Development of the head collar and collar spines during the larval stages of Isthmiophora hortensis (Digenea: Echinostomatidae).

It is uncertain when the head collar and collar spines of Isthmiophora hortensis (Digenea: Echinostomatidae), a zoonotic echinostome species in Far Eastern Asia, develop during its larval stages. In this study, the appearance of the head collar and collar spines was studied using light and scanning electron microscopy in cercariae and metacercariae experimentally obtained from freshwater snails (Lymnaea pervia) and tadpoles (Rana nigromaculata), respectively. The cercariae were shed from the snail on day 30 after exposure to laboratory-hatched miracidia. Metacercariae were obtained from the experimental tadpoles at 3, 6, 12, 15, 20, 24, 26, and 30 h after exposure to the cercariae. The head collar was already visible in the cercarial stage, although its degree of development was weak. However, collar spines did not appear in the cercarial stage and even in the early metacercarial stage less than 24 h postinfection in tadpoles. Collar spines became visible in the metacercariae when they grew older than 24 h. It was concluded that the head collar of I. hortensis developed early in the cercarial stage, but the development of collar spines did not occur until the worms became 24-h-old metacercariae in our experimental setting. Counting the number of collar spines was concluded as an unfeasible diagnostic method for I. hortensis cercariae when they are shed from the snail host.

Morphological and molecular characteristics of Paralecithodendrium longiforme (Digenea: Lecithodendriidae) adults and cercariae from Chinese pipistrelle bats and viviparid snails in Thailand.

This study aimed to describe the morphological and molecular characteristics of Paralecithodendrium longiforme (Digenea: Lecithodendriidae) adults and cercariae isolated in Thailand. Adult flukes were isolated from the Chinese pipistrelle bat (Hypsugo sp.), and cercariae were detected in the viviparid snail (Filopaludina martensi martensi) from Chiang Mai province. The morphological characteristics were observed and described using conventional methods, and the molecular characteristics with internal transcribed spacer 2 (ITS2) and 28S rDNA gene sequences. The adult flukes were fusiform, 0.84-0.98 mm in length, and 0.37-0.49 mm in width, and were distinguishable from other species by the presence of longitudinal uterine coils. The cercariae were nonvirgulate xiphidiocercariae, with the oral sucker bigger than the acetabulum, the tail without fin fold, a body size of 117.5-138.3 × 48.3-52.2 µm, and a tail size of 100.7-103.7 × 15.0-18.9 µm. Molecular studies revealed that the adults and cercariae shared 99.3% (ITS2) and 99.6% (28S rDNA) homology with each other. They were phylogenetically close to P. longiforme with an identity of 94.5% for ITS2 and 98.7% for 28S rDNA. This study provides new information on the natural definitive host and first intermediate host of P. longiforme in Thailand. The discovery of its cercarial stage in Filopaludina snails highlights the importance of monitoring the associated second intermediate host and prevention and control of this potentially zoonotic trematode.

High Prevalence of Echinostoma mekongi Infection in Schoolchildren and Adults, Kandal Province, Cambodia.

A high prevalence of Echinostoma mekongi infection (13.9%; 260/1,876) was found among schoolchildren and adults in Kandal Province, Cambodia, by fecal examination, worm expulsion, and molecular analysis of cox1 and nd1 genes. The source of infection was consumption of Pila sp. snails, a finding confirmed morphologically and molecularly.

Paleoparasitology research on ancient helminth eggs and larvae in the Republic of Korea.

Paleoparasitology is a discipline that applies existing conventional and molecular techniques to study parasites found in ancient ruins. This review focuses on the history of the discovery of parasites (mostly helminth eggs and larvae) in archaeological soil samples and mummies in Korea from the Three Kingdoms Period to the Joseon Dynasty (100 BCE-1910 CE). We also briefly review important milestones in global paleoparasitology. The helminth species reported so far in Korea included Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis (larva), Trichostrongylus sp. (larva), Paracapillaria philippinensis (syn. Capillaria philippinensis), Enterobius vermicularis, Fasciola hepatica, dicrocoeliids, Paragonimus westermani, Clonorchis sinensis, Metagonimus yokogawai, Pygidiopsis summa, Gymnophalloides seoi, Isthmiophora hortensis, Dibothriocephalus nihonkaiensis (syn. Diphyllobothrium nihonkaiense), and Taenia spp. tapeworms. The findings obtained by Korean paleoparasitologists/archaeologists have brought about deep insight into the status of helminthic infections in Korea's past populations. Continued paleoparasitological research is essential for further understanding of ancient parasites and parasitic diseases in Korea.

Standard- and large-sized eggs of Trichuris trichiura in the feces of schoolchildren in the Yangon Region, Myanmar: Morphological and molecular analyses.

Standard- and large-sized eggs of Trichuris trichiura were found in the feces of schoolchildren in Yangon, Myanmar during epidemiological surveys and mass deworming with albendazole in 2017-2019. The standard-sized eggs were identified as those of T. trichiura, but it was necessary to exclude the possibility of the large-sized eggs belonging to Trichuris vulpis, a dog whipworm. We conducted morphological and molecular studies to determine the species of the 2 types of Trichuris eggs. Individual eggs of both sizes were isolated from Kato-Katz fecal smears (n=20) and mechanically destroyed using a 23G injection needle. Nuclear DNA was extracted, and the 18S rRNA region was sequenced in 15 standard-sized eggs and 15 large-sized eggs. The average size of standard-sized eggs (T. trichiura) was 55.2×26.1 µm (range: 51.7-57.6×21.3-28.0 µm; n=97), whereas the size of large-sized eggs was 69.3×32.0 µm (range: 65.1-76.4×30.1-34.5 µm; n=20), slightly smaller than the known size of T. vulpis. Regarding standard-sized eggs, the 18S rRNA nucleotide sequences exhibited 100% homology with T. trichiura deposited in GenBank and 88.6-90.5% homology with T. vulpis. Regarding large-sized eggs, the nucleotide sequences showed 99.8-100% homology with T. trichiura in GenBank and 89.6-90.7% homology with T. vulpis. Both standard- and large-sized eggs of Trichuris spp. found in Myanmar schoolchildren during 2017-2019 were morphologically and molecularly confirmed to belong to T. trichiura. The conversion of eggs from smaller to large sizes might be due to anthelmintic treatments with albendazole.

Gut microbiota differences induced by Toxoplasma gondii seropositivity in stray cats in South Korea.

T. gondii is a highly prevalent parasite worldwide, with cats serving as its final host. However, few studies have investigated the impact of T. gondii infection on cat gut microbiota. Therefore, this study examined the influence of T. gondii infection on the gut microbiota of stray cats and identified potential pathogens in their feces. This study examined T. gondii infection through blood of stray cats and the influence of microbiota in their feces using 16S rRNA gene amplicon sequencing. The results revealed significant differences in gut microbiota composition and diversity between the T. gondii seropositive and seronegative groups. Seropositive samples displayed a lower number of operational taxonomic units and reduced Shannon index than the seronegative samples. The seropositive and seronegative groups exhibited enrichment of taxa, including Escherichia and Enterobacteriaceae and Collinsella, Bifidobacterium, and Roseburia, respectively. Furthermore, potential pathogen species, including Campylobacter, Escherichia, and Streptococcus, were identified in the fecal samples. These findings suggest that T. gondii infection significantly impacts gut microbiota composition and diversity in stray cats. Additionally, an increased potential pathogen load, represented by Escherichia spp., was observed. These results underscore the importance of monitoring the prevalence of zoonotic pathogens in stray cats, as they can serve as reservoirs for zoonotic diseases.

High endemicity of Opisthorchis viverrini infection among people in northern Cambodia confirmed by adult worm expulsion.

Opisthorchis viverrini infection is an emerging disease in Cambodia, especially in central and southeastern areas. However, its status in northern areas bordering Lao PDR has been relatively unknown. The present study was performed to investigate the status of O. viverrini infection among people in Preah Vihear and Stung Treng provinces through fecal examination to detect eggs and recovery of adult flukes from some of the egg-positive cases. Fecal examinations were performed on a total of 1101 people from 10 villages in the 2 provinces using the Kato-Katz thick smear technique. For recovery of adult flukes and other helminth parasites 10 volunteers positive for eggs of Opisthorchis viverrini and/or minute intestinal flukes (Ov/MIF), in Kampong Sangkae village, Preah Vihear province, were administered a single oral dose of 40 mg/kg praziquantel plus 5-10 mg/kg of pyrantel pamoate and purged with 40-50 g magnesium salts. Adult trematodes, together with nematodes and cestodes expelled in diarrheic stools were collected under a stereomicroscope or with the naked eye. The proportion of egg-positive cases for overall liver and intestinal helminths was high but not notably different between the 2 provinces, 65.5% in Preah Vihear versus 64.7% in Stung Treng. The average proportion of Ov/MIF egg-positive cases was 59.8%. A total of 315 adult specimens of O. viverrini were recovered from the 10 volunteers (4-98 specimens per individual; mean, 32). A smaller number of Haplorchis taichui adults, an intestinal fluke, were found mixed-infected in 7 (103 specimens in total; 1-31 per individual; mean, 15) of the 10 volunteers. Adult specimens of hookworms, Enterobius vermicularis, Trichostrongylus sp., and a Taenia tapeworm strobila were recovered in some cases. Based on the results, it has been confirmed that the surveyed areas in Preah Vihear and Stung Treng provinces, Cambodia, are highly endemic areas of O. viverrini infection with a low-grade mixed infection with H. taichui.

Prevalence of head louse infestation among primary schoolchildren in the Republic of Korea: nationwide observation of trends in 2011-2019.

Head louse infestation is a significant public health problem across the world, particularly among preschool and primary schoolchildren. This study investigated the trends of head louse infestation in the Republic of Korea over a 9-year period (2011-2019), targeting primary schoolchildren in 3 areas of Seoul, 4 other large cities, and 9 provinces. A survey was administered annually by the health staff of each regional office (n= 16) of the Korea Association of Health Promotion (KAHP). The branch offices of KAHP examined a total of 51,508 primary schoolchildren, comprising 26,532 boys and 24,976 girls. Over the 9-year survey, a total of 1,107 (2.1%) schoolchildren tested positive for adults and/or nits of Pediculus humanus capitis. The prevalence was 2.8% (133/4,727) in 2011-2012 and gradually decreased to 0.8% (49/6,461) in 2019 (P< 0.05). Head lice were found more frequently in girls (3.0%; 746/24,976) than in boys (1.4%; 361/26,532) (P< 0.05). In terms of geographic localities, the highest infestation rate, 4.7% (average prevalence over 9 years), was observed in southern Seoul (Gangnam branch of KAHP), whereas the lowest infestation rate, 0.7%, was seen in Gyeongsang (north and south provinces) and western Seoul. Although the prevalence decreased significantly during the 9-year period, head louse infestation remains a health and hygiene issue among primary schoolchildren in the Republic of Korea. Regular surveys along with health education are needed to further improve children's hair hygiene.

Updates on parasite infection prevalence in the Joseon period based on parasitological studies of human coprolites isolated from archaeological sites in the cities of Euijeongbu, Gumi, and Wonju.

Parasite infection rates estimated by examining ancient coprolites can provide insights into parasitism in Joseon society. Using newly discovered Joseon period cases is essential to regularly update the parasite infection rates and reinforce the reliability of our previous estimations. In the present study, we investigated parasite infections in Joseon coprolites newly isolated from the cities of Euijeongbu, Gumi, and Wonju. We then updated the overall parasite infection rates of Joseon period samples (n= 30) as follows: 86.7% (26/30) for Trichuris trichiura, 56.7% (17/30) for Ascaris lumbricoides, 30.0% (9/30) for Clonorchis sinensis, and 30.0% (9/30) for Paragonimus westermani. The parasite infection rates in the Joseon society, estimated through coprolite examination, were very similar to those determined previously despite the addition of new cases to the existing data pool.

Trematode metacercariae and adults in cyprinoid fish from Khun Thale Swamp in Surat Thani province, Thailand.

The present study aimed to determine the infection status of trematode metacercariae and adults in cyprinoid fish from the Khun Thale Swamp in Surat Thani, Southern Thailand, with epidemiologic and faunistic viewpoints. In 2020, 577 fish in 15 species were collected in the summer (February-April) and rainy (September-November) seasons. Fish were individually examined for trematode metacercariae in the whole body and adults in the gastrointestinal tract using a stereomicroscope. Three species of digenetic trematode metacercariae, i.e., Haplorchis taichui, Haplorchoides mehrai, and Centrocestus formosanus, were detected in the muscle, fin, and/or scale of fish. Two species of adult flukes, including Rohdella siamensis and Helostomatis cyprinorum, were collected in the intestines. The prevalence of overall trematode infections was 32.4% (187/577 fish), which was higher in the rainy season (41.4%; 118/285) than in the summer season (23.6%; 69/292). The metacercariae of H. taichui and H. mehrai were detected in 7 fish species each, and those of C. formosanus were found only in Rasbora toneri. The aspidogastrean trematode R. siamensis (adult) was detected in Babonymus gonionotus. A digenean species, H. cyprinorum (adult), was found in Labiobarbus siamensis and Osteochilus vittatus. The present study has first confirmed that the metacercariae of heterophyid flukes, including H. taichui, H. mehrai, and C. formosanus, and adults of R. siamensis (Aspidogastrea) and H. cyprinorum (Digenea) are infected in some species of the cyprinoid fish from the Khun Thale Swamp in Surat Thani, Thailand.

A parasitological study on the possible toilet ruins of the Japanese colonial period in Korea.

In the past decade, experts have conducted parasitological research on archaeological specimens in Korea to collect historical parasite infection data. In these studies, parasitologists successfully described the infection pattern of each parasite species in history. However, in the first half of the 20th century, archaeoparasitological reports have been scant. In 2021, we conducted a parasitological examination of a toilet-like structure that emerged in the early 20th century. This structure was built by stacking 2 wooden barrels; and in the study samples, we found ancient Trichuris trichiura, Ascaris lumbricoides (unfertilized), and Taenia spp. eggs and therefore proposed a higher possibility that the barrels could have been used as a toilet at the time. To understand how the antihelminthic campaign since the 1960s helped reduce parasite infection rates in Korea, more research should focus on early-20th-century toilet ruins.

The spatial-temporal risk profiling of Clonorchis sinensis infection over 50 years implies the effectiveness of control programs in South Korea: a geostatistical modeling study.

Background: Over the past 50 years, two national control programs on Clonorchis sinensis infection have been conducted in South Korea. Spatial-temporal profiles of infection risk provide useful information on assessing the effectiveness of the programs and planning spatial-targeted control strategies. Methods: Advanced Bayesian geostatistical joint models with spatial-temporal random effects were developed to analyze disease data collecting by a systematic review with potential influencing factors, and to handle issues of preferential sampling and data heterogeneities. Changes of the infection risk were analyzed. Findings: We presented the first spatial-temporal risk maps of C. sinensis infection at 5 × 5 km2 resolution from 1970 to 2020 in South Korea. Moderate-to-high risk areas were shrunk, but temporal variances were shown in different areas. The population-adjusted estimated prevalence across the country was 5.99% (95% BCI: 5.09-7.01%) in 1970, when the first national deworming campaign began. It declined to 3.95% (95% BCI: 2.88-3.95%) in 1995, when the campaign suspended, and increased to 4.73% (95% BCI: 4.00-5.42%) in 2004, just before the Clonorchiasis Eradication Program (CEP). The population-adjusted prevalence was estimated at 2.77% (95% BCI: 1.67-4.34%) in 2020, 15 years after CEP started, corresponding to 1.42 (95% BCI: 0.85-2.23) million infected people. Interpretation: The first nationwide campaign and the CEP showed effectiveness on control of C. sinensis infection. Moderate-to-high risk areas identified by risk maps should be prioritized for control and intervention. Funding: The National Natural Science Foundation of China (project no. 82073665) and the Natural Science Foundation of Guangdong Province (project no. 2022A1515010042).

Molecular Detection of Haplorchis pumilio Eggs in Schoolchildren, Kome Island, Lake Victoria, Tanzania.

A survey of intestinal helminths targeting 1,440 schoolchildren in 12 primary schools on Kome Island (Lake Victoria), Tanzania, revealed small trematode eggs in 19 children (1.3%), seemingly of a species of Haplorchis or Heterophyes. The eggs were molecularly confirmed to be Haplorchis pumilio on the basis of 18S and 28S rDNA sequences.

The notable global heterogeneity in the distribution of COVID-19 cases and the association with pre-existing parasitic diseases.

BACKGROUND: The coronavirus Disease 2019 (COVID-19) is a respiratory disease that has caused extensive ravages worldwide since being declared a pandemic by the World Health Organization (WHO). Unlike initially predicted by WHO, the incidence and severity of COVID-19 appeared milder in many Low-to-Middle-Income Countries (LMIC). To explain this noticeable disparity between countries, many hypotheses, including socio-demographic and geographic factors, have been put forward. This study aimed to estimate the possible association of parasitic diseases with COVID-19 as either protective agents or potential risk factors. METHODS/PRINCIPAL FINDINGS: A country-level ecological study using publicly available data of countries was conducted. We conceptualized the true number of COVID-19 infections based on a function of test positivity rate (TPR) and employed linear regression analysis to assess the association between the outcome and parasitic diseases. We considered demographic, socioeconomic, and geographic confounders previously suggested. A notable heterogeneity was observed across WHO regions. The countries in Africa (AFRO) showed the lowest rates of COVID-19 incidence, and the countries in the Americas (AMRO) presented the highest. The multivariable model results were computed using 165 countries, excluding missing values. In the models analyzed, lower COVID-19 incidence rates were consistently observed in malaria-endemic countries, even accounting for potential confounding variables, Gross Domestic Product (GDP) per capita, the population aged 65 and above, and differences in the duration of COVID-19. However, the other parasitic diseases were not significantly associated with the spread of the pandemic. CONCLUSIONS/SIGNIFICANCE: This study suggests that malaria prevalence is an essential factor that explains variability in the observed incidence of COVID-19 cases at the national level. Potential associations of COVID-19 with schistosomiasis and soil-transmitted helminthiases (STHs) are worthy of further investigation but appeared unlikely, based on this analysis, to be critical factors of the variability in COVID-19 epidemic trends. The quality of publicly accessible data and its ecological design constrained our research, with fundamental disparities in monitoring and testing capabilities between countries. Research at the subnational or individual level should be conducted to explore hypotheses further.

Exosomal miRNA-21 from Toxoplasma gondii-infected microglial cells induces the growth of U87 glioma cells by inhibiting tumor suppressor genes.

Toxoplasma gondii is an intracellular protozoan parasite that can modulate the microenvironment of infected hosts and is known to be associated with the incidence of brain tumor growth. In this study, we suggested that the exosomal microRNA-21 derived from Toxoplasma infection would contribute to the growth of brain tumors. Exosomes of BV2 microglial cells infected with Toxoplasma were characterized and confirmed internalization to U87 glioma cells. Exosomal miRNA expression profiles were analyzed using microRNA array and miR-21A-5p associated with Toxoplasma and tumor sorted. We also examined the mRNA level of tumor-associated genes in U87 glioma cells by changing the level of miR-21 within exosomes and the effects of exosomes on the proliferation of human U87 glioma cells. Expression of miRNA-21 was increased and anti-tumorigenic genes (FoxO1, PTEN, and PDCD4) were decreased in exosomes within T. gondii-infected U87 glioma cells. Toxoplasma-infected BV2-derived exosomes induced proliferation of U87 glioma cells. The exosomes induced the growth of U87 cells in a mouse tumor model. We suggest that the increased exosomal miR-21 from Toxoplasma-infected BV2 microglial cells may play an important role as a cell growth promotor of U87 glioma cells through a down-regulation of anti-tumorigenic genes.

Molecular Diagnosis of Pseudoterranova decipiens Sensu Stricto Infections, South Korea, 2002‒2020.

Human Pseudoterranova decipiens larval infections were diagnosed by molecular analysis of mitochondrial cox1 and nd1 genes in 12 health check-up patients in South Korea during 2002-2020. Based on high genetic identity (99.3%-100% for cox1 and 96.7%-98.0% for nd1), we identified all 12 larvae as P. decipiens sensu stricto.

General overview of the current status of human foodborne trematodiasis.

Foodborne trematodes (FBT) of public health significance include liver flukes (Clonorchis sinensis, Opisthorchis viverrini, O. felineus, Fasciola hepatica and F. gigantica), lung flukes (Paragonimus westermani and several other Paragonimus spp.) and intestinal flukes, which include heterophyids (Metagonimus yokogawai, Heterophyes nocens and Haplorchis taichui), echinostomes (Echinostoma revolutum, Isthmiophora hortensis, Echinochasmus japonicus and Artyfechinostomum malayanum) and miscellaneous species, including Fasciolopsis buski and Gymnophalloides seoi. These trematode infections are distributed worldwide but occur most commonly in Asia. The global burden of FBT diseases has been estimated at about 80 million, however, this seems to be a considerable underestimate. Their life cycle involves a molluscan first intermediate host, and a second intermediate host, including freshwater fish, crustaceans, aquatic vegetables and freshwater or brackish water gastropods and bivalves. The mode of human infection is the consumption of the second intermediate host under raw or improperly cooked conditions. The major pathogenesis of C. sinensis and Opisthorchis spp. infection includes inflammation of the bile duct which leads to cholangitis and cholecystitis, and in a substantial number of patients, serious complications, such as liver cirrhosis and cholangiocarcinoma, may develop. In lung fluke infections, cough, bloody sputum and bronchiectasis are the most common clinical manifestations. However, lung flukes often migrate to extrapulmonary sites, including the brain, spinal cord, skin, subcutaneous tissues and abdominal organs. Intestinal flukes can induce inflammation in the intestinal mucosa, and they may at times undergo extraintestinal migration, in particular, in immunocompromised patients. In order to control FBT infections, eating foods after proper cooking is strongly recommended.

Ancient DNA of Metagonimus yokogawai Recovered from Joseon Period Human Remains Newly Discovered at Goryeong County in South Korea.

PURPOSE: Metagonimiasis, commonly seen in East Asian countries, is a parasitic disorder caused by definitive hosts' ingestion of undercooked freshwater fishes. Recently, genetic analysis has proved 28S rRNA and cytochrome c oxidase subunit I (COI) mtDNA gene to be a successful marker differentiating species of the genus Metagonimus. In the present study, using specimens from the newly discovered Joseon Dynasty human remains of Goryeong, we obtained updated genetic data on genus Metagonimus, which was also prevalent during the Joseon period. METHODS: The ancient DNA (aDNA) was retrieved from the coprolite sample of the seventeenth century, half-mummified individual discovered at Goryeong Country, South Korea. Cloning and sequencing were performed on PCR-amplified amplicons for M. yokogawai 28S rRNA and COI mtDNA gene. The consensus sequences were used for species identification and phylogenetic analysis using NCBI/BLAST and MEGA X software. RESULTS: Based on the COI mtDNA gene region, the Goryeong sequence was confirmed as belonging to M. yokogawai, as it was shown to form a separate cluster with other M. yokogawai taxa that are distinct also from M. takahashii and M. miyatai. CONCLUSION: In a series of our genetic analyses on genus Metagonimus using samples retrieved from Joseon-period cases, aDNA sequences of genus Metagonimus revealed in South Korea thus far are those of M. yokogawai, but not of M. miyatai or M. takahashii yet.

Detection of Gnathostoma spinigerum Advanced 3rd-Stage Larvae in the Chinese Edible Frog, Hoplobatrachus rugulosus, from Local Markets in Phnom Penh, Cambodia.

The Chinese edible frogs, Hoplobatrachus rugulosus (n=20), and the striped snakehead fish, Channa striata (n=34), were purchased from local markets in 3 administrative regions of Cambodia (Phnom Penh, Pursat, and Takeo Provinces) from May 2017 to April 2019, and their infection status with Gnathostoma sp. larvae was investigated. The frogs and fish were transported to the laboratory with ice and examined using the artificial digestion method. Advanced 3rd-stage larvae (AdL3) of Gnathostoma spinigerum, 24 in total number (1-6 larvae/frog), were detected from 6 (60.0%) out of 10 frogs purchased from Phnom Penh. No gnathostome larvae were detected in 10 frogs purchased from Takeo Province and 34 snakeheads from Phnom Penh, Pursat, and Takeo Provinces. AdL3 isolated from the frogs were 2.55- 3.90 mm long and 0.31-0.36 mm wide. They had a characteristic head bulb (0.081×0.191 mm in average size) with 4 rows of hooklets, a muscular long esophagus (0.950-1.230 mm long), and 2 pairs of cervical sacs (0.530-0.890 mm long). The average number of hooklets in the 1st, 2nd, 3rd, and 4th rows was 41, 45, 48, and 51, respectively. These features were consistent with G. spinigerum AdL3. By the present study, it has been first confirmed that the Chinese edible frog, H. rugulosus, from Phnom Penh serves as a second intermediate host for G. spinigerum, although their intensity of infection was not so high compared to other previously reported localities.