Hoang Van Hien

Current status of fish-borne zoonotic trematode infections in Gia Vien district, Ninh Binh province, Vietnam

BackgroundNinh Binh province is known as an endemic area of fish-borne zoonotic trematode (FZT) transmission in Vietnam. A cross-sectional study was conducted in Gia Minh and Gia Thinh communes of Gia Vien district, Ninh Binh province to investigate the infections with different stages of FZT in various host species.MethodsFaecal samples from 1,857 humans were examined for trematode eggs using the Kato-Katz method, while faecal samples from 104 dogs, 100 cats, and 100 pigs were examined using the Formalin-ethyl acetate concentration technique (FECT). A total of 483 specimens of freshwater fish, representing 9 species, were examined for metacercariae using the artificial digestion method. Three methods of cercarial detection (shedding, crushing and cutting) were applied for examination of 3,972 specimens of freshwater snails, representing 7 species. All relevant data e.g. location, sex, age group, animal species, and habitat were recorded for statistical analyses.ResultsHelminth eggs were found in 65.5% of the human faecal samples, including 20.5% of faecal samples containing small trematode eggs. Infection with small trematodes differed among communes, age groups and sexes. Eggs of small trematodes were found in 32.7% of faecal samples from dogs, 49.0% from cats and 13.0% from pigs. The difference in prevalences and intensities were significant among species of animals but did not differ between the two communes. All fish species were infected with FZT, with an average prevalence of 56.1% and a mean intensity of 33.245 metacercariae per gram. Prevalence and intensity in fish differed significantly among cummunes and fish groups. Six species of zoonotic trematodes were identified. Metacercariae of the small liver fluke, Clonorchis sinensis, was only found in Hemiculter leucisculus. A total of 9 specimens from two snail species, Melanoides tuberculata and Bithynia fuchsiana, were infected with trematodes and four cercarial types were detected in the study sites.ConclusionsWe conclude that Gia Minh and Gia Thinh communes are continuing to be hot-spot endemic areas of FZT and other helminths infections where the habit of eating raw fish by the local people is still present.

Co-existence of Paragonimus harinasutai and Paragonimus bangkokensis metacercariae in fresh water crab hosts in central Viet Nam with special emphasis on their close phylogenetic relationship.

During our epidemiological surveys for Paragonimus species in central Viet Nam, we found four morphologically different Paragonimus metacercariae in mountainous crabs. They were identified as metacercariae of Paragonimus westermani, P. bangkokensis, P. proliferus, and P. harinasutai in the order of their prevalence in crab hosts. This is the first discovery of P. harinasutai in Viet Nam, co-inhabiting with P. bangkokensis and other species. Metacercariae of P. harinasutai were given orally to a cat to obtain adult worms. Then, ITS2 and CO1 sequences of metacercariae and adults of P. harinasutai, and metacercariae of P. bangkokensis collected from the same place were determined for analyses of phylogenetic relationships to other P. harinasutai and P. bangkokensis populations as well as related species. The results of molecular analyses showed that P. harinasutai from Quang Binh province of central Viet Nam was almost completely identical with those from Vientiane, Lao PDR; P. bangkokensis from Quang Binh, Viet Nam was also almost completely identical with those from Lao PDR and from Quang Ninh province, Viet Nam. Except for one P. harinasutai isolate from China, all populations of P. harinasutai and P. bangkokensis from Thailand, Lao and Viet Nam make a single clade in both ITS2 and CO1 trees. In ITS2 sequences, AT deletion and ATC insertion were observed in some isolates of both species, indicating recent gene flow between P. harinasutai and P. bangkokensis. Moreover, because of their extremely high genetic similarities and their co-inhabitation in the same crab hosts found in Thailand, Lao PDR and Viet Nam, they should be considered as the sister species at the early stage of divergence. In addition, P. microrchis previously described from Yunnan, China should be placed as the synonym of P. harinasutai, because of their morphological and molecular similarities.

Discovery of Paragonimus skrjabini in Vietnam and its phylogenetic status in the Paragonimus skrjabini complex.

Two members of the Paragonimus skrjabini complex, P. skrjabini and P. miyazakii, are now considered as two sub-species, P. skrjabini skrjabini and P. skrjabini miyazakii. They are well known as important pathogens for human paragonimiasis in China and Japan. Recently, members of this species complex have been reported from India. Here we report the first discovery of P. skrjabini from freshwater crab hosts in Thanh Hoa province, Vietnam. For morphological and molecular phylogenetic studies, adult worms were obtained by experimental infection in cats and dogs. Molecular analyses of metacercariae and adults revealed that the P. skrjabini population from Thanh Hoa, Vietnam was almost completely identical with that from Yunnan province, China. Those populations from Thanh Hoa, Vietnam and Yunnan, China and those from Manipur, India were significantly different from P. skrjabini populations reported from other localities of China in cytochrome oxidase subunit 1 (CO1) gene sequences, indicating considerable genetic variation within the P. skrjabini complex. Moreover, low bootstrap values in the CO1 tree suggested that more variant genotypes belonging to P. skrjabini complex may be found in other Asian countries in between Vietnam and India, such as Myanmar, Laos and Thailand. Since P. skrjabini is known as a pathogen for humans, paragonimiasis cases caused by P. skrjabini might be found in Vietnam and other Asian countries.

Genetically variant populations of Paragonimus proliferus Hsia & Chen, 1964 from central Vietnam

Among about 50 nominal Paragonimus species, Paragonimus proliferus is rather a rare species, found only in Yunnan province, China, until our recent discovery of this species in Lai Chau province, northern Vietnam close to Yunnan, China. Here we add Quang Binh province, central Vietnam as a new endemic area of P. proliferus. Large excysted metacercariae found in mountainous crabs, Potamiscus tannanti, were morphologically identified as P. proliferus, which was confirmed further by molecular analyses. Second internal transcribed spacer (ITS2) sequences of the P. proliferus population in Quang Binh province were completely (100%) identical with those of P. proliferus populations in Lai Chau province, northern Vietnam and Yunnan province, China. However, cytochrome oxidase subunit 1 (CO1) gene sequences of Quang Binh population were significantly different (5.6%) from that of previously reported northern Vietnam and Yunnan, China populations. A phylogenetic tree revealed that all CO1 sequences of P. proliferus Quang Binh population formed a distinct group, which was clustered with northern Vietnam and Yunnan, China populations with the bootstrap value of 75%. This is the first record of the genetically variant population of P. proliferus, distribution of which is geographically remote from the previously reported endemic areas in the border between northern Vietnam and Yunnan, China, suggesting that P. proliferus may be much more widely distributed in the Indochina peninsula (or South-East Asia) than expected.

Molecular identification of the trematode Paragonimus in faecal samples from the wild cat Prionailurus bengalensis in the Da Krong Nature Reserve, Vietnam.

Conventional identification of Paragonimus species and their natural definitive hosts is based on the morphological features of adult parasites isolated from the lungs of wild mammalian hosts. However, wild animals are protected by strict regulations and sampling is not always possible. Recently, molecular techniques have been developed to identify the internal transcribed spacer (ITS) sequences of Paragonimus eggs in faeces/sputum of human patients. Also, mammalian hosts can be identified using the D-loop sequence of mitochondrial DNA in faecal samples. In this study, we used molecular techniques on faeces from wild animals collected in Da Krong Nature Reserve, Quang Tri province, central Vietnam, where Paragonimus metacercariae are highly prevalent in mountain crabs, to identify Paragonimus species and their natural definitive hosts. The results indicated that wild cats, Prionailurus bengalensis, were infected with at least three different Paragonimus species, P. westermani, P. skrjabini and P. heterotremus. Because all of these species can infect humans in Asian countries, human paragonimiasis should be considered in this area.

First intermediate hosts of Paragonimus spp. in Vietnam and identification of intramolluscan stages of different Paragonimus species

BackgroundMembers of the genus Paragonimus require at least three hosts in their life-cycles. The obligatory first intermediate hosts are freshwater snails. In Vietnam, although seven Paragonimus species have been recorded, the natural first intermediate hosts of almost all species have not been confirmed. The aim of this study was, therefore, to investigate snail hosts of Paragonimus species in Vietnam, and to identify Paragonimus species at intramolluscan stages.MethodsFreshwater snails were collected from streams in Yen Bai and Quang Tri Provinces, where high prevalences of Paragonimus metacercariae in crab hosts have been reported. Snails were morphologically identified and then examined individually for Paragonimus cercariae using shedding and crushing methods. Chaetomicrocercous cercariae, the morphological class to which Paragonimus cercariae belong, were collected for morphological description and molecular species identification by analyses of ITS2 sequences. The infected snail species were identified based on analyses of nucleotide sequences of the cox1 gene.ResultsThree snail species were found to be infected with Paragonimus cercariae at low infection rates, ranging between 0.07–1.0%. The molecular analyses identified them as Sulcospira quangtriensis and 2 species of subfamily Triculinae. In a phylogenetic tree, these two triculine snails were related to the genera Gammatricula and Tricula with low posterior probabilities. Thus we named them as Triculinae sp. 1 and Triculinae sp. 2. Cercariae from the three snail species, Sulcospira quangtriensis, Triculinae sp. 1 and Triculinae sp. 2, were molecularly identified as Paragonimus westermani, P. heterotremus and P. proliferus, respectively. The cercariae of the three species are morphologically similar to each other, but their daughter rediae can be distinguished by the length of the intestine and the number of cercariae per redia. The rediae of P. westermani have a long intestine and each contain 6–8 cercariae. In contrast, those of P. heterotremus and P. proliferus have a short intestine and each redia contain 10–12 and 5–6 cercariae, respectively.ConclusionsThree snail species, Sulcospira quangtriensis, Triculinae sp. 1 and Triculinae sp. 2, serve as the first intermediate hosts of P. westermani, P. heterotremus and P. proliferus, respectively, in Vietnam. The length of the intestine of rediae and the number of cercariae per redia are valuable characteristics for distinguishing between larvae of these Paragonimus species.

Morphological and molecular characteristics of Angiostrongylus cantonensis Chen et al., 1935 from Bac Ninh and Hung Yen provinces, Vietnam

The rat lungworm, Angiostrongylus cantonensis, is a nematode that parasites the lungs of rats - the natural definitive hosts of the nematodes. This nematode can cause Angiostrongyliasis for human when people eat uncooked/undercooked snails infected with infective larvae (L3). In Vietnam, although some suspected Angiostrongyliasis cases have been reported, there has been no investigation for A. cantonensis in natural definitive hosts (rats). This is the first record of A. cantonensis in rats collected from Bac Ninh and Hung Yen provinces, Vietnam based on morphological and molecular identification. A total of 234 rats, including 147 R. norvegicus and 87 R. losea , were examined. The rat lungworms were found in both rat species. The overall infection rates in Hung Yen and Bac Ninh provinces were 40.4% and 21.4%, respectively. The infection rates in R. norvegicus (60.0 and 26.3%) were higher than that of R. losea (14.4% and 2.2%). The description of A. cantonensis and the molecular phylogenetic relationship among Angiostrongylus genus were given and discussed herein.

Infection situatus of parasites in freshwater fishs collected in Lam river, Do Luong district, Nghe An province

The results obtained from our research about the parasitic worm, which parasitized freshwater fishs of Lam river (Do Luong district, Nghe An province) provide us the basic knowledge about parasites of freshwater fishs in North Central Vietnam. Total 217 individuals of freshwater fish were carried out an autopsy for checking fish parasites. Among them, 118 fishes (occupied 54.4% total number of surgical fish) belonged to 13 species of Cypriniformes (occupied 54.2% total number of fish species). Of the total 832 samples of fish parasites collected, there were 504 samples of Monogenea, 212 Trematodes, 25 Cestodes, 16 Acanthocephala, 44 Nematoda and 31 Copepoda. Infected rate of fish in study site was not too high, the prevalence was 39.2%. and the density of parasites per each infected host was 9,8 individuals/host. Monogenea was the highest infected group (26.3%), while other groups were lower: from 1.4% to 7.4%. The infected rate was highest in cranoglanis 90%, the next was hemicultus 54.5%, tilapia 50%, red - eyed carp and major carp 40%, mudfish 28.0%. In case of 14 fish species, with 10 surgical fish individuals, the infection rate fluctuated between 16.7% and 90%. The infected rate was observed as the highest in Cranoglanis sinensis (90%) and the lowest in Tilapia (16.7%). Parasite infection intensity in not too high, one individual of fish infected with 9.8 worms in average (min - max: 1 - 84). The highest infected intensity was found in common carp 33.0 worms/fish (1 - 64), the next is red - eyed carp 29.0worm/fish and cranoglanis 13.9worms/fish; the lowest infection intensity was found in hemicultur 12.3 worms/fish. The certain host are normally infected with 2 or 3 parasite groups their prevalence and density is not too high.

A preliminary stury on ectoparasite composition of wild birds in Ba Be national park

The knowledge and published paper the ectoparasites of wild birds in Vietnam are very little. In fact, there are some specialized references that provided biological characteristics, ecology and the distribution of this group in several parts of Vietnam, but the conditions as well as research purposes set out, only focus of interest on livestocks and animals, which live near the human i.e. mice (Rodentia). This paper reported that 12 individuals of the total 45 birds captured in Ba Be national park infected with ectoparasites. The infectious intensity of ectoparasites on those birds was from 1 to 38 samples. Two ectoparasite species were determined as Myrsidea ochracei and Brueelia alophoixi , five suggested ectoparasites species are undetermined.