Exceptionally High Prevalence of Infection of Bithynia siamensis goniomphalos with Opisthorchis viverrini Cercariae in Different Wetlands in Thailand and Lao PDR

Nadda Kiatsopit 0 1 2 Paiboon Sithithaworn 0 1 2 Weerachai Saijuntha 0 1 2 Thidarut Boonmars 0 1 2 Smarn Tesana 0 1 2 Jiraporn Sithithaworn 0 1 2 Trevor N. Petney 0 1 2 Ross H. Andrews rhandrews@gmail 0 1 2 0 Parasitology and Liver Fluke and Cholangiocarcinoma Research Cen- ter, Faculty of Medicine, Khon Kaen University , Khon Kaen 40002 , Thailand 1 Department of Parasitology, Faculty of Medicine, Khon Kaen University , Khon Kaen , Thailand; Liver Fluke and Cholangiocarcinoma Research Centre (LFCRC), Faculty of Medicine, Khon Kaen University , Khon Kaen , Thailand; Walai Rukhavej Botanical Research Institute, Mahasarakham University , Mahasarakham , Thailand; Faculty of Associated Medical Sciences, Khon Kaen University , Khon Kaen , Thailand; Department of Ecology and Parasitology, Karlsruhe Institute of Technology , Karlsruhe , Germany 2 Authors' addresses: Nadda Kiatsopit , Paiboon Sithithaworn, Thidarut Boonmars, Smarn Tesana, and Ross H. Andrews , Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand and Liver Fluke and Cholangiocarcinoma Research Center The carcinogenic liver fluke, Opisthorchis viverrini, requires Bithynia snail intermediate hosts in its life cycle. However, the prevalence of O. viverrini in snail intermediate hosts is typically low (< 1%). Here, we examined B. siamensis goniomphalos from 48 localities in Thailand and The Lao People's Democratic Republic (Lao PDR) and reported highprevalence levels of O. viverrini. The highest-prevalence levels per locality were 6.93% (mean = 3.04%) in Thailand and 8.37% (mean = 2.01%) in Lao PDR; 4 of 13 localities examined showed prevalence higher than any prevalence previously recorded. The number of cercariae infecting snails and their prevalence were positively correlated with the size of the snails. High prevalence occurred in the Songkram River wetland (Thailand) and the Nam Ngum River wetland (Lao PDR). Our results show that transmission of O. viverrini from humans as well as animal reservoir hosts to snail intermediate hosts is ongoing and potentially increasing in endemic areas across Thailand and Lao PDR. - The liver fluke Opisthorchis viverrini is a food-borne trematode pathogen in the Mekong Region in Southeast Asia, where it infects at least 9 million individuals.1 Not only is O. viverrini itself pathogenic, it is classified as a type one carcinogen and is the major causative agent for cholangiocarcinoma (CCA) in the area.2 In northeast Thailand and The Lao Peoples Democratic Republic (Lao PDR), O. viverrini is a major medical problem, with prevalence rates commonly reaching 30% or more in rural populations in Thailand and over 80% in Lao PDR.3,4 The life cycle of O. viverrini includes the freshwater snails Bithynia funiculata, B. siamensis siamensis, and B. s. goniomphalos as first intermediate hosts, with the latter occurring in northeast Thailand and Lao PDR. A wide variety of freshwater cyprinid fishes act as second intermediate hosts. Humans are the most important final hosts, although cats and dogs can harbor adult worms.1 The prevalence of O. viverrini cercariae in Bithynia snail hosts is reportedly very low, ranging from 0.03% to 2.47%.511 To date, the prevalence of O. viverrini infection in B. s. goniomphalos has been found to range between 0.03% and 1.3% in Thailand69 and 0.5% and 2.47% in Lao PDR.1113 This finding is in contrast to the very high prevalence in cyprinid fish (9095%) and humans.1417 The three Bithynia taxa, which are sexually reproducing, are the critical amplifying components in the transmission of O. viverrini, and they are a controlling factor for the potential spread of opisthorchiasis and CCA. Here, we examine the prevalence and cercarial shedding of O. viverrini in B. s. goniomphalos in Thailand and Lao PDR and determine their association with snail size. MATERIALS AND METHODS Samples of B. s. goniomphalos were collected from five major wetlands: the Mun, Chi, and Songkram Rivers basins in Thailand and the Nam Ngum and Sae Bang Heang Rivers basins in Lao PDR. A total of 5,790 snails from 25 localities were collected in Thailand from nine northeastern provinces (Figure 1). In Lao PDR, a total of 5,848 snails from 23 localities were collected in Vientiane and Savannakhet provinces. The snails were collected by handpicking and scooping, and they were identified morphologically according to the works by Brandt,18 Upatham and others,19 and Chitramvong.20 The shell size of each snail was measured under a dissecting microscope. Snail samples were placed individually into plastic containers for cercarial shedding. To estimate the number of cercariae released per day, O. viverrini-positive snails were kept in the dark for 12 hours and then exposed to light for 12 hours. Cercariae released from each snail during both the dark and light phases were counted under a dissecting microscope to calculate the number of cercariae per snail per day. The cercariae from infected snails were identified by light microscopy and confirmed by polymerase chain reaction (PCR).21 A random sample of cercariae from snails collected from Thailand and Lao PDR were passed to fish (silver barb; Barbonymus gonionotus) to obtain O. viverrini metacercariae, which were fed to hamsters to produce adult worms for definitive identification. Snail samples were collected between 2008 and 2011 when snails were available, especially during winter months (November to January). Four of five wetlands contained B. s. goniomphalos infected with O. viverrini cercariae (Figure 1). In Thailand, infected snails were found at four (16%) localities, with an average of 3.04%; in Lao PDR, infected snails were found at nine (39.13%) localities, with an average of 2.01%. In the Nam Ngum wetland, B. s. goniomphalos was found to be infected at eight localities: one locality in the Sae Bang Heang wetland in Lao PDR and three localities in the Songkram wetland: one locality in the Chi wetland in Thailand. The difference in the proportion of infected sites between Thailand and Lao PDR approached significance (Fishers exact test, one-tailed P = 0.069). Of the total number of 48 localities examined, 13 contained snails infected with O. viverrini. For infected snails, prevalence in Lao PDR ranged from 0.37% to 8.37% (Table 1). Of the nine localities with snails positive for O. viverrini, two localities from the Nam Ngum wetland showed higher prevalence than any previously recorded. In Thailand, prevalence ranged from 0.22% to 6.93%, and of the four positive localities, two localities from the Songkram wetland showed higher prevalence than any previously recorded. All localities with exceptionally high prevalence were rice fields with very shallow water. As shown in Figure 2 , large B. s. goniomphalos had a prevalence of 9.80% and 9.77% for length (> 10.0 mm) versus width (> 6.0 mm), respectively. Medium-sized snails (length = 8.1 10.0 mm, width = 5.16.0 mm) had prevalence of 3.59 (...truncated)