Morphological and Molecular Characterization of Paragonimus skrjabini Complex from Yunnan, China: A Brief Report.

PURPOSE: To perform environmental sampling and molecular identification of Paragonimus in endemic regions, which may help in minimizing transmission among humans. METHODS: Mountain crabs from the genus Potamiscus were collected and the encysted metacercariae were extracted and subjected to morphological identification, followed by animal inoculation in Sprague-Dawley (SD) rats. After 112 days of infection, animals were killed and adult worms were extracted from lungs and muscles. The morphology of adult worms was characterized by microscopy and molecular identification was done by polymerase chain reaction, followed by sequencing of cox1 and ITS2 genes. Phylogenetic analysis was done by maximum parsimony method. RESULTS: A total of 447 crabs were captured from the streams of Tongchang Town, Jinping County, Yunnan Province, China. The infection rate was found to be 41% (186 out of 447 crabs). The metacercariae of Paragonimus skrjabini was identified by the characteristics round or spherical encysted form measuring 410 to 460 × 400 to 460 µm. After animal infection in SD rats, adults were presumptively confirmed to be P. skrjabini, which was also confirmed by gene amplification and sequence analysis of cox1 and ITS2 regions. Paragonimus skrjabini clustered with previously reported P. skrjabini from Yunnan and Vietnam. The confidence values of their branches were > 95%. Phylogenetic analysis of the ITS2 region revealed two distinct clusters with distinct geographical grouping. Phylogenetic analysis with the combined data sets reiterated the geographical grouping with P. skrjabini from Yunnan clustering with strains from Vietnam. CONCLUSION: Metacercariae of P. skrjabini was discovered in freshwater crabs in Yunnan province, China, and the strains were phylogenetically related to P. skrjabini from Vietnam.

Morphological and molecular characterization of Paragonimus caliensis Little, 1968 (Trematoda: Paragonimidae) from Medellin and Pichinde, Colombia.

Paragonimiasis is a subacute to chronic inflammatory granulomatous lung disease caused by the genus Paragonimus. In Latin America Paragonimus mexicanus Miyazaki & Ishii, 1968 is the only confirmed species to cause human infections. Paragonimus caliensis Little, 1968 is an uncommon species often regarded as a synonym of P. mexicanus. Recently, the study of two types of Paragonimus metacercariae from Costa Rica has provided new molecular and morphological evidence that P. caliensis is a separate species from P. mexicanus. In the present study, molecular, morphological and phylogenetic tools have been used to characterize two populations of Paragonimus located at west of Medellin, Antioquia and at Pichinde, Valle del Cauca (type locality of P. caliensis), Colombia. Adults and metacercariae obtained from Medellin, and metacercariae from Pichinde were analyzed. For morphological observations we used light microscopy and scanning electron microscopy (SEM). Morphology of metacercariae and adults matched with the holotype of P. caliensis. The number and arrangement of sensory papillae in the acetabulum region differs from the morphotypes reported for P. caliensis in Costa Rica. Two morphotypes in branching patterns of ovary and two morphotypes in branching patterns of testes were identified. The main morphological differences between P. caliensis and P. mexicanus corresponded to the size of gonads and their relative positions in the body, and the occasional presence of a cyst wall in P. caliensis metacercariae. The molecular and phylogenetic analyses (using nuclear ribosomal ITS2 and partial cytochrome c oxidase subunit 1 CO1 sequences) confirmed that P. caliensis from the type locality is the same species from Medellin and Costa Rica. Furthermore, these analyses also suggest genetic as well as geographical separation of P. caliensis populations between Colombia and Costa Rica. Currently, P. mexicanus and P. caliensis are sympatric in the Colombian Pacific bioregion, and specific diagnosis based on their egg size is not possible. Therefore, it is necessary to determine the biogeographic distribution ranges of both species and to implement molecular techniques to establish the role of P. caliensis in human paragonimiasis in Colombia.

Detection of Paragonimus mexicanus (Trematoda) metacercariae in crabs from Oaxaca, Mexico.

Metacercariae of Paragonimus mexicanus were collected in crabs Tehuana guerreroensis (Rathbun, 1933) in the municipality of Putla, Oaxaca, Mexico. Metacercariae were found in 20.8% of the crabs collected, with an average of 1.9 metacercarie per crab. Stained metacercariae showed the specific characteristics of P. mexicanus by morphology and sequencing a fragment of the 28S ribosomal gene obtained by PCR. These findings reveal that T. guerreroensis is an intermediate host for P. mexicanus; this new report is relevant considering the potential risk of transmission in the states of Oaxaca and Guerrero, Mexico.

Detection of multiple species of human Paragonimus from Mexico using morphological data and molecular barcodes.

Paragonimus mexicanus is the causal agent of human paragonimiasis in several countries of the Americas. It is considered to be the only species of the genus present in Mexico, where it is responsible for human infection. Through the investigation of P. mexicanus specimens from several places throughout Mexico, we provide morphological, molecular and geographical evidence that strongly suggests the presence of at least three species from this genus in Mexico. These results raise questions regarding the diagnosis, treatment, prophylaxis and control of human paragonimiasis in Mexico. We also provide a brief discussion regarding biodiversity inventories and the convenience of providing molecular and morphological information in biodiversity studies.

Natural hybridization between Paragonimus bangkokensis and Paragonimus harinasutai.

Paragonimus bangkokensis and Paragonimus harinasutai, which are morphologically distinguishable species, often co-infect in the same crab intermediate hosts. Recent molecular phylogenetic analyses revealed that these two species are genetically close to each other and are considered as the sister species. While we have been studying Paragonimus adult worms obtained from the lungs of a cat experimentally infected with Paragonimus metacercariae which were morphologically identified as P. harinasutai collected from central Viet Nam, one out of 6 adult worms has grouped cuticular spines, which is a feature of P. bangkokensis. By molecular analyses, the CO1 sequence of this specimen was identical with that of P. bangkokensis, but the ITS2 and the D2 region of 28S rDNA sequences showed a two peak pattern. Then, PCR products of the ITS2 and the D2 region of 28S rDNA sequences were ligated to TOPO vector and subcloned to determine the heterozygosity. Two types of sequences were obtained from each ITS2 and D2 region of 28S; one was identical with P. harinasutai and the other with P. bangkokensis. Taking all these morphological and molecular data together, we identified this adult worm as a hybrid specimen of P. bangkokensis and P. harinasutai.

Paragonimus worm from a New Guinea native in 1926.

OBJECTIVE: To reobserve and research the specimen of Paragonimus worm found in the left lung of a New Guinea native in 1926, which was previously identified as Paragonimus westermani Kerbert or Paragonimus ringeri Cobbold. METHODS: Using reconstructive software and microscopy to observe some organs of the worm, and compared with other species of paragonimus. RESULTS: The three dimensional (3D) views of ovary and two testes of New Guinea specimen showed that the ovary was clearly divided into six lobes. These two testes were situated oppositely in the body. One teste was divided into four branches, while another was divided into five. The cuticular spines were arranged in groups over the entire skin covered in a slide, each group was consisted of two to four single spine. CONCLUSIONS: Based on 3D views and measurements, we reclassified it as Paragonimus siamensis. This was also the first report of human case infected by Paragonimus siamensis.

Indochinamon ou (Crustacea: Potamidae) as a new second intermediate host for Paragonimus harinasutai in Luang Prabang Province, Lao PDR.

Paragonimus harinasutai metacercariae were found in a species of freshwater crab, Indochinamon ou, collected in a small stream of Namback District, Luang Prabang Province, Lao PDR. Adult flukes were recovered after experimental infection of the metacercariae to dogs. Metacercariae were round or slightly elliptical, 0.666 x 0.626 mm in average size, and had a thin cyst wall of about 20 microm in thickness, a black excretory bladder, convoluted ceca, and some pinkish materials in the body. Adults were somewhat elongated, 95.2 x 36.5 mm in average size, covered with single-tipped tegumental spines, had a smaller oral sucker than the ventral sucker, a moderately branched ovary, and 5-6 lobulated testes. Eggs were ovoid and bilaterally symmetrical in shape, 79 x 45 microm in average size, and had a uniformly thickened shell. By the present study, it has been confirmed that I. ou is a new second intermediate host for P. harinasutai.

Morphological differences and molecular similarities between Paragonimus bangkokensis and P. harinasutai.

Paragonimus bangkokensis and Paragonimus harinasutai were found one after another in the same crab host, Potamon smithianus, in Thailand in 1967 and 1968. Both species were also recently found in China and Lao People's Democratic Republic (PDR). Those two Paragonimus spp. are distinguishable from each other by morphological features of metacercariae and adults. However, recently, the DNA sequences of second internal transcribed spacer region (ITS2) and cytochrome oxidase subunit 1 (CO1) genes of those two species in Thailand were reported to be highly similar to each other. In the present study, we collected P. bangkokensis in two provinces in Vietnam (the first record in Vietnam) and both P. bangkokensis and P. harinasutai in Lao PDR for the morphological and molecular phylogenetic analyses to clarify the mutual relationship between the two species. The results show that P. bangkokensis and P. harinasutai were distinguishable from each other by morphology such as the size of metacercariae and the arrangement of cuticular spines of adult worms. However, the molecular phylogenetic analyses of ITS2 and CO1 genes clearly indicate that P. bangkokensis and P. harinasutai make a monophyletic group.

[Distribution of Paragonimus (Digenea: Troglotrematidae) in Antioquia Province, Colombia, based on metacercariae counts in freshwater crabs]. / Distribución parcial de Paragonimus (Digenea: Troglotrematidae) en Antioquia, por presencia de metacercarias en cangrejos dulciacuícolas.

INTRODUCTION: Paragonimosis or lung fluke disease courses with signs similar to those seen in tuberculosis. The causative agent is a parasite of the genus Paragonimus (Digenea: Troglotrematidae). People become infected by ingesting raw or partially cooked crabs containing metacercariae. The first focus of human paragonimosis in Colombia was recorded in the county of Urrao, where two species of crabs infected with Paragonimus were found. In 2005, crabs with Paragonimus' metacercariae were captured near Medellín, western Colombia. This prompted a search for the parasite in other locations through its presence in the crabs. OBJECTIVE: To establish the distribution of Paragonimus in Antioquia, we evaluated the presence of metacercaria in freshwater brachyuran crabs. MATERIALS AND METHODS: From 2005 to 2007, crabs were captured in 13 counties of Antioquia. The crabs were relaxed and dissected to determine presence of trematodes and then to make the taxonomic identifications. RESULTS: From 52 crabs captured in 9 counties, 42 (80.8%) were found with Paragonimus metacercariae. The crabs were identified as Pseudothelphusidae in 2 genera--Hypolobocera and Strengeriana--and were assigned to four species. Three of the species were recorded for the first time as hosts of Paragonimus. CONCLUSIONS: A Paragonimus' distribution map was constructed for Antioquia; for the first time urban zones were included. Because of the high rate of infection, the handling and consumption of raw and poorly cooked crabs pose risk factors for human infection. Because crabs are affordable and provide means of easy diagnosis, crabs are targeted as primary agents of and diagnostic tools for paragonmosis.

[Persistence of a paragonimosis focus in the department of Lakota, Côte d'Ivoire (West Africa)]. / Persistance d'un foyer de paragonimose dans le département de Lakota, Côte d'Ivoire (Afrique de l'Ouest).

A field survey was carried out from March to August 2006 in three villages around the town of Lakota (Côte d'Ivoire) to verify the presence of paragonimosis in local people, wild vertebrates, and crabs. Out of the 92 patients who were recruited because of their chronic cough, haemoptysis and/or epilepsy, 3 had Paragonimus eggs in their stools and/or sputa. Examination of stools belonging to 24 wild mammals and a reptile revealed the presence of eggs in three civets (Viverra civetta) and a mongoose (Crossarchus obscurus). Six local crabs (out of the 30 Liberonautes latidactylus dissected) harboured Paragonimus metacercariae having low diameters (299 to 315 pm). The presence of several paragonimid species (at least 2) in the district of Lakota was hypothesized. However, the existence of quantitative variations in metacercarial diameters for the same species of Paragonimus cannot be excluded.

Description of a new lung fluke species, Paragonimus vietnamensis sp. nov. (Trematoda, Paragonimidae), found in northern Vietnam.

Based on morphology of metacercariae and adult worms together with molecular data from our previous study, we describe herein a new lung fluke species named Paragonimus vietnamensis sp. nov. Metacercariae of this new species is round and extremely large in size (nearly 800 mum in diameter) having a fragile outer cyst of variable thickness and a thin fragile inner cyst. There are little or no space between metacercaria and the inner cyst wall. These and other morphological features of metacercariae of P. vietnamensis sp. nov. are not completely identical with but have some similarities with those of P. microrchis, P. proliferus, or P. menglaensis. On the other hand, adult worms obtained by experimental infection of these large metacercariae are oval in shape, having a ventral sucker slightly larger than the oral one, and having singly arranged relatively short cuticular spines. These morphological features of adult worms are partly similar to but not identical with those of P. microrchis, P. skrjabini, P. yunnanenis, P. xiangshanensis, or P. harinasutai. Taken these morphological data and our previous molecular phylogenetic analyses of ITS2 and CO1 sequences of metacercariae and adult worms together, P. vietnamensis sp. nov. is different from any other known Paragonimus spp.

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.

A new species of Paragonimus (Trematoda: Troglotrematidae) from a cat infected with metacercariae from mountain crabs Larnaudia larnaudii.

The identification of Paragonimus spp. lung flukes is based on the morphology of both the metacercaria and the adult. A very small Paragonimus sp. metacercaria was found in a freshwater crab caught in Kanchanaburi Province, West Thailand, an area where metacercariae of Paragonimus heterotremus had not been found. The metacercariae cysts were 180-204 microm in diameter, which was smaller than metacercariae of P. heterotremus. The coefficient of difference in body size between this metacercaria and P. heterotremus was 1.69, which was greater than a subspecific difference. Adults recovered from the lungs of a cat fed with the metacercariae were morphologically similar to, but smaller than, P. heterotremus dissected from the lungs of a feline experimental host. The tegumental spines of the worm in this study were singly spaced in arrangement, which is similar to, but larger than, the spines of P. heterotremus. Therefore, Paragonimus pseudoheterotremus is proposed as a new species.

Paragonimus skrjabini Chen, 1959 (Digenea: Paragonimidae) and related species in eastern Asia: a combined molecular and morphological approach to identification and taxonomy.

A molecular and morphometric investigation is reported on the species complex of mammalian lungflukes of which Paragonimus skrjabini Chen, 1959 and P. miyazakii Kamo, Nishida, Hatsushika & Tomimura, 1961 are the best-known examples. This species complex (here called the P. skrjabini complex) is shown to be monophyletic using DNA sequences from the nuclear ITS2 region and the mitochondrial cox1 gene. The latter marker permits the discrimination of populations, some previously named as distinct species, from various geographical locations in China and Japan. Morphometric analysis of a number of variables accords remarkably closely with the molecular results. Main findings are that (1) nominal P.skrjabini from Fujian Province in eastern China is phylogenetically very close to P. miyazakii from Japan. It is proposed that both taxa should be referred to the same subspecies as P. krjabini miyazakii. (2) Populations from Guangdong Province, Guangxi Zhuang Autonomous Region, Sichuan and Hubei should be referred to P. skrjabini skrjabini. Specimens of P. skrjabini from the Yangtze basin in Sichuan and Hubei are remarkably similar to one another according to genetic and morphometric data, but are not so different from Guangdong populations that they should be referred to a different subspecies at this stage. Specimens of P. skrjabini from Yunnan have not been assigned to a subspecies at this stage. (3) Partly following earlier studies, we regard the following as synonyms of P. skrjabini: P. miyazakii (reduced to subspecific status); P. szechuanensis Chung & Tsao, 1962 (probably belongs to P. s. skrjabini); and P. hueitungensis Chung, Hsu, Ho, Kao, Shao, Chiu, Pi, Liu, Ouyang, Shen, Yi & Yao, 1975 (probably belongs to P.s.skrjabini ); P. veocularis (Chen & Li, 1979) (specimens from Fujian regarded as belonging to this species should be referred to P.skrjabini miyazakii: the subspecific status of specimens from the type-locality in northern Sichuan is unclear). A number of questions remain unresolved. The name P. hokuoensis Ho & Chung, 1964 was proposed for two individual metacercariae of distinctive appearance from southern Yunnan. DNA sequences from very similar metacercariae from the same locality place this nominal species within, or sister to, the P. skrjabini complex. As yet, nothing is known regarding adult morphology or biology of this taxon and we retain it here as a distinct species. P. heterorchis (Zhou, Pang & Hsiang, 1982) might be a synonym of P. skrjabini: the form of the metacercaria provides evidence against this view and further work is required. P. macrorchis Chen, 1962 has probably been confused with P. skrjabiniin China. Within China, the former probably occurs only on Hainan Island, although P. fukienensis Tang & Tang, 1962 from Fujian Province could be a synonym.

Paragonimus ohirai metacercariae in crabs collected along the Arakawa River in Tokyo, Japan.

Brackish water crabs infected with Paragonimus ohirai metacercariae have been reported in various regions in Japan. However, infected crabs have not been identified in Tokyo. We therefore collected the crab, Chiromantes dehaani, between August 2002 and July 2003 from 12 locations along the Arakawa River that flows through Tokyo. Of the 922 captured crabs, 177 (19%) from 6 locations were infected with Paragonimus metacercariae. The prevalence of metacercariae at these 6 locations ranged from 5 to 89%. The number of metacercariae per infected crab ranged from 1 to 190, with an average of 13.1. The morphological features of the metacercariae and of adult worms recovered from test rats infected with metacercariae showed that the metacercariae in the infected crabs were P. ohirai Miyazaki, 1939. The ITS2 sequence data support this conclusion. This paper is the first description of P. ohirai infection of crabs in Tokyo.

[Studies on Paragonimus proliferus].

OBJECTIVE: To make identification between Paragonimus menglaensis and Paragonimus proliferus. METHODS: Crabs were collected from same area where P. proliferus and P. menglaensi were reported, metacercariae and excysted metacercariae were separated. Adult worms were collected from experimental infection and identified. RESULTS: The metacercaria is large, with an average size of (1.23 +/- 0.087) mm x (1.10 +/- 0.073) mm, covered with a thin and fragile cyst wall; the size of excysted metacercariae is (2.01 +/- 0.71) mm x (0.62 +/- 0.12) mm, with irregular bough-like wrinkles excretory bladder resembling in front of ventral sucker, two pointed and slim distal ends of gut locate at 1/6 of the body from the tail end; the adult worm has large uterine mass, with an average length of 1/4.2 of the whole body. The natural definitive host for P. proliferus is not monkeys, dogs, and cats, but rats. The metacercaria of the reported P. menglaensis has been mixed up with that of P. microrchis from the same crab, excysted metacercaria has been same to that of P. proliferus, and an immature worm has been mistakenly identified as its adult worm. CONCLUSION: P. proliferu is a valid independent species, while P. menglaensis is a mis-identified, invalid one.

Morphological description and life cycle of Paragonimus sp. (Trematoda: Troglotrematidae): causal agent of human paragonimiasis in Colombia.

The first morphological description is made of all stages of the life cycle of a Paragonimus species infecting humans in Colombia. Larval stages were obtained both in vitro and from field collections. Adult Paragonimus spp. are described. The aquatic snail Aroapyrgus sp. serves as an intermediate host of this species, both naturally and experimentally, yielding rediae and cercariae. Crabs (Hypolobocera bouvieri monticola and H. emberarum) were found to be the natural second intermediate hosts, and individuals of another crab species (Strengeria sp.) were also infected in the laboratory.

Occurrence of the lung fluke, Paragonimus heterotremus in Manipur, India.

OBJECTIVES: To determine the natural crustacean host, the Paragonimus species, and to investigate the potential host-parasite relationship between Manipur Paragonimus and some of the laboratory animals. METHODS: The laboratory animals such as puppies, albino rats and Swiss mice were infected orally with metacercariae isolated from the fresh water crabs, Potamiscus manipurensis. The fecal specimens of the experimentally infected animals were examined microscopically for Paragonimus eggs at regular intervals. The animals were autopsied on days 35 to approximately 328 after infection and the isolated worms were flattened between glass slides and fixed in 70% alcohol. The worms were stained with carmine and mounted with Canada balsam for morphological studies. The eggs were collected in 5% formol saline solution for microscopy. The flukes were classified into 4 developmental stages. RESULTS: A total of 11 worms, 5 mature, 5 immature and 1 pre-adult were recovered. The morphological features of the metacercariae, worms and eggs were similar to those of Paragonimus heterotremus. CONCLUSION: Manipur is one of the rare areas in the world where Paragonimus heterotremus is prevalent and the puppies are ideal experimental animal host. This species may be one of the important causes of paragonimiasis in animals and humans in Manipur.

Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae.

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.

Occurrence of a diploid type and a new first intermediate host of a human lung fluke, Paragonimus westermani, in Korea.

The biology, chromosome number, and karyotype of a lung fluke, Paragonimus westermani (Kerbert, 1878) collected in Haenam, Haenam-gun Chollanam-do, Korea were analyzed. We compared the size of metacercariae from Haenam with those taken from a crayfish collected at Youngam, Youngam-gun, Chollanam-do, Korea. The mean length of P. westermani metacercariae from Haenam was 300.3 microm and that from Youngam was 362.0 microm. Adult worms were recovered from the lungs of experimentally infected dogs. The mean egg sizes obtained from adult flukes were 72.1 x 46.8 microm from Haenam and 93.5 x 54.2 microm from Youngam. Semisulcospira tegulata collected in the Youngam area were found to be infected with cercariae of P. westermani, one of the snail-borne human lung fluke trematodes in Korea. Of 4218 snails studied, 5 (0.12%) harbored P. westrermani larvae. This is the first report of S. tegulata serving as the initial intermediate host of P. westermani. The chromosome numbers of P. westermani from Haenam and Youngam were 2n = 22 and 3n = 33. The diploid type of P. westermani has not been previously reported in Korea.