth membrane structures, which are thought to be the sites for flavivirus replication, were not observed. Moreover, different TBEV strains exhibited a different interaction with the host macrophages. The inability of strain 263 to replicate in mouse macrophages as the first site of significant viral replication in vivo could be associated with the inability of this strain to establish a serious infection in mice.

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.