Prevalence, morphology, and molecular characteristics of Sarcocystis spp. in domestic goats (Capra hircus) from Kunming, China.

Despite the importance of worldwide goat production, little is known about the prevalence of Sarcocystis spp. in domestic goats (Capra hircus) in China. The aims of the present study were to determine prevalence of Sarcocystis spp. in domestic goats in Kunming, China, as well as to identify parasite species based on morphological characteristics and DNA sequence analysis. Only microscopic sarcocysts of Sarcocystis spp. were detected in 174 of 225 goats (77.3 %). By light and transmission electron microscopy, two species, i.e., Sarcocystis capracanis and Sarcocystis hircicanis, were identified. Two sarcocysts from each of the two species were randomly selected for DNA extraction; the 18S rRNA gene (18S rRNA), the 28S rRNA gene (28S rRNA), and the mitochondrial cytochrome c oxidase subunit 1 (cox1) were amplified by the polymerase chain reaction (PCR) and subsequently sequenced. The results were compared with other previously sequenced Sarcocystis species retrieved from GenBank. There was little sequence variation between two isolates of the same species. S. capracanis was most closely related with Sarcocystis tenella; 18S rRNA, 28S rRNA, and mitochondrial cox1 sequences shared identities of 95.7-99.1, 95.3, and 92.3-93.2 % with those of S. tenella, respectively. Thus, mitochondrial cox1 sequences seem to perform better than 18S rRNA sequences or 28S rRNA sequences for identification of the two species. S. hircicanis was most closely related to Sarcocystis arieticanis, i.e., 18S rRNA and 28S rRNA sequences of the former species shared 97.2-97.4 and 95.6-96.1 % identities with those of latter, respectively. Phylogenetic analysis inferred from the three genetic markers yielded similar results and indicated the two species were within a group of Sarcocystis species with canines as known, or presumed, definitive hosts.

OCCURRENCE AND GENETIC CHARACTERIZATION OF GRA6 AND SAG2 FROM TOXOPLASMA GONDII OOCYSTS IN CAT FECES, KUNMING, CHINA.

Toxoplasma gondii-like oocysts were detected in 4% of cat feces using light microscopy in Kunming, China. Amplicon was generated from these oocysts using a T. gondii- but not Hammondia hammondi-specific primer set. Cat and KM mouse were susceptible to infection by these T. gondii-like oocysts. Tissue cysts were found in the brain of the experimentally infected animals and were infective to mouse, indicating that the T. gondii-like oocysts isolated from naturally infected cats had a facultative secondary host life cycle. Comparison of GRA6 and SAG2 sequences with those of reference strains indicated that the cat-derived T. gondii (KM isolate) were typical of T. gondii genotype II. This is the first report of isolation, identification, and genotyping of T. gondii from feline feces in China. The occurrence of oocyst shedding in the cat population studied was higher than that found in most cat populations world-wide, which strongly implies the need for further studies of population genetic structure of T. gondii, as well as for prevention and control of T. gondii infection in cats in China.

Sarcocystis clethrionomyelaphis Matuschka, 1986 (Apicomplexa: Sarcocystidae) infecting the large oriental vole Eothenomys miletus (Thomas) (Cricetidae: Microtinae) and its phylogenetic relationships with other species of Sarcocystis Lankester, 1882.

Sarcocystis clethrionomyelaphis Matuschka, 1986 was first identified in skeletal muscles of 47 (75.8%) of 62 large oriental voles Eothenomys miletus (Thomas) captured between March 2012 and May 2014 in Anning Prefecture of Yunnan Province (China). Sarcocyst walls were thick and possessed villous protrusions measuring 3.5-5.5 µm in length. Beauty rat snakes Elaphe taeniura (Cope) fed sarcocysts of the species shed sporulated oöcysts measuring 13-18×9-13 (16×12) µm with a prepatent period of 16 to 17 days. Phylogenetic analysis based on 18S rRNA gene sequences revealed a close relationship between S. clethrionomyelaphis and other colubrid-transmitted species of Sarcocystis Lankester, 1882. This is the first report identifying S. clethrionomyelaphis from its natural intermediate host.

Quantitative estimation of the cost of parasitic castration in a Helisoma anceps population using a matrix population model.

Larval trematodes frequently castrate their snail intermediate hosts. When castrated, the snails do not contribute offspring to the population, yet they persist and compete with the uninfected individuals for the available food resources. Parasitic castration should reduce the population growth rate lambda, but the magnitude of this decrease is unknown. The present study attempted to quantify the cost of parasitic castration at the level of the population by mathematically modeling the population of the planorbid snail Helisoma anceps in Charlie's Pond, North Carolina. Analysis of the model identified the life-history trait that most affects lambda, and the degree to which parasitic castration can lower lambda. A period matrix product model was constructed with estimates of fecundity, survival, growth rates, and infection probabilities calculated in a previous study. Elasticity analysis was performed by increasing the values of the life-history traits by 10% and recording the percentage change in lambda. Parasitic castration resulted in a 40% decrease in lambda of H. anceps. Analysis of the model suggests that decreasing the size at maturity was more effective at reducing the cost of castration than increasing survival or growth rates of the snails. The current matrix model was the first to mathematically describe a snail population, and the predictions of the model are in agreement with published research.

Life history cost of trematode infection in Helisoma anceps using mark-recapture in Charlie's Pond.

Parasitism has the potential to affect key life history traits of an infected host. Perhaps the most studied interactions are in snail-trematode systems, where infection can result in altered growth rates, survival, and/or fecundity of the individual. Positive correlations between host size and parasite prevalence are often attributed to changes in growth rates or mortality, which have been observed in the laboratory. Extending lab-based conclusions to the natural setting is problematic, especially when environmental conditions differ between the laboratory and the field. The present study uses reproduction experiments and mark-recapture methods to directly measure key life history traits of the pulmonate snail Helisoma anceps in Charlie's Pond. Based on previous laboratory and field experiments on H. anceps, we predict a significant reduction in fecundity, but not growth rate or survival, of infected snails. Individual capture histories were analyzed with multistate models to obtain estimates of survival and infection probabilities throughout the year. Recaptured individuals were used to calculate specific growth rates. Trematode infection resulted in complete castration of the host. However, neither survival nor growth rates were found to differ between infected and uninfected individuals. The probability of infection exhibited seasonal variation, but it did not vary with size of the snail. These results suggest that the correlation between host size and trematode prevalence is not due to differential mortality or changes in growth rates. Instead, the infection accumulates in large snails via the growth of smaller, infected individuals.

Long-term analysis of Charlie's Pond: fecundity and trematode communities of Helisoma anceps.

Charlie's Pond (North Carolina) harbors a diverse community of trematodes that infect the planorbid snail Helisoma anceps. Research at the Pond began in 1984 and serves as a foundation on which to investigate long-term changes in trematode communities. In 2002, 2005, and 2006 average size and fecundity of H. anceps were calculated each month, and seasonal trends analyzed with randomization tests. Concomitantly, trematode infections were recorded, and the community composition compared to those from previous studies. Helisoma anceps in 2002, 2005, and 2006 were smaller and less fecund than snails in 1984. The trematode community was consistently diverse, with 11 species recovered in 2006 versus 7 in 1984. However, the prevalence of Halipegus occidualis was much lower than previously observed (60% in 1984) and never exceeded 20% during the latter years. The decline of emergent vegetation is likely contributing to these changes. Aquatic macrophytes increase the surface area for growth of periphyton, the food source of these snails. Limited food supplies result in lower snail growth rates and fecundity. Similarly, emergent vegetation creates foci of transmission for H. occidualis between the frog definitive host and the snail intermediate host. When these areas are lost from the Pond, probability of transmission is reduced, and prevalence in the snail declines.

Morphology, Molecular Characteristics, and Demonstration of a Definitive Host for Sarcocystis rommeli from Cattle (Bos taurus) in China.

Sarcocysts of Sarcocystis rommeli were found for the first time in 6 of 34 (17.6%) cattle (Bos taurus) in China. With light microscopy, sarcocysts of S. rommeli were up to 1,130 µm long, with a striated, 4-8-µm-thick cyst wall. Using transmission electron microscopy, the villar protrusions (vp) were 4.7-5.2 × 0.2-0.3 µm, and 0.3-0.5 µm apart from each other. The vp contained microtubules extending from the top of the vp to the middle of the ground substance layer (gsl). A BLAST search of the near full-length 18S rRNA and partial mitochondrial cox1 sequences of S. rommeli revealed 98.7% identity and 99.2% identity with sequences of Sarcocystis bovini in GenBank, respectively. Two domestic cats (Felis catus) fed sarcocysts of S. rommeli shed oocysts/sporocysts in their feces with a prepatent period of 14 to 15 days; the partial mitochondrial cox1 sequences of these oocysts/sporocysts shared the high identities, that is, 99.4% and 99.5%, with cox1 sequences of S. rommeli sarcocysts and S. bovini sarcocysts, respectively. This is the first demonstration of a definitive host for S. rommeli.

An examination of the infracommunities and component communities from impala (Aepyceros melampus) in the Kruger National Park, South Africa.

The intestinal helminth parasites of the impala from the Kruger National Park, South Africa, were examined to describe the parasite community structure. Demographic variation and the associated differences in behavior were used to further investigate the patterns of community composition. Monte Carlo simulations were performed to test for differences in species richness and mean abundance between the various demographic groups, and nonmetric multidimensional scaling ordination was used to compare community composition. Seventeen species of nematodes, totaling more than 1.3 million worms, were recovered. Males harbored a greater number of nematode species than did females, but adult females were more heavily infected than their male counterparts. Lambs acquired infections early in life, and their parasite community composition rapidly approached that of the older animals. The parasite community in the juvenile and adult males was significantly different from the community of the adult females. These data suggest that social and feeding behavior of the different age-sex classes structure the parasite component community of impala. Additionally, the distinction between common and rare parasites, and their classification in other herbivores, implies complex transmission dynamics that includes extensive species sharing within the Kruger National Park.

Prevalance, Morphology, and Molecular Characterization of Sarcocystis heydorni Sarcocysts from Cattle (Bos Taurus) in China.

Cattle are intermediate hosts for 2 zoonotic species of Sarcocystis, Sarcocystis hominis and Sarcocystis heydorni. Here we report S. heydorni from cattle for the first time in China. Sarcocysts of S. heydorni were found in muscle from 173 of 1,630 (10.6%) cattle in abattoirs (9.7% in skeletal muscles, 3.4% esophagus, 2.5% diaphragm, and 0.1% tongue; heart muscle was negative). By means of light microscopy, S. heydorni sarcocysts were thin-walled (<1 µm). Using transmission electron microscopy, the sarcocyst wall had short (0.3-0.5 × 0.5-0.9 µm) stubby protrusions, the tips of which contained electron-dense, disk-shaped plaques, similar to the sarcocyst wall type 29b. In preliminary transmission attempts, a human volunteer did not excrete sporocysts in feces after ingesting 579 sarcocysts S. heydorni isolated from cattle. Phylogenetic analysis using the 2 molecular markers (18S rRNA gene and mitochondrial cox1 gene) indicated S. heydorni shared the closest affinity with species of Sarcocystis, which employ ruminants as intermediate hosts and canids as definitive hosts.

Temperature effects on survival and excystment of cercariae of Halipegus occidualis (Trematoda).

Survival of the cystophorous cercariae of Halipegus occidualis (Trematoda: Hemiuridae) from naturally infected Helisoma anceps (Gastropoda: Planorbidae) was determined following constant temperature storage at 4, 16, 22, 26 and 30 degrees C. At 4 degrees C, 90% of cercariae survived for 14 weeks and 50% survived for 30 weeks. Survival varied inversely with temperature, and at 30 degrees, 90% survived for 1.5 weeks and 50% survived for 2.5 weeks. At the time of release from snails, cercariae excysted within 0.5 s of stimulation. Excystment times ultimately increased to 1-3 s at all temperatures as length of storage increased, but no connection was detected between excystment time and the activity of the cercaria. Excystment times began increasing while survival was still above 90%. Temperature specific mortality rates of cercariae were estimated, and used to simulate the survival of cercariae released naturally at various times of the year. The results of these simulations suggest that cercariae of H. occidualis are present in the environment at all times of the year, but that cercariae present in late winter (January to mid-March) may have an impaired excystment mechanism that reduces their infectivity.

Relationship between structure and stability of a Halipegus occidualis component population in green frogs: a test of selective treatment.

The aggregated nature of helminth parasite populations has led to the suggestion that selectively treating heavily infected hosts can efficiently reduce parasite abundance and morbidity within a host population. Moreover, it has been postulated that a selective treatment protocol might have long-term effects on parasite transmission by disrupting the stability attributed to aggregated parasite population distributions by theoretical models. Long-term investigation has demonstrated year-to-year consistency in the population dynamics of Halipegus occidualis in green frogs from Charlie's Pond, North Carolina. In 1996, removal of all but 1 worm from each frog with > or =15 worms reduced the estimated component worm population by 45%, thereby decreasing mean intensity and aggregation (variance-to-mean ratio) of H. occidualis in the frogs by 85% and 63%, respectively. The following year, mean intensity, aggregation, and host colonization trends returned to pretreatment levels, indicating no effect of worm removal and demonstrating the stability of this host-parasite system. Although this result might be attributable to inefficient treatment or the presence of infection reservoirs, it is suggested that parasite population stability in this system might be governed by prevalence rather than intensity of adult worms. Therefore, repeated selective treatment might effectively modify intensity-dependent morbidity in similar host-parasite systems but should not affect further parasite transmission.

Plagioporus sinitsini (Digenea : Opecoelidae): a one-host life cycle.

Adult Plagioporus sinitsini occur within daughter sporocysts voided with the feces of prosobranch snails Elimia symmetrica in Basin Creek, North Carolina. These worms produced eggs containing active miracidia while still in the snail. Adults in snails and adults in rosyside dace, Clinostomus funduloides, collected from the same stream were indistinguishable morphometrically. Adults in snails develop from cotylocercous cercariae sequestered in daughter sporocysts that pass through the metacercaria stage. These observations, and previous study in Michigan, suggest that the life cycle of P. sinitsini has 3 potential pathways, i.e., a 3-host life cycle involving molluscan, arthropod, and piscine hosts, a 2-host life cycle involving only molluscan and piscine hosts, and a 1-host life cycle involving only the snail host. The truncated life cycles do not appear to be the result of paedomorphosis.

Snail size as a determinant of prepatent period duration in Helisoma anceps infected with Halipegus occidualis (Digenea: Hemiuridae).

Pulmonate snails (Helisoma anceps) of varying sizes were exposed to 2, 4, 8, or 16 eggs of Halipegus occidualis and observed over several months to determine the duration of the prepatent period. Infection probability was positively correlated with number of eggs ingested. The number of eggs ingested did not, however, have a significant effect on the duration of the prepatent period, presumably because low infectivity of the eggs (15%) dictated that most patent infections arose from a single miracidium. There was a significant, positive correlation between snail length (measured at time of exposure) and duration of prepatent period, suggesting that density-dependent effects are, in part, responsible for the initiation of cercariae production in H. occidualis.

The effect of diet and reproductive maturity on the growth and reproduction of Helisoma anceps (Pulmonata) infected by Halipegus occidualis (Trematoda).

Inhibition of snail reproductive activity by larval trematodes often results in increased growth of the snail, termed gigantism, during which the snail is hypothesized to allocate excess energy normally used for reproduction to somatic growth. Using the planorbid snail Helisoma anceps and the hemiurid trematode Halipegus occidualis, this hypothesis was examined by raising snails on low and high quality diets under 3 infection conditions: uninfected (UNI) and infected prior to (IPR) and after (IAR) reproductive maturity. Snails exhibited gigantism as an increase in wet mass only in the IPR condition on a high quality diet; snails in the IAR condition were not significantly different in size from UNI controls. Throughout the experiment, snails raised on a high quality diet were larger than those on the low quality diet, regardless of infection status, primarily because of increased growth rates prior to the onset of reproduction. Egg production by UNI snails on the high quality diet was 20 times higher than snails on the low diet, even though diet quality only differed by a factor of 4, and infected snails (both IPR and IAR) showed significantly reduced reproductive activity compared to UNI controls. The number of cercariae shed by each snail on a daily basis was not significantly different between IPR and IAR conditions when each diet quality was examined separately; more cercariae were produced by snails raised on the high quality diet. The prevalence of infection at the end of the experiment was lowest among snails raised on the low quality diet, suggesting the diet, i.e., energy intake, influences the loss of infections. Snail mortality did not differ among the various treatment conditions. These data suggest that gigantism is dependent upon snail reproductive maturity at the time of infection and food quality, and comparison to growth rates and fecundity data from previous field studies indicates that H. anceps in the field are probably protein deprived. Gigantism does not appear to be adaptive for the snail because survival was not increased, or for the parasite because cercariae production was the same for snails exhibiting gigantism or not. These results also stress the need for caution when examining the fecundity and growth rates of both uninfected and infected snails in the laboratory, especially when comparing these data to other laboratory or field studies.

Guild structure of larval trematodes in the snail Helisoma anceps: patterns and processes at the individual host level.

Factors that influenced the infracommunity structure of trematodes parasitizing the pulmonate snail Helisoma anceps were studied over a 15-mo period; the guild included 8 species of parasites. Infracommunities were depauperate, with double patent infections observed in only 7 of 1,485 infected snails; a total of 4,899 was examined. Halipegus occidualis-Haematoloechus longiplexus was the most common dual infection. Both species share the same definitive host and, in both cases, eggs are the infective stage for the snail. Switches and losses of infections in individual snails were observed, suggesting the occurrence of dynamic interactions within the guild. A dominance hierarchy was constructed based on field observations and experimental infections. Echinostomatids were dominant; species without rediae in their life cycles were subordinates. Halipegus occidualis (which has rediae) was intermediate in dominance. Spatial and temporal heterogeneity in the distribution and abundance of trematode infective stages indicate that not all the snails have the same probability of becoming infected. Habitat structure, behavior of the definitive host, the nature of the infective stages, and snail population dynamics (mortality, recruitment, and size structure) generated spatial and temporal heterogeneity in this system. As a consequence, predictions of the probabilities of interspecific interactions based on an analysis of observed and expected frequencies of multiple infections could be inappropriate unless the potential sources of heterogeneity are considered.

The component community structure of larval trematodes in the pulmonate snail Helisoma anceps.

Factors that affected the component community structure of larval trematodes in the pulmonate snail Helisoma anceps in Charlie's Pond, North Carolina, were studied over a 15-mo period using a multiple mark-recapture protocol. Patent infections of 8 species were observed in 1,485 of 4,899 snails examined. Reproductive activity, population size, and survival rate of the snail population were estimated to evaluate the extent of resource availability for the parasites. Antagonistic interactions between trematode species that occurred at the infracommunity level had a neglible effect on the composition and structure of the component community. The patterns observed at this level were related to temporal heterogeneity in the abundance of infective stages (mostly miracidia), differential responses of trematode species to the diverse and constantly changing distribution of snail size and abundance, differential mortality of snails infected with certain trematode species, constant recruitment of 1 trematode species over time, and the existence of predictable disturbances such as the complete mortality of the host population and recruitment of a replacement population during a 6-8 wk period. The last factor operated as a reset mechanism for this snail-trematode system once each year. A model of patch dynamics, with snails as patch resources, best explains the organization and dynamics of this system.

Allopodocotyle chiliticorum n. sp. (Digenea: Opecoelidae) from redlip shiners, Notropis chiliticus, in Basin Creek, North Carolina.

Allopodocotyle chiliticorum n. sp. is described from the intestines of redlip shiners (Notropis chiliticus) from Basin Creek, North Carolina. The new species is characterized by circumcecal vitelline fields that are not confluent in the post-testicular space, which distinguishes it from the 3 previously known species of this genus described from freshwater fishes. The new species is characterized further by an elongate vitelline reservoir lying dorsal to the ovary, an ovary as large or larger than the testes, and an excretory vesicle not reaching the posterior testis. A. chiliticorum n. sp. most closely resembles Allopodocotyle lepomis (Dobrovolny, 1939) in body shape, testes shape, and terminal genitalia but is distinguished further from this species by the extent of the intestinal ceca. The new species is the fourth species of Allopodocotyle known from freshwater fishes in North America.

Seasonal population dynamics of Halipegus occidualis and Halipegus eccentricus (Digenea:Hemiuridae) in their amphibian host, Rana clamitans.

The seasonal population dynamics of Halipegus occidualis and Halipegus eccentricus (Hemiuridae) in their amphibian host, the green frog (Rana clamitans), were examined for 3 yr (weekly, April through October). Frogs were caught, marked, and examined for H. occidualis and H. eccentricus, both of which occur as immatures and adults in the buccal cavity. Frogs were then released and allowed to continue natural recruitment and loss of the parasites. It was thus possible to monitor individual infrapopulations over successive time periods. One-hundred and forty-nine frogs were caught and released, with a total of 328 observations. Overall, the levels of both parasites among male and female frogs were similar, as were levels of infection among adult and juvenile hosts. There was, however, no correlation between the total number of H. occidualis and the body size (snout-vent length) of R. clamitans. In contrast, there was a significant correlation between the total number of H. eccentricus and frog size. Recruitment of both species began in May, peaked in June/July, and ended in July (H. eccentricus) or August (H. occidualis). In general, both the prevalence and relative density of H. occidualis was greater than that of H. eccentricus and may be related to space constraints in the buccal cavity of R. clamitans. Adults of both species were observed from April through October. Following increases in parasite recruitment, infrapopulation sizes declined in September 1992 and in August of 1993 and 1994. Examination of variance to mean ratios indicated that both species were overdispersed in the frogs. Large declines in the variance to mean ratios for H. occidualis after periods of greatest recruitment are most likely associated with the loss of larger infrapopulations, suggesting that there may be density-dependent regulation of infrapopulation size. By monitoring individual hosts using the mark-release-recapture protocol, dynamic changes in parasite infrapopulations were observed, e.g., there were losses of immature worms and rapid changes in infrapopulation sizes, observations that would not be made with typical host-parasite systems.

The suprapopulation dynamics of Bothriocephalus acheilognathi in a North Carolina reservoir: abundance, dispersion, and prevalence.

A 2 1/2-year study (September 1980-March 1983) of abundance, dispersion, and prevalence of the pseudophyllidean cestode, Bothriocephalus acheilognathi, in 3 species of fish (Gambusia affinis, Notropis lutrensis, and Pimephales promelas) was conducted in 3 ecologically distinct areas of a North Carolina cooling pond. Mean infrapopulation density and prevalence differed by site, season, and species and size of hosts. Degree of aggregation and abundance and prevalence of gravid worms differed by species of host. Abundance of gravid worms was significantly lower in metapopulations from localities that received power plant effluents. The differences in infrapopulation density, prevalence, and aggregation appeared to be related to predator-prey interactions, which varied with season and local community structure. Differences in abundance of gravid worms, on the other hand, were probably caused by differential suitability of hosts and by local variation in selenium concentration in the water column. Thus, it appears that both biotic and abiotic components of the host community determined the suprapopulation dynamics of B. acheilognathi in Belews Lake.

Trematode community structure in the pulmonate snail Physa gyrina.

Trematode community structure was examined in the pulmonate snail Physa gyrina over a 12-mo period. A total of 1,181 snails was collected from Charlie's Pond, North Carolina, and was found to be parasitized by 6 trematode species. Infracommunities were relatively species rich when compared to previous studies, with 18.4% of infected snails having multi-species infections. Halipegus eccentricus was found in 88.4% of multiple infections, usually with 1 of 2 other autogenic, egg-transmitted species (Haematoloechus complexus and Glypthelmins quieta). Neither negative interspecific interactions nor a dominance hierarchy were apparent among the trematodes. These factors, along with high snail vagility and the temporal heterogeneity of infective stages in the environment, are suggested to have contributed to the high number of multiple infections. Halipegus eccentricus was also the most prevalent parasite throughout the study, occurring in nearly 50% of all snails during May, June, and July. Apparent parasite-induced host mortality caused by this trematode species contributed to increasing species diversity in larger snails. The component community was also affected by the continuous recruitment of new snails into the host population and by changes in the number of infective stages present over time.