Bee Lee Ong

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.

Tuberculosis in captive Asian elephants ( Elephas maximus ) in Peninsular Malaysia

A cross-sectional study was conducted from 10 January to 9 April 2012, to determine the seroprevalence of tuberculosis (TB) of all captive Asian elephants and their handlers in six locations in Peninsular Malaysia. In addition, trunk-wash samples were examined for tubercle bacillus by culture and polymerase chain reaction (PCR). For 63 elephants and 149 elephant handlers, TB seroprevalence was estimated at 20.4% and 24.8%, respectively. From 151 trunkwash samples, 24 acid-fast isolates were obtained, 23 of which were identified by hsp65-based sequencing as non-tuberculous mycobacteria. The Mycobacterium tuberculosis-specific PCR was positive in the trunk-wash samples from three elephants which were also seropositive. Conversely, the trunk wash from seven seropositive elephants were PCR negative. Hence, there was evidence of active and latent TB in the elephants and the high seroprevalence in the elephants and their handlers suggests frequent, close contact, two-way transmission between animals and humans within confined workplaces.

Identification of New Genomospecies in the Mycobacterium terrae Complex

Members of the Mycobacterium terrae complex are slow-growing, non-chromogenic acid-fast bacilli found in the natural environment and occasionally in clinical material. These genetically closely-related members are difficult to differentiate by conventional phenotypic and molecular tests. In this paper we describe the use of whole genome data for the identification of four strains genetically similar to Mycobacterium sp. JDM601, a newly identified member of the M. terrae complex. Phylogenetic information from the alignment of genome-wide orthologous genes and single nucleotide polymorphisms show consistent clustering of the four strains together with M. sp. JDM601 into a distinct clade separate from other rapid and slow growing mycobacterial species. More detailed inter-strain comparisons using average nucleotide identity, tetra-nucleotide frequencies and analysis of synteny indicate that our strains are closely related to but not of the same species as M. sp. JDM601. Besides the 16S rRNA signature described previously for the M. terrae complex, five more hypothetical proteins were found that are potentially useful for the rapid identification of mycobacterial species belonging to the M. terrae complex. This paper illustrates the versatile utilization of whole genome data for the delineation of new bacterial species and introduces four new genomospecies to add to current members in the M. terrae complex.

Multiphasic strain differentiation of atypical mycobacteria from elephant trunk wash

Background. Two non-tuberculous mycobacterial strains, UM_3 and UM_11, were isolated from the trunk wash of captive elephants in Malaysia. As they appeared to be identical phenotypes, they were investigated further by conventional and whole genome sequence-based methods of strain differentiation. Methods. Multiphasic investigations on the isolates included species identification with hsp65 PCR-sequencing, conventional biochemical tests, rapid biochemical profiling using API strips and the Biolog Phenotype Microarray analysis, protein profiling with liquid chromatography-mass spectrometry, repetitive sequence-based PCR typing and whole genome sequencing followed by phylogenomic analyses. Results. The isolates were shown to be possibly novel slow-growing schotochromogens with highly similar biological and genotypic characteristics. Both strains have a genome size of 5.2 Mbp, G+C content of 68.8%, one rRNA operon and 52 tRNAs each. They qualified for classification into the same species with their average nucleotide identity of 99.98% and tetranucleotide correlation coefficient of 0.99999. At the subspecies level, both strains showed 98.8% band similarity in the Diversilab automated repetitive sequence-based PCR typing system, 96.2% similarity in protein profiles obtained by liquid chromatography mass spectrometry, and a genomic distance that is close to zero in the phylogenomic tree constructed with conserved orthologs. Detailed epidemiological tracking revealed that the elephants shared a common habitat eight years apart, thus, strengthening the possibility of a clonal relationship between the two strains.

Evidence and potential risk factors of tuberculosis among captive Asian elephants and wildlife staff in Peninsular Malaysia

Elephant tuberculosis (TB) caused by Mycobacterium tuberculosis is an important re-emerging zoonosis with considerable conservation and public health risk. We conducted prospective cohort and cross-sectional studies in elephants and wildlife staff respectively in order to identify potential risk factors associated with TB in captive Asian elephants and their handlers in Peninsular Malaysia. Sixty elephants in six different facilities were screened for TB longitudinally using the ElephantTB STAT-PAK and DPP VetTB assays from February 2012 to May 2014, and 149 wildlife staff were examined for tuberculosis infection using the QuantiFERON-TB Gold In-tube (QFT) assay from January to April, 2012. Information on potential risk factors associated with infection in both elephants and staff were collected using questionnaires and facility records. The overall seroprevalence of TB amongst the elephants was 23.3% (95% CI: 13.8-36.3) and the risk of seroconversion was significantly higher among elephants with assigned mahouts [p=0.022, OR=4.9 (95% CI: 1.3-18.2)]. The percentage of QFT responders among wildlife staff was 24.8% (95% CI: 18.3-32.7) and the risk of infection was observed to be significantly associated with being a zoo employee [p=0.018, OR=2.7 (95% CI: 1.2-6.3)] or elephant handler [p=0.035, OR=4.1 (95% CI: 1.1-15.5)]. These findings revealed a potential risk of TB infection in captive elephants and handlers in Malaysia, and emphasize the need for TB screening of newly acquired elephants, isolating sero-positive elephants and performing further diagnostic tests to determine their infection status, and screening elephant handlers for TB, pre- and post-employment.

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.

Prevalence and risk factors of Japanese encephalitis virus (JEV) in livestock and companion animal in high-risk areas in Malaysia

Japanese encephalitis (JE) is vector-borne zoonotic disease which causes encephalitis in humans and horses. Clinical signs for Japanese encephalitis virus (JEV) infection are not clearly evident in the majority of affected animals. In Malaysia, information on the prevalence of JEV infection has not been established. Thus, a cross-sectional study was conducted during two periods, December 2015 to January 2016 and March to August in 2016, to determine the prevalence and risk factors in JEV infections among animals and birds in Peninsular Malaysia. Serum samples were harvested from the 416 samples which were collected from the dogs, cats, water birds, village chicken, jungle fowls, long-tailed macaques, domestic pigs, and cattle in the states of Selangor, Perak, Perlis, Kelantan, and Pahang. The serum samples were screened for JEV antibodies by commercial IgG ELISA kits. A questionnaire was also distributed to obtain information on the animals, birds, and the environmental factors of sampling areas. The results showed that dogs had the highest seropositive rate of 80% (95% CI: ± 11.69) followed by pigs at 44.4% (95% CI: ± 1.715), cattle at 32.2% (95% CI: ± 1.058), birds at 28.9% (95% CI: ± 5.757), cats at 15.6% (95% CI: ± 7.38), and monkeys at 14.3% (95% CI: ± 1.882). The study also showed that JEV seropositivity was high in young animals and in areas where mosquito vectors and migrating birds were prevalent.

First molecular detection of porcine bocavirus in Malaysia

Several strains of porcine bocaviruses have been reported worldwide since their first detection in Sweden in 2009. Subsequently, the virus has been reported to be associated with gastrointestinal and respiratory signs in weaner and grower pigs. Although Malaysia is host to a self-sufficient swine livestock industry, there is no study that describes porcine bocavirus in the country. This report is the first to describe porcine bocavirus (PBoV) in Malaysian swine herds. PBoV was identified in various tissues from sick and runt pigs using the conventional PCR method with primers targeting conserved regions encoding for the nonstructural protein (NS1) gene. Out of 103 samples tested from 17 pigs, 32 samples from 15 pigs were positive for porcine bocavirus. In addition, a higher detection rate was identified from mesenteric lymph nodes (52.9%), followed by tonsil (37.0%), and lungs (33.3%). Pairwise comparison and phylogenetic analyses based on a 658-bp fragment of NS1 gene revealed that the Malaysian PBoV strains are highly similar to PBoV3 isolated in Minnesota, USA. The presence of porcine bocavirus in Malaysia and their phylogenetic bond was marked for the first time by this study. Further studies will establish the molecular epidemiology of PBoV in Malaysia and clarify pathogenicity of the local isolates.

Japanese encephalitis in Malaysia: An overview and timeline

Japanese encephalitis (JE) is a vector-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). It causes encephalitis in human and horses, and may lead to reproductive failure in sows. The first human encephalitis case in Malaya (now Malaysia) was reported during World War II in a British prison in 1942. Later, encephalitis was observed among race horses in Singapore. In 1951, the first JEV was isolated from the brain of an encephalitis patient. The true storyline of JE exposure among humans and animals has not been documented in Malaysia. In some places such as Sarawak, JEV has been isolated from mosquitoes before an outbreak in 1992. JE is an epidemic in Malaysia except Sarawak. There are four major outbreaks reported in Pulau Langkawi (1974), Penang (1988), Perak and Negeri Sembilan (1998-1999), and Sarawak (1992). JE is considered endemic only in Sarawak. Initially, both adults and children were victims of JE in Malaysia, however, according to the current reports; JE infection is only lethal to children in Malaysia. This paper describes a timeline of JE cases (background of each case) from first detection to current status, vaccination programs against JE, diagnostic methods used in hospitals and factors which may contribute to the transmission of JE among humans and animals in Malaysia.