This review brings together decades of work on elasmobranch tapeworms. The field has advanced significantly over the past 20 yr, with an emphasis on the discovery and description of novel taxa, and the establishment of phylogenetic frameworks for individual orders and their interrelationships. Tapeworms parasitizing elasmobranchs represent 9 orders and include 977 species and 201 genera—over 250 species and 50 genera are new within the last 2 decades. The 9 orders are treated individually, highlighting recent assessments of phylogenetic relationships informed by molecular sequence data. All but the “Tetraphyllidea” are monophyletic. Although much remains to be learned about their interrelationships, existing phylogenetic hypotheses suggest that elasmobranch tapeworms have played a key role in the evolution of the cestodes of essentially all other vertebrate groups. The apical organ is a defining feature (i.e., a synapomorphy) of a clade consisting of acetabulate taxa and Litobothriidea. Novel hook amino acid composition data support the independent origin of hooks in the various groups of hooked tapeworms. Cestode records exist for representatives of most of the major groups of elasmobranchs, however skates (Rajiformes) and catsharks (“Scyliorhinidae”) are particularly neglected in terms of species sampled. The majority of tapeworm species are extremely host-specific exhibiting species-specific (i.e., oioxenous) associations with their hosts. Rapid advancements in elasmobranch taxonomy, with over 300 of the 1,200 species appearing new in the past 20 yr, signal the need for careful attention to be paid to host identifications; such identifications are best documented using a combination of specimen, photographic, and molecular data. Above the species level, many cestode taxa are restricted to host orders, families, or even genera. Documentation of these affiliations allows robust predictions to be made regarding the cestode faunas of unexplored elasmobranchs. Trypanorhynchs are the notable exceptions. Life cycles remain poorly known. Recent applications of molecular methods to larval identifications have reinvigorated this area of research. Tapeworms are more diverse in elasmobranchs of tropical and subtropical waters, but they occur globally not only at the poles and in deep waters, but also in freshwaters of South America and Southeast Asia. The cestode faunas of batoids are much more speciose and complex than those of sharks. The faunas of deeper water sharks are particularly depauperate. The tapeworms of elasmobranchs and their hosts are now among the most well documented host-parasite systems in existence. This system has not yet reached its potential as a resource for investigations of basic ecological and evolutionary principles.

Tissue cyst stages are an intriguing aspect of the developmental cycle and transmission of species of Sarcocystidae. Tissue-cyst stages of Toxoplasma, Hammondia, Neospora, Besnoitia, and Sarcocystis contain many infectious stages (bradyzoites). The tissue cyst stage of Cystoisospora (syn. Isospora) possesses only 1 infectious stage (zoite), and is therefore referred to as a monozoic tissue cyst (MZTC). No tissue cyst stages are presently known for members of Nephroisospora. The present report examines the developmental biology of MZTC stages of Cystoisospora Frenkel, 1977. These parasites cause intestinal coccidiosis in cats, dogs, pigs, and humans. The MZTC stages of C. belli are believed to be associated with reoccurrence of clinical disease in humans.

The mechanisms underlying parasite-altered host behavior and fitness remain largely unanswered. The purpose of this review is to provide a perspective that has not been fully incorporated into the debate on how parasites manipulate their hosts. We argue that performance capacity is an important target of parasitic manipulation, and we aim to integrate the study of performance with that of parasitic manipulations of host behavior and fitness. We performed a meta-analysis from the published literature of 101 measures of the effect of parasites on host performance capacity to address the following questions. (1) Do parasites exert an important effect on host performance capacity? (2) Is that effect routinely to decrease or enhance performance capacity? And, (3) what factors explain variation in the effect sizes that have been quantified? Although negligible–small effect sizes were detected in 40/101 measures, host performance capacity was overall affected by parasitic infection, with a negative direction and medium–large magnitude in 58/101 measures and an increase in performance capacity in 3/101 measures. Host age, type of host performance, the host tissue infected by the parasite, and whether the study was experimental or based on natural infections each explained a significant amount of the variation in effect size. The significance of each factor is briefly discussed in light of the potential adaptive character of host manipulations by parasites.

The nervous system (NS) of the cestodes Diphyllobothrium dendriticum (Diphyllobothriidea) and Caryophyllaeus laticeps (Caryophyllidea) was investigated using immunocytochemistry. The GABA neurotransmitter was identified in the NS of both species; GABAergic neurons were detected in the main nerve cords (MC). GABA-like immunoreactive neurons were predominantly unipolar and exhibited more intensive immunoreactivity in the neurite than in the perikaryon. In C. laticeps, GABA-like immunoreactive somas are located in both the MCs and peripheral NS near the longitudinal muscles. The innervation of the body musculature was studied using a combination of antibodies against GABA, serotonin (5-HT), and FMRFamide and with complementary staining of F-actin. In both species, the location of GABAergic neurites is associated with all muscle layers including the subtegumental, longitudinal, transverse, and dorsoventral muscles. The cytomorphology of 5-HTergic motoneurons in the MCs of both species is described and differences in muscle innervation between D. dendriticum and C. laticeps are demonstrated. No evidence for co-localization of GABA with 5-HT or FMRFamide neurotransmitters at any particular neuron was found. Neuropiles in MCs and peripheral NS had separate sets of immunoreactive neurites. A functional role for GABA in muscle innervation is discussed.

Ambush foragers must employ a long-distance dispersal strategy to maximize reproductive success in the absence of hosts. This hypothesis was tested by comparing lateral dispersal of the ambusher, Steinernema carpocapsae, and the cruiser, Heterorhabditis bacteriophora, nematodes from infected host cadavers in autoclaved, silt-loam soil in large microcosms (0.05–1.5 m²) with or without vegetation in the absence of hosts. Dispersal was estimated by taking soil cores (5 × 2 cm in diameter) from the microcosms at different intervals (6–240 hr) and distances (3.8–61 cm) from the infected host cadavers and baiting with Galleria mellonella larvae. The numbers of baited larvae killed and the numbers of infective juveniles (IJs) penetrated in dead baits were counted to compute the percentage of IJs dispersed from the source cadavers, based on the emergence potential and penetration efficiency of the 2 species, and analyzed. Vegetation enhanced dispersal of both species but more so for H. bacteriophora. Although the pattern of dispersal differed spatio-temporally for the 2 species, average population displacement was similar (∼6 cm/day). A majority of the S. carpocapsae population ambushed close to the source cadaver (<3.8 cm), whereas a majority of H. bacteriophora population dispersed between 7–12 cm away from the source cadaver. About 4% of the S. carpocapsae population dispersed faster than the fastest H. bacteriophora, reaching 30–61 cm, compared to only 2% of the H. bacteriophora population dispersing this far. This use of ‘sprinters' for long-distance dispersal may represent an adaptive dispersal strategy by the otherwise ambush forager S. carpocapsae in the absence of hosts.

Temperature variation on the host is known to influence ectoparasite distributions. Ectoparasites may also use temperature gradients between host regions when moving on the host; however, tests are rare. Feather-feeding wing lice (Phthiraptera: Ischnocera) spend the majority of their time on the flight feathers of their avian hosts where they insert their bodies between feather barbs to escape host preening. However, because wing lice feed on downy abdominal feathers, they must repeatedly migrate between the flight feathers and body regions of their hosts. We performed a series of experiments that tested thermo-orientation in wing lice and evaluated its potential use during louse migrations between host regions. We found that wing lice can rapidly and accurately locate nearby heat targets that approximate host temperatures (37 C), demonstrating a capacity for directed thermo-orientation. We next tested the preference of wing lice for temperatures found along migration routes between bird flight feathers and their body regions. Wing lice could distinguish between temperatures found within distinct bird regions, and lice that had recently fed preferred the cooler temperatures (32 C), similar to those within bird flight feathers where they typically reside. However, when starved for 18–20 hr, wing lice shifted their preferences toward temperatures typical of bird body regions where they feed (36 C), demonstrating an ability to use thermal cues when moving between bird regions. We discuss the use of thermal cues during louse migration and microhabitat selection, as well as other potential impacts of thermo-orientation on host–parasite interactions.

Fecundity reduction is a well-recognized phenomenon of parasite infection in insects. Reduced production of eggs might increase longevity of a host and release nutrients to both host and parasite that would otherwise be used for oogenesis. The objective of this study was to assess effects on fecundity caused by Hepatozoon sipedon, an apicomplexan blood parasite of snakes, in its invertebrate host, the mosquito Culex pipiens. In the first gonotrophic cycle, the mean number of eggs laid by mosquitoes infected with H. sipedon did not differ significantly from those laid by uninfected mosquitoes. However, in the second gonotrophic cycle infected mosquitoes laid significantly fewer eggs than did uninfected mosquitoes, and fecundity was reduced by 100% in mosquitoes with parasite burdens of more than 60 oocysts. There was a significant negative correlation between parasite burden, or the number of oocysts, and the number of eggs produced in the second gonotrophic cycle. Significantly fewer viable larvae hatched from eggs laid by infected compared to uninfected mosquitoes in the second gonotrophic cycle. These data indicate that fecundity reduction occurs in this system, although the physiological mechanisms driving this phenotype are not yet known.

The rhizocephalan barnacle Loxothylacus panopaei is a parasitic castrator of xanthid crabs that has invaded the U.S. Atlantic coast. It was transported to the Chesapeake Bay in the mid-1960s with mud crabs associated with Gulf coast oysters and has since spread north to Long Island Sound, New York, and south to Cape Canaveral, Florida. Here we report parasite prevalence at 3 South Carolina sites—2 from which the parasite had not been previously reported—and examine the genetic relationships of North and South Carolina L. panopaei populations relative to Gulf of Mexico and other Atlantic coast parasite populations. Total L. panopaei prevalence was 24.2% among all 3 sites, with monthly prevalence as high as 51.6% at Waties Island, South Carolina. Sequence analyses of North and South Carolina specimens revealed the presence of 4 cytochrome c oxidase subunit I haplotypes—3 commonly found in other invasive populations and 1 new haplotype found in a single specimen from the Rachel Carson Reserve in Carteret County, North Carolina—and indicate that the Carolina populations are a result of range expansion from the original Atlantic coast invasion.

Plasmodium reticulum, the causative agent for avian malaria (a protozoan), and Dirofilaria immitis, the causative agent for canine heartworm (a filarial nematode), are 2 obligate parasites transmitted by mosquitoes. The objective of this project was to identify whether either parasite was present in Tennessee mosquitoes and to illustrate the need for collecting spatial and temporal vector–parasite data. During 2012, mosquitoes were collected from the East Tennessee Research and Education Center (ETREC) in eastern Tennessee and the Ames Plantation Research and Education Center (AMES) in western Tennessee using CO₂ traps and gravid traps. Once mosquitoes were identified to species, their heads and thoraces were pooled in groups of ≤10, and the entire pool underwent DNA extraction and parasite amplification via polymerase chain reaction (PCR) for Plasmodium and Haemoproteus parasite DNA (cytochrome b) and for Dirofilaria species DNA (internal transcribed spacer-2 ribosomal DNA). All positive PCR amplicons were bidirectionally sequenced to confirm positivity and to identify the potential parasite genotype. This approach resulted in 762 mosquito pools, 150 pools from AMES and 612 pools from ETREC. In total, 3,260 mosquitoes were collected, representing 28 mosquito species. The 3 abundant genera were Culex (2,440 specimens, 74.8%), Aedes (720, 22.1%), and Anopheles (85, 2.6%). The remaining specimens included 13 Psorophora, 1 Orthopodomyia signiferia, and 1 Coquillettidia perturbans. Of the 150 pools from AMES, 1 pool (0.7%) was positive for avian malaria and 12 pools (8.0%) were positive for canine heartworm. Of the 612 pools from ETREC, 61 pools (10.0%) were positive for avian malaria and 8 pools (1.3%) were positive for canine heartworm. Positive pools for both Plasmodium and Dirofilaria were primarily Culex pipiens and occurred later in the season. The confirmation of the agents for avian malaria and canine heartworm illustrates the need for concurrent spatial and temporal studies using different trapping methods. The confirmation of avian malaria and canine heartworm in Tennessee illustrates the need for concurrent spatial and temporal studies. Future studies incriminating the potential vector populations will begin to unravel the complex relationships that intimately tie together hosts, vectors, and parasites. Results provide a significant contribution to the knowledge of the diversity of mosquito parasites present in Tennessee, and the presence of positive field populations warrants additional research exploring the environmental factors contributing to transmission.

Schistosomes infect around 280 million people worldwide. The worms survive in the veins of the final host, where thioredoxin glutathione reductase (TGR) activity helps the parasites to survive in the aerobic environment. In the present study, we synthesized 4 small interfering RNAs (siRNA S1, S2, S3, and S4) targeting the Schistosoma japonicum (Sj) TGR gene and used them to knockdown the TGR gene. The knockdown effects of the siRNAs on SjTGR, and the thioredoxin reductase (TrxR) activity of SjTGR, were evaluated in vitro. The results of transfection with the siRNAs via the soaking method in vitro were confirmed by flow cytometry. S2 siRNA at a final concentration of 200 nM partially inhibited the expression of SjTGR at both the transcript and protein levels in vitro. TrxR-activity was lower in worms in the S2 siRNA-treated group compared with the control groups. Further analysis revealed that purified recombinant SjTGR could remove oxygen free radicals but not H₂O₂ directly, which may explain the incomplete effects of RNA interference on SjTGR. The results of this study indicate that SjTGR may play an important role in the clearance of oxygen free radicals and protection of S. japonicum parasites against oxidative damage.

Infection of pigs with Toxoplasma gondii is a common source of human toxoplasmosis and causes serious economic losses. In vivo-induced antigen technology (IVIAT) is an effective immunological technique to identify the antigens that a pathogen specifically expressed during infection. To discover the genes that are important in T. gondii infection of pigs, we employed IVIAT using sera from infected pigs. Fourteen antigens were identified including microneme protein 11 (MIC11), dense granule protein 5 (GRA5), 18 kDa cyclophilin (C-18), serine proteinase inhibitor (PI), calmodulin (CaM), leucine-rich repeat protein ( LRRP), D-3-phosphoglycerate dehydrogenase (D3PD), elongation factor 1-gamma (EF1), and 6 hypothetical proteins. The increased transcription levels of 5 (MIC11, GRA5, C-18, PI, and CaM) of the 14 molecules identified by IVIAT were confirmed by real-time PCR. The full length or partial proteins encoded by these 5 genes were expressed in Escherichia coli, and their immunogenicity was confirmed by Western blot analysis with positive porcine sera. Further functional studies were conducted with CaM. Suppression of CaM expression by RNA interference decreased T. gondii tachyzoites cell attachment, invasion, and egress but did not influence their replication. The proteins identified in this study are predicted to be involved in cell invasion, ion–protein binding, protein folding, biosynthesis, and metabolism. The results of the functional analysis support the hypothesis that CaM contributes to parasite pathogenesis during infection. These results may have significant implications for the discovery of candidate molecules for the development of potential therapies and preventive measures against toxoplasmosis in pigs.

Between April 2012 and September 2013, feces from 18 green anoles, Anolis carolinensis from Arkansas (n = 14), Louisiana (n = 1), and Oklahoma (n = 3) were examined for coccidia. Two species of coccidians were found, including a new caryosporan and a new eimerian. Oocysts of Caryospora natchitochesensis n. sp. from a single A. carolinensis from Louisiana were subspheroidal to ovoidal with a smooth, yellow-to-brown–pigmented, bilayered wall of equal thickness (∼0.3–0.7) and measured (L × W) 13.1 × 12.3 μm, with a length/width (L/W) ratio of 1.1. A micropyle and oocyst residuum were absent, but a polar granule was present. Sporocysts were ovoidal and measured 10.1 × 7.4 μm, L/W was 1.4. A Stieda body (∼1.0 μm) was present, but substieda and parastieda bodies were absent. The sporocyst residuum was composed of dispersed granules or globules among sporozoites. Oocysts of Eimeria robisoni n. sp. from 1 of 12 (8%) green anoles from Arkansas were ellipsoidal with a smooth, unilayered wall (∼0.4–0.5) and measured (L × W) 14.5 × 10.5 μm, with L/W ratio of 1.4. A micropyle and oocyst residuum were absent, but 1–4 (usually 2) polar granules were present. Sporocysts were subspheroidal to ovoidal and measured 5.8 × 4.9 μm, L/W was 1.2. Stieda, substieda, and parastieda bodies were absent. The sporocyst residuum was composed of dispersed granules between sporozoites. None of the anoles from Oklahoma was found to be passing oocysts. This is the second time an eimerian and a caryosporan have been reported from green anoles. A summary of the coccidians of lizards of the family Dactyloidae is provided, with special emphasis on the Anolis of the United States.

Floriparicapitus n. gen. (Cestoda: Lecanicephalidea), with Floriparicapitus euzeti n. gen., n. sp. as its type, is erected to house 3 new tapeworm species and 2 known species that are transferred to the new genus, all parasitizing sawfishes and guitarfishes (order Rhinopristiformes) in Indo-Pacific waters. The new genus differs from the 21 valid lecanicephalidean genera in its possession of a large scolex bearing a laterally expanded apical organ in the form of a rugose sheet in combination with a cirrus conspicuously armed with spinitriches and 3 pairs of excretory vessels. It most closely resembles Lecanicephalum, but differs conspicuously in its possession of 3, rather than 1, pair of excretory vessels. Two new species are described from sawfishes: Floriparicapitus euzeti n. sp., from Pristis clavata and Floriparicapitus juliani n. sp. from Pristis pristis, both from Australia. Floriparicapitus plicatilis n. sp. is described from the guitarfish Glaucostegus typus in Australia and the guitarfish Glaucostegus thouin in Malaysian Borneo. Two species formerly assigned to Cephalobothrium are transferred to the new genus; Floriparicapitus variabilis (Southwell, 1911) n. comb. from the sawfish Anoxypristis cuspidata in Sri Lanka and Floriparicapitus rhinobatidis (Subhapradha, 1955) n. comb. from the guitarfish Glaucostegus granulatus in India. The species from guitarfish differ conspicuously from those parasitizing sawfish in their possession of only 4 (F. plicatilis n. sp.) or 5 (F. rhinobatidis n. comb.) testes per proglottid versus 9 or more in the 3 sawfish-parasitizing species. The latter 3 species differ from one another in scolex width, acetabular size, number of proglottids, and cirrus sac size. As it stands, the new genus appears to be restricted to a subclade of the Rhinopristiformes consisting of the sawfishes and species of Glaucostegus.

Moennigia celinae n. sp. collected from the small intestine of Chaetophractus vellerosus and Chaetophractus villosus (Xenarthra, Dasypodidae) from Argentina is herein described. This new species belongs to the genus Moennigia because it possesses a short uterus with few eggs, atrophied distal branch of the ovejector, vulva near the anus, and a conical tail. The new species has a synlophe with 17 symmetrical ridges and slight ventro–dorsal orientation. The spicule length:body length ratio is similar to that of the other species parasitic of Dasypodidae; however, Moennigia celinae n. sp. differs from Moennigia pintoi and Moennigia lutzi because the latter lack a gubernaculum, and from Moennigia complexus, Moennigia moennigi, Moennigia filamentosus, Moennigia intrusa, Moennigia littlei, Moennigia pulchra and Moennigia dessetae by the latter having very complex spicules with 2 or 3 points at the distal extremity. Moreover, Moennigia celinae n. sp. differs from Moennigia virilis by the length and shape of its spicules. Moennigia celinae n. sp. can be distinguished from Moennigia travassosi by the shape of the dorsal ray of the caudal bursa. Moennigia celinae n. sp. resembles Moennigia pseudopulchra but the gubernaculum of the latter is V-shaped. This is the second report of a species of Moennigia in Argentina and the first for the genus Chaetophractus.

Four new species are described in the lecanicephalidean genus Hornellobothrium from the ocellated eagle ray, Aetobatus ocellatus, from Australia and Indonesian Borneo. The species possess flat, laterally expanded immature proglottids and a small internal, glandular apical organ diagnostic of the genus. To date, only 2 valid species are recognized in the genus: Hornellobothrium cobraformis Shipley & Hornell, 1906, from “Aetobatus narinari” from Sri Lanka, and Hornellobothrium extensivum Jensen, 2005, from A. ocellatus, also from Australia. The new species differ from H. cobraformis in the shape of the apical organ and mature proglottids, and from H. extensivum in testes number and scolex dimensions. The new species can be distinguished from one another based on the following combination of features: Hornellobothrium gerdaae n. sp. possesses testes arranged in 2 layers and lacks post-ovarian vitelline follicles; Hornellobothrium iotakotta n. sp. has a single column of 4 testes and lacks spiniform microtriches on the distal bothridial surface; both Hornellobothrium kolossakotta n. sp. and Hornellobothrium najaforme n. sp. possess a single column of 6 testes, but H. najaforme n. sp. is a longer worm with a greater number of laterally expanded immature proglottids. Host associations suggest this genus is potentially limited to A. ocellatus. While reports of up to 9 lecanicephalidean congeners in a single host species exist, they are considered dubious and in need of verification, mainly because of host identification issues. This study demonstrates the presence of at least 5 species of Hornellobothrium in A. ocellatus. Insufficient sampling of this host species across the Indo-Pacific and at each locality may account for the apparent restricted distribution of species of Hornellobothrium.

Toxoplasmosis continues to be a public health problem, causing significant morbidity worldwide. Currently available medications, effective for acute toxoplasmosis, are nonetheless problematic due to adverse side effects in many patients. In addition, no medication is able to completely eradicate the parasite cysts, rendering infected individuals at risk for reactivation upon becoming immunocompromised. We examined the anti–T. gondii activity of 2 derivatives of artemisinin. In vitro metabolic stability tests revealed that both derivatives are stable in mouse plasma but only the thiazole CPH4-136 is stable in the presence of mouse microsomes. When tested in a mouse model of acute toxoplasmosis, both derivatives showed modest efficacy dependent upon the compound dose and the solvent vehicle. Finally, in a mouse model of chronic T. gondii infection, CPH4-136 at 3 mg/kg once per day for 32 days moderately but significantly decreased mouse brain cyst burden. Collectively, our findings suggest that artemisinin derivatives are partially effective in treating experimental T. gondii infections.

The resistance of Babesia parasites to current anti-babesiosis drugs is an issue of major concern. The inosine 5′-monophosphate dehydrogenase (IMPDH) of Babesia gibsoni has been identified and characterized as a molecular drug target in our previous studies. In the present study, inhibitory effects of IMPDH inhibitors (mycophenolate mofetil, mizoribine, ribavirin, 7-nitroindole, and mycophenolic acid) were evaluated in vitro or in vivo. In the inhibition assay of recombinant B. gibsoni IMPDH activity, mycophenolate mofetil was the most potent inhibitor (IC₅₀ = 2.58 ± 1.32 μM) while ribavirin was the least potent. The inhibitory effects of mycophenolate mofetil, mizoribine, ribavirin, and 7-nitroindole on the in vitro growths of B. gibsoni and Babesia bovis were also assessed. The results revealed that mycophenolate mofetil was the most potent inhibitor of the multiplications of both B. gibsoni (IC₅₀ = 0.13 ± 0.05 μM) and B. bovis (IC₅₀ = 0.97 ± 0.49 μM). Ribavirin was also the least potent for both B. gibsoni and B. bovis in vitro. Mycophenolic acid, a metabolite of mycophenolate mofetil, caused an inhibition of Babesia microti in mice with noticeable improvement in hematological parameters of the infected mice (ED₅₀ = 44.15 ± 12.53 mg/kg). Although the report provides a non-exhaustive view of potential treatment strategy without addressing the potential adverse effect of immune suppression on infections, these results indicated that the IMPDH might be a molecular target of MPA for B. microti. Altogether, we provide a basis for development of antibabesia prodrugs by targeting IMPDH of the parasites in the treatment of babesiosis.

Previous studies comparing the genome sequences of Cryptosporidium parvum with Cryptosporidium hominis identified a number of highly divergent genes that might reflect positive selection for host specificity. In the present study, the C. parvum DNA sequence cgd8-5370, which encodes a protein whose amino acid sequence differs appreciably from its homologue in C. hominis, was cloned by PCR and expressed as a recombinant protein in Escherichia coli. Antisera raised against the recombinant cgd8-5370 antigen strongly recognized a unique 33 kDa protein in immunoblots from reducing and non-reducing SDS-PAGE of native C. parvum protein. However, anti-Cp33 sera did not recognize the native 33 kDa homologue in C. hominis. In an immunofluorescence assay (IFA), anti-Cp33 serum recognized an antigen in the anterior end of air-dried C. parvum sporozoites but failed to bind at any sites in C. hominis sporozoites, indicating its specificity for C. parvum. IFA staining of live C. parvum sporozoites with anti-Cp33 serum failed to bind to the parasite, indicating that the CP33 antigen is not on the sporozoite surface, which is consistent with topology predictions based on the encoded amino acid sequence. RT-PCR analysis of cgd8-5370 mRNA before or during C. parvum oocyst excystation revealed transcripts only in excysting sporozoites. Thus, Cp33 represents one of a small number of proteins shown to differentiate C. parvum from C. hominis sporozoites and oocysts.

Given the paucity of literature available on rabbits infected with Cryptosporidium in Sichuan Province (China), 290 fecal samples were collected from rabbits in the animal house of Sichuan Agricultural University, China and examined for Cryptosporidium oocysts using the Sheather's sucrose flotation technique and a modified acid-fast staining method. Three samples tested positive (prevalence = 1.03%). The positive isolates were genotyped by sequence analysis of the 18S rRNA, HSP70, COWP, and Cp135 genes and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene. Phylogenetic analysis was established using the neighbor-joining (NJ) method. All the isolates were identified as Cryptosporidium cuniculus. Further subtyping of the positive isolates was performed by DNA sequencing of the 60-kDa glycoprotein (gp60) gene. Only 1 subtype family was detected, Va, which was proposed to be a new subtype, VaA31. This study is the first report about the prevalence, genetic identification, and Cp135 gene of C. cuniculus in rabbits in Sichuan Province, China. The obtained results indicate that the C. cuniculus subtype in rabbits in Sichuan Province is unique.

The genus Paracreptotrema Choudhury, Pérez-Ponce de León, Brooks and Daverdin, 2006 (Digenea), comprises 4 species that parasitize poeciliid and cyprinodontid fishes in Middle America. Based on morphological characters exhibited by adults of the type species (Paracreptotrema blancoi Choudhury, Pérez-Ponce de León, Brooks and Daverdin, 2006) the genus was tentatively assigned to Allocreadiidae Looss, 1902. We sequenced domains D1–D3 of the 28S rRNA gene of several specimens of Paracreptotrema heterandriae Salgado-Maldonado, Caspeta-Mandujano and Vázquez, 2012, and analyzed these in a phylogenetic context along with 10 allocreadiid taxa plus several xiphidiatan and 2 monorchiatan digeneans, in order to test the proposed inclusion of P. heterandriae in Allocreadiidae. Maximum likelihood and Bayesian inference analyses of 28S rRNA gene sequences confirmed the classification of Paracreptotrema as an allocreadiid. In the phylogenetic trees, Paracreptotrema appears to be the sister taxon of Creptotrematina aguirrepequenoi Jiménez-Guzmán, 1973, and both are closely related to Auriculostoma Scholz, Aguirre-Macedo, and Choudhury, 2004, with Wallinia Pearse, 1920, as the sister taxon of all these genera; nevertheless, the interrelationships between these 4 genera were not totally resolved. ITS2 sequences of several specimens of P. heterandriae showed null intraspecific variation. Scanning electron microscopy microphotograph demonstrated the presence of 13 and 4 dome-like papillae arranged around the outer and inner edges of the oral sucker, respectively.

The family Taeniidae comprises many parasitic species, which cause serious zoonoses. However, effective identification of Taeniidae species is a long-standing problem, especially in samples from wild hosts with mixed infections of different Taeniidae species. DNA barcoding analysis of small fragments of the cytochrome c oxidase subunit I (COI) gene has been confirmed as an effective and useful method for identifying Taenia species. We therefore performed DNA barcoding analysis using a 351-bp region of the COI gene to identify 27 taeniid species including 9 in the genus Echinococcus, 2 in Hydatigera, 15 in Taenia, and 1 in Versteria. A total of 484 COI sequences were used to calculate genetic divergence expressed by the Kimura 2-parameter (K2P) distance. The mean intra-specific K2P distance in the family Taeniidae was 0.71 ± 0.17% (±SE), while inter-specific divergences were considerably higher. We found that, generally, a 2.0% optimal barcoding threshold could be set to distinguish taeniid species. Taenia polyacantha and Hydatigera taeniaeformis were the only 2 false-positive species identification cases in this study for their intra-specific divergences above the 2.0% optimal threshold. Their high intra-specific divergences coincided with fact that cryptic divergences exist in these 2 species, to which new taxa were recommended. On the other hand, sister species T. asiatica and T. saginata showed a 2.48 ± 0.83% inter-specific divergence, which was the smallest among all the taeniid species. Although fitting the 2.0% optimal species barcoding threshold, the close genetic relationship between T. asiatica and T. saginata implies that longer mitochondrial DNA sequences like the complete COI sequence are needed to strictly distinguish them. Therefore, we concluded that the barcoding technique based on a 351-bp region of the COI gene is able to distinguish taeniid species except for cryptic T. polyacantha and H. taeniaeformis and should be carefully used in distinguishing the closely related species T. asiatica and T. saginata.