Local outbreak of spontaneous pneumothorax secondary to paragonimosis in southwestern Ontario dogs.

Three dogs were diagnosed with spontaneous pneumothorax and referred to the Ontario Veterinary College Health Sciences Centre for management. The 3 dogs were diagnosed with secondary spontaneous pneumothorax due to paragonimosis. The diagnosis was made by visualization of adult trematodes during surgical exploration with histopathological confirmation in 1 dog, and detection of trematode eggs via fecal sedimentation in the other 2 dogs. Two of the dogs developed unusual additional lesions, including hemoabdomen, muscle abscess, and abdominal adhesions. These were suspected to be secondary to aberrant fluke larval migration. All 3 dogs lived within a relatively small geographical area of Ontario and were hospitalized between December 2021 and March 2022. Each dog survived to discharge with surgical or medical management of the pneumothorax and treatment with a prolonged course of fenbendazole. Key clinical message: Paragonimosis should be considered as a differential diagnosis for canine spontaneous pneumothorax in areas where Paragonimus kellicotti is, or may be, endemic, or in dogs that have travelled to endemic areas - particularly if the patient has a history of cough or potential exposure to freshwater crayfish. Routine anthelmintic treatment does not prevent infection and standard fecal floatation methods may not detect the eggs. Therefore, diagnostic testing should include a fecal sedimentation test and thoracic radiographs to screen for P. kellicotti.

Éclosion locale de pneumothorax spontané secondaire à la paragonimose chez des chiens du sud-ouest de l'Ontario. Trois chiens ont reçu un diagnostic de pneumothorax spontané et ont été référés au Ontario Veterinary College Health Sciences Centre pour prise en charge. Les 3 chiens ont été diagnostiqués avec un pneumothorax spontané secondaire dû à la paragonimose. Le diagnostic a été posé par la visualisation des trématodes adultes lors de l'exploration chirurgicale avec confirmation histopathologique chez 1 chien, et la détection des oeufs de trématodes par sédimentation fécale chez les 2 autres chiens. Deux des chiens ont développé des lésions supplémentaires inhabituelles, notamment un hémoabdomen, un abcès musculaire et des adhérences abdominales. Ces lésions étaient soupçonnées d'être secondaires à une migration larvaire aberrante de la douve. Les 3 chiens vivaient dans une zone géographique relativement restreinte de l'Ontario et ont été hospitalisés entre décembre 2021 et mars 2022. Chaque chien a survécu jusqu'à sa sortie avec une prise en charge chirurgicale ou médicale du pneumothorax et un traitement avec un traitement prolongé au fenbendazole.Message clinique clé :La paragonimose doit être considérée comme un diagnostic différentiel du pneumothorax spontané canin dans les zones où Paragonimus kellicotti est, ou peut être, endémique, ou chez les chiens qui ont voyagé dans des zones endémiques ­ en particulier si le patient a des antécédents de toux ou une exposition potentielle aux écrevisses d'eau douce. Le traitement vermifuge de routine n'empêche pas l'infection et les méthodes de flottaison fécale standard peuvent ne pas détecter les oeufs. Par conséquent, les tests de diagnostic doivent inclure un test de sédimentation fécale et des radiographies thoraciques pour dépister P. kellicotti.(Traduit par Dr Serge Messier).

A case of a dog with paragonimiasis after consumption of raw deer meat.

A 6-year-old castrated male Cavalier King Charles Spaniel was referred to the Animal Medical Center, Tokyo University of Agriculture and Technology, for examination and treatment of recurrent pneumothorax. Chest radiography and computed tomography showed multiple cavitary lesions in the caudal right posterior lobe. These lesions were surgically excised via thoracotomy. Subsequent histopathological examination revealed paragonimiasis. In the postoperative review, we found that the owner had fed raw deer meat to the dog four months earlier. Deer meat has attracted attention as a source of Paragonimus in humans. To our knowledge, this is the first report of Paragonimus infection in a dog due to deer meat consumption.

Sporadic endemicity of zoonotic Paragonimus in raccoon dogs and Japanese badgers from Miyazaki Prefecture, Japan.

Paragonimiasis is a zoonotic trematode infection caused by Paragonimus spp. To determine the recent status of Paragonimus infections in wild animals, this study investigated Paragonimus spp. in 39 raccoon dogs and 54 Japanese badgers from March 2019 to January 2021 in Miyazaki Prefecture, and examined metacercariae in freshwater crabs. Triploid P. westermani was found in one raccoon dog (2.6%), and metacercariae were recovered from Eriocheir japonica captured near the infected animal collected. One Japanese badger (1.9%) harbored P. skrjabini miyazakii; this prevalence was lower than the approximately 30% that was reported in the 1970s. Results indicated that zoonotic Paragonimus was sporadically prevalent in wild animals. Further investigation in various animals is awaited to elucidate current wildlife reservoirs for those Paragonimus.

A record of juvenile Paragonimus skrjabini miyazakii from the urinary bladder of the Japanese toad, Bufo japonicus formosus.

We found juveniles of Paragonimus in the urinary bladder of a Japanese toad (Bufo japonicus formosus) captured in Kyoto. These were molecularly identified as Paragonimus skrjabini miyazakii Kamo, Nishida, Hatsushika et Tomimura 1961. This is the first report of P. s. miyazakii found in anuran hosts in Japan, indicating that anurans can be paratenic hosts of P. s. miyazakii, as is also the case for Paragonimus skrjabini skrjabini in China. This finding suggests that definitive hosts of P. s. miyazakii can be infected by eating not only crabs or mammal paratenic hosts, but also anurans.

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.

Distribution of Pseudorabies Virus Antigen in Hunting Dogs with Concurrent Paragonimus westermani Infection.

Following isolation of pseudorabies virus (PRV) from two hunting dogs in Oita prefecture, Japan, we investigated the PRV antigen distribution in the tissues of the infected animals. At necropsy, PRV-associated lesions included facial oedema, tonsillar and meningeal congestion, blotchy haemorrhages on the pericardium and mitral valves, and incomplete splenic contraction in one dog, with less prominent findings in the other dog. Multiple pulmonary nodules were seen in both cases, caused by the diploid form of Paragonimus westermani lung flukes, as confirmed by PCR-restriction fragment length polymorphism and gene sequencing analyses. Histological examination revealed that the PRV infection was associated with lesions of non-suppurative encephalitis in the brainstem. PRV antigen was detected in the cerebrum, cerebellum, brainstem, submucosal and myenteric plexuses, and mononuclear cells, mainly in the bone marrow, lymph nodes, tonsils and spleen tissues. There was evidence of PRV dissemination to the brain via the trigeminal or olfactory routes, in addition to possible spread to lymphoid organs via infected mononuclear cells.

First report of Paragonimus skrjabini miyazakii metacercariae in Geothelphusa dehaani (Sawagani) occurring in Iwate Prefecture, Japan.

Paragonimiasis is an important food-borne zoonosis caused by Paragonimus flukes and is endemic to western Japan. However, there have been few epidemiological studies in the Tohoku district of northeastern Japan. In this study, Paragonimus metacercariae (mc) was detected in Geothelphusa dehaani (Japanese freshwater crab or Sawagani) in Iwate Prefecture. Out of the 207 Sawagani collected from 35 localities, 12 individuals from six localities were infected with Paragonimus mc. The mc were identified as P. skrjabini miyazakii based on the sequences of nuclear ribosomal internal transcribed spacer 2 and mitochondrial cytochrome c oxidase I. This is the first report of P. s. miyazakii mc infection in Sawagani in Iwate Prefecture.

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.

[Investigation on epidemic foci of paragonimiasis in eastern part of Zhenghe County, Fujian Province].

OBJECTIVE: To investigate the species and hosts of Paragonimus and its infection rate in eastern part of Zhenghe County, Fujian Province, so as to determine the local foci of Paragonimus. METHODS: The snails, crabs and stools of wild cats were collected for the examinations of cercariae, metacercariae and eggs of Paragonimus. The geographical and environmental conditions of the areas were also investigated. RESULTS: A total of 4 890 Pseudobythinella jianouensis snails and 1 035 Semisulcospira liberlina snails were examined, and the cercariae of Paragonimus were only found in P. jianouensis, with an infection rate of 0.10% (5/4 890). Bottapotamon zhengheensis sp. nov. as the second intermediate host of P. skrjabini, were examined, and the infection rate was 85.29% (29/34) and the average numbers of metacercariae per crab and per gram of crab tissues were 3.85 and 0.62, respectively. Thirty-six Sinopotamun fujianensis crabs, as the second intermediate host of P. westermani, were examined, and the infection rate was 38.89% (14/36) and the average numbers of metacercariae per crab and per gram of crab tissues were 6.43 and 0.03, respectively. The eggs of Paragonimus were detected in 1 of 2 muck specimens of wild cats. CONCLUSIONS: The data suggest that there is a focus of middle-to-high level of infection caused by P. westermani and P. skrjabini in the eastern part of Zhenghe County.

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.

Paragonimosis in a cat and the temporal progression of pulmonary radiographic lesions following treatment.

A 16 mo old cat presented with a 5 mo history of dyspnea, coughing, and gagging. Radiographic findings revealed seven nodules measuring 1-3 cm distributed multifocally in the lungs. Examination of feces revealed large numbers of eggs of Paragonimus kellicotti. Two fenbendazole treatment regimens (28 mg/kg per os q 12 hr for 21 days) and prednisone were required to eliminate the infection. Resolution of pulmonary nodules was monitored for 8 mo following successful treatment, and four lesions were still partially visible at 8 mo.

Paragonimiasis in KwaZulu-Natal Province, South Africa.

Nine cases of paragonimiasis have been reported from cats (4), dogs (2) and children (3) in South Africa, with an additional suspected case in an adult female patient. Details of these cases are reviewed. All nine cases, and perhaps the adult case as well, were from the province of KwaZulu-Natal but locality data are only available for six of them. These six cases represent four localities which all lie below 100 m above sea level in the province's lowlands, suggesting that there may be a focus of transmission here. The molluscan first intermediate host must be one of the two prosobranch snail species present in the area, Melanoides tuberculata or Tomichia natalensis, and the decapod second intermediate host the common river crab Potamonautes sidneyi. All infected cats and dogs had pulmonary infections, while two human cases for which there is sufficient information had extrapulmonary infections. Transmission appears to be ongoing but the invasive snail Tarebia granifera may be competing with both M. tuberculata and T. natalensis. If so, this may bring transmission to an end.

Application of a real-time fluorescence resonance energy transfer polymerase chain reaction assay with melting curve analysis for the detection of Paragonimus heterotremus eggs in the feces of experimentally infected cats.

Paragonimus heterotremus is a medically important lung fluke that causes human and animal paragonimiasis in Southeast Asia, including Thailand. In the current study, a real-time fluorescence resonance energy transfer polymerase chain reaction (real-time FRET PCR) with melting curve analysis was developed and evaluated to detect P. heterotremus eggs in the feces of experimentally infected cats. The detection limit of this method for the P. heterotremus DNA sequence was 3 × 10(2) copies of the positive control plasmid and 10(-3) ng of P. heterotremus genomic DNA. The assay system could detect 10 eggs of P. heterotremus per gram of cat feces. No fluorescence signal was observed when DNA purified from 16 other organisms or genomic DNA from cats and human beings were tested. Real-time FRET PCR yielded positive results for all fecal samples from 17 P. heterotremus-infected cats and showed a negative relationship (r = -0.852, P < 0.001) between the number of parasite eggs in feces and the number of PCR cycles. The assay could detect genomic DNA from P. heterotremus, P. westermani, P. macrorchis, P. siamensis, P. harinasutai, and P. bangkokensis and can differentiate P. heterotremus from the other 5 species. The 6 Paragonimus species examined were divided into 4 groups by melting peak analysis. This assay can be useful for the detection of, and epidemiological studies on, P. heterotremus infection in endemic areas.

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.

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.

Sensitive and rapid detection of Paragonimus westermani infection in humans and animals by loop-mediated isothermal amplification (LAMP).

In the present study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of Paragonimus westermani adults, metacercariae, and eggs in human and animal samples. The LAMP amplification can be finished in 45 min under isothermal condition at 60°C by employing a set of four species-specific primer mixtures and the results can be checked by naked-eye visualization. No amplification products were detected with deoxyribunucleic acid (DNA) of related trematode species including Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma mansoni, and Schistosoma japonicum. The method was further validated by examining P. westermani DNA in intermediate hosts including freshwater crabs and crayfish, as well as in sputum and pleural fluid samples from patients of paragonimiasis. These results indicated that the LAMP assay was highly specific, sensitive, and rapid, and it was approximately 100 times more sensitive than conventional specific PCR. The LAMP assay established in this study provides a rapid and sensitive tool for the detection of P. westermani DNA in freshwater crabs, crayfish, sputum, and pleural fluid samples, which has important implications for effective control of human paragonimiasis.