Redescription, molecular characterisation and Wolbachia endosymbionts of Mansonella (Tupainema) dunni (Mullin & Orihel, 1972) (Spirurida: Onchocercidae) from the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia) in Peninsular Malaysia.

The genus Mansonella Faust, 1929 includes 29 species, mainly parasites of platyrrhine monkeys in South America and anthropoid apes in Africa. In Malaysia, Mansonella (Tupainema) dunni (Mullin & Orihel, 1972) was described from the common treeshrew Tupaia glis Diard & Duvaucel (Scandentia). In a recent classification of the genus Mansonella, seven subgenera were proposed, with M. (Tup.) dunni as a monotypic species in the subgenus Tupainema. In this study, we collected new material of M. (Tup.) dunni from common treeshrews in Peninsular Malaysia and redescribed the morphological features of this species. We found that M. (Tup.) dunni differs from M. (Cutifilaria) perforata Uni et al., 2004 from sika deer Cervus nippon (Cetartiodactyla) in Japan, with regards to morphological features and predilection sites in their respective hosts. Based on multi-locus sequence analyses, we examined the molecular phylogeny of M. (Tup.) dunni and its Wolbachia genotype. Species of the genus Mansonella grouped monophyletically in clade ONC5 and M. (Tup.) dunni was placed in the most derived position within this genus. Mansonella (Tup.) dunni was closely related to M. (M.) ozzardi (Manson, 1897) from humans in Central and South America, and most distant from M. (C.) perforata. The calculated p-distances between the cox1 gene sequences for M. (Tup.) dunni and its congeners were 13.09% for M. (M.) ozzardi and 15.6-16.15% for M. (C.) perforata. The molecular phylogeny of Mansonella spp. thus corroborates their morphological differences. We determined that M. (Tup.) dunni harbours Wolbachia endosymbionts of the supergroup F genotype, in keeping with all other Mansonella species screened to date.

Description and molecular characteristics of Morishitium polonicum malayense Urabe, Nor Hashim & Uni, n. subsp. (Trematoda: Cyclocoelidae) from the Asian glossy starling, Aplonis panayensis strigata (Passeriformes: Sturnidae) in Peninsular Malaysia.

We describe Morishitium polonicum malayense n. subsp. from Asian glossy starlings (Aplonis panayensis strigata) (Horsfield, 1821) (Passeriformis: Sturnidae) caught in Malaysia. The trematodes had parasitized the air sacs and the thoracic and body cavities of 40 out of 67 (59.7%) birds examined. The specimens each had an oral sucker, a postpharyngeal genital pore, and tandem testes, but lacked a ventral sucker. The morphological characteristics of our specimens were similar to those of M. polonicum polonicum (Machalska, 1980) from Poland. However, the anterior extremity of vitelline follicles of the present specimens sometimes extended to the level of pharynx. The oral sucker width, oral sucker width/pharynx width ratio, and intertesticular space metrics differed from those of M. p. polonicum. The maximum-likelihood trees based on the cytochrome c oxidase subunit I (COI) and the internal transcribed spacer 2 (ITS2) sequences indicated that the species from the present study formed a sister group with M. p. polonicum from the Czech Republic. The p-distances of COI and ITS2 sequences between the present specimens and M. p. polonicum from the Czech Republic were 6.9-7.5% and 0.6%, respectively. These genetic divergences indicate the border for intra- or interspecific variation of digeneans. The definitive host species and geographical distribution of the current specimens were distinct from those of M. p. polonicum from Europe. We thus concluded that the present specimens are ranked as a new subspecies of M. polonicum, namely M. polonicum malayense n. subsp.

Description, molecular characteristics and Wolbachia endosymbionts of Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. (Nematoda: Filarioidea) from the Bornean bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae) of Sarawak, Malaysia.

BACKGROUND: The genus Onchocerca Diesing, 1841 includes species of medical importance, such as O. volvulus (Leuckart, 1893), which causes river blindness in the tropics. Recently, zoonotic onchocercosis has been reported in humans worldwide. In Japan, O. dewittei japonica Uni, Bain & Takaoka, 2001 from wild boars is a causative agent for this zoonosis. Many filarioid nematodes are infected with Wolbachia endosymbionts which exhibit various evolutionary relationships with their hosts. While investigating the filarial fauna of Borneo, we discovered an undescribed Onchocerca species in the bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae). METHODS: We isolated Onchocerca specimens from bearded pigs and examined their morphology. For comparative material, we collected fresh specimens of O. d. dewittei Bain, Ramachandran, Petter & Mak, 1977 from banded pigs (S. scrofa vittatus Boie) in Peninsular Malaysia. Partial sequences of three different genes (two mitochondrial genes, cox1 and 12S rRNA, and one nuclear ITS region) of these filarioids were analysed. By multi-locus sequence analyses based on six genes (16S rDNA, ftsZ, dnaA, coxA, fbpA and gatB) of Wolbachia, we determined the supergroups in the specimens from bearded pigs and those of O. d. dewittei. RESULTS: Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. is described on the basis of morphological characteristics and its genetic divergence from congeners. Molecular characteristics of the new species revealed its close evolutionary relationship with O. d. dewittei. Calculated p-distance for the cox1 gene sequences between O. borneensis n. sp. and O. d. dewittei was 5.9%, while that between O. d. dewittei and O. d. japonica was 7.6%. No intraspecific genetic variation was found for the new species. Wolbachia strains identified in the new species and O. d. dewittei belonged to supergroup C and are closely related. CONCLUSIONS: Our molecular analyses of filarioids from Asian suids indicate that the new species is sister to O. d. dewittei. On the basis of its morphological and molecular characteristics, we propose to elevate O. d. japonica to species level as O. japonica Uni, Bain & Takaoka, 2001. Coevolutionary relationships exist between the Wolbachia strains and their filarial hosts in Borneo and Peninsular Malaysia.

Zoonotic infection with Onchocerca dewittei japonica in an 11-year-old boy in Kansai Region, Western Honshu, Japan.

An 11-year-old boy living in Otsu City, Shiga Prefecture, Kansai Region, Western Honshu, Japan had zoonotic onchocercosis. The patient developed a painful swelling on the little finger of his left hand. The worm detected in the excised mass had external transverse ridges but did not have inner striae in the cuticle. On the basis of the parasite's histopathological characteristics, the causative agent was identified as a female Onchocerca dewittei japonica (Spirurida: Onchocercidae). The species of the filarial parasite was confirmed by sequencing the cox1 gene of the parasite. The Japanese wild boar Sus scrofa leucomystax is a definitive host for O. dewittei japonica, which is then transmitted by blackflies as the vector to humans. The current case described occurred in the Kansai Region, Western Honshu, where such infections were previously not reported.

Morphological and molecular characteristics of Malayfilaria sofiani Uni, Mat Udin & Takaoka n. g., n. sp. (Nematoda: Filarioidea) from the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia) in Peninsular Malaysia.

BACKGROUND: The filarial nematodes Wuchereria bancrofti (Cobbold, 1877), Brugia malayi (Brug, 1927) and B. timori Partono, Purnomo, Dennis, Atmosoedjono, Oemijati & Cross, 1977 cause lymphatic diseases in humans in the tropics, while B. pahangi (Buckley & Edeson, 1956) infects carnivores and causes zoonotic diseases in humans in Malaysia. Wuchereria bancrofti, W. kalimantani Palmieri, Pulnomo, Dennis & Marwoto, 1980 and six out of ten Brugia spp. have been described from Australia, Southeast Asia, Sri Lanka and India. However, the origin and evolution of the species in the Wuchereria-Brugia clade remain unclear. While investigating the diversity of filarial parasites in Malaysia, we discovered an undescribed species in the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia). METHODS: We examined 81 common treeshrews from 14 areas in nine states and the Federal Territory of Peninsular Malaysia for filarial parasites. Once any filariae that were found had been isolated, we examined their morphological characteristics and determined the partial sequences of their mitochondrial cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes. Polymerase chain reaction (PCR) products of the internal transcribed spacer 1 (ITS1) region were then cloned into the pGEM-T vector, and the recombinant plasmids were used as templates for sequencing. RESULTS: Malayfilaria sofiani Uni, Mat Udin & Takaoka, n. g., n. sp. is described based on the morphological characteristics of adults and microfilariae found in common treeshrews from Jeram Pasu, Kelantan, Malaysia. The Kimura 2-parameter distance between the cox1 gene sequences of the new species and W. bancrofti was 11.8%. Based on the three gene sequences, the new species forms a monophyletic clade with W. bancrofti and Brugia spp. The adult parasites were found in tissues surrounding the lymph nodes of the neck of common treeshrews. CONCLUSIONS: The newly described species appears most closely related to Wuchereria spp. and Brugia spp., but differs from these in several morphological characteristics. Molecular analyses based on the cox1 and 12S rRNA genes and the ITS1 region indicated that this species differs from both W. bancrofti and Brugia spp. at the genus level. We thus propose a new genus, Malayfilaria, along with the new species M. sofiani.