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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.

Fishborne zoonotic trematodes (FZT), including Opisthorchis viverrini, are public health problems in Southeast Asian countries, i.e., Thailand, Lao People's Democratic Republic (Lao PDR), Vietnam, and Cambodia. O. viverrini can provoke remarkable pathology and morbidity, such as cholangiocarcinoma, among residents of riverside endemic areas [1,2]. Infections with this liver fluke in humans are usually caused by habitual consumption of raw freshwater fish containing metacercariae. This infection is highly prevalent among riparian populations having the food habit of eating raw fish. Many riverside areas along Khieu et al. [10] broadly analyzed the information regarding the epidemiological situation of O. viverrini infection in Cambodia.
Several workers previously reported the infection status of zoonotic trematode metacercariae (ZTM), in particular, O. viverrini, in freshwater fish purchased from Phnom Penh, Pursat, Takeo, and Kratie Provinces [8,9,[17][18][19][20]. However, studies on the infection status of freshwater fish with ZTM need to be continuously performed in different localities of Cambodia. Therefore, in this study, we surveyed the prevalence and intensity of O. viverrini metacercarial infection in fish purchased from local markets in 3 southern administrative regions, i.e., Phnom Penh, Takeo, and Kandal Provinces.
We purchased a total of 295 fish specimens (24 species) from local markets in 3 southern regions, i.e., Phnom Penh (86 fish in 8 species in May 2018), Takeo Province (99 fish in 16 species in November 2017), and Kandal Province (110 fish in 13 species in January-February 2020), Cambodia. The collected fish were transported to the Department of Parasitology and Tropical Medicine, Gyeongsang National University College of Medicine, Jinju, Korea with ice. The fish species were identified with the aid of the FishBase website (http://www.fishbase.org/ search.php) and by the help of a Korean ichthyologist (Choi SH). Individual fish was finely ground in a mortar with a pestle, the ground fish meat was mixed with artificial gastric juice, and the mixture was incubated at 36˚C for about 2 hr. The digested material was filtered through a 1× 1 mm mesh, and washed with 0.85% saline until the supernatant became clear. The sediment was carefully examined for metacercariae under a stereomicroscope. The metacercariae of each species (only ZTM) were collected viewing from the general features and were counted to get hold of the prevalence (%) and intensity of infection (no. of ZTM per fish infected) by fish species. The metacercariae of 175-210 (av. 195)×138-163 (150) 50 µm in size with characteristic morphologies of the oral and ventral suckers and the excretory bladder were regarded as the O. viverrini metacercaria (OvMc) (Fig. 1A).
A few other ZTM that belong to the family Heterophyidae or the Echinostomatidae were also detected in this study. In Takeo    In our study, the prevalence and intensity of OvMc in fish were relatively low compared with our previous studies (by the Korean team) in Cambodia [8,9,20], with the exception of Kandal Province which revealed higher values. The endemicity indices of OvMc (mean no. of metacercariae per fish x prevalence/100) in this study were 1.0 (Phnom Penh), 5.5 (Takeo), and 21.6 (Kandal). Chai et al. [20] detected a total of 690 OvMc (18.6 metacercariae per fish) in 37 (50.0%) out of 74 fish examined in Phnom Penh, where the endemicity index was 9.3. Sohn et al. [9] detected a total of 50 OvMc (10.0/fish) in 5 (100%) of 5 P. proctozysron from Takeo province, and the endemicity index was 10.0. Sohn et al. [8] also detected a total of 367 OvMc (19.3/fish) in 19 (63.3%) out of 30 fish examined in Kratie Province, and the endemicity index was 12.2.
In Kandal and Takeo Provinces, Touch et al. [18] reported lower prevalence and lower intensity of OvMc; they detected a total of 789 OvMc (4.8/fish) in 163 (17.5%) out of 929 fish, and the endemicity index was only 0.8. From the present and previous studies, we could confirm that the endemicity of OvMc has been continuously maintained in southern localities of Cambodia along the Mekong River, i.e., Phnom Penh, Takeo, Kratie, and Kandal Provinces. However, the endemicity was more or less different by survey localities. The endemicity index of OvMc was the highest (21.6) in Kandal Province as depicted in this study.
Conclusively, it has been confirmed in this study that OvMc are more or less prevalent in a variety of fish species commercially available in Phnom Penh, Takeo, and Kandal Provinces, Cambodia along the Mekong River. However, with the exception of Kandal Province, their endemicity was not so high compared with our previous reports in Cambodia. There were some limitations in this study, such as a small number of fish specimens and a small number of fish species examined.

ACKNOWLEDGMENTS
We thank Jung-A Kim and Hee-Ju Kim in the Department of Parasitology, Gyeongsang National University College of Medicine, Jinju, Korea, for their help in fish examinations. We also thank the staff of the Korea Association of Health Promotion, Seoul, Republic of Korea, who participated in the Korea-Cambodia Cooperation Project on Parasite Control in Cambodia (2017, 2018, and 2020).