Efficacy assessment of miltefosine and curcumin against Clonorchis sinensis infection.

Praziquantel (PZQ) is currently the only approved drug for treating clonorchiasis, but its poor efficacy against Clonorchis sinensis larvae has highlighted the need to develop newer drugs. In this study, to address this challenge, we investigated the anti-parasitic efficacy of miltefosine (MLT), curcumin (CUR), and PZQ against C. sinensis metacercariae (CsMC), newly excysted juvenile worms (CsNEJs), and adults. Larvicidal effects of MLT and CUR surpassed those elicited by PZQ in vitro. These two drugs exerted their effect against both CsMC and CsNEJs in a dose- and time-dependent manner. To confirm the effect of these drugs in vivo, Syrian golden hamsters were orally infected with 100 CsMC and subsequently treated with MLT, CUR, or PZQ at 1 and 4 weeks post-infection (wpi). MLT and CUR reduced the worm recoveries at 1 and 4 wpi, indicating that these drugs were efficacious against both larvae and adult C. sinensis. PZQ was only efficacious against adult worms. Interestingly, both MLT and CUR showed lower levels of C. sinensis-specific IgG responses than the infection control group, implying that worm burden and bile IgG responses could be correlated. These results indicate that MLT and CUR are efficacious against both larval and adult stages of C. sinensis, thereby highlighting their potential for further development as alternative therapeutic options for clonorchiasis.

Pearl formation associated with gymnophallid metacercariae in Mytilus galloprovincialis from the Northwestern Adriatic coast: Preliminary observations.

Gymnophallids of the genus Parvatrema are small trematodes infecting waterbirds in their adult stage. Several species of clams and mussels have been found to act as first and second intermediate hosts, in which the trematode larval stages induce the formation of pearls. In this study, a wild population of Mytilus galloprovincialis was sampled along the Northwestern coast of the Adriatic Sea to evaluate the origin and extent of visible pearls. Parasitological investigations, including morphological and molecular analyses, and histopathology were carried out on a representative sample of mussels (n = 158) from June to September 2021. The overall prevalence of infection reached 75.3 %, and the intensity of infection ranged from a few trematodes to thousands per mussel, mostly occurring in the mantle and surrounded by variable numbers of conchiolin layers. Morphological studies allowed classification of the metacercariae as belonging to the genus Parvatrema, and the pairwise comparison of the obtained sequences, encompassing the internal transcribed spacer (ITS) region, revealed a genetic similarity (96.8 %) to Parvatrema duboisi. However, the phylogenetic analysis demonstrated the independent clustering of the obtained ITS sequences compared to other available Parvatrema species. For the relevant commercial impact that pearl formation may have on farmed mussels, ecological and epidemiological aspects of this infection would deserve further investigation in the area.

Survey of the Prosthogonimus spp. metacercariae infection in the second intermediate host dragonfly in Heilongjiang Province, China.

Prosthogonimiasis poses a threat to the reproductive system of poultry and wild birds, which are the definitive hosts of the parasite causing this disease. However, the parasite infection of the second intermediate host (dragonfly), the primary vector of this pathogen, is rarely reported. In this study, the prevalence of Prosthogonimus infection in dragonflies was investigated from June 2019 to October 2022 in Heilongjiang Province, northeast China. The species of metacercariae isolated from dragonfly were identified by morphological characteristics, molecular biology techniques, and animal infection experiments. The results showed that 11 species of dragonflies and one damselfly were identified and among six of the dragonflies infected by Prosthogonimus metacercariae, Sympetrum depressiusculum (28.53%) had the highest infection rate among all positive dragonflies, followed by Sympetrum vulgatum (27.86%) and Sympetrum frequens (20.99%), which are preferred hosts, and the total prevalence was 20.39% (2061/10,110) in Heilongjiang Province. Three species of Prosthogoniumus metacercariae were isolated, including Prosthogonimus cuneatus, Prosthogonimus pullucidus, and Prosthogonimus sp., among which P. cuneatus was the dominant species in dragonflies in Heilongjiang Province. This is the first report on the prevalence of Prosthogonimus in dragonflies in China, which provides baseline data for the control of prosthogonimiasis in Heilongjiang Province and a reference for the prevention of prosthogonimiasis in other areas of China.

Genetic characterization and phylogenetic relationships of Phyllodistomum parasites in Indian subcontinent: insights from freshwater fish and shrimp hosts.

Phyllodistomum is the large digenean group of fish parasites, with 25 species described so far in the Indian subcontinent. Here, we redescribed two adult species of Phyllodistomum (P. srivastava Rai 1964 and P. parorchium Jaiswal 1957) collected from freshwater fish Heteropneustes fossilis Bloch, 1974 and Glossogobius giuris Ham, 1822, respectively, and an unknown Phyllodistomum metacercaria from shrimp (Macrobrachium dayanum Henderson, 1893). These parasites were genetically characterized using 28S and first and second internal transcribed spacers (ITS1 and ITS2) regions of the nuclear ribosomal DNA and CoxI region of the mitochondrial (mt) DNA to establish the link between metacercaria and adult. Morphologically, both the unknown metacercaria in shrimp and adult Phyllodistomum srivastava in fish, resembled in terms of crenulated margin of hind body, arrangement of diagonal testes, bipartite seminal vesicle, and compact paired vitelline masses. The two adult parasite species, P. srivastava from P. parorchium, were different in terms of shape and size of the body, ratio of suckers, the absence of crenulated margin of hind body, a single chambered seminal vesicle, and deeply lobed paired vitelline masses, in the former species. Comparison of the 28S, ITS, and mtCoxI sequence data suggested P. srivastava and Phyllodistomum metacercaria belong to the same species, and supported the distinction between P. srivastava and P. parorchium. Exploring the potential impact of Phyllodistomum infection on host behaviour and health would be prospective areas for future research.

Description of a metacercaria of a zoogonid trematode Steganoderma cf. eamiqtrema Blend and Racz, 2020 (Microphalloidea: Zoogonidae), with notes on the phylogenetic position of the genus Steganoderma Stafford, 1904, and resurrection of the subfamily Lecithostaphylinae Odhner, 1911.

Metacercariae of the zoogonid trematode Steganoderma cf. eamiqtrema ex crab Chionoecetes bairdi caught in the Sea of Okhotsk were described using morphological and molecular-genetic (ITS2 region, 28S rRNA and nd1 genes) data. These are the first molecular-genetic data for the genus Steganoderma. The studied trematodes differed from S. eamiqtrema in having a much larger body size. The phylogenetic analysis based on the 28S rRNA gene supported neither the current taxonomic hypothesis that Steganoderma belongs to the subfamily Lepidophyllinae nor the earlier views that the Steganodermatinae and the Lecithostaphylinae are synonymous. The topology of the phylogenetic tree shows that the Steganodermatinae and the Lecithostaphylinae are independent subfamilies. However, morphological differences between them are obscure. Until morphological evidence for the Steganodermatinae is found, we propose to distinguish the subfamily Lepidophyllinae sensu stricto with the genera Lepidophyllus and Urinatrema, and the subfamily Lecithostaphylinae sensu lato uniting all the other former lepidophyllines. Thus, for now, we propose to consider the Steganodermatinae as a conditional synonym for Lecithostaphylinae sensu lato.

Prevalence and Intensity of Opisthorchis viverrini Metacercarial Infection in Fish from Phnom Penh, Takeo, and Kandal Provinces, Cambodia.

The prevalence and intensity of Opisthorchis viverrini metacercariae (OvMc) were investigated in fish from 3 southern administrative regions along the Mekong River in Cambodia, i.e., Phnom Penh, Takeo, and Kandal Provinces from 2017 to 2020. A total of 295 freshwater fish (24 species) were transported to our laboratory with ice and examined using the artificial digestion method. In Phnom Penh, among 4 fish species positive for OvMc, 9 (23.7%) of 38 specimens examined were infected, and their intensity of infection averaged 4.3 metacercariae per infected fish. In Takeo Province, among 10 fish species positive for OvMc, 24 (38.1%) out of 63 fish examined were infected, and their intensity of infection was av. 14.4 metacercariae per infected fish. In particular, all of 3 Osteochilus schlegelii fish examined were infected, and their infection intensity was high, 34.7 metacercariae per fish. In Kandal Province, among 6 fish species positive for OvMc, 46 (90.2%) out of 51 specimens examined were infected, and their infection intensity was 24.0 metacercaraie per infected fish. All fish of Systomus orphoides (n=17), Barbonymus altus (n=14), and Rasbora aurotaenia (n=2) were infected, and their intensity of infection averaged 37.7, 21.6, and 18.5 metacercariae per fish, respectively. Metacercariae of Haplochis yokogawai, Haplorchis taichui, and Centrocestus formosanus were detected in fish from Takeo and Kandal Provinces. From these results, it has been confirmed that a variety of fish species from Phnom Penh, Takeo, and Kandal Provinces are commonly infected with OvMc, and preventive measures to avoid human O. viverrini infection should be performed in Cambodia.

Echinostoma mekongi: Discovery of Its Metacercarial Stage in Snails, Filopaludina martensi cambodjensis, in Pursat Province, Cambodia.

Echinostoma mekongi was reported as a new species in 2020 based on specimens collected from humans in Kratie and Takeo Province, Cambodia. In the present study, its metacercarial stage has been discovered in Filopaludina martensi cambodjensis snails purchased from a local market nearby the Tonle Sap Lake, Pursat Province, Cambodia. The metacercariae were fed orally to an experimental hamster, and adult flukes were recovered at day 20 post-infection. They were morphologically examined using light and scanning electron microscopes and molecularly analyzed by sequencing of their mitochondrial cox1 and nad1 genes. A total of 115 metacercariae (1-8 per snail) were detected in 60 (60.0%) out of 100 Filopaludina snails examined. The metacercariae were round, 174 µm in average diameter (163-190 µm in range), having a thin cyst wall, a head collar armed with 37 collar spines, and characteristic excretory granules. The adult flukes were elongated, ventrally curved, 7.3 (6.4-8.2)×1.4 (1.1-1.7) mm in size, and equipped with 37 collar spines on the head collar (dorsal spines in 2 alternating rows), being consistent with E. mekongi. In phylogenetic analyses, the adult flukes showed 99.0-100% homology based on cox1 sequences and 98.9-99.7% homology based on nad1 sequences with E. mekongi. The results evidenced that F. martensi cambodjensis snails act as the second intermediate host of E. mekongi, and hamsters can be used as a suitable experimental definitive host. As local people favor to eat undercooked snails, these snails seem to be an important source of human infection with E. mekongi in Cambodia.

Parvatrema duboisi (Digenea: Gymnophallidae) Life Cycle Stages in Manila Clams, Ruditapes philippinarum, from Aphae-do (Island), Shinan-gun, Korea.

Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphaedo (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307-570 (av. 395) µm long and 101-213 (av. 157) µm wide. Most of the daughter sporocysts contained 15-20 furcocercous cercariae each. The cercariae measured 112-146 (av. 134) µm in total length and 35-46 (av. 40) µm in width, with 69-92 (av. 85) µm long body and 39-54 (av. 49) µm long tail. The metacercariae were 210-250 (av. 231) µm in length and 170-195 (av. 185) µm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1-5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8-99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.

Survey of Zoonotic Trematode Metacercariae in Fish from Water Systems of Geum-gang (River) in Republic of Korea.

The infection status of zoonotic trematode metacercariae (ZTM) was surveyed in freshwater fishes from the water systems of Geum-gang (River) in the Republic of Korea (Korea). A total of 1,161 freshwater fishes from 6 local sites of Geum-gang were examined with the artificial digestion method for 4 years (2012-2015). Clonorchis sinensis metacercariae were detected in 122 (37.2%) out of 328 fishes in the positive fish species from 4 surveyed areas, and their mean intensity was 43 per fish infected. Metagonimus spp. metacercariae were found in 432 (51.7%) out of 835 fishes in the positive fish species from all 6 surveyed areas, and their mean intensity was 30 per fish infected. Centrocestus armatus metacercariae were detected in 285 (75.0%) out of 380 fishes in the positive fish species from 6 surveyed areas, and their mean intensity was 2,100 per fish infected. Echinostoma spp. metacercariae were found in 56 (19.7%) out of 284 fishes in the positive fish species from 5 surveyed areas, and their mean intensity was 10 per fish infected. Clinostomum complanatum metacercariae were detected in 98 (57.3%) out of 171 fishes in the positive fish species from only 2 surveyed areas, and their mean intensity was 11 per fish infected. Conclusively, the endemicity of ZTM is not so high in fishes from water systems of Geum-gang in Korea although it is more or less different by fish species, surveyed areas and ZTM species.

High Endemicity with Clonorchis sinensis Metacercariae in Fish from Yongjeon-cheon (Stream) in Cheongsong-gun, Gyeongsangbuk-do, Korea.

The infection status with Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fishes from Yongjeon-cheon (a branch of Nakdong-gang) located in Cheongsong-gun, Gyeongsangbuk-do, the Republic of Korea (Korea). A total of 750 fishes in 19 species were examined by the artificial digestion method for 2 years (2019 and 2020). CsMc were detected in 378 (51.4%) out of 735 fishes in 14 species (73.7%), and the infection intensity was 666 per fish infected. In 2019, CsMc were found in 172 (68.0%) out of 253 fishes in 10 species, and the infection intensity was 565 per fish infected. In 2020, CsMc were detected in 206 (62.2%) out of 331 fishes in 10 species, and the infection intensity was 751 per fish infected. The other zoonotic trematode, ie. Metagonimus spp., Centrocestus armatus, Echinostoma spp. and Clinostomum complanatum, metacercariae were also detected in fishes from the survey streams, but their endemicities were relatively low. Conclusively, it was first confirmed that CsMc are highly endemic in fishes from Yongjeon-cheon in Cheongsong-gun, Gyeongsangbuk-do, Korea.

Prevalence and Infection Intensity of Zoonotic Trematode Metacercariae in Fish from Soyang-cheon (Stream), in Wanju-gun, Jeollabuk-do, Korea.

We surveyed on the infection status of zoonotic trematode metacercariae (ZTM) in freshwater fishes from Soyang-cheon (a branch stream of Mangyeong-gang) in Wanju-gun, Jeollabuk-do, the Republic of Korea. A total of 927 fishes were individually examined with the artificial digestion method during 2013-2015 (462 fish in 15 spp.) and 2018-2019 (465 fish in 25 spp.). Clonorchis sinensis metacercariae were detected in 207 (31.4%) out of 659 fishes in 14 positive fish species (PFS), and their mean intensity was 114 per fish infected (PFI). Metagonimus spp. metacercariae were found in 302 (37.4%) out of 808 fishes in 21 PFS, and their mean intensity was 12 PFI. Centrocestus armatus metacercariae were detected in 222 (59.0%) out of 376 fishes in 12 PFS, and their mean intensity was 383 PFI. Echinostoma spp. metacercariae were found in 139 (22.1%) out of 628 fishes in 10 PFS, and their mean intensity was 7 PFI. Clinostomum complanatum metacercariae were detected in 14 (6.5%) out of 214 fishes in 4 PFS, and their mean intensity was 2.4 PFI. Metorchis orientalis metacercariae were detected in 36 (13.5%) out of 267 fishes in 5 PFS, and their mean intensity was 4.3 PFI. Conclusively, the prevalence and infection intensity of ZTM is generally not so high in fishes from Soyang-cheon. However, those of C. sinensis metacercariae are more or less higher in 2 fish species, Pungtungia herzi and Sarcocheilichthys variegatus wakiyae.

Survey of Zoonotic Trematode Metacercariae in Fish from Irrigation Canal of Togyo-jeosuji (Reservoir) in Cheorwon-gun, Gangwon-do, Republic of Korea.

The infection status of zoonotic trematode metacercariae (ZTM) was investigated in total 568 freshwater fishes (19 species) from the irrigation canal of Togyo-jeosuji (Reservoir) in Cheorwon-gun, Gangwon-do, the Republic of Korea for 3 years (2018-2020). All fishes were examined using the artificial digestion method. The metacercariae of Clonorchis sinensis (CsMc) were detected in 180 (43.8%) out of 411 fish of positive species, and their infection intensity was 38 per fish infected (PFI). Especially, in 2 fish species, i.e., Pseudorasbora parva and Puntungia herzi, the prevalence was 82.1% and 31.3%, and the infection intensity with CsMc was 88 and 290 PFI, respectively. Metagonimus spp. metacercariae (MsMc) were found in 403 (74.1%) out of 544 fish of positive species, and their infection intensity was 62 PFI. In the pale chub, Zacco platypus, the prevalence of MsMc was 98.6%, and their infection intensity was 144 PFI. Centrocestus armatus metacercariae were detected in 171 (38.9%) out of 440 fish of positive species, and their infection intensity was 1,844 PFI. Echinostoma spp. metacercariae were found in 94 (19.6%) out of 479 fish of positive species, and their infection intensity was 3 PFI. Metorchis orientalis metacercariae were detected in 43 (29.3%) out of 147 fish of positive species, and their infection intensity was 4 PFI. By the present study, it has been confirmed that some species of ZTM, including CsMc and MsMc, are prevalent in fishes from the irrigation canal of Togyo-jeosuji in Cheorwon-gun, Gangwon-do, Korea.

Endemicity of Zoonotic Trematode Metacercariae in Fish from Deokcheon-gang (River) in Sancheong-gun, Gyeongsangnam-do, Republic of Korea.

The endemicity of zoonotic trematode metacercariae (ZTM) was investigated with total 871 freshwater fishes (19 species) from Deokcheon-gang (a branch stream of Gyeongho-gang) in Sancheong-gun, Gyeongsangnam-do, Korea for 3 years (2018-2020). All fishes were examined with the artificial digestion method. The metacercariae of Clonorchis sinensis (CsMc) were detected in 233 (36.3%) out of 642 fish in 11 positive fish species (PFS), and their infection intensity was 27 per fish infected (PFI). Especially, in index fish, Puntungia herzi, of CsMc infection, prevalence was 64.2% and infection intensity was 37 PFI. Metagonimus spp. metacercariae (MsMc) were found in 760 (87.5%) out of 869 fish in 18 PFS and their infection intensity was 228 PFI. In sweet smelt, Plecoglossus altivelis, the prevalence of MsMc was 97.6% and their infection intensity was 3,570 PFI. Centrocestus armatus metacercariae were detected in 209 (29.4%) out of 710 fish in 8 PFS and their infection intensity was 1,361 PFI. Echinostoma spp. metacercariae were found in 293 (42.6%) out of 688 fish in 15 PFS and their infection intensity was 5 PFI. Metacercariae of Clinostomum complanatum and Metorchis orientalis were also detected in 2.7% and 21.2% fish in 4 PFS and their infection intensities were 3.1 and 3.4 PFI respectively. By the present study, it was confirmed that some species of ZTM including CsMc and MsMc are more or less prevalent in fishes from Deokcheon-gang in Sancheong-gun, Gyeongsangnam-do, Korea.

A month for the mission: using a sentinel approach to determine the transmission window of digenean cercariae in the subarctic White Sea.

In the digenean life cycle the cercaria ensures an important transmission stage, from the first intermediate host to the second or the definitive host. In regions with pronounced seasonality, this process occurs within a certain interval, the transmission window. In high latitudes, the size of transmission window has previously been determined only by comparing data on seasonal dynamics of infection level in various categories of hosts or extrapolating the results of laboratory experiments on cercarial biology to natural conditions. In this study, we evaluated the dynamics of infection of the second intermediate hosts (mussels Mytilus edulis) with cercariae of two digenean species, Himasthla elongata (Himasthlidae) and Cercaria parvicaudata (Renicolidae), at a littoral site at the White Sea by exposing cages with uninfected mussels during the warm season. This is the first such study in a subarctic sea. Mussel infection was observed from May to mid-September, but its intensity was the greatest only for approximately a month, from some moment after 10 July to mid-August, when water temperature was within the optimal range for cercarial emergence in both studied species (15‒20°C). During this time, the mussels accumulated 66.3 ± 6.2% metacercariae of H. elongata and 79.7 ± 5.3% metacercariae of C. parvicaudata out of the total number accumulated during the experimental period. We suggest that climate warming at high latitudes may prolong the period when the water temperatures are optimal for cercariae emergence, thereby intensifying digenean transmission in coastal ecosystems.

Morphological and Molecular Characteristics of Clinostomid Metacercariae from Korea and Myanmar.

Morphological and molecular characterization of clinostomid metacercariae (CMc) was performed with the specimens collected in fish from Korea and Myanmar. Total 6 batches of clinostomid specimens by the fish species and geographical localities, 5 Korean and 1 Myanmar isolates, were analyzed with morphological (light microscopy and SEM) and molecular methods (the cytochrome c oxidase 1 gene and internal transcribed spacer 1/5.8S rRNA sequence). There were some morphological variations among CMc specimens from Korea. However, some morphometrics, i.e., the size of worm body and each organ, ratio of body length to body width, and morphology of cecal lumens, were considerably different between the specimens from Korea and Myanmar. The surface ultrastructures were somewhat different between the specimens from Korea and Myanmar. The CO1 sequences of 5 Korean specimens ranging 728-736 bp showed 99.6-100% identity with Clinostomum complanatum (GenBank no. KM923964). They also showed 99.9-100% identity with C. complanatum (FJ609420) in the ITS1 sequences ranging 692-698 bp. Meanwhile, the ITS1 sequences of Myanmar specimen showed 99.9% identity with Euclinostomum heterostomum (KY312847). Five sequences from Korean specimens clustered with the C. complanatum genes, but not clustered with Myanmar specimens. Conclusively, it was confirmed that CMc from Korea were morphologically and molecularly identical with C. complanatum and those from Myanmar were E. heterostomum.

High Prevalence of Clonorchis sinensis and Other Zoonotic Trematode Metacercariae in Fish from a Local Market in Yen Bai Province, Northern Vietnam.

A small survey was performed to investigate the recent infection status of Clonorchis sinensis and other zoonotic trematode metacercariae in freshwater fish from a local market of Yen Bai city, Yen Bai province, northern Vietnam. A total of 118 fish in 7 species were examined by the artificial digestion method on March 2016. The metacercariae of 4 species of zoonotic trematodes, i.e., C. sinensis, Haplorchis pumilio, Haplorchis taichui, and Centrocestus formosanus, were detected. The metacercariae of C. sinensis were found in 62 (69.7%) out of 89 fish (5 species), and their intensity of infection was very high, 81.2 per fish infected. Prevalences of 3 intestinal flukes, H. pumilio, H. taichui and C. formosanus, were 75.0%, 47.6%, and 31.7% in positive fish species, respectively, with the metacercarial intensities of 15.5, 10.3, and 2.2 per fish infected. From the above results, it has been confirmed that various species of freshwater fish continue to play the role of the infection source of C. sinensis and other zoonotic trematodes in Yen Bai city, Yen Bai province, northern Vietnam. It is of particular note that the prevalence and intensity of C. sinensis metacercariae are much higher than those reported in previous studies in fish in northern Vietnam.

Low-Grade Endemicity of Opisthorchiasis, Yangon, Myanmar.

We performed an epidemiologic survey of opisthorchiasis in Yangon, Myanmar. The fecal egg-positive rate of residents was 0.7%, and we recovered an adult fluke after chemotherapy and purging of an egg-positive resident. We detected Opisthorchis viverrini metacercariae in freshwater fish. We found the Yangon area to have low-grade endemicity of opisthorchiasis.

Artyfechinostomum sufrartyfex Trematode Infections in Children, Bihar, India.

Eating raw or insufficiently cooked mollusks is a known risk factor for human echinostomiasis. We confirmed identification of Artyfechinostomum sufrartyfex trematodes as the causative agent of disease among 170 children in northern Bihar, India. We also identified the snail Pila globosa as a potential source of infections in the study area.

Is species identification of Echinostoma revolutum using mitochondrial DNA barcoding feasible with high-resolution melting analysis?

The taxonomic evaluation of Echinostoma species is controversial. Echinostoma species are recognized as complex, leading to problems associated with accurate identification of these species. The aim of this study was to test the feasibility of using DNA barcoding of cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) conjugated with high-resolution melting (HRM) analysis to identify Echinostoma revolutum. HRM using COI and ND1 was unable to differentiate between species in the "revolutum complex" but did distinguish between two isolates of 37-collar-spined echinostome species, including E. revolutum (Asian lineage) and Echinostoma sp. A from different genera, e.g., Hypoderaeum conoideum, Haplorchoides mehrai, Fasciola gigantica, and Thapariella anastomusa, based on the Tm values derived from HRM analysis. Through phylogenetic analysis, a new clade of the cryptic species known as Echinostoma sp. A was identified. In addition, we found that the E. revolutum clade of ND1 phylogeny obtained from the Thailand strain was from a different lineage than the Eurasian lineage. These findings reveal the complexity of the clade, which is composed of 37-collar-spined echinostome species found in Southeast Asia. Taken together, the systematic aspects of the complex revolutum group are in need of extensive investigation by integrating morphological, biological, and molecular features in order to clarify them, particularly in Southeast Asia.

Evaluation and Microanalysis of Parasitic and Bacterial Agents of Egyptian Fresh Sushi, Salmo salar.

The present study aimed to evaluate the quality of fresh sushi in Egypt. Fifty samples of sushi (Salmo salar) were collected from restaurants in Alexandria, Egypt. Paraffin, semi-thin and ultra-thin sections were used for parasitological analysis by light and transmission electron microscopy. Bacteria were isolated by the dilution plate and direct plate methods and identified by a Vitek system. Twenty (40%) of the total examined samples showed microsporidia and helminth metacercariae infections. Histochemical stains showed distinct pinkish-red pyriform microspores embedded in muscular tissue stained with Gram, periodic acid-Schiff (PAS), and Ziehl-Neelsen (ZN) stains. Semi-thin sections showed double membrane xenoma-inducing granulomas containing spores at different developmental stages. Empty sporophorous vesicles and free spores were observed in the electron microscopic images. A bacteriological assay showed forty samples (80%) contaminated with human pathogenic bacteria with the average total bacterial counts ranging from 32 to 526 CFU/g. Four species of human pathogenic bacteria were identified in the examined samples, namely Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Serratia plymuthica in 40, 38, 11, and 6 samples, respectively. These constitute the first record of fresh sushi product in Egypt and indicate the potential pathogenicity associated with raw seafood products.