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  1. Vector-Borne Diseases in Stray Dogs in Peninsular Malaysia and Molecular Detection of Anaplasma and Ehrlichia spp. from Rhipicephalus sanguineus (Acari: Ixodidae) Ticks
    Koh FX, Panchadcharam C, Tay ST
    J Med Entomol, 2016 Jan;53(1):183-7.
    PMID: 26494821 DOI: 10.1093/jme/tjv153
    Little data are available on the prevalence and transmission of vector-borne diseases in stray dogs in Peninsular Malaysia. This study was designed to determine the occurrence of vector-borne pathogens in Malaysian stray dogs using serological and molecular approaches. In total, 48 dog blood samples were subjected to serological analysis using SNAP 4Dx kit (IDEXX Laboratories, Westbrook, ME). The presence of Ehrlichia and Anaplasma DNA in the dog blood samples and Rhipicephalus sanguineus (Latreille) ticks was detected using nested polymerase chain reaction assays. Positive serological findings against Ehrlichia canis and Anaplasma phagocytophilum were obtained in 17 (39.5%) and four (9.3%) of 43 dog samples, respectively. None of the dog blood samples were positive for Borrelia burgdorferi and Dirofilaria immitis. DNA of E. canis and A. phagocytophilum was detected in 12 (25.5%) and two (4.3%) of 47 dog blood samples, and 17 (51.5%) and one (3.0%) of 33 R. sanguineus ticks, respectively. Additionally, DNA of Ehrlichia spp. closely related to Ehrlichia chaffeensis was detected in two (6.1%) R. sanguineus ticks. This study highlights the prevalence of anaplasmosis and ehrlichiosis in dogs in Malaysia. Due to the zoonotic potential of Ehrlichia and Anaplasma spp., appropriate measures should be instituted for prevention and control of vector-borne diseases in dogs.
  2. Fulltext Molecular evidence of potential novel spotted fever group rickettsiae, Anaplasma and Ehrlichia species in Amblyomma ticks parasitizing wild snakes
    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. Molecular investigation of Anaplasma spp. in domestic and wildlife animals in Peninsular Malaysia
    Koh FX, Panchadcharam C, Sitam FT, Tay ST
    Vet Parasitol Reg Stud Reports, 2018 08;13:141-147.
    PMID: 31014863 DOI: 10.1016/j.vprsr.2018.05.006
    Anaplasma spp. are Gram-negative obligate intracellular, tick-borne bacteria which are of medical and veterinary importance. Little information is available on Anaplasma infection affecting domestic and wildlife animals in Malaysia. This study investigated the presence of Anaplasma spp. in the blood samples of domestic and wildlife animals in Peninsular Malaysia, using polymerase chain reaction (EHR-PCR) assays targeting the 16S rRNA gene of Anaplasmataceae. High detection rates (60.7% and 59.0%, respectively) of Anaplasma DNA were noted in 224 cattle (Bos taurus) and 78 deer (77 Rusa timorensis and one Rusa unicolor) investigated in this study. Of the 60 amplified fragments obtained for sequence analysis, Anaplasma marginale was exclusively detected in cattle while Anaplasma platys/Anaplasma phagocytophilum was predominantly detected in the deer. Based on sequence analyses of the longer fragment of the 16S rRNA gene (approximately 1000 bp), the occurrence of A. marginale, Anaplasma capra and Candidatus Anaplasma camelii in cattle, Candidatus A. camelii in deer and Anaplasma bovis in a goat was identified in this study. To assess whether animals were infected with more than one species of Anaplasma, nested amplification of A. phagocytophilum, A. bovis and Ehrlichia chaffeensis DNA was performed for 33 animal samples initially screened positive for Anaplasmataceae. No amplification of E. chaffeensis DNA was obtained from animals investigated. BLAST analyses of the 16S rDNA sequences from three deer (R. timorensis), a buffalo (Bubalus bubalis) and a cow (B. taurus) reveal similarity with that of Candidatus Anaplasma boleense strain (GenBank accession no.: KX987335). Sequence analyses of the partial gene fragments of major surface protein (msp4) gene from two deer (R. timorensis) and a monitor lizard (Varanus salvator) show the detection of a strain highly similar (99%) to that of A. phagocytophilum strain ZJ-China (EU008082). The findings in this study show the occurrence of various Anaplasma species including those newly reported species in Malaysian domestic and wildlife animals. The role of these animals as reservoirs/maintenance hosts for Anaplasma infection are yet to be determined.
  4. Fulltext Molecular survey and sequence analysis of Anaplasma spp. in cattle and ticks in a Malaysian farm
    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.
  5. Fulltext Rickettsial infections in monkeys, Malaysia
    Tay ST, Koh FX, Kho KL, Sitam FT
    Emerg Infect Dis, 2015 Mar;21(3):545-7.
    PMID: 25695615 DOI: 10.3201/eid2103.141457
  6. Fulltext Prevalence and molecular heterogeneity of Bartonella bovis in cattle and Haemaphysalis bispinosa ticks in Peninsular Malaysia
    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.
  7. Francisella spp. detected in Dermacentor ticks in Malaysian forest reserve areas
    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.
  8. The first molecular survey of theileriosis in Malaysian cattle, sheep and goats
    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.
  9. Spotted Fever Group Rickettsioses and Murine Typhus in a Malaysian Teaching Hospital
    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.
  10. Molecular detection of Anaplasma spp. in pangolins (Manis javanica) and wild boars (Sus scrofa) in Peninsular Malaysia
    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.
  11. Fulltext Rickettsial seropositivity in the indigenous community and animal farm workers, and vector surveillance in Peninsular Malaysia
    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.
  12. Fulltext Factors Associated with Tick Bite Preventive Practices among Farmworkers in Malaysia
    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.

  13. Fulltext Molecular characterisation of the tick Rhipicephalus microplus in Malaysia: new insights into the cryptic diversity and distinct genetic assemblages throughout the world
    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.
  14. Ehrlichia and Anaplasma Infections: Serological Evidence and Tick Surveillance in Peninsular Malaysia
    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.
  15. Detection of Schistosoma spindale ova and associated risk factors among Malaysian cattle through coprological survey
    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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