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  1. Kho KL, Tay ST
    J Med Entomol, 2019 02 25;56(2):526-532.
    PMID: 30312440 DOI: 10.1093/jme/tjy169
    Rickettsia felis (Rickettsiales: Rickettsiaceae) is an emergent human pathogen that causes febrile illnesses in various parts of the world. This study describes the identification and growth characteristics of a R. felis-like organism (designated as Rickettsia sp. TH2014) cultured from Ctenocephalides orientis fleas in rural Malaysia. In this study, culturing of rickettsiae from filtered triturated flea lysates was performed in Aedes albopictus C6/36 cells. Cytopathic effects were observed from one of the samples 4 d post-inoculation. Electron microscopy revealed actively replicating intracytosolic coccobacillary organisms in the rickettsia-infected cells. Sequence analysis of amplified citrate synthase (gltA) gene fragment shows complete match of the rickettsia with Rickettsia sp. Rf31 in Southeast Asia, and 'Candidatus Rickettsia senegalensis' strain PU01-02 in Africa. The whole-genome sequence of Rickettsia sp. TH2014 was determined and assembled. The estimated genome size and guanine + cytosine content of the rickettsia are 1.37 Mb and 32.9%, respectively. The high values of average nucleotide identity and tetra-nucleotide signature correlation index obtained from pairwise genome comparison study suggest the identification of the rickettsia as R. felis. The whole-genome single-nucleotide polymorphism analysis demonstrates close genetic relatedness of the rickettsia with R. felis and Rickettsia asemboensis. However, based on sequence analyses of rickettsial genes (16S rDNA, gltA, ompB, and sca4), Rickettsia sp. TH2014 is found to be distinct from R. felis and R. asemboensis. The sequence analyses reveal that Rickettsia sp. TH2014 is highly similar to 'Ca. Rickettsia senegalensis' detected in fleas from Africa, Asia, and North America. Further investigation to provide insights on pathogenic potential and transmission dynamics of the rickettsia is warranted.
  2. Kho KL, Koh FX, Tay ST
    Parasit Vectors, 2015;8:112.
    PMID: 25889376 DOI: 10.1186/s13071-015-0719-3
    Amblyomma 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.
  3. Kho KL, Tan PE, Tay ST
    J Med Entomol, 2019 02 25;56(2):547-552.
    PMID: 30304529 DOI: 10.1093/jme/tjy168
    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.
  4. Tay ST, Kho KL, Wee WY, Choo SW
    Acta Trop, 2016 Mar;155:25-33.
    PMID: 26658020 DOI: 10.1016/j.actatropica.2015.11.019
    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.
  5. Tay ST, Kho KL, Lye SF, Ngeow YF
    J Vet Med Sci, 2018 Apr 18;80(4):653-661.
    PMID: 29311425 DOI: 10.1292/jvms.17-0448
    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.
  6. Tay ST, Koh FX, Kho KL, Ong BL
    Trop Biomed, 2014 Dec;31(4):769-76.
    PMID: 25776603 MyJurnal
    This study was conducted to determine the occurrence of Anaplasma spp. in the blood samples of cattle, goats, deer and ticks in a Malaysian farm. Using polymerase chain reaction (PCR) and sequencing approach, Anaplasma spp. was detected from 81(84.4%) of 96 cattle blood samples. All blood samples from 23 goats and 22 deer tested were negative. Based on the analysis of the Anaplasma partial 16S ribosomal RNA gene, four sequence types (genotypes 1 to 4) were identified in this study. Genotypes 1-3 showed high sequence similarity to those of Anaplasma platys/ Anaplasma phagocytophilum, whilst genotype 4 was identical to those of Anaplasma marginale/ Anaplasma centrale/ Anaplasma ovis. Anaplasma DNA was detected from six (5.5%) of 109 ticks which were identified as Rhipicephalus (formely known as Boophilus) microplus ticks collected from the cattle. This study reported for the first time the detection of four Anaplasma sequence types circulating in the cattle population in a farm in Malaysia. The detection of Anaplasma DNA in R. microplus ticks in this study provides evidence that the ticks are one of the potential vectors for transmission of anaplasmosis in the cattle.
  7. Kho KL, Koh FX, Jaafar T, Nizam QN, Tay ST
    BMC Vet Res, 2015;11:153.
    PMID: 26179499 DOI: 10.1186/s12917-015-0470-1
    Bartonellosis is an emerging zoonotic infection responsible for a variety of clinical syndromes in humans and animals. Members of the genus Bartonella exhibit high degrees of genetic diversity and ecologic plasticity. The infection is usually transmitted to animals and humans through blood-feeding arthropod vectors such as fleas, lice, ticks and sandflies. This study was conducted to investigate the prevalence of Bartonella species in 184 beef cattle, 40 dairy cattle, 40 sheep and 40 goats in eight animal farms across Peninsular Malaysia. Bartonella-specific PCR assays and sequence analysis of partial fragments of the citrate synthase gene were used for detection and identification of B. bovis. Isolation of B. bovis was attempted from PCR-positive blood samples. Molecular heterogeneity of the isolates was investigated based on sequence analysis of gltA, ITS, rpoB genes, ERIC-PCR, as well as using an established multilocus sequence typing (MLST) method. The carriage rate of B. bovis in ticks was also determined in this study.
  8. Koh FX, Nurhidayah MN, Tan PE, Kho KL, Tay ST
    Vet Parasitol Reg Stud Reports, 2019 08;17:100315.
    PMID: 31303231 DOI: 10.1016/j.vprsr.2019.100315
    Limited information is available on tropical ticks and tick-borne bacteria affecting the health of humans and animals in the Southeast Asia region. Francisella tularensis is a tick-borne bacterium which causes a potentially life-threatening disease known as tularemia. This study was conducted to determine the occurrence of Francisella spp. in questing ticks collected from Malaysian forest reserve areas. A total of 106 ticks (mainly Dermacentor and Haemaphysalis spp.) were examined for Francisella DNA using a Polymerase chain reaction (PCR) assay targeting the bacterial 16S rDNA. Francisella DNA was detected from 12 Dermacentor ticks. Sequence analysis of the amplified 16S rDNA sequences (1035 bp) show >99% identity with that of Francisella endosymbiont reported in a tick from Thailand. A dendrogram constructed based on the bacterial 16S rDNA shows that the Francisella spp. were distantly related to the pathogenic strains of F. tularensis. Three Francisella-positive ticks were identified as Dermacentor atrosignatus, based on sequence analysis of the tick mitochondrial 16S rRNA gene. Further screening of cattle and sheep ticks (Haemaphysalis bispinosa and Rhipicephalus microplus) and animal samples (cattle, sheep, and goats) did not yield any positive findings. Our findings provide the first molecular data on the occurrence of a Francisella strain with unknown pathogenicity in Dermacentor questing ticks in Malaysia.
  9. Kho KL, Amarajothi ADG, Koh FX, Panchadcharam C, Hassan Nizam QN, Tay ST
    Vet Parasitol Reg Stud Reports, 2017 12;10:149-153.
    PMID: 31014589 DOI: 10.1016/j.vprsr.2017.08.003
    This study reports the molecular detection of Theileria spp. from six cattle farms, a sheep farm and a goat farm located at different states in Peninsular Malaysia. Animal blood samples were screened for the presence of Theileria DNA using a conventional polymerase chain reaction (PCR) assay. A total of 155 (69.2%) of 224 cattle investigated were PCR-positive for Theileria DNA. The occurrences of Theileria spp. ranged from 17.5% to 100.0% across six cattle farms. Theileria DNA was detected from 90.0% of 40 sheep but none of 40 goats examined in this study. Sequence analyses of amplified 18S rRNA partial fragments (335-338bp) confirmed the identification of Theileria buffeli, Theileria sergenti, and Theileria sinensis in representative samples of cattle and ticks. T. luwenshuni was identified in the infected sheep. The high occurrences of Theileria spp. in our farm animals highlight the needs for appropriate control and preventive measures for theileriosis.
  10. Kho KL, Koh FX, Singh HK, Zan HA, Kukreja A, Ponnampalavanar S, et al.
    Am J Trop Med Hyg, 2016 10 05;95(4):765-768.
    PMID: 27402519
    Limited information is available on the etiological agents of rickettsioses in southeast Asia. Herein, we report the molecular investigation of rickettsioses in four patients attending a teaching hospital in Malaysia. DNA of Rickettsia sp. RF2125, Rickettsia typhi, and a rickettsia closely related to Rickettsia raoultii was detected in the blood samples of the patients. Spotted fever group rickettsioses and murine typhus should be considered in the diagnosis of patients with nonspecific febrile illness in this region.
  11. Koh FX, Kho KL, Panchadcharam C, Sitam FT, Tay ST
    Vet Parasitol, 2016 Aug 30;227:73-6.
    PMID: 27523941 DOI: 10.1016/j.vetpar.2016.05.025
    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.
  12. Tay ST, Mokhtar AS, Low KC, Mohd Zain SN, Jeffery J, Abdul Aziz N, et al.
    Med Vet Entomol, 2014 Aug;28 Suppl 1:104-8.
    PMID: 25171613 DOI: 10.1111/mve.12075
    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.
  13. Khoo JJ, Chen F, Kho KL, Ahmad Shanizza AI, Lim FS, Tan KK, et al.
    Ticks Tick Borne Dis, 2016 07;7(5):929-937.
    PMID: 27132518 DOI: 10.1016/j.ttbdis.2016.04.013
    Ticks are vectors in the transmission of many important infectious diseases in human and animals. Ticks can be readily found in the semi-forested areas such as the settlements of the indigenous people in Malaysia, the Orang Asli. There is still minimal information available on the bacterial agents associated with ticks found in Malaysia. We performed a survey of the bacterial communities associated with ticks collected from domestic animals found in two Orang Asli villages in Malaysia. We collected 62 ticks, microscopically and molecularly identified as related to Haemaphysalis wellingtoni, Haemaphysalis hystricis and Haemaphysalis bispinosa. Bacterial 16s rRNA hypervariable region (V6) amplicon libraries prepared from the tick samples were sequenced on the Ion Torrent PGM platform. We detected a total of 392 possible bacterial genera after pooling and sequencing 20 samples, indicating a diverse bacterial community profile. Dominant taxa include the potential tick endosymbiont, Coxiella. Other dominant taxa include the tick-associated pathogen, Rickettsia, and environmental bacteria such as Bacillus, Mycobacterium, Sphingomonas and Pseudomonas. Other known tick-associated bacteria were also detected, including Anaplasma, Ehrlichia, Rickettsiella and Wolbachia, albeit at very low abundance. Specific PCR was performed on selected samples to identify Rickettsia and Coxiella. Sequence of Rickettsia felis, which causes spotted fever in human and cats, was identified in one sample. Coxiella endosymbionts were detected in three samples. This study provides the baseline knowledge of the microbiome of ticks in Malaysia, focusing on tick-associated bacteria affecting the Orang Asli communities. The role of the herein found Coxiella and Rickettsia in tick physiology or disease transmission merits further investigation.
  14. Kho KL, Koh FX, Hasan LI, Wong LP, Kisomi MG, Bulgiba A, et al.
    Emerg Microbes Infect, 2017 Apr 12;6(4):e18.
    PMID: 28400593 DOI: 10.1038/emi.2017.4
    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.
  15. Ghane Kisomi M, Wong LP, Tay ST, Bulgiba A, Zandi K, Kho KL, et al.
    PLoS One, 2016;11(6):e0157987.
    PMID: 27341678 DOI: 10.1371/journal.pone.0157987
    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.

  16. Low VL, Tay ST, Kho KL, Koh FX, Tan TK, Lim YA, et al.
    Parasit Vectors, 2015;8:341.
    PMID: 26104478 DOI: 10.1186/s13071-015-0956-5
    The 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.
  17. Koh FX, Kho KL, Kisomi MG, Wong LP, Bulgiba A, Tan PE, et al.
    J Med Entomol, 2018 02 28;55(2):269-276.
    PMID: 29202206 DOI: 10.1093/jme/tjx204
    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.
  18. Tan TK, Low VL, Lee SC, Panchadcharam C, Tay ST, Ngui R, et al.
    Jpn. J. Vet. Res., 2015 May;63(2):63-71.
    PMID: 26164875
    The present study was conducted to determine the occurrence of Schistosoma spindale ova and its associated risk factors in Malaysian cattle through a coprological survey. A total of 266 rectal fecal samples were collected from six farms in Peninsular Malaysia. The overall infection rate of S. spindale was 6% (16 of 266). Schistosoma spindale infection was observed in two farms, with a prevalence of 5.4% and 51.9%, respectively. This trematode was more likely to co-occur with other gastro-intestinal parasites (i.e., Dicrocoelium spp., Paramphistomum spp., strongyle, Eimeria spp. and Entamoeba spp.). Chi-square analysis revealed that female cattle are less likely to get S. spindale infection as compared to male cattle (OR = 0.3; 95% CI = 0.08-1.06; p < 0.05), and cattle weighing lower than 200 kg, were significantly at higher risk than those higher than 200 kg (OR = 5; 95% CI = 1.07-24.79; p < 0.05) to the infection. Multivariate analysis confirmed that among the cattle in Malaysia, the age (cattle with two year old and higher: OR = 21; 95% CI = 2.48-179.44; p < 0.05) and weight (weighing 200 kg and lower: OR = 17; 95% CI = 3.38-87.19; p < 0.05) were risk factors for S. spindale infection among Malaysian cattle.
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