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.

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.

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.

First isolation of Hammondia heydorni from dogs in China.

Fecal samples of 945 dogs were examined microscopically in 2 refuge facilities in China from March 2010 to November 2011. In 8 dogs, oocysts, 9-14 µm in size, were found. Their morphology was similar to those of Hammondia heydorni and Neospora caninum. Sporulated Hammondia/Neospora-like oocysts were fed to 2 dogs, 2 gerbils, 2 guinea pigs, and 2 KM mice; tissues from these inoculated animals were then fed to coccidia-free dogs to determine species susceptibility to these oocysts. Oocysts were not excreted in the feces of dogs or rodents inoculated with oocysts. However, the dogs fed the tissues of gerbils or guinea pigs that were inoculated orally with oocysts excreted fresh oocysts. Dogs fed tissues from guinea pigs inoculated with brain and muscular homogenate from guinea pigs that were fed sporulated Hammondia/Neospora-like oocysts did not excrete oocysts. These findings indicated that the oocysts from naturally infected dogs had an obligatory 2-host life cycle, with gerbils and guinea pigs as intermediate hosts. DNA isolated from these oocysts could not be amplified using N. caninum- and Toxoplasma gondii-specific primers. However, positive amplification with the H. heydorni-specific primers confirmed the presence of H. heydorni DNA in the samples. A comparison of the intron 1 sequence of the alpha tubulin gene with those from H. heydorni from dogs and H. triffittae from foxes showed that dog-derived oocysts possessed a different alpha tubulin gene. Both our dog-derived sequence and 2 previous alpha tubulin gene sequences from H. triffittae from foxes contained a 9-bp insertion relative to 3 sequences of H. heydorni from dogs. However, when the 9-bp insertion from H. triffittae sequences were compared, the 9-bp insertion in our dog-derived sequence had a nucleotide substitution. The present study, therefore, provides new evidence of genetic diversity among isolates from dogs. This is the first survey for H. heydorni in dogs from China.

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.

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.

Community structure and seasonal dynamics of helminth parasites in Lepomis cyanellus and L. macrochirus from Charlie's Pond, North Carolina: host size and species as determinants of community structure.

The community structure and seasonal dynamics of 16 helminth species infecting green (Lepomis cyanellus) and bluegill (L. macrochirus) sunfishes in Charlie's Pond, North Carolina, was examined. One hundred and fifty-four fishes including 90 green sunfish and 64 bluegill sunfish were collected between March and November 2000 and examined for the presence of helminth parasites. Five of these species underwent significant changes in abundance in green sunfish infracommunities, 3 of which also displayed seasonal changes in prevalence. Three of the 16 species fluctuated seasonally in bluegill infrapopulations; 2 also underwent changes in prevalence. Species richness and diversity varied across the 9-mo period for both host species, whereas total helminth abundance remained constant. Analysis of component communities revealed differences in community structure for the 2 host species. Bluegills were found to harbor larger and more diverse communities. Bluegills also contained larger infrapopulations of 5 species, whereas green sunfish had greater abundance of 2 species. Interpretation of these data suggests that host species and size are strongly associated with the predictability of community structure.

Patterns of association, nestedness, and species co-occurrence of helminth parasites in the greater kudu, Tragelaphus strepsiceros, in the Kruger National Park, South Africa, and the Etosha National Park, Namibia.

The helminth parasites of the greater kudu from the Kruger National Park (KNP), South Africa, and the Etosha National Park (ENP), Namibia, were examined to determine the major patterns of spatial and demographic variation in community structure and to evaluate nonrandomness in parasite community assembly. Nonmetric multidimensional scaling ordination procedures were used to test for differences in parasite community composition between hosts of the 2 parks and between hosts of different demographic groups within KNP. Infracommunities within KNP were also examined for patterns of nonrandomness using 2 null models, i.e., nestedness and species co-occurrence. Infracommunities of KNP and ENP were significantly different from each other, as were infracommunities of different host demographic groups within KNP. Parasite species in the greater kudu from KNP displayed significant levels of nestedness and were found to co-occur less frequently than expected by chance; however, this lack of co-occurrence was significant only when all demographic groups were considered. When restricted to any particular age class, co-occurrence patterns could not be distinguished from random. Overall, these data suggest that biogeography and host demographics are important factors in determining community organization of helminth parasites in the greater kudu.

Downstream changes in the composition of the parasite community of fishes in an Appalachian stream.

The spatial distribution of 6 parasite species (Myxobolus sp., Dactylogyrus sp., Sterliadochona ephemeridarum, Plagioporus sinitsini, Allopodocotyle chiliticorum, Allocreadium lucyae) was studied in 5 species of fishes (Oncorhynchus mykiss, Clinostomus funduloides, Notropis chiliticus, Rhinichthys atratulus, Semotilus atromaculatus) in Basin Creek, an Appalachian stream in North Carolina. Nonmetric multidimensional scaling and vector fitting were used to determine if the proximity of sampling sites was related to community similarity. Position along Basin Creek was significantly related to parasite community structure. Breaks in parasite community composition were imposed by waterfalls at upstream areas of Basin Creek that restricted distributions of C. funduloides, N. chiliticus, and S. atromaculatus and at the downstream limit of the study area by a break in the distribution of S. ephemeridarum coincident with the existence of a dam but were independent of suitable piscine host distributions. These discontinuities in parasite community composition imply that the relationship between proximity of sites and community similarity is predictive because distance between sites is related to the probability that fish at different sampling sites recruit parasites from different species pools. This relationship is not the same for all component communities.

A perspective on the ecology of trematode communities in snails.

This paper presents a perspective on the ecology of trematodes in snail hosts based on recent evidence. Because flukes use snails almost obligatorily as first intermediate hosts, we highlight the role of gastropods as keystone species for trematodes and their communities. After reviewing recent developments in the transmission of trematodes to and from snails, we discuss trematode communities within individual snails (infracommunities) and in snail populations (component communities). Results garnered using various protocols at the infracommunity level are reviewed. The few data available, all from marine systems, indicate that low colonization rates characterize infracommunities, suggesting that trematode infracommunities tend to be isolationist in character rather than interactive. The variety of trematode species present in a component community seems to be determined by spatial overlap of definitive hosts. Relative abundance of species in a component community shows little dependence on negative interspecific interactions at the level of the infracommunity. Temporal aspects of trematode communities are related to the life history of the host snail. The component communities of long-lived snails (mostly marine) integrate many infection episodes whereas shorter-lived snails (mostly freshwater) acquire new component communities each time host cohorts turnover.

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.

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.

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.

Robust estimation of parasite component community richness.

The performance of 2 nonparametric estimators of species richness, the bootstrap (S(B)) and kth-order jackknife (S(Jk)), are compared using simulated parasite communities. The parameters of the simulation match those of an earlier comparison that favored S(B) as an estimator but did not include S(Jk). S(Jk) is the least biased of the 2 estimators. Whereas the bias of S(B) is significantly affected by true species richness and the proportion of rare species, the bias of S(Jk) is relatively insensitive to changes in these parameters, and is, therefore, recommended as a robust estimator of species richness.

The role of habitat in structuring Halipegus occidualis metapopulations in the green frog.

The transmission dynamics of the trematode Halipegus occidualis in its definitive host, Rana clamitans, have been examined over a 5-yr period in a North Carolina pond. The breeding season of green frogs coincides with the period of worm recruitment, during which time male frogs are territorial and females show strong site fidelity. This site fidelity allows inferences to be made regarding the suitability of a particular habitat for worm transmission based on frog infection intensities within that habitat. Four foci of infection were identified in the pond by plotting worm infrapopulation size against site of host capture. Sites within infection foci are characterized by shallow water and emergent vegetation, factors favorable for overlapping distributions of the 4 hosts in the life cycle of H. occidualis. Consistent year-to-year worm prevalences and intensities, despite fluctuations in frog population size, are thought to be the result of a relatively constant proportion of the frog population being present in infection foci each year. Removal of worms from heavily infected frogs in the fifth year resulted in further heavy worm recruitment by treated frogs suggesting that site selection can predispose a frog to heavy infection. Further, the sum of removed parasites and those recruited after parasite removal by treated frog hosts was higher than worm infrapopulations observed in previous years, indicating that worm density regulates parasite infrapopulation size in heavily infected frogs.