Trichuris trichiura (whipworm) are soil-transmitted helminths (STHs) that causing trichuriasis in human. Trichuris vulpis, a canine whipworm has also been reported occasionally in humans. However, an overlapping dimension in the morphology and due to limited external characters between both species may lead to the potential for misidentification. Although there has been an extensive study on the distribution of whipworm in both human and animal hosts, little is known about the molecular epidemiology of Trichuris species in both hosts. To investigate to characterize the whipworm species and to determine the genetic relationship between species infecting both humans and animals, we sequenced the small subunit ribosomal RNA (SSU rRNA) regions of Trichuris egg isolated from humans, dogs and cats in a rural community in Malaysia. A total of 524 fresh fecal samples were collected from humans and animals. The overall prevalence of Trichuris was 59.9% as determined by microscopy examination. The molecular analysis showed that 98.7% were identified as T. trichiura in the human fecal sample. Interestingly, 1.3% were identified as T. vulpis. As for animal fecal sample, 56.8% and 43.2% were identified as T. trichiura and T. vulpis, respectively. Phylogenetic and sequence analysis demonstrated that T. trichiura isolates were genetically distinct from T. vulpis isolates from both hosts. This finding implies that companion animals can be a reservoir and mechanical transmitter for T. trichiura infection in human and also highlighting the possible zoonotic potential of T. vulpis. This finding may also suggest that cross-transmission between humans and animal hosts in sympatric setting may be a source of infection in both hosts. More studies are needed to better understand the transmission dynamic and public health significance of Trichuris infection in both hosts.
Village chicken production, a traditional, small-scale, and extensive backyard poultry industry, has been profitable for local farmers in Myanmar. However, there is scanty information available concerning the infection of these chickens with avian pathogens, including haemoprotozoan parasites. In the present study, we provide the first report of microscopic detection and molecular identification of Leucocytozoon and Plasmodium parasites from seven different areas of Myanmar. Leucocytozoon gametocytes were detected in 17.6% (81/461) of the blood smears from village chickens. The nested polymerase chain reaction (PCR) for targeting Leucocytozoon mitochondrial cytochrome b (cyt b) genes had a 17.6% positive rate. Although the positive rate of nested PCR targeting Plasmodium/Haemoproteus cyt b was 34.3%, the PCR protocol was observed to possibly amplify DNA of a certain species of Leucocytozoon. There were no obvious clinical signs in the infected birds. Statistical analysis of the microscopic detection and PCR detection rates using the age and sex of birds as internal factors revealed that the statistical significances differed according to the study area. The sequencing of 32 PCR products obtained from each study area revealed infection by Leucocytozoon caulleryi in three birds, Leucocytozoon sabrazesi in two birds, Leucocytozoon schoutedeni in two birds, Leucocytozoon sp. in eighteen birds, and Plasmodium juxtanucleare in seven birds; however, Haemoproteus infection was not detected. While L. sabrazesi was detected in chickens from the central region of Myanmar, the other haemosporidians were detected in those from different areas. In the haplotype analysis, we detected 17 haemosporidian cyt b haplotypes, including two for L. caulleryi, one for L. sabrazesi, two for L. schoutedeni, nine for Leucocytozoon sp., and three for P. juxtanucleare. Phylogenetic analysis of the cyt b haplotypes revealed a considerably close genetic relationship among chicken haemosporidians detected in Myanmar, Thailand, and Malaysia. These results indicate that well-recognized widespread species of chicken Leucocytozoon and Plasmodium are distributed nationwide in Myanmar, providing new insights into the ecosystem and control strategies of haemosporidian parasites in domesticated chickens in Myanmar.
A recently described species of black fly, Simulium wayani Takaoka and Chen, from the island of Timor was chromosomally mapped to provide insights into its evolutionary and biogeographic history. The morphologically based species status of S. wayani is supported by a suite of fixed chromosomal rearrangements and unique sex chromosomes derived primarily from a large pool of polymorphisms in the S. ornatipes complex in Australia. The banding patterns of its polytene chromosomes indicate that S. wayani is closely related to a pair of homosequential cryptic species (S. norfolkense Dumbleton and S. ornatipes cytoform A2) in the S. ornatipes Skuse complex on mainland Australia; all three species uniquely share the same amplified band in their chromosomal complement. The low level of polymorphism and heterozygosity in S. wayani, relative to Australian populations of the S. ornatipes complex, suggests few colonization events from the larger land mass.
Black flies (Simuliidae) are important biting insects and vectors of diseases agents of humans and livestock. Thus, understanding the taxonomy and biodiversity of these insects is crucial for control and management of these diseases. In this study, we used mitochondrial cytochrome c oxidase I sequences to examine genetic diversity of three human-biting and possible vector black fly taxa; the Simulium asakoae species-complex, S. chamlongi and S. nigrogilvum. High levels of genetic diversity (>3.5% intraspecific genetic divergence) were found in all three taxa. Phylogenetic analyses indicated that the S. asakoae complex can be divided into seven groups with the largest group consisting of specimens from Thailand, Malaysia and Myanmar. This group most likely represents true S. asakoae. The remaining haplotypes formed groups with conspecific haplotypes or with other closely related species. Among these groups, one including S. monglaense and another including S. myanmarense suggest that certain specimens identified as S. asakoae most likely belong to those species. Therefore, they constitute new locality records for Thailand and also represent new records of anthropophily. Members of S. chamlongi are not monophyletic as its clade also included S. hackeri. A median joining network revealed strong geographic associations of the haplotypes of S. nigrogilvum suggesting limitation of gene flow. Because this species occurs mainly in high elevation habitats, low land areas could present a barrier to gene flow.
During a study on the quality of the indoor environment, Acanthamoeba spp. were detected in 20 out of 87 dust samples collected from air-conditioners installed in a four-story campus building located in Kuala Lumpur, Malaysia. Twenty-one cloned Acanthamoeba isolates designated as IMU1 to IMU21 were established from the positive primary cultures. Five species were identified from the 16 isolates according to the morphological criteria of Pussard and Pons; i.e. A. castellanii, A. culbertsoni, A. griffini, A. hatchetti and A. polyphaga. Species identities for the remaining five isolates (IMU4, IMU5, IMU15, IMU20 and IMU21), however, could not be determined morphologically. At genotypic characterization, these isolates were placed into T3 (IMU14); T5 (IMU16 and IMU17) and T4 (all the remaining isolates). To predict the potential pathogenicity of these Acanthamoeba isolates, thermo- and osmotolerance tests were employed; many isolates were predicted as potential human pathogens based on the outcome of these tests. This is the first time potentially pathogenic Acanthamoeba have been isolated from air-conditioners in Malaysia.
Three new species of black flies from Thailand, Simulium wangkwaiense, S. tadtonense and S. maeklongkeense, are described based on their adults, pupal exuviae and cocoons. All three new species are assigned to the Simulium (Simulium) striatum species-group, bringing its total number in Thailand to seven. Simulium wangkwaiense sp. nov. is the species formerly called S. quinquestriatum in Thailand. Certain male and pupal morphological characteristics are shown to separate all seven Thai species of this species-group. All of the three new species have been analyzed genetically for their phylogenetic relationships, with three known related species (except for S. thailandicum), by using the fast-evolving nuclear big zinc finger (BZF) gene.
A new black fly species, Simulium (Gomphostilbia) kiewlomense, is described from females, males, pupae and mature larvae in Thailand. This new species is placed in the S. asakoae species-group and is characterized by having a combination of the elongate female sensory vesicle, widened male hind basitarsus, which is much wider than the hind femur, small pupal terminal hooks, and light greenish larval abdominal segments 1-3. Taxonomic notes are given to separate this new species from other related species. A DNA analysis using the COI gene shows that this new species has two genoforms with 1.21% difference. This is the 28th species of the S. asakoae species-group in Thailand, strengthening the evidence for high species diversity of this species-group.
Most female black flies in the genus Simulium are blood-sucking flies and they can cause various parasitic diseases in human and animal. A total of 94 species of black flies have been reported in Malaysia, however, their biting behavior and role as vector of infectious agents remain understudied. To fill in this knowledge gap, we attempted to survey adult black flies from field populations in Peninsular Malaysia. In a survey carried out in 2017 at Tasik Kenyir, Terengganu, three females were caught while attracted and landed on human skin. Further morphological and molecular analyses showed that the specimens were identical to Simulium (Gomphostilbia) aziruni Takaoka, Hashim & Chen of the Simulium gombakense species-group. This is the first report on a black fly species attracted to human in Malaysia which serves as a steppingstone towards in-depth studies for black flies in this region.
Ongoing urbanisation of the working population as well as cross-border migration of workers particularly into large cities has contributed to the development and growth of urban slums. These deprived areas are conducive for the transmission of intestinal pathogens including hookworm. The aim of this study was to determine both the prevalence and species identity of hookworm infections among the migrant worker community in Malaysia. A total of 388 faecal samples were collected from migrant workers between September 2014 and August 2015, representing workers from five employment sectors: construction, manufacturing, agriculture and plantations, food services and domestic services. Faecal samples were examined by microscopy and positive samples were subjected to molecular analysis. A total of 51 samples (13.1%) were positive by microscopy for hookworm infections. A two-step PCR based method amplifying a fragment of the 28S rRNA-ITS2 region was used to identify infections by Necator americanus and Ancylostoma spp. PCR products positive for Ancylostoma spp. were sequenced bidirectionally, and sequences analysed through BLAST and phylogenetic analysis. Samples containing Ancylostoma duodenale were further characterized by amplification and sequencing a fragment of cytochrome c oxidase subunit 1 (cox1) gene. PCR amplicons were successfully obtained from 42 (82.4%) of 51 samples, with 81.0% (34 of 42) identified as Necator americanus, 16.7% (7 of 42) as Ancylostoma spp. and 2.4% (1 of 42) as mixed infections of both species. All eight Ancylostoma spp. were confirmed to be Ancylostoma duodenale and this is the first time A. duodenale was reported in Malaysia. Samples containing A. duodenale from Nepalese and Indonesian workers shared high-similarity and were distinct compared to sequences from other countries. This study highlights the prevalence of hookworm infections among migrant workers living in Malaysia. Our findings underscore the necessity of screening migrant workers for hookworm infections, particularly those working in food-related services and industries.
Two new species of Simulium (Gomphostilbia), S. (G.) myanmarense and S. (G.) monglaense, are described from females, males, pupae and larvae from Myanmar. The two new species are placed in the S. asakoae species-group, and are similar to each other in the female and male but distinguished in the pupa by the presence or absence of an anterodorsal projection of the cocoon, and in the larva by a unique pattern of colored markings on the abdomen. Taxonomic notes are given to separate these species from related species. The COI gene sequences of both species are compared with those of eight species of the S. asakoae species-group and three species of the S. ceylonicum species-group. Both new species are most closely related to each other, further supporting their morphological classification in the S. asakoae species-group.
The rat lungworm Angiostrongylus cantonensis is a food-borne zoonotic parasite of public health importance worldwide. It is the primary etiologic agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans in many countries. It is highly endemic in Thailand especially in the northeast region. In this study, A. cantonensis adult worms recovered from the lungs of wild rats in different geographical regions/provinces in Thailand were used to determine their haplotype by means of the mitochondrial partial cytochrome c oxidase subunit I (COI) gene sequence. The results revealed three additional COI haplotypes of A. cantonensis. The geographical isolates of A. cantonensis from Thailand and other countries formed a monophyletic clade distinct from the closely related A. malaysiensis. In the present study, distinct haplotypes were identified in seven regions of Thailand - AC10 in Phitsanulok (northern region), AC11 in Nakhon Phanom (northeastern region), AC15 in Trat (eastern region), AC16 in Chantaburi (eastern region), AC4 in Samut Prakan (central region), AC14 in Kanchanaburi (western region), and AC13 in Ranong (southern region). Phylogenetic analysis revealed that these haplotypes formed distinct lineages. In general, the COI sequences did not differentiate the worldwide geographical isolates of A. cantonensis. This study has further confirmed the presence of COI haplotype diversity in various geographical isolates of A. cantonensis. The COI gene sequence will be a suitable marker for studying population structure, phylogeography and genetic diversity of the rat lungworm.
Zoonotic onchocerciasis is a human infection caused by Onchocerca species of animal origins and transmitted by black fly vectors. The reported incidence of this disease has increased throughout the world. This study aims to clarify the vectorial roles of black fly species in zoonotic filarial transmission in Tak province, western Thailand. The integrated approach of morphological and DNA sequence-based analyses was used to identify species of both wild-caught female black flies and infective filarial larvae found in the infected black flies. All of 494 female black flies captured were identified as Simulium nigrogilvum, through scanning electron microscopy (SEM) and DNA sequence analyses based on the cytochrome c oxidase subunit I (COI) and subunit II (COII), and the fast-evolving nuclear elongation complex protein 1 (ECP1) genes. Four females of S. nigrogilvum harbored one to three third-stage larvae (infective larvae) in their thoraces, with an infection rate of 0.81% (4/494). All infective larvae were similar in morphology and size to one another, being identified as Onchocerca species type I (= O. sp. type A), a bovine filaria, originally reported from Japan, and also as O. sp. found in S. nodosum in Thailand, based on their body lengths and widths being 1,068-1,346 µm long by 25-28 µm wide, and morphological characters. Comparisons of cytochrome c oxidase subunit I (COI) and 12S rRNA sequences of mitochondrial DNA (mtDNA) and phylogenetic analyses with those of previous reports strongly supported that all larvae were O. sp. type I. This report is the first indicating the presence of O. sp. type I in Thailand and its vector being S. nigrogilvum.
A new species of black fly, Simulium (Gomphostilbia) rampae, is described, based on adult male, its pupal exuviae and mature larvae collected from Doi Inthanon National Park, northern Thailand. This new species is placed in the Simulium asakoae species-group, and is characterized in the male by the high number of upper-eye facets in 17 vertical columns and 18 horizontal rows, in the pupa by the gill with a long common basal stalk, cone-shaped terminal hook, and cocoon with an anterodorsal projection, and in the larva by the medium-long postgenal cleft. A DNA analysis using COI gene supported its assignment to the S. asakoae species-group and showed its close relationship to S. (G.) udomi Takaoka & Choochote and S. (G.) chiangdaoense Takaoka & Srisuka. This is the fourth member of the S. asakoae species-group recorded from Thailand.
Simulium (Gomphostilbia) omutaense Ogata & Sasa, 1954 is the only named species in the Simulium batoense species-group of the subgenus Gomphostilbia Enderlein recorded from Honshu and Kyushu, Japan. It represents the northernmost distribution of this species-group, of which most members are distributed in the Oriental region. This species, the only member of the Simulium omutaense subgroup, is unique among the seven subgroups of the S. batoense species-group by having the pupal gill with one long filament and seven short filaments, similar to the arrangement of the pupal gill filaments in the S. zonatum subgroup of the S. epistum species-group in the same subgenus. This species is fully redescribed based on adults, pupal exuviae and mature larvae, and is most similar to species of the S. decuplum subgroup, based on adult morphological characteristics, although the pupal gill of the latter subgroup is markedly different by having 10 or 12 short filaments. Its close relationship to the S. decuplum subgroup is supported by a DNA analysis using COI gene sequences, with genetic distances of 9.30-11.02%. On the other hand, genetic distances between S. (G.) omutaense and species of the S. zonatum subgroup were 16.32-16.93%. Our study shows that a similar arrangement of the pupal gills in two different species-groups, which is rarely seen, has evolved independently and its occurrence does not necessarily reflect phylogenetic relationships.
A new species of black fly, Simulium (Gomphostilbia) isanense, is described based on females, males, pupae and mature larvae from Thailand. This new species is placed in the Simulium epistum species-group of the subgenus Gomphostilbia Enderlein. It is characterized by the pupal gill with eight filaments arranged as 3+3+2 from dorsal to ventral, of which an inner filament of the ventral pair is slightly longer than its counter filament. Taxonomic notes are provided to distinguish this new species from S. (G.) angulistylum Takaoka & Davies from Peninsular Malaysia, and three other related species. The difference between this new species and S. (G.) angulistylum is supported by genetic distances using the mitochondrial COI gene.
Malaria is a notorious disease which causes major global morbidity and mortality. This study aims to investigate the genetic and haplotype differences of Plasmodium knowlesi (P. knowlesi) isolates in Malaysian Borneo and Peninsular Malaysia based on the molecular analysis of the cytochrome b (cyt b) gene. The cyt b gene of 49 P. knowlesi isolates collected from Sabah, Malaysian Borneo and Peninsular Malaysia was amplified using PCR, cloned into a commercialized vector and sequenced. In addition, 45 cyt b sequences were retrieved from humans and macaques bringing to a total of 94 cyt b gene nucleotide sequences for phylogenetic analysis. Genetic and haplotype analyses of the cyt b were analyzed using MEGA6 and DnaSP ver. 5.10.01. The haplotype genealogical linkage of cyt b was generated using NETWORK ver. 4.6.1.3. Our phylogenetic tree revealed the conservation of the cyt b coding sequences with no distinct cluster across different geographic regions. Nucleotide analysis of cyt b showed that the P. knowlesi isolates underwent purifying selection with population expansion, which was further supported by extensive haplotype sharing between the macaques and humans from Malaysian Borneo and Peninsular Malaysia in the median-joining network analysis. This study expands knowledge on conservation of the zoonotic P. knowlesi cyt b gene between Malaysian Borneo and Peninsular Malaysia.
Five new species of black flies, Simulium (Gomphostilbia) chiangraiense, S. (G.) huaikaeoense, S. (G.) khaokhoense, S. (G.) maeklangense, and S. (G.) pamiangense, are described based on adults, pupae and mature larvae from Thailand. These five species are similar to one another and are placed in the S. decuplum subgroup of the S. batoense species-group by having the male fore coxae darkened, ventral plate flat and transverse; pupal gill with 10 short filaments; and larval postgenal cleft deep approaching or reaching the posterior margin of the hypostoma. Simulium (G.) chiangraiense sp. nov. and S. (G.) pamiangense sp. nov. differ from the three other new species by the much longer pupal gill filaments and darkened dorsal surface of abdominal segments 1 and 2. Taxonomic notes are provided to distinguish these five new species from five other related species. The phylogenetic positions of these new species in the S. decuplum subgroup are presented based on the mitochondrial COI gene. Simulium (G.) pamiangense sp. nov. and S. (G.) huaikaeoense sp. nov. are highly similar to each other genetically, showing a sister relationship, though they are clearly different morphologically. On the contrary, S. (G.) khaokhoense sp. nov. is distantly positioned from S. (G.) huaikaeoense nov., though it is almost indistinguishable morphologically from the latter.
The female, male, pupa and mature larva of Simulium (Simulium) poolpholi sp. nov. collected from a large and seasonal stream in northeastern Thailand are described. This new species is placed in the S. striatum species-group by the female sternite 8 concave posteromedially in form of the inverted-U shape, ovipositor valve with a ventrally produced process along its inner margin, male ventral plate saddle-shaped, style with a basal protuberance, and pupal gill with 10 filaments. It is morphologically similar to S. (S.) phraense Takaoka, Srisuka & Saeung from northern Thailand by lacking annular ridges on the surface of pupal gill filaments but it is distinguished from the latter species by the arrangement of pupal gill filaments. Molecular analysis using the fast-evolving nuclear big zinc finger (BZF) gene shows that S. (S.) poolpholi sp. nov. formed a distinct clade, being separated from the seven other Thai species of this species-group by a genetic distance of 1.84-4.55%. Our discovery increases the number of species in the S. striatum species-group in Thailand to nine.
Bartonella can infect a variety of mammals including humans and has been detected in the Americas, Europe, Africa, and Asia. Roughly two-thirds of identified Bartonella species are found and maintained in rodent reservoirs, with some of these species linked to human infections. Rodents (N=236) were caught from the Sahiwal division of Punjab, Pakistan and tested for Bartonella using PCR targeting gltA and rpoB genes, followed by sequencing of rpoB-positive samples. Genetic relatedness to other published Bartonella spp. rpoB gene sequences were examined using BLAST and phylogenetic analysis. Overall, 7.62% (18/236) of rodents were positive for both gltA and rpoB fragments. Rattus rattus and R. norvegicus had 7.94% (12/151) and 7.05% (6/85) positivity rates for Bartonella DNA, respectively. Phylogenetic analysis revealed a close relatedness between Bartonella spp. from Pakistan to Bartonella spp. from China, Nepal, and Malaysia. This study is the first reported detection of Bartonella spp. in R. rattus and R. norvegicus from the Sahiwal area of Punjab, Pakistan.
A new black fly species, Simulium (Gomphostilbia) wijiti, is described based on adult females, males, pupal exuviae and mature larvae from Mae Hong Son Province, Thailand. This new species is placed in the Simulium ceylonicum species-group. It is distinguished from four Thai members of the S. ceylonicum species-group [S. (G.) curtatum Jitklang et al., S. (G.) pangsidaense Takaoka, Srisuka & Saeung, S. (G.) sheilae Takaoka & Davies, and S. (G.) trangense Jitklang et al.], in the female by the short to medium long sensory vesicle; in the male by the large number of upper-eye (large) facets in 15 vertical columns and 15 or 16 horizontal rows; in the pupa by the dorsum of abdominal segments darkened; and in the larva by the antenna as long as or slightly shorter than the stem of the labral fan (longer than the stem of the labral fan in four other species). Phylogenetic analysis based on the COI gene sequences revealed that this new species is genetically closely related to S. leparense of the S. ceylonicum species-group, but is clearly separated from the latter species, and also from the three Thai related species (S. curtatum, S. sheilae and S. trangense) of the same species-group with interspecific genetic distances ranging from 9.65% to 12.67%. This is the fifth member of the S. ceylonicum species-group recorded from Thailand.