Prevalence and genetic characterization of eimeriid coccidia from feces of black-necked cranes, Grus nigricollis.

Disseminated visceral coccidiosis (DVC) is a widely distributed intestinal and extraintestinal disease of cranes caused by eimeriid coccidia and has lethal pathogenicity to several crane species. Here, feces of 164 black-necked cranes collected in Dashanbao Black-necked Crane National Nature Reserve, China, were examined to determine the prevalence of coccidial oocysts. Of the 164 fecal samples, 76 (46.3%) were positive for oocysts of Eimeria, including E. gruis in 59 (35.9%), E. reichenowi in 52 (31.7%), and E. bosquei in 47 (28.7%) by microscopic observation. Sixty-eight (89.5%) of these positive samples included two or more morphologically identifiable species of Eimeria. The nearly full length 18S rRNA gene (18S rRNA; about 1.8 kb) and partial mitochondrial cytochrome c oxidase I gene (COX1; about 1.3 kb) from oocysts of each morphologically distinct species of Eimeria were amplified, sequenced, and analyzed. BLAST searches using these new 18S rRNA sequences for E. gruis, E. reichenowi, or E. bosquei showed the most similar sequences were those of E. gruis (98.7-99.7% identity), E. reichenowi (97.9-100% identity), or E. gruis (98.6-99.6% identity) isolated from different species of Grus. BLAST searches using the new COX1 sequences for the three species of Eimeria showed that no nucleotide sequences of Eimeria and Isospora coccidia in GenBank have more than 83.0% identity with these species. Identities among the new COX1 sequences were 91.8% for E. gruis and E. reichenowi, 94.5% for E. gruis and E. bosquei, and 91.3% for E. reichenowi and E. bosquei. Phylogenetic analysis based on 18S rRNA or COX1 sequences indicated that Eimeria spp. in black-necked cranes were clustered together with other previously identified Eimeria species from different cranes.

Sarcocystis dehongensis n. sp. (Apicomplexa: Sarcocystidae) from water buffalo (Bubalus bubalis) in China.

Water buffalo (Bubalus bubalis) is the intermediate host for at least four species of Sarcocystis: S. fusiformis, S. buffalonis, S. levinei, and S. sinensis/S. dubeyi. Here, a new species, Sarcocystis dehongensis, is reported in 51 of 756 (6.7%) water buffaloes in China. By light microscopy, the cysts of S. dehongensis were macroscopic, up to 18.5 mm long and 95 µm in diameter; 4.9-11.9 µm villous protrusions extended beyond the sarcocyst wall. Using transmission electron microscopy, the sarcocyst wall had lancet- or leaf-like protrusions in longitudinal section, but the cross section showed that the protrusions appeared as mushroom-like in shape with a core of tightly packed microtubules, similar to "type 24." BLAST searches revealed that S. dehongensis shared the most similarities with the 18S rDNA sequence of S. hardangeri (92.4%) and mitochondrial cox1 gene sequence of S. ovalis (81.0%), whereas no sequences in GenBank were found to be significantly similar to the ITS-1 region of S. dehongensis. A phylogenetic analysis based on 18S rDNA and mitochondrial cox1 gene sequences suggested that S. dehongensis was closely related to Sarcocystis species from cervids that employ corvids as definitive hosts.

Sarcocystis menglaensis n. sp. (Apicomplexa: Sarcocystidae) from Williamson's mouse deer Tuagulus williamsoni (Kloss) (Artiodactyla: Tragulidae).

Williamson's mouse deer, Tuagulus williamsoni (Kloss), is one of the smallest ungulates among tragulid species found in northern Thailand, and Yunnan Province, China. Here we describe Sarcocystis menglaensis n. sp., infecting two of 14 (14.3%) Williamson's mouse deer from south-western China. By light microscopy, sarcocysts of S. menglaensis are microscopic, up to 2,170 µm in length, and have a striated sarcocyst wall with 1.5-3.6 µm long palisade-like protrusions. Transmission electron microscopy observations revealed that sarcocyst wall is of "type 10f", and has numerous villar protrusions folded over the cyst wall. The villar protrusions contained microtubules dispersed throughout the protrusions. Phylogenetic analysis based on 18S rDNA and mitochondrial cox1 gene sequences indicated that S. menglaensis shared a close affinity with species of Sarcocystis Lankester, 1982 from ruminants, which utilise felids as definitive hosts.

Sarcocystis eothenomysi n. sp. (Apicomplexa: Sarcocystidae) from the large oriental vole Eothenomys miletus (Thomas) (Cricetidae: Microtinae) from Anning, China.

Fifty-six oriental voles, Eothenomys miletus (Thomas), were collected in Anning prefecture of Yunnan Province (China) between March 2012 and December 2013 and examined for the presence of sarcocysts. Sarcosysts of a new species, Sarcocystis eothenomysi n. sp., were found in 14 out of 56 E. miletus (25%); they possessed a striated cyst wall, c.1-2 µm thick. Under transmission electron microscopy the cysts of S. eothenomysi exhibited numerous small, irregular protrusions, which may appear T-shaped in some sections. A phylogenetic analysis based on 18S rRNA gene sequences indicated that S. eothenomysi shares closest affinity with those species of Sarcocystis Lankester, 1982, which use cobra or viperid snakes as definitive hosts. We therefore, hypothesise that a venomous snake may serve as the definitive host for S. eothenomysi. This is the first species of Sarcocystis reported from Eothenomys spp.

Snail species diversity impacts the infection patterns of Echinostoma spp.: Examples from field collected data.

Rapid losses of biodiversity due to the changing landscape have spurred increased interest in the role of species diversity and disease risk. A leading hypothesis for the importance of biodiversity in disease reduction is the dilution effect, which suggests that increasing species diversity within a system decreases the risk of disease among the organisms inhabiting it. The role of species diversity in trematode infection was investigated using field studies from sites across the U.S. to examine the impact of snail diversity in the infection dynamics of both first and second intermediate larval stages of Echinostoma spp. parasites. The prevalence of Echinostoma spp. sporocysts/rediae infection was not affected by increases in snail diversity, but significant negative correlations in metacercariae prevalence and intensity with snail diversity were observed. Additionally, varying effectiveness of the diluting hosts was found, i.e., snail species that were incompatible first intermediate hosts for Echinostoma spp. were more successful at diluting the echinostome parasites in the focal species, while H. trivolvis, a snail species that can harbor the first intermediate larval stages, amplified infection. These findings have important implications not only on the role of species diversity in reducing disease risk, but the success of the parasites in completing their life cycles and maintaining their abundance within an aquatic system.

Sarcocystis spp. in domestic sheep in Kunming City, China: prevalence, morphology, and molecular characteristics.

Sheep (Ovis aries) are intermediate hosts for at least six named species of Sarcocystis: S. tenella, S. arieticanis, S. gigantea, S. medusiformis, S. mihoensis, and S. microps. Here, only two species, S. tenella and S. arieticanis, were found in 79 of 86 sheep (91.9%) in Kunming, China, based on their morphological characteristics. Four genetic markers, i.e., 18S rRNA gene, 28S rRNA gene, mitochondrial cox1 gene, and ITS-1 region, were sequenced and characterized for the two species of Sarcocystis. Sequences of the three former markers for S. tenella shared high identities with those of S. capracanis in goats, i.e., 99.0%, 98.3%, and 93.6%, respectively; the same three marker sequences of S. arieticanis shared high identities with those of S. hircicanis in goats, i.e., 98.5%, 96.5%, and 92.5%, respectively. No sequences in GenBank were found to significantly resemble the ITS-1 regions of S. tenella and S. arieticanis. Identities of the four genetic markers for S. tenella and S. arieticanis were 96.3%, 95.4%, 82.5%, and 66.2%, respectively.

Microhabitat Differences in the Benthic Substrata Affect Parasitism in a Pulmonate Snail Host, Helisoma anceps.

The microhabitats in which hosts live can potentially influence the ability and success of parasites in finding and infecting these hosts. The infection dynamics of both digenetic trematode parasites and a nematode parasite (Daubaylia potomaca) infecting a pulmonate snail, Helisoma anceps , were observed in a small North Carolina lake using 3 different classifications of substratum type based on percent coverage by leaves and debris. There were no differences in snail site occupancy or density between substratum types, but small-scale differences in microhabitat impacted parasite prevalence and intensity in their gastropod hosts. Snails inhabiting substrata covered in more leaf litter and debris had a lower prevalence and intensity of infection by all of the trematode species and life stages when compared to H. anceps inhabiting other substrata types, while only the intensity of infection was impacted in D. potomaca infections. These findings emphasize the importance of microhabitat, specifically its physical components, in influencing parasite infection in intermediate hosts and that small-scale differences may significantly affect the patterns of infection.

Tranmission pattern differences of miracidia and cercariae larval stages of digenetic trematode parasites.

Digenetic trematodes have complex life cycles involving multiple hosts and free-living larval stages. Some species have 2 lar-val stages that infect snails, with miracidia and cercariae using these molluscs as first and second intermediate hosts, respec-tively. Although both larval stages may infect the same snail species, this is accomplished using different chemical cues and may be influenced by different biotic and abiotic factors. Significant differences in the infection patterns of these parasitic stages regarding host size and density were observed in 2 separate field studies. The prevalence of sporocysts/rediae and mean abundance of Echinostoma spp. metacercariae infection were positively correlated with host size, while the prevalence of Echinostoma spp. cercariae infection was positively correlated with host density across 5 different pulmonate snail species. Larger snails within a given species tend to be older and the increased exposure time may be responsible for the positive correlations with host size. Additionally, infection by miracidia in more vagile snail hosts was influenced by trematode species richness at a sample site, which may be attributed to increased encounter rate as a result of increased movement by the snail hosts. Echinostoma spp. metacercariae prevalence was influenced by host density, possibly due to high abundances of larval clones and their response to more generalized chemical cues attributed to low host specificity by cercariae. Although they can infect the same gastropod hosts, miracidia and cercariae infection are dependent on different factors at both the individual and population level of their snail hosts.

Autogenic-allogenic status affects interpond community similarity and species area relationship of macroparasites in the bluegill sunfish, Lepomis macrochirus, from a series of freshwater ponds in the Piedmont area of North Carolina.

The effects of autogenic-allogenic status on the species-area relationship and the relationship between geographic distance and intercommunity dissimilarity were investigated in macroparasite communities of the bluegill sunfish Lepomis macrochirus. Rank correlation analyses were used to examine the relationship between pond surface area and species richness of all species collectively and of autogenic species and allogenic species separately. A positive relationship was found for allogenic species, whereas there was no association for all species, nor was there an association when the study was restricted to autogenic species. Mantel tests were used to determine the relationship between geographic distance and community dissimilarity for all species and for autogenic and allogenic species independently. Total community dissimilarity and allogenic dissimilarity were found to be influenced by geographic distance, whereas autogenic dissimilarity was random with regard to interpond distances. These findings serve to illustrate the importance of the autogenic-allogenic dichotomy and demonstrate that dispersal ability can influence commonly observed ecological patterns.

Variation in life cycle affects the distance decay of similarity among bluegill sunfish parasite communities.

Rates of community similarity decay were examined among parasite communities of the bluegill sunfish from isolated populations located throughout the midwestern and southeastern United States. Rates of decay were compared for assemblages composed of all species, autogenic species only, and allogenic species only. Rates of similarity decay were calculated by regressing a matrix of intercommunity distance against a matrix of intercommunity Sorenson similarity for each group of species. Significance was determined with the use of a Mantel test using 1,000 permutations. Significant rates of decay were found for all groups; however, allogenic similarity decayed according to an exponential function, whereas autogenic similarity decayed according to a linear function. Overall, the results suggest that autogenic-allogenic status is an important factor determining the rate at which similarity decays, but illustrate the need for more insight regarding the factors that affect parasite dispersal ability.

Auto-infection by Echinostoma spp. cercariae in Helisoma anceps.

Auto-infection is a life history strategy used by many parasitic organisms, including digenetic trematodes. The process of autoinfection most frequently involves the transfer of a life cycle stage of the parasite from one site to another inside the same host, usually accompanied by morphological transformation. Moreover, among trematodes, the stage being transferred may also move from one host to another in completing the life cycle, i.e., an indirect cycle. Echinostoma spp. parasites offer the opportunity to study auto-infection because they utilize gastropods as both first and second intermediate hosts. Rejection of a null model predicting independent infection of first and second intermediate larval stages coupled with the presence of rediae being the best predictor of metacercariae prevalence and intensity suggests that auto-infection by Echinostoma spp. cercariae is occurring in their molluscan hosts. Shell length was also found to be a significant predictor of metacercariae intensity in the snails hosts, but this is most likely attributed to larger snails being more commonly infected with Echinostoma spp. rediae as opposed to an increased likelihood of cercariae infection. Auto-infection as a life history strategy increases transmission success of the parasite, but may also have negative consequences for the parasite that necessitate auto-infection coupled with the release of cercariae to maximize transmission success and host survival.

Host specificity and the distribution-abundance relationship in a community of parasites infecting fishes in streams of North Carolina.

A positive relationship between distribution and local abundance is often observed among species in a community. The resource-breadth hypothesis suggests that this pattern is the result of differential abilities among species to utilize available resources, such that generalists are widely distributed and locally abundant, and specialists are narrowly distributed and locally sparse. This hypothesis was tested in a community consisting of 22 species or morphospecies of parasites infecting members of 18 species of fish among 14 sites in 7 small streams in the Appalachian Mountains of North Carolina. A positive relationship between distribution (fraction of sites occupied) and abundance (average local abundance) was evident among parasite species. The number of host species infected by each parasite species was positively related to both distribution and average local abundance; both relationships held after statistical removal of the distribution and abundance of the hosts, respectively. These results support the resource-breadth hypothesis as an explanation for the distribution-abundance relationship in this system.

Differences in snail ecology lead to infection pattern variation of Echinostoma spp. larval stages.

The infection patterns of parasites are often tied to host behavior. Although most studies have investigated definitive hosts and their parasites, intermediate host behavior may play a role in shaping the distribution and accumulation of parasites, particularly the larval stages. In an attempt to answer this question, more than 4,500 pulmonate snails were collected from 11 states in the mid-Atlantic and Midwestern United States in the summer of 2012. These snails were necropsied and echinostome metecercariae were commonly observed infecting the snails as 2(nd) intermediate hosts (20.0%). The snails included species of 3 genera with distinct differences in the infection patterns of Echinostoma spp. metacercariae among them. Physa spp. (comprising of P. acuta and P. gyrina) snails exhibited a significantly higher prevalence of infection (23.5%) than both Lymnaea columella (11.6%) and Helisoma spp. (comprising of H. anceps and H. trivolvis) (14.2%; P < 0.05), with no difference in prevalence observed between the latter 2 genera (P > 0.05). The intensity of metacercariae within the snail hosts was significantly different between the 3 genera (P < 0.05), with L. columella having the highest intensity (24.3 ± 5.6), followed by Physa spp. (15.2 ± 1.5) and Helisoma spp. (5.0 ± 0.9). Differences in prevalence and intensity were also observed when the different snail families co-habited the same body of water. The disparities in infection patterns are likely due to distinct differences in the behavioral and feeding ecology of the snail hosts.

Shedding patterns of Daubaylia potomaca (Nematoda: Rhabditida).

Daubaylia potomaca is a nematode parasite that exhibits an unusual direct life cycle in planorbid snails in which adult females are the infective stage, after being shed from a definitive host. The present study examined the shedding patterns of this nematode to determine what cues or mechanisms might lead to the parasite leaving its host. A correlation was found between host death and the frequency and number of D. potomaca shed, suggesting that the nematodes can detect that the host is dying and may leave in search of a new host. Furthermore, elevated intensities of D. potomaca in the snail induce shedding earlier, suggesting that competition for space and resources may also play a role in the shedding patterns of the nematode, but not when time to death is controlled. Finally, nematodes shed a longer time before host death were significantly longer and more likely to be gravid than those shed as time to snail death approached, implying that the nematode reaching maturity or being inseminated might also be cues for D. potomaca to leave its snail host. In summary, the shedding patterns of D. potomaca appear to be a complex mix of host death detection, competition, and nematode maturation.

Hemophagocytic syndrome as uncommon presentation of disseminated toxoplasmosis in an immunocompetent adult from Chinese Kunming.

Hemophagocytic syndrome is a rare disease that is often fatal, despite treatment. An immunocompetent patient was presented with fever, hepatosplenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia, which conformed to a hemophagocytic syndrome diagnosis. Despite broad antibiotic treatment, the patient's clinical condition rapidly deteriorated and he died within 8 days of admission. Blood cultures and a serology test were negative; however, based on morphological characteristics, tissue cysts Toxoplasma gondii were found in the bone marrow. Based on polymerase chain reaction analysis, identity of the parasite was confirmed. Although very rare, T. gondii-associated hemophagocytic syndrome should be suspected in the case of cytopenia or multiorgan failure symptoms. To our knowledge, this is the first fatal toxoplasmosis case reported from China.

Complex interactions among a nematode parasite (Daubaylia potomaca), a commensalistic annelid (Chaetogaster limnaei limnaei), and trematode parasites in a snail host (Helisoma anceps).

Many biotic interactions can affect the prevalence and intensity of parasite infections in aquatic snails. Historically, these studies have centered on interactions between trematode parasites or between trematodes and other organisms. The present investigation focuses on the nematode parasite Daubaylia potomaca and its interactions with a commensal, Chaetogaster limnaei limnaei , and a variety of trematode species. It was found that the presence of C. l. limnaei indirectly increased the mean intensity of D. potomaca infections, apparently by acting as a restraint for various trematode parasites, particularly the rediae of Echinostoma sp. In turn, Echinostoma sp. rediae adversely affected the mean intensity of D. potomaca by their consumption of both juvenile and adult nematodes present in tissues of the snail. These organisms not only belong to 3 different phyla but occupy distinct trophic levels as well. The complex interactions among these 3 organisms in the snail host provide an excellent example of biotic interactions influencing the infection dynamics of parasites in aquatic snails.