Marine fish parasites in the Cat Ba Archipelago, Vietnam: the results of 2010-2023 field surveys.

Between 2010 and 2023, a longitudinal study was undertaken to uncover the diversity of the parasite fauna of marine fishes in the Cat Ba Archipelago, a world biosphere reserve, in Vietnam. A total of 1,042 specimens representing 80 different fish species were collected and examined. Of these, 68 fish species, represented by 994 specimens (95.39%), were infected with parasites. A total of 162 parasitic species were discovered, including 54 trematodes, 37 monogeneans, 27 crustaceans, 15 myxozoans, 10 acanthocephalans, 10 nematodes, 7 cestodes, and 2 hirudineans. Over the course of the survey, twenty new species were described, including 7 acanthocephalans and 13 trematodes. Additionally, twenty species were recorded for the first time from the Cat Ba Archipelago and twenty-two species had new host records reported. The prevalence and mean intensity of parasite infection were found to be unaffected by season. These data on the parasitic fauna of Cat Ba Archipelago not only expand our knowledge of the diversity of Vietnam, but also provide strong baseline data for measuring future change resulting from environmental perturbations.

First morphological and molecular identification of third-stage larvae of Anisakis typica (Nematoda: Anisakidae) from marine fishes in Vietnamese water.

Anisakid nematodes are parasites of cetaceans, their larval stages live in marine fishes. The third-stage larvae of some Anisakis species are also the etiological agents of human anisakiasis caused by consumption of raw or undercooked infected fish. Thus, identification of Anisakis larvae at the species level is crucial for their ecology and epidemiology. In Vietnam, although Anisakis larvae have been reported, they have not been identified to the species level. The aim of this study was, therefore, to identify third-stage larvae of Anisakis collected from marine fishes in Vietnamese water, based on morphological characteristics and molecular analysis. All Anisakis larvae found in this study were morphologically similar to each other and identical to Anisakis typica. In addition, molecular analysis based on ITS1-5.8S-ITS2 sequences confirmed them as A. typica. Vietnamese A. typica population was genetically close to those from Asian countries and Australia. The third-stage larvae of A. typica were collected from eight fish species from three localities in the South of Vietnam. Among them, seven were recorded as new intermediate hosts of A. typica. This is the first identification of A. typica larvae in Vietnamese water with records of new fish hosts.

Are Melanoides tuberculata and Tarebia granifera (Gastropoda, Thiaridae), suitable first intermediate hosts of Clonorchis sinensis in Vietnam?

BACKGROUND: Two thiarid snail species, Melanoides tuberculata and Tarebia granifera have been reported as first intermediate hosts of the liver fluke Clonorchis sinensis; however, their role as true first intermediate hosts has not been verified. Thus, the present study aimed to clarify the suitability of these two snail species as first intermediate hosts of C. sinensis. This was accomplished by collecting snails from a highly endemic area for C. sinensis in Vietnam, the Thac Ba reservoir, and identifying shed cercariae using molecular techniques. We also conducted experimental infections of five snail species including M. tuberculata and T. granifera with eggs of C. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: A total of 11,985 snails, representing 10 species were sampled. Five snail species, M. tuberculata, T. granifera, Lymnaea swinhoei, Parafossarulus manchouricus, and Bithynia fuchsiana were found shedding cercariae with an overall prevalence of infection ranging from 0.7% to 11.5%. Seven cercarial types were recorded. Cercariae of C. sinensis were only found in Parafossarulus manchouricus. Using a multiplex PCR approach for detecting C. sinensis infection, the prevalence in P. manchouricus was 4.2%. Additionally, all five snail species were experimentally exposed to C. sinensis eggs, however only P. manchouricus was successfully infected with an infection rate of 7.87%. CONCLUSIONS/SIGNIFICANCE: We confirmed that in the Thac Ba reservoir, Vietnam, the two thiarids, M. tuberculata and T. granifera are not suitable first intermediate hosts of C. sinensis. Only P. manchouricus was found infected by C. sinensis in nature, and was the only species that became infected experimentally.

Molecular identification of morphologically similar microcercous cercariae of two trematode families, Paragonimidae and Troglotrematidae, concurrently found in the same snail species of the subfamily Triculinae.

Microcercous cercariae possess a very short tail and are produced by digenean species of several families including medically important species, such as members of the genera Paragonimus Braun, 1899, Nanophyetus Chapin, 1927 and Troglotrema Odhner, 1914. During our survey of cercariae of Paragonimus spp. in Vietnam, we found microcercous cercariae from ten (0.29%) out of 3,400 snails of Triculinae gen. sp. 2. They were morphologically and molecularly analysed for species identification. The molecular analysis, based on ITS2 sequences, revealed two distinct species: four specimens were identical to Paragonimus proliferus Hsia et Chen, 1964 (Paragonimidae Dollfus, 1939), and the other six specimens were closest to members of the family Troglotrematidae Odhner, 1914 and were temporarily named Troglotrematidae gen. sp. Morphologically, cercariae of the two species found in this study are similar to each other in their gross characteristics but can be distinguished from one another by subtle morphological details. The cercaria of P. proliferus has an I-shaped excretory bladder and does not have mucous gland cells. In contrast, that of Troglotrematidae gen. sp. has a Y-shaped excretory bladder and mucous gland cells. Besides, the redia of P. proliferus is elongate with a short intestine and contains 5-6 cercariae whereas that of Troglotrematidae gen. sp. is more round with a longer intestine and harbours 3-4 cercariae. Our results have shown the importance of the shape of the excretory bladder and the presence/absence of mucous gland cells of the cercaria as well as the shape and size of the redia, and its intestinal length as valuable taxonomic characters of intramolluscan trematode larvae. In addition, the finding of similar microcercous cercariae of different species in the same snail species suggests that careful attention to morphological details is required in the differentiation of Paragonimus cercariae and those of closely related species.

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

BACKGROUND: Members 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. METHODS: Freshwater 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. RESULTS: Three 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. CONCLUSIONS: Three 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.

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

BACKGROUND: Ninh 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. METHODS: Faecal 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. RESULTS: Helminth 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. CONCLUSIONS: We 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.

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis.

We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.