Infection with Paragonimus westermani of boar-hunting dogs in Western Japan maintained via artificial feeding with wild boar meat by hunters.

Infection of boar-hunting dogs with Paragonimus westermani was investigated in Western Japan. Blood and rectal feces were collected from 441 dogs in the three districts (205 in Kinki, 131 in Chugoku and 105 in Shikoku District). In a screening ELISA for serum antibody against P. westermani antigen, 195 dogs (44.2%) showed positive reaction. In the 195 dogs, 8 dogs were found excreting P. westermani eggs after molecular analysis of fecal eggs, and additional 7 were identified serologically for the parasite infection because of their stronger reactivity against P. westermani antigen than against antigens of other species of Paragonimus. A spatial analysis showed that all of the P. westermani infections were found in Kinki and Chugoku Districts. In this area, dogs' experience of being fed with raw boar meat showed high odds ratio (3.35) to the sero-positivity in the screening ELISA, and the frequency of such experiences was significantly higher in sero-positive dogs. While clear relationship was not obtained between predation of boars by dogs during hunting and their sero-positivity. Therefore, it is suggested that human activity of feeding with wild boar meat is the risk factor for P. westermani infection in boar-hunting dogs. Considering that hunting dogs could play as a major definitive host and maintain the present distribution of P. westermani in Western Japan, control measures for the infection in hunting dogs, such as prohibition of raw meat feeding and regular deworming, should be undertaken.

Depressor effect of chymase inhibitor in mice with high salt-induced moderate hypertension.

The aim of the present study was to determine whether long-term high salt intake in the drinking water induces hypertension in wild-type (WT) mice and whether a chymase inhibitor or other antihypertensive drugs could reverse the increase of blood pressure. Eight-week-old male WT mice were supplied with drinking water containing 2% salt for 12 wk (high-salt group) or high-salt drinking water plus an oral chymase inhibitor (TPC-806) at four different doses (25, 50, 75, or 100 mg/kg), captopril (75 mg/kg), losartan (100 mg/kg), hydrochlorothiazide (3 mg/kg), eplerenone (200 mg/kg), or amlodipine (6 mg/kg). Control groups were given normal water with or without the chymase inhibitor. Blood pressure and heart rate gradually showed a significant increase in the high-salt group, whereas a dose-dependent depressor effect of the chymase inhibitor was observed. There was also partial improvement of hypertension in the losartan- and eplerenone-treated groups but not in the captopril-, hydrochlorothiazide-, and amlodipine-treated groups. A high salt load significantly increased chymase-dependent ANG II-forming activity in the alimentary tract. In addition, the relative contribution of chymase to ANG II formation, but not actual average activity, showed a significant increase in skin and skeletal muscle, whereas angiotensin-converting enzyme-dependent ANG II-forming activity and its relative contribution were reduced by high salt intake. Plasma and urinary renin-angiotensin system components were significantly increased in the high-salt group but were significantly suppressed in the chymase inhibitor-treated group. In conclusion, 2% salt water drinking for 12 wk caused moderate hypertension and activated the renin-angiotensin system in WT mice. A chymase inhibitor suppressed both the elevation of blood pressure and heart rate, indicating a definite involvement of chymase in salt-sensitive hypertension.

Evaluation of the MGL method to detect Paragonimus eggs and its improvement.

Dog feces containing 500 Paragonimus westermani eggs per gram were examined by the Medical General Laboratory (MGL), the simple sedimentation (SS), and the Army Medical School III (AMS III) methods. The number of eggs per gram of feces (EPG) obtained by the MGL method was 17.2 and was significantly lower than those obtained by the SS method (324.0) and the AMS III method (505.6). When isolated P. westermani eggs were processed by the MGL method and four layers (ether, ether-fecal, formalin layers, and sediment) of the final centrifugation product were separately examined, almost 100% of eggs were found at the ether-fecal layer. Similarly, when fecal samples containing P. westermani, Paragonimus skrjabini miyazakii, Paragonimus ohirai, or Paragonimus harinasutai eggs were processed by the MGL method, more than 95% of the eggs were found in the supernatant layers. The formalin-ethyl acetate (FEA) method showed a similar tendency as the MGL method and over 90% of eggs remained in the supernatant layers. Contrary to Paragonimus eggs, 63 and 96% of Clonorchis and Metagonimus eggs were found in the sediment in the MGL method, respectively. When surfactant (Tween 80) was added to fecal solution, most of Paragonimus eggs spun down in the sediment in the MGL and FEA methods, suggesting that Paragonimus eggs have hydrophobic components on their surface. It is suggested that surfactant addition to the fecal solution should be considered when the MGL method is used for detection of Paragonimus eggs.

Paragonimus paishuihoensis metacercariae in freshwater crabs, Potamon lipkei, in Vientiane Province, Lao PDR.

Among Paragonimus species, P. paishuihoensis is one of the most mysterious and poorly understood species. Metacercariae are characterized by having a unique dendritically branched excretory bladder. However, the morphology of the adult worm remains unknown. To date, metacercariae of this species have been reported only in China and Thailand. In this study, we first found P. paishuihoensis metacercariae in freshwater crabs, Potamon lipkei, in Hinheub District, Vientiane, Lao PDR, with a prevalence of 77.7% and the average intensity of 10.3 (range 1-28) metacercariae per crab. The molecular data based on ITS2 and CO1 markers indicated that P. paishuihoensis from Laos and Thailand were almost completely identical and were close to members of the Paragonimus bangkokensis/Paragonimus harinasutai complex. Attempts to infect experimental animals (cats, dogs, and rats) with P. paishuihoensis were unsuccessful, suggesting that these animals might be unsuitable definitive hosts for the species. Further studies are necessary to elucidate the taxonomic status and life cycle of P. paishuihoensis.

Rapid identification of paragonimiasis foci by lay informants in Lao People's Democratic Republic.

BACKGROUND: Paragonimiasis is a food-borne trematodiasis leading to lung disease. Worldwide, an estimated 21 million people are infected. Foci of ongoing transmission remain often unnoticed. We evaluated a simple questionnaire approach using lay-informants at the village level to identify paragonimiasis foci and suspected paragonimiasis cases. METHODOLOGY/PRINCIPAL FINDINGS: The study was carried out in an endemic area of Lao People's Democratic Republic. Leaders of 49 remote villages in northern Vientiane Province were asked to notify suspected paragonimiasis patients using a four-item questionnaire sent through administrative channels: persons responding positively for having chronic cough (more than 3 weeks) and/or blood in sputum with or without fever. We validated the village leaders' reports in ten representative villages with a door-to-door survey. We examined three sputa of suspected patients for the presence of Paragonimus eggs and acid fast bacilli. 91.8% of village leaders participated and notified a total of 220 suspected patients; 76.2% were eventually confirmed; an additional 138 suspected cases were found in the survey. Sensitivity of village leaders' notice for "chronic cough" and "blood in sputum" was 100%; "blood in sputum" alone reached a sensitivity of 85.7%. SIGNIFICANCE: Our approach led to the identification of three previously unknown foci of transmission. A rapid and simple lay-informant questionnaire approach is a promising low-cost community diagnostic tool of paragonimiasis control programs.

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.

Discovery of Paragonimus westermani in Vietnam and its molecular phylogenetic status in P. westermani complex.

Paragonimus westermani is the most well-known species among the genus Paragonimus. It is widely distributed in Asia with considerable genetic diversity to form P. westermani species complex. While P. westermani distributed in Japan, Korea, China, and Taiwan are genetically homogeneous to form the East Asia group, those found in other geographic areas are heterogeneous and would be divided into several groups. Recent discoveries of P. westermani in India and Sri Lanka highlighted new insights on molecular phylogenetic relationship of geographic isolates of this species complex. Since Vietnam is located at the east end of Southeast Asia, the intermediate position between South and East Asia, it is of interest to see whether P. westermani is distributed in this country. Here, we report that P. westermani metacercariae were found in mountainous crabs, Potamiscus sp., collected in Quangtri province in the central Vietnam. Adult worms were successfully obtained by experimental infection in cats. Molecular phylogenetic analyses revealed that P. westermani of Vietnamese isolates have high similarities with those of East Asia group.

Discovery of Paragonimus proliferus in Northern Vietnam and their molecular phylogenetic status among genus Paragonimus.

During an epidemiological survey for Paragonimus and paragonimosis in northern Vietnam, we found extremely large excysted metacercariae (2.50 +/- 0.14 mm in length and 0.72 +/- 0.08 mm in width; mean +/- standard deviation of 20 samples) in mountainous crabs, Potamiscus mieni. Adult worms were successfully obtained by intraperitoneal injection with those large excysted metacercariae in a cat. Morphological and morphometric data of those large excysted metacercariae and the adult worms derived from them are identical to those of Paragonimus proliferus found in Yunnan province, China. However, when second internal transcribed spacer region and cytochrome oxidase subunit 1 gene sequences of those metacercariae and adult worms were compared with those of known Paragonimus spp. deposited in the GenBank, they were almost completely identical to those of Paragonimus hokuoensis metacercariae in China, of which adult worms have never been reported. This is the first record of P. proliferus in Vietnam and the first record from outside of China. Phylogenetic relationship between P. proliferus and P. hokuoensis is discussed.

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.

Molecular cloning and analysis of stage and tissue-specific expression of cathepsin B encoding genes from Fasciola gigantica.

The transcriptional products of Fasciola gigantica genes encoding cathepsin B proteases were cloned from adult, newly excysted juvenile (NEJ), and metacercarial stages. The obtained cDNAs were named FG cat-B1, FG cat-B2, and FG cat-B3. The deduced amino acid sequences of the encoded proteases have identities ranging from 64 to 79%. Sequence comparison with homologous proteins showed that all functional important residues formerly described for cathepsin B are conserved. Southern analysis confirmed the presence of a family of related cathepsin B genes in the genome of F. gigantica. Northern analysis revealed a common transcript size of 1400 nucleotides with abundant cathepsin B transcripts detected in metacercarial and NEJ stages. Cathepsin B transcripts were located by RNA in situ hybridization in the caecal epithelial cells, in cells underlining the proximal part of the digestive tract, and in the tegumental cells underlining the surface tegument. Furthermore, transcripts were detected in the tissues of the reproductive system including cells of prostate, Mehlis, and vitelline glands, testis, and eggs. Stage-specific gene expression was investigated by RT-PCR using gene-specific primers and hybridization with a labeled cathepsin B probe. FG cat-B1 transcripts were detected in all stages, whereas FG cat-B2 and FG cat-B3 transcripts were expressed in metacercariae, NEJ, and juvenile parasites only. The switching off of the cat-B2 and cat-B3 genes during the maturation of the parasites implicates that these enzymes may be involved in digesting host tissues during penetration and migration to the liver, whereas cat-B1 present in all stages may perform general digestive function.