Temperature dependence of Opisthorchis felineus infection in the first intermediate host snail, Bithynia troschelii.

Opisthorchiasis is one of the most serious trematodiases in Russia, where the world's largest focus is located in the Ob basin. Temperature is an important factor affecting the metabolism of cold-blooded animals. It determines the development of the causative agent of opisthorchiasis, Opisthorchis felineus, and the success of infection of an intermediate host, the snail Bithynia troschelii. In the present study, the effect of water temperature on the development of the liver fluke O. felineus in the host snail was assessed, as was the temperature threshold at which B. troschelii hibernation initiates. Adult uninfected B. troschelii individuals collected from natural bodies of water were infected with O. felineus and maintained at different temperatures of water (18-30 °C, intervals of 3 °C) in the laboratory. Each snail was fed with embryonated uterine eggs of O. felineus at 24 °C. O. felineus infection in snails was detected using polymerase chain reaction (PCR) using specific primers. The prevalence of O. felineus infection in B. troschelii depends on the water temperature in which the snails are maintained. The highest infection rate of 45.2 % ± 12.1 % was observed at 27 °C (p ≥ 0.1). The longest lifespan of infected and uninfected B. troschelii was recorded at water temperatures of 24 and 27 °C. The snails were more successfully infected at the beginning of the warm season. Among the infected individuals, the majority (up to 85 %) were large snails. Cercarial shedding was not detected in experimentally infected snails. Apparently, this is due to the natural physiological state of Bithynia snails during the autumn-winter diapause, when opisthorchiids development in snails stops. At 10 °C, complete hibernation of all B. troschelii snails was observed, and infection by the trematodes became impossible. The highest prevalence of infection was recorded at 27 °C, suggesting that during climate warming, an increase in opisthorchiid infection of snails may occur, which must be considered when epidemiological measures are planned.

Molecular genetic species definition of Opisthorchiidae-like cercariae from Bithyniidae snails of Chany Lake (South-Western Siberia, Russia).

The studies of opisthorchiids larval stages associated with Bithyniidae snails can provide important and the most reliable data for opisthorchiidoses foci characterization due to the low mobility of such opisthorchiid's host species as Bithyniidae snails. The foci of opisthorchiosis (caused by Opisthorchis felineus) and metorchiosis (caused by Metorchis bilis) are overlapping in the basins of the Ob and Irtysh Rivers. Thus, difficulties with determining the species of cercariae significantly reduce the accuracy of epidemiological conclusions regarding opisthorchiosis, which has a much higher medical significance. Moreover, M. bilis cercariae identification is complicated by the fact that the focus of metorchiosis caused by Metorchis xanthosomus (infecting fish-eating predatory birds) occurs on the same territory. In this study, we for the first time carried out the molecular genetic identification of West-Siberian opisthorchiid cercariae to verify morphological identification and confirmed O. felineus's main association with Bithynia troschelii snail and M. bilis'-with B. tentaculata snail. Thus, our study applied a two-stage approach: the morphological identification of any opisthorchiid-like cercariae in Bithynia snails and subsequent molecular genetic precise species determination of cercariae in the reduced samples.

DESCRIPTION AND PHYLOGENETIC AFFINITIES OF A NEW SPECIES OF NEOPSILOTREMA (DIGENEA: PSILOSTOMIDAE) FROM LESSER SCAUP, AYTHYA AFFINIS (ANSERIFORMES: ANATIDAE).

Neopsilotrema is a small genus of psilostomid digeneans parasitic in the intestine of birds in the Palearctic and Nearctic. At present, the genus includes 4 species: Neopsilotrema lisitsynae from the Palearctic and Neopsilotrema affine, Neopsilotrema lakotae, and Neopsilotrema marilae from the Nearctic. Herein, we describe a new species, Neopsilotrema itascae n. sp., from lesser scaup Aythya affinis collected in Minnesota. The species can be distinguished from congeners on the basis of the ventral sucker:oral sucker width ratio, body width:length ratio, and cirrus sac size, along with other characters. We generated new 28S ribosomal deoxyribonucleic acid (DNA) and NADH dehydrogenase (ND1) mitochondrial DNA sequence data of a variety of psilostomids from the Palearctic and Nearctic along with sequences of the ribosomal internal transcribed spacer (ITS) region (ITS1 + 5.8S + ITS2) from 3 Neopsilotrema species. The molecular phylogenetic affinities of a variety of psilostomid taxa were studied using 28S sequence data. The 28S sequences of psilostomids demonstrated 1-7.9% intergeneric divergence, whereas the sequences of ND1 had 17.7-34.1% intergeneric divergence. The interspecific divergence among members of Neopsilotrema was somewhat lower (0.2-0.5% in 28S; 0.3-0.4% in ITS; 12-15.7% in ND1). Our comparison of DNA sequences along with morphologic study suggests Holarctic distribution of N. lisitsynae.

A study of tribendimidine effects in vitro and in vivo on the liver fluke Opisthorchis felineus.

BACKGROUND: The food-borne liver fluke Opisthorchis felineus is an epidemiologically important species and the causative agent of opisthorchiasis across an extensive territory of Eurasia. For decades, treatment of opisthorchiasis has been based on praziquantel. Tribendimidine could be an alternative drug that has been successfully tested for Opisthorchis viverrini and Clonorchis sinensis infections. We aimed to assess tribendimidine effects in comparison with praziquantel in vivo and in vitro against the liver fluke Opisthorchis felineus. RESULTS: In this study we (i) calculated half-maximal inhibitory concentrations (IC50) by motility tests against O. felineus adults and newly excysted metacercarie after tribendimidine treatment in vitro; (ii) determined whether tribendimidine and PZQ effects on adult liver flukes are dependent on or mediated by white blood cells; and (iii) tested in vivo the anthelmintic activity of tribendimidine on juvenile and adult worms. We found that the efficiency of tribendimidine in vitro was similar (IC50 = 0.23 µM for newly excysted metacercariae and 0.19 µM for adult worms) to that of praziquantel (IC50 0.98 µM for newly excysted metacercariae and 0.47 µM for adult worms). The treatment of adult worms in vivo with praziquantel or tribendimidine at 400 mg/kg resulted in a 76% and 77.2% reduction, respectively, in the worm burden during chronic infection. CONCLUSIONS: The differences between WBR values after PZQ and TBN treatment were not significant, thus tribendimidine was as effective as praziquantel against O. felineus liver flukes. Given the broad-spectrum activity of tribendimidine and efficacy against O. felineus, this drug may be a promising candidate for the treatment of opisthorchiasis felinea and other liver fluke infections.

The effect of trematode parthenites on the individual fecundity of Bithynia troscheli (Prosobranchia: Bithyniidae).

We studied the long-term infection of Bithynia troscheli (Paasch, 1842) snails with trematodes and estimated the influence of trematode parthenites on the individual fecundity of female snails from the Kargat River (Chany Lake, Russia). The prevalence of B. troscheli females infected by trematode parthenites varied from 7.12% to 17.35% in different years. Eleven redioid species from 5 families and 9 sporocystoid species from 5 families of trematodes were found during this investigation. Snails' fecundity was analysed in relation to the type of infection (redioid or sporocystoid species). Fecund females of B. troscheli were uninfected or they had pre-patent infections from 5 families of trematodes (Psilostomidae, Notocotylidae, Prosthogonimidae, Lecithodendriidae, and Cyathocotylidae). 89.9% of infected B. troscheli females were infertile. Moreover, 13.57% and 1.29% egg capsules (of infected and uninfected females, respectively) were without embryos (χ² = 323.24, p<0.001). The results of the two-way analysis of variability confirmed that trematode parthenites influenced significantly the individual fecundity of B. troscheli. The age of B. troscheli females alone did not alter the individual fecundity, however age in combination with infection by tremathode parthenites influenced significantly the number of normal egg capsules. We also found that under unfavorable environmental conditions the proportion of fertile females increased by 23.7% among uninfected and by 219% among infected females.

A novel nuclear marker, Pm-int9, for phylogenetic studies of Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis (Opisthorchiidae, Trematoda).

Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, the trematodes of the family Opisthorchiidae, are important human parasites. Two previous studies (Kang et al. Parasitol Int 57:191-197, 2008; Katokhin et al. Dokl Biochem Biophys 421:214-217, 2008) have provided evidence using ribosomal and mitochondrial sequences that O. viverrini, O. felineus, and C. sinensis are closely related. We developed a novel nuclear marker, Pm-int9, which included the ninth intron of the paramyosin gene and flanking exon sequences. Samples of O. felineus from four localities of West Siberia, C. sinensis from the Russian Far East, and O. viverrini from Thailand were genotyped by Pm-int9. Little variation was detected in exon sequences, however, intron sequences turned out to be more variable than ribosomal internal transcribed spacers. We can conclude that Pm-int9 is valuable for interspecific variation studies. Phylogenetic analysis based on Pm-int9 revealed that O. viverrini and C. sinensis were closer to each other than either of them to O. felineus, supporting the opinion that C. sinensis should be considered the sister species of Opisthorchis spp.

Temporal variation in prevalence and abundance of metacercariae in the pulmonate snail Lymnaea stagnalis in Chany Lake, West Siberia, Russia: long-term patterns and environmental covariates.

Infrapopulations of trematode metacercariae were monitored in the snail Lymnaea stagnalis over 17 yr (1982-1999) at Chany Lake, Novosibirskaya Oblast', Russia. Eighteen trematode species were recorded. Patterns of occurrence varied from 4 species (Echinoparyphium aconiatum, Echinoparyphium recurvatum, Moliniella anceps, and Cotylurus cornutus) that persisted at relatively high prevalence (> 60% of samples) across sites, seasons, and years, to species that were very rare and sporadic in occurrence. The stability of the 4 common species was probably because of their occurrence either in a wide range of definitive hosts or in a host adapted to the extreme abiotic changes that occurred from year to year in these wetlands. The prevalence and mean abundance of C. cornutus were negatively correlated with water level in the wetlands; its prevalence was also correlated with water temperature. The mean abundance of M. anceps was positively correlated with water level. The most probable explanation for the cyclic dynamics of infections of the common species is change in population sizes and densities of definitive and intermediate hosts, which mediated cyclic alterations in water levels.