Kai Ling Kho

Molecular characterisation of the tick Rhipicephalus microplus in Malaysia: new insights into the cryptic diversity and distinct genetic assemblages throughout the world

BackgroundThe morphotaxonomy of Rhipicephalus microplus complex has been challenged in the last few years and prompted many biologists to adopt a DNA-based method for distinguishing the members of this group. In the present study, we used a mitochondrial DNA analysis to characterise the genetic assemblages, population structure and dispersal pattern of R. microplus from Southeast Asia, the region where the species originated.MethodsA phylogeographic analysis inferred from the 16S rRNA and cytochrome oxidase subunit I (COI) genes was performed with five populations of R. microplus collected from cattle in Malaysia. Malaysian R. microplus sequences were compared with existing COI and 16S rRNA haplotypes reported globally in NCBI GenBank.ResultsA total of seven and 12 unique haplotypes were recovered by the 16S rRNA and COI genes, respectively. The concatenated sequences of both 16S rRNA and COI revealed 18 haplotypes. Haplotype network and phylogenetic analyses based on COI+16S rRNA sequences revealed four genetically divergent groups among Malaysian R. microplus. The significantly low genetic differentiation and high gene flow among Malaysian R. microplus populations supports the occurrence of genetic admixture. In a broader context, the 16S rRNA phylogenetic tree assigned all isolates of Malaysian R. microplus into the previously described African/the Americas assemblage. However, the COI phylogenetic tree provides higher resolution of R. microplus with the identification of three main assemblages: clade A sensu Burger et al. (2014) comprises ticks from Southeast Asia, the Americas and China; clade B sensu Burger et al. (2014) is restricted to ticks that originated from China; and clade C sensu Low et al. (2015) is a new genetic assemblage discovered in this study comprising ticks from India and Malaysia.ConclusionsWe conclude that the R. microplus complex consisting of at least five taxa: R. australis, R. annulatus, R. microplus clade A sensu Burger et al. (2014), R. microplus clade B sensu Burger et al. (2014) and the new taxon, R. microplus clade C sensu Low et al. (2015). The use of COI as the standard genetic marker in discerning the genetic assemblages of R. microplus from a broad range of biogeographical regions is proposed.

Identification of rickettsiae from wild rats and cat fleas in Malaysia

Rickettsioses are emerging zoonotic diseases reported worldwide. In spite of the serological evidence of spotted fever group rickettsioses in febrile patients in Malaysia, limited studies have been conducted to identify the animal reservoirs and vectors of rickettsioses. This study investigated the presence of rickettsiae in the tissue homogenates of 95 wild rats and 589 animal ectoparasites. Using PCR assays targeting the citrate synthase gene (gltA), rickettsial DNA was detected in the tissue homogenates of 13 (13.7%) wild rats. Sequence analysis of the gltA amplicons showed 98.6–100% similarity with those of Rickettsia honei/R. conorii/R. raoultii (Rickettsiales: Rickettsiaceae). Sequence analysis of outer membrane protein A gene (ompA) identified Rickettsia sp. TCM1 strain from two rats. No rickettsia was detected from Laelaps mites, Rhipicephalus sanguineus and Haemaphysalis bispinosa ticks, and Felicola subrostratus lice in this study. R. felis was identified from 32.2% of 177 Ctenocephalides felis fleas. Sequence analysis of the gltA amplicons revealed two genotypes of R. felis (Rf31 and RF2125) in the fleas. As wild rats and cat fleas play an important role in the enzoonotic maintenance of rickettsiae, control of rodent and flea populations may be able to reduce transmission of rickettsioses in the local setting.

Molecular evidence of potential novel spotted fever group rickettsiae, Anaplasma and Ehrlichia species in Amblyomma ticks parasitizing wild snakes

BackgroundAmblyomma ticks parasitize a wide range of animals in tropical regions. This study describes the identification of Amblyomma ticks from wild snakes in Malaysia and the detection of potential human pathogens such as Rickettsia, Anaplasma, Ehrlichia and bartonellae in the ticks.FindingsTwenty one adult ticks (twelve A. varanense and nine Amblyomma helvolum ticks) identified from seven Python molurus snakes in Sepang and a pool of six A. helvolum ticks from a Naja sumatrana snake in Johore, Malaysia were investigated in this study. Amplification of the citrate synthase (gltA), 190-kDa surface antigen gene (ompA), 135-kDa surface antigen (ompB) and surface cell antigen (sca4) genes followed by sequence analysis confirmed the presence of two potential novel spotted fever group rickettsiae in the ticks. Candidatus Rickettsia sepangensis from an engorged A. varanense tick demonstrated high sequence similarity to Rickettsia tamurae; while Candidatus Rickettsia johorensis from two samples (individual and pooled) of A. helvolum and two A. varanense ticks were closely related to Rickettsia raoultii. Anaplasma and Ehrlichia DNA were detected from seven and two ticks, respectively. No bartonellae was detected from any of the ticks.ConclusionThe finding in this study suggests that Amblyomma ticks parasitizing wild snakes may serve as reservoir hosts and carriers for rickettsioses, anaplasmosis and ehrlichiosis in this region.

Molecular detection of Anaplasma spp. in pangolins (Manis javanica) and wild boars (Sus scrofa) in Peninsular Malaysia.

Anaplasma spp. infects a wide variety of wildlife and domestic animals. This study describes the identification of a novel species of Anaplasma (Candidatus Anaplasma pangolinii) from pangolins (Manis javanica) and Anaplasma bovis from wild boars (Sus scrofa) in Malaysia. Based on 16S rRNA gene sequences, Candidatus Anaplasma pangolinii is identified in a distinct branch within the family Anaplasmataceae, exhibiting the closest sequence similarity with the type strains of Anaplasma bovis (97.7%) and Anaplasma phagocytophilum (97.6%). The sequence also aligned closely (99.9%) with that of an Anaplasma spp. (strain AnAj360) detected from Amblyomma javanense ticks. The nearly full length sequence of the 16S rRNA gene derived from two wild boars in this study demonstrated the highest sequence similarity (99.7%) to the A. bovis type strain. Partial 16S rRNA gene fragments of A. bovis were also detected from a small population of Haemaphysalis bispinosa cattle ticks in this study. Our finding suggests a possible spread of two Anaplasma species in the Malaysian wildlife and ticks. The zoonotic potential of the Anaplasma species identified in this study is yet to be determined.

Ehrlichia and Anaplasma Infections: Serological Evidence and Tick Surveillance in Peninsular Malaysia

Abstract Little information is available on human anaplasmosis and ehrlichiosis in Southeast Asia despite increasing reports of the detection of Anaplasma spp. and Ehrlichia spp. in the ticks. We report herein the serological findings against the tick-borne pathogens in a group of animal farm workers (n = 87) and indigenous people (n = 102) in Peninsular Malaysia. IgG antibodies against Ehrlichia chaffeensis were detected from 29.9% and 34.3% of farm workers and indigenous people, respectively, using commercial indirect immunofluorescence assays. Comparatively, only 6.9% of the indigenous people but none of the animal farm workers were seropositive to Anaplasma phagocytophilum. A polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Anaplasmataceae was used to identify Anaplastamataceae in ticks collected from various locations adjacent to the areas where the serological survey was conducted. In this study, a total of 61.5% of ticks infesting farm animals, 37.5% of ticks infesting peri-domestic animals in rural villages, 27.3% of ticks collected from wildlife animals, and 29.1% of questing ticks collected from forest vegetation were positive for Anaplasmataceae DNA. Sequence analyses of 16S rRNA gene region (238 bp) provide the identification for Anaplasma marginale, Anaplasma bovis, Anaplasma platys, A. phagocytophilum, and Anaplasma spp. closely related to Candidatus Cryptoplasma californiense in ticks. E. chaffeensis DNA was not detected from any ticks, instead, Ehrlichia sp. strain EBm52, Ehrlichia mineirensis and Candidatus Ehrlichia shimanensis are the only Ehrlichia sp. identified from cattle ticks in this study. Further investigation is required to ascertain the occurrence of zoonotic transmission of Ehrlichia and Anaplasma infections in Peninsular Malaysia.

Factors Associated with Tick Bite Preventive Practices among Farmworkers in Malaysia.

Background Farmworkers are at high-risk for tick bites, which potentially transmit various tick-borne diseases. Previous studies show that personal prevention against tick bites is key, and certain factors namely, knowledge, experience of tick bites, and health beliefs influence compliance with tick bites preventive behaviour. This study aimed to assess these factors and their associations with tick bite preventive practices among Malaysian farmworkers. Methods A total of eight cattle, goat and sheep farms in six states in Peninsular Malaysia participated in a cross-sectional survey between August and October 2013 Results A total of 151 (72.2%) out of 209 farmworkers answered the questionnaire. More than half of the farmworkers (n = 91) reported an experience of tick bites. Farms with monthly acaricide treatment had significantly (P<0.05) a low report of tick bites. Tick bite exposure rates did not differ significantly among field workers and administrative workers. The mean total knowledge score of ticks for the overall farmworkers was 13.6 (SD±3.2) from 20. The mean total tick bite preventive practices score for all farmworkers was 8.3 (SD±3.1) from 15. Fixed effect model showed the effects of four factors on tick bite prevention: (1) farms, (2) job categories (administrative workers vs. field workers), (3) perceived severity of tick bites, and (4) perceived barriers to tick bite prevention. Conclusions A high proportion of farmworkers, including administrative workers, reported an experience of tick bites. The effectiveness of monthly acaricide treatment was declared by low reports of tick bites on these farms. Tick bite preventive practices were insufficient, particularly in certain farms and for administrative workers. Our findings emphasise the need to have education programmes for all farmworkers and targeting farms with low prevention practices. Education and health programmes should increase the perception of the risk of tick bites and remove perceived barriers of tick bite prevention.

Whole-genome sequence analysis and exploration of the zoonotic potential of a rat-borne Bartonella elizabethae.

Bartonella elizabethae has been known to cause endocarditis and neuroretinitis in humans. The genomic features and virulence profiles of a B. elizabethae strain (designated as BeUM) isolated from the spleen of a wild rat in Kuala Lumpur, Malaysia are described in this study. The BeUM strain has a genome size of 1,932,479bp and GC content of 38.3%. There is a high degree of conservation between the genomes of strain BeUM with B. elizabethae type strains (ATCC 49927 and F9251) and a rat-borne strain, Re6043vi. Of 2137 gene clusters identified from B. elizabethae strains, 2064 (96.6%) are indicated as the core gene clusters. Comparative genome analysis of B. elizabethae strains reveals virulence genes which are known in other pathogenic Bartonella species, including VirB2-11, vbhB2-B11, VirD4, trw, vapA2-5, hbpA-E, bepA-F, bepH, badA/vomp/brp, ialB, omp43/89 and korA-B. A putative intact prophage has been identified in the strain BeUM, in addition to a 8kb pathogenicity island. The whole genome analysis supports the zoonotic potential of the rodent-borne B. elizabethae, and provides basis for future functional and pathogenicity studies of B. elizabethae.

Rickettsial seropositivity in the indigenous community and animal farm workers, and vector surveillance in Peninsular Malaysia

Rickettsioses are emerging zoonotic diseases that are often neglected in many countries in Southeast Asia. Rickettsial agents are transmitted to humans through exposure to infected arthropods. Limited data are available on the exposure of indigenous community and animal farm workers to the aetiological agents and arthropod vectors of rickettsioses in Peninsular Malaysia. Serological analysis of Rickettsia conorii and Rickettsia felis was performed for 102 individuals from the indigenous community at six rural villages and 87 workers from eight animal farms in Peninsular Malaysia in a cross-sectional study. The indigenous community had significantly higher seropositivity rates for R. conorii (P<0.001) and R. felis (P<0.001), as compared to blood donors from urban (n=61). Similarly, higher seropositivity rates for R. conorii (P=0.046) and R. felis (P<0.001) were noted for animal farm workers, as compared to urban blood donors. On the basis of the sequence analysis of gltA, ompA and ompB, various spotted fever group rickettsiae closely related to R. raoultii, R. heilongjiangensis, R. felis-like organisms, R. tamurae, Rickettsia sp. TCM1, R. felis, Rickettsia sp. LON13 and R. hulinensis were identified from tick/flea samples in animal farms, indigenous villages and urban areas. This study describes rickettsial seropositivity of the Malaysian indigenous community and animal farm workers, and provides molecular evidence regarding the presence of rickettsial agents in ticks/fleas infesting domestic animals in Peninsular Malaysia.

Diversity of Rickettsiae in Feeding and Questing Ticks Collected From a Malaysian Forest Reserve Area

Abstract High seropositivity to Rickettsia conorii and Rickettsia felis has been reported in Malaysian indigenous community living in settlements adjacent to forest areas. The current study was conducted to determine the type and distribution of rickettsiae in feeding and questing ticks that were collected from a forest reserve area at Kuala Lompat in Pahang, Malaysia. Using PCR assays targeting citrate synthase (gltA), outer membrane protein A (ompA) and B (ompB) genes, rickettsiae were detected from approximately one-third of 98 ticks (mainly Dermacentor and Haemaphysalis spp.) collected from the forest reserve. BLAST analysis reveals the predominance of Rickettsia sp. RF2125 in both feeding and questing ticks and Rickettsia sp. TCM1 in the questing ticks. Sequences exhibiting close genetic relationship with Rickettsia raoultii, Rickettsia tamurae, Rickettsia heilongjiangensis, and Rickettsia asiatica were also detected from the ticks. This study highlights the diversity of rickettsial species and potential tick vectors which may contribute to the high seropositivity observed among the local communities.

Phylogeny and putative virulence gene analysis of Bartonella bovis

Bartonella bovis is a small Gram-negative bacterium recognized as an etiological agent for bacteremia and endocarditis in cattle. As few reports are available on the taxonomic position of B. bovis and its mechanism of virulence, this study aims to resolve the phylogeny of B. bovis and investigate putative virulence genes based on whole genome sequence analysis. Genome-wide comparisons based on single nucleotide polymorphisms (SNP) and orthologous genes were performed in this study for phylogenetic inference of 27 Bartonella species. Rapid Annotation using Subsystem Technology (RAST) analysis was used for annotation of putative virulence genes. The phylogenetic tree generated from the genome-wide comparison of orthologous genes exhibited a topology almost similar to that of the tree generated from SNP-based comparison, indicating a high concordance in the nucleotide and amino acid sequences of Bartonella spp. The analyses show consistent grouping of B. bovis in a cluster related to ruminant-associated species, including Bartonella australis, Bartonella melophagi and Bartonella schoenbuchensis. RAST analysis revealed genes encoding flagellar components, in corroboration with the observation of flagella-like structure of BbUM strain under negative straining. Genes associated with virulence, disease and defence, prophages, membrane transport, iron acquisition, motility and chemotaxis are annotated in B. bovis genome. The flagellin (flaA) gene of B. bovis is closely related to Bartonella bacilliformis and Bartonella clarridgeiae but distinct from other Gram-negative bacteria. The absence of type IV secretion systems, the bona fide pathogenicity factors of bartonellae, in B. bovis suggests that it may have a different mechanism of pathogenicity.