Correction: A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam.

[This corrects the article DOI: 10.1371/journal.pone.0198565.].

A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam.

Rhinoceros (rhinos) have suffered a dramatic increase in poaching over the past decade due to the growing demand for rhino horn products in Asia. One way to reverse this trend is to enhance enforcement and intelligence gathering tools used for species identification of horns, in particular making them fast, inexpensive and accurate. Traditionally, species identification tests are based on DNA sequence data, which, depending on laboratory resources, can be either time or cost prohibitive. This study presents a rapid rhino species identification test, utilizing species-specific primers within the cytochrome b gene multiplexed in a single reaction, with a presumptive species identification based on the length of the resultant amplicon. This multiplex PCR assay can provide a presumptive species identification result in less than 24 hours. Sequence-based definitive testing can be conducted if/when required (e.g. court purposes). This work also presents an actual casework scenario in which the presumptive test was successfully utlitised, in concert with sequence-based definitive testing. The test was carried out on seized suspected rhino horns tested at the Institute of Ecology and Biological Resources, the CITES mandated laboratory in Vietnam, a country that is known to be a major source of demand for rhino horns. This test represents the basis for which future 'rapid species identification tests' can be trialed.

An internationally standardized species identification test for use on suspected seized rhinoceros horn in the illegal wildlife trade.

Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common 'rhino horn' substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing.

The role of rice fields, fish ponds and water canals for transmission of fish-borne zoonotic trematodes in aquaculture ponds in Nam Dinh Province, Vietnam.

BACKGROUND: Fish-borne zoonotic trematodes (FZT), such as Clonorchis sinensis, Opistorchis viverini (Opisthorchiidae) and intestinal trematodes of the family Heterophyidae, constitute a public health hazard in Vietnam and infections with these trematodes has been linked to consumption of raw or undercooked fish from aquaculture. The FZT transmission pathways, however, are more complicated than just the presence of intermediate snail hosts in aquaculture ponds as ponds may exchange water with surrounding habitats such as rice fields and irrigation canals and thereby these surrounding habitats may be a source of snails and cercariae and contribute to FZT infection in cultured fish. METHODS: This is a longitudinal descriptive study on selected farms (n = 30) in Nam Dinh Province which is endemic for FZT. At each farm, we sampled one pond, a small irrigation canal used to supply the pond with water, and a nearby rice field. At each of these three sites, we estimated the density of the FZT intermediate snail hosts and determined their trematode infection status. Comparative analysis was performed for the prevalence and density of FZT infections in fish and snails. RESULTS: Species of the Thiaridae, and most notably Melanoides tuberculata, the most important host species for FZT belonging to the Heterophyidae, were particularly abundant in ponds and small canals, i.e. M. tuberculata was found in 27 ponds and 13 small canals. Bithynia fuchsiana, a potential host for both Heterophyidae and Opisthorchiidae, was rarely found in fish ponds but common in rice fields. A total of 12 types of cercariae were found in the snails and pleurolophocercous cercariae, primarily FZT, constituted about 40 % of all cercarial infections. The fish species cultured were mainly carp species and Haplorchis pumilio was the dominating trematode species infecting fish. Clonorchis spp. were not recorded in any of the ponds. FZT transmission to fish was intense during the summer period (May-June to November) but less intense during the winter months (December-January) partly because cercarial emergence ceases due to the low temperature. CONCLUSION: Our findings highlight the complexity of FZT transmission within aquaculture farm settings and suggest that efforts to control these infections must take a holistic approach using interventions against all stages of the transmission cycle.

Morphological and molecular characterization of lymnaeid snails and their potential role in transmission of Fasciola spp. in Vietnam.

Freshwater snails of the family Lymnaeidae play an important role in the transmission of fascioliasis worldwide. In Vietnam, 2 common lymnaeid species, Lymnaea swinhoei and Lymnaea viridis, can be recognized on the basis of morphology, and a third species, Lymnaea sp., is known to exist. Recent studies have raised controversy about their role in transmission of Fasciola spp. because of confusion in identification of the snail hosts. The aim of this study is, therefore, to clarify the identities of lymnaeid snails in Vietnam by a combination of morphological and molecular approaches. The molecular analyses using the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA clearly showed that lymnaeids in Vietnam include 3 species, Austropeplea viridis (morphologically identified as L. viridis), Radix auricularia (morphologically identified as L. swinhoei) and Radix rubiginosa (morphologically identified as Lymnaea sp.). R. rubiginosa is a new record for Vietnam. Among them, only A. viridis was found to be infected with Fasciola spp. These results provide a new insight into lymnaeid snails in Vietnam. Identification of lymnaeid snails in Vietnam and their role in the liver fluke transmission should be further investigated.

Distribution of freshwater snails in family-based VAC ponds and associated waterbodies with special reference to intermediate hosts of fish-borne zoonotic trematodes in Nam Dinh Province, Vietnam.

Fish-borne zoonotic trematodes, such as Clonorchis sinensis, heterophyids and others, constitute a public health concern in parts of northern Vietnam and infections with these trematodes are often thought to be linked to fish culture. One common fish culture system is the integrated fish-livestock (VAC) ponds where individual households have 1 or more ponds. Fish fry, mainly of various carp species, produced in hatcheries, not necessarily local, are introduced into nursery ponds and after approximately 6 weeks, juvenile fishes are transferred to household ponds, referred to as grow-out ponds. Grow-out ponds are usually fertilized with organic debris, including animal excreta, to stimulate algal growth and subsequently fish growth. This paper describes the distribution of freshwater snails and occurrence of trematode infections in these in VAC ponds and associated habitats as part of a major study on risk factors of FZT infections in cultured fish in two communes, Nghia Lac and Nghia Phu, Nghia Hung District, Nam Dinh Province. The area is under intense rice cultivation with an extensive canal network supplying fields and also household VAC ponds. A total of 16 snail species was found and four were widely distributed i.e. Angulyagra polyzonata, Melanoides tuberculata, Bithynia fuchsiana and Pomacea insularum. Snail diversity and counts were higher in nursery ponds than in grow-out ponds. Species of the families Thiaridae and Viviparidae were more abundant than other species in VAC ponds while species of the Bithyniidae, Stenothyridae and Planorbidae dominated in rice fields and small canals. Trematode infections were found in eight snail species and among these M. tuberculata had the highest overall prevalence of infection (13.28%). No trematode infections were found in species of the Viviparidae and Ampullaridae except for metacercariae. Parapleurolophocercous and pleurolophocercous cercariae constituted the most common type of cercariae recovered, contributing 40.6% of all infections followed by echinostome cercariae (35.0%) and xiphidiocercariae (17.3%). Bithynia fuschiana and M. tuberculata had the most diverse trematode fauna. C. sinensis was not recorded in this study. The VAC pond system in this area, is very important for transmission of minute intestinal trematodes while they play little role in transmission of C. sinensis as its intermediate hosts, bithynid snails, rarely occur in these ponds. From a public health perspective this is positive as the effects of infections with intestinal trematodes are considered mild. On the other hand it is possible that even such subtle effects could have importance in public health as transmission is very intense in the area. And this in combination with the aquaculture importance, reduced marketability of fishes with high metacercariae loads, warrants that control efforts against these trematodes are initiated to reduce transmission in this production system.

Morphological and molecular identification of two Paragonimus spp., of which metacercariae concurrently found in a land crab, Potamiscus tannanti, collected in Yenbai Province, Vietnam.

Paragonimosis is an important food-borne zoonosis especially in Asian countries. Among Paragonimus species, Paragonimus westermani followed by P. skrjabini complex are the major pathogens for human paragonimosis in Asia. In addition, P. heterotremus is an important pathogen in southern China and the Indochina Peninsula and is the only proven species to cause human paragonimosis in Vietnam. During a recent survey in Yenbai Province in northern Vietnam, we found small and large types of Paragonimus metacercariae often concurrently in mountainous crabs, Potamiscus tannanti. Adult worms from those small and large metacercariae were obtained separately by experimental infection in dogs and cats. Morphological and molecular phylogenetic study based on sequences of ITS2 and a part of CO1 genes were performed for the identification of small and large metacercariae and their adults. The results showed that small metacercariae and their adults are completely identical with P. heterotremus in morphology and molecular genetic profiles. In contrast, large metacercariae and their adults have some morphological similarities with P. skrjabini and P. harinasutai, but are unidentifiable from each other by morphology alone. Molecular phylogenetic tree analyses on ITS2 and CO1 genes revealed that large metacercariae and their adults were grouped in the same clade and different from any known Paragonimus species. Although they share the same ancestor with P. skrjabini complex, their genetic distance was considerably different from two other known subspecies, P. skrjabini skrjabini and P. skrjabini miyazakii. Our results provide a new insight on the phylogeny of the genus Paragonimus.