First molecular identification and genetic diversity of Strongyloides stercoralis and Strongyloides fuelleborni in human communities having contact with long-tailed macaques in Thailand.

The parasitic nematodes, Strongyloides stercoralis and Strongyloides fuelleborni, can infect humans and non-human primates. We amplified and sequenced a portion of the 18S ribosomal RNA gene (rRNA) and of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of Strongyloides from humans in the study area in Thailand, where people have frequent contact with long-tailed macaques. Fresh stool samples were obtained from 213 people and were examined using the agar plate culture method. The overall prevalence of Strongyloides infection was 8.92% (19/213). From a total of 19 worms (one per infected person), 18 adult males had 18S rRNA sequences identical with that of S. stercoralis and one adult female had a sequence almost identical with that of S. fuelleborni. A median-joining network of cox1 sequences revealed nine new haplotypes from S. stercoralis, and an overall haplotype diversity (Hd) of 0.9309. The single haplotype of S. fuelleborni was also new and contributed to an overall haplotype diversity for that species of 0.9842. This is the first molecular identification of S. stercoralis and S. fuelleborni in a human community having contact with long-tailed macaques in Thailand. It is also the first report of S. fuelleborni infecting a human in Thailand.

TREMATODE INFECTION OF FRESHWATER SNAIL, FAMILY BITHYNIIDAE IN THAILAND.

Opisthorchis viverrini is restricted to and requires for its aquatic life cycle only Bithynia snail as first intermediate host but many species of cyprinid fish as second intermediate hosts. A survey in Thailand of trematode infection in freshwater snails of the family Bithyniidae carried out during October 2008 - July 2009 found a total of 5,492 snails, classified into ten species distributed in various geographic areas. Bithyniafuniculata and Gabbia pygmaea were localized to the north, B. s. goniomphalos, Wattebledia siamensis and W. crosseana to northeast and B. s. siamensis, Hydrobioides nassa and G. wykoffi to central region. W. baschi and G. erawanensis was found only in the south and Erawan waterfall, Kanchanaburi Province, respectively. Trematode infection rate was 3.15%. Cercariae were identified as belonging to six types, namely, amartae , monostome, mutabile, O. viverrini, virgulate, and unknown. The prevalence of cercarial infection in B. s. goniomphalos of amartae, mutabile, O. viverrini, virgulate, and unknown type cercaria was 0.55%, 0.74%, 1.07%, 2.87%, and 0.37%, respectively, and in B. s. siamensis monostome (1.10%) and virgulate (0.55%). Only virgulate cercariae were shed from W. crosseana (3.85%) and W. siamensis (5.19%). Cercariae of the unknown type were found in G. wykoffi (1.69%). No infection of O. viverrini cercariae was detected in the other species.

Temperature dependence of Opisthorchis viverrini infection in first intermediate host snail, Bithynia siamensis goniomphalos.

Determining of the success of a parasite's infectiveness in its snail host clearly depends on environmental conditions. Temperature, one of the most influential factors impinging on metabolism of cold-blooded animals, is believed to be an important factor in parasitic infection in snails. In order to elucidate the influence of temperature, sex and size of snails on infectivity of Opisthorchis viverrini to its first intermediate host, Bithynia siamensis goniomphalos, 960 snails were divided into 2 groups by sex. Each group was subdivided by their size into small and medium sub-groups. Each snail was fed with embryonated uterine-eggs of O. viverrini at different temperatures (16-37°C, 3°C intervals). Dissections were carried out 1, 7, 14, 28 and 56 days thereafter and detection of O. viverrini infection was undertaken by PCR using specific primers. Infection was strongly temperature-dependent, as temperature increases of 1°C resulted in increased odds of infection 5.4% (P<0.01). A temperature of 34°C gave the highest rate of infection of 44.14%. We also found that the odds of infection in small sized snails was 39.8% higher relative to medium sized snails (P<0.05). Relative to day 1, the decrease in the odds of infection was detected when the day post infection was longer (P<0.01). Proportion of infection in female was not different to male significantly.

Light and electron microscopy observations of embryogenesis and egg development in the human liver fluke, Opisthorchis viverrini (Platyhelminthes, Digenea).

Eggs of most species digenean flukes hatch in the external environment to liberate larvae that seek and penetrate a snail intermediate host. Those of the human liver flukes, Opisthorchis viverrini, hatch within the gastrointestinal canal of their snail hosts. While adult parasites are primarily responsible for the pathology in cases of human opisthorchiasis, their eggs also contribute by inducing granulomata and in serving as nidi for gallstone formation. In view of the peculiar biology of O. viverrini eggs and their contribution to pathology, we investigated embryogenesis in this species by light and transmission electron microscopy. Egg development was traced from earliest stages of coalescence in the ootype until full embryonation in the distal region of the uterus. Fully mature eggs were generally impermeable to resin and could not be examined by conventional electron microscopy methods. However, the use of high-pressure freezing and freeze-substitution fixation of previously fixed eggs enabled the internal structure of mature eggs, particularly the subshell envelopes, to be elucidated. Fertilization occurs in the ootype, and the large zygote is seen therein with a single spermatozoon wrapped around its plasma membrane. As the zygote begins to divide, the spent vitellocytes are pushed to the periphery of the eggs, where they progressively degrade. The early eggshell is formed in the ootype by coalescing eggshell precursor material released by approximately six vitelline cells. The early eggs have a thinner eggshell and are larger than, but lack the characteristic shape of, mature eggs. Characteristic shell ornamentation, the "muskmelon" appearance of eggs, appears after eggshell polymerization in the ootype. Pores are not present in the shell of O. viverrini eggs. The inner and outer envelopes are poorly formed in this species, with the outer envelope evident beneath the eggshell at the opercular pole of the mature egg. The miracidium has a conical anterior end that lacks the distinctive lamellar appearance of the terebratorium of other digeneans, such as the schistosomes. The miracidium is richly glandular, containing an apical gland in the anterior end, large cephalic gland, and posterior secretory glands. Each gland contains a secretory product with different structure. The paucity of vitelline cells associating with eggs, the reduced size of eggs, and reduced complexity of the extraembryonic envelopes are interpreted as adaptations to the peculiar hatching biology of the miracidia.

Effect of soil surface salt on the density and distribution of the snail Bithynia siamensis goniomphalos in northeast Thailand.

Opisthorchis viverrini infection is associated with human cholangiocarcinoma and northeast Thailand has the highest incidence of this disease in the world. Bithynia siamensis goniomphalos is the major freshwater snail intermediate host of O. viverrini in this area and an analysis based on geographical information systems was used to determine the effect of variation in soil surface salt on the density and distribution of this snail. A malacological survey was carried out in 56 water bodies in the Khorat basin, northeast Thailand at locations with various soil surface salt levels. Mollusk samples were collected from 10 ecologically representative water body sites with 10-20 sampling stations in each. The shoreline of clear, shallow water bodies was found to be the preferred B. s. goniomphalos habitat. The snails were exclusively found in water with salinity levels ranging between 0.05 and 22.11 parts per thousand (ppt), which supports the notion that B. s. goniomphalos prefers water with some saline content over pure, freshwater. The highest snail population densities were in rice fields, ponds, road-side ditches and canals within a water salinity range of 2.5-5.0 ppt. However, the presence of B. s. goniomphalos was negatively correlated with water salinity (P ≤0.05), both with regard to density and distribution. The areas with the highest density of B. s. goniomphalos were those with less than 1% soil surface salt (potential index = 0.314), while the lowest densities were found in areas exceeding 50% soil surface salt (potential index = 0.015).