In March and April 2016, 150 white perch (Morone americana) were collected from various localities in Chesapeake Bay and examined for coccidia. A previously undescribed species of coccidia was observed in the hepatic bile ducts and gallbladder of all white perch (100%) examined. We describe this species using morphological characteristics, histology, and gene sequences of the small-subunit ribosomal DNA (rDNA), large-subunit rDNA, and mitochondrial genes cytochrome oxidase 1 (COI), cytochrome oxidase b (Cytb), and cytochrome oxidase 3 (COIII). Oocysts of Goussia bayae n. sp. were subspherical with a single-layered smooth wall and measured (length [L] × width [W]) 26.2 × 21.8 µm, with a L/W ratio of 1.2. A micropyle was present but a micropyle cap, polar granules, and oocyst residuum were absent. Each oocyst contained 4 sporocysts that were ellipsoidal and measured (L × W) 12.6 × 7.8 µm, with a L/W ratio of 1.6. A pair of sporozoites was present, but sporocysts lacked a Stieda body and residuua. Meronts and gamonts were epicellular in biliary epithelial cells and oocysts were coelozoic in hepatic and common bile ducts and gallbladder. This is the first report of Goussia spp. from white perch and the first mitochondrial DNA sequence reported from a Goussia species. Phylogenetic analysis indicates basal placement of G. bayae to Eimeriidae, Choleoeimeria, and Sarcocystidae.

The life cycle of Sarcocystis species is heteroxenous (2-host), with carnivores being the definitive host and herbivores serving as intermediate hosts in predator-prey relationships. Raptors (eagles, hawks, falcons, and owls) are apex predators and are not consumed routinely by other carnivores, making the occurrence of sarcocysts in their muscles unusual. Recent reports of sarcocysts in eagles and owls with Sarcocystis encephalitis suggests that this condition may be becoming more frequent, and Sarcocystis falcatula has been implicated as the agent of encephalitis in golden (Aquila chrysaetos) and bald eagles (Haliaeetus leucocephalus) as well as great horned owls (Bubo virginianus). The present study was done to determine the prevalence of sarcocysts of Sarcocystis species in the muscles of raptors from the southeastern United States. Pectoral and heart muscle from 204 raptor patients from the Carolina Raptor Center, Huntersville, North Carolina were tested for the presence of Sarcocystis species using histology. Only a few sarcocysts were seen in sections of pectoral muscle from 39 of 204 raptors (19.1%) and heart muscle from 9 that also had sarcocysts in their pectoral muscle. Two structural types of sarcocysts, thin-walled (1 µm; 62%) or thick-walled (>2 µm, 38%), were seen. Statistical analysis of raptor age and gender was done by Fisher's exact test on samples from raptors with 20 or more samples per group. The prevalence of sarcocysts by age (2 yr or more) was significant for red-shouldered hawks (Buteo lineatus) (P = 0.022) and Cooper's hawks (Accipitercooperii) (P = 0.028). Sarcocyst prevalence in male raptors from these groups evaluated statistically were always less than in females. Prevalence in female red-tailed hawks (Buteo jamaicensis) (42.1%) was significantly greater than in males (6.7%) using Fisher's exact test (P = 0.047). Examination of case histories from the 39 sarcocyst-positive raptors did not reveal an association with sarcocysts in raptor pectoral or heart muscle and in a diagnosis of encephalitis. Additional studies are needed to determine the epidemiology and relationships of Sarcocystis spp. that use raptors as intermediate hosts and the importance of Sarcocystis spp. in the overall wellbeing of raptors in their natural environments.

The relationship between a parasite and its host will have an energy cost for the host at some point; however, this basic hypothesis has seldom been evaluated. Using Emerita analoga as a model species, we investigated the aerobic metabolism, ammonia excretion rate, and locomotor performance patterns of crabs both uninfected and infected with the acanthocephalan Profilicollis altmani. Our results show that infected Emerita had a lower metabolic rate compared to uninfected ones. Whether or not this decrease is a result of the pathology of the parasite infection or due to manipulation of the host by the parasite is still unknown. We discuss the importance of using anaerobic and aerobic metabolism and more-complex scenarios in order to understand the effects on host fitness and not only on the immediate response of the host.

The coccidian parasite Cystoisospora canis (syn. Isospora canis) can cause clinical disease in dogs. Three generation of schizonts have been reported in the small intestine of dogs before oocysts are excreted 9–11 days post inoculation (PI). Here, we re-evaluated asexual development of C. canis in 2 dogs necropsied 10 days after oral inoculation with 100,000 C. canis oocysts; both dogs had excreted oocysts 9 days PI. Asexual and sexual stages were seen in the lamina propria throughout the small intestine. Merozoites of different sizes were present, often in the same vacuole. They were arranged singly, in pairs, and many within a single parasitophorous vacuole. The maximum number of nuclei within developing merozoites in a group was 8, but it could not be discerned if they were individual nuclei or parts of merozoites. Findings of abundant asexual stages 1 day after dogs had started excreting oocysts indicated continued asexual multiplication beyond the prepatent period. The stages found resemble the 3 generations reported previously. The mode of division of the asexual generations remains unclear. The results of the present study indicate that there are many generations that are difficult to determine because of the multiplication of merozoites in the original host cell without leaving it to enter new host cells. From the literature, it is evident that cat and dog coccidia (Cystoisospora spp.) divide by more than 1 type of division, including endodyogeny. In the past, the schizont/meront groups containing more than 1 generation have been called “cysts.” However, cyst is not an accurate term because it is best used for an orally infective stage of coccidia; monozoic tissue cysts of C. canis can occur in paratenic hosts in extraintestinal organs. We recommend the term “types” as originally proposed for intestinal stages of Toxoplasma gondii and used for the original description of the life cycle of C. suis of swine when describing endogenous stages of the Sarcocystidae. Ultrastructural studies are needed to determine the precise form of multiplication of canine Cystoisospora species.

Species in the genus Klossiella Smith and Johnson, 1902 are unique among the suborder Adeleorina because they are monoxenous in mammals exclusively, whereas all other reported members of the Adeleorina use invertebrates as definitive hosts. Unlike other coccidia, all members of the Adeleorina undergo syzygy, the association of microgamonts and macrogamonts before maturation to gametes and syngamy. After fertilization, many members of the Adeleorina produce thin-walled polysporocystic oocysts. Despite being biologically similar to other members of the Adeleorina, the phylogenetic placement of the genus Klossiella has been questioned based on its unique host affinity. In the present study, 2 cases of Klossiella equi were reported from the kidneys of horses in Ontario. Details of the life cycle as well as mitochondrial and nuclear 18S ribosomal DNA (18S rDNA) sequences were analyzed to provide both morphological and molecular evidence for the phylogenetic placement of K.equi. Initially, various stages of the life cycle were identified in histological slides prepared from the kidney tissue, and DNA was isolated from the infected tissue. Polymerase chain reaction and Sanger sequencing were used to generate a complete mitochondrial genome sequence (6,569 bp) and a partial 18S rDNA sequence (1,443 bp). The K. equi 18S rDNA sequence was aligned with various publicly available apicomplexan 18S rDNA sequences. This alignment was used to generate a phylogenetic tree based on Bayesian inference. Multiple K. equi stages were identified including meronts, microgamonts, and macrogamonts associating in syzygy as well as thin-walled oocysts in various stages of sporogonic development. The 18S rDNA sequence of K. equi positioned within the monophyletic Adeleorina clade. The mitochondrial genome of K. equi contained 3 coding sequences for cytochrome c oxidase I, cytochrome c oxidase III, and cytochrome b as well as various fragmented ribosomal sequences. These components were arranged in a unique order that has not been observed in other apicomplexan mitochondrial genomes sequenced to date. Overall, it was concluded that there were sufficient morphological and molecular data to confirm the placement of K. equi and the genus Klossiella among the Adeleorina. The biological and molecular data obtained from these cases may assist with future studies evaluating the prevalence and life history of this seemingly underreported parasite and better define the impact of K. equi on the health of domestic and wild equids.

Coiling patterns of heligmonellid nematodes were examined for 520, 208, and 33 individuals of Nippostrongylus brasiliensis, Orientostrongylus tenorai, and Sabanema sp., respectively, collected from murine rodents of Indonesia. Besides typical sinistral coiling, complete dextral coiling was found in 3.3% of N. brasiliensis and 12.1% of Sabanema sp. Mixed coiling with partial sinistral and dextral patterns was also observed in 38.8% of N. brasiliensis, 60.7% of Sabanema sp., and 3.4% of O. tenorai. In dextral coils, the left ventral area with large ridges was located inside as in sinistral coils, keeping the ability to cling to intestinal villi. The cuticular dilatation at left to left dorsal area was located caudally in sinistral coils but rostrally in dextral coils. Presence of mixed coiling indicates that the coiling patterns can change. As the transition of coiling pattern accompanies a change in direction of coil axis, it is surmised that the dextral coiling may be chosen when a worm leaves a villus to move to another villus.

In this paper 4 species of bat ectoparasites are recorded from the island of Sint Eustatius, Dutch Caribbean. One species of true bug (Hemiptera: Polyctenidae) as well as 3 species of bat flies (Diptera: Hippoboscidae: Streblinae) are recorded. All species are photographed. The first DNA barcodes for 3 bat ectoparasite species (Trichobius frequens, Trichobius intermedius, and Hesperoctenes fumarius) have been posted to the BOLD database; DNA barcode sequences for a fourth species (Megistopoda aranea) are the first from a Caribbean island.

Toxoplasmosis, caused by Toxoplasma gondii, is associated with several clinical syndromes, including encephalitis, chorioretinitis, and congenital infection. Toxoplasma gondii is a ubiquitous apicomplexan parasite found in both humans and animals. Mongolian gerbils, which are more susceptible to both high- and low-virulence Toxoplasma strains compared with mice, are considered useful models for assessing diagnosis and treatment methods for toxoplasmosis, as well as infection by and host defense to this organism. Here we established a quantitative real-time polymerase chain reaction (qPCR) method targeting the B1 gene for early and specific detection of T. gondii infection in Mongolian gerbil. The detection limit of the developed qPCR was approximately 1 T. gondii tachyzoite. This method was also applied to detect T. gondii genomic DNA in experimentally infected Mongolian gerbils, with positive results in blood (66.7%), liver (73.3%), lung (80.0%), spleen (80.0%), and peritoneal fluid (66.7%) samples as early as 1 day postinfection. Specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Cryptosporidium parvum, Eimeria tenella, Trypanosoma evansi, Schistosoma japonicum, Angiostrongylus cantonensis, and Strongyloides stercoralis. This study first reports the use of Mongolian gerbils as an animal model for early diagnosis of toxoplasmosis by qPCR.

Gastroenteritis caused by single or multiple pathogens remains a major diagnostic challenge for the laboratory, as diagnosis is achieved using different techniques with variable sensitivity and specificity. The aim of this study was to evaluate the EasyScreen^™ Enteric Protozoa Detection Kit, a multiplex PCR assay for the detection and identification of the 5 most common protozoan parasites in fecal samples. A total of 632 fecal samples, submitted for routine screening to 2 centers in northeastern Italy, was included in the study. The results of the molecular assay were compared to those of the standard diagnostic procedures, represented by microscopy and immunoassays. Out of 32 samples testing positive by conventional tools, 31 were detected as concordantly positive using the EasyScreen Kit. Additionally, 91 out of 632 samples only tested positive by the molecular test, therefore increasing the positive detection rate by 275%. Finally, the EasyScreen assay detected 14 co-infections compared to 3 co-infections identified by conventional methods. The EasyScreen Kit provided a rapid and sensitive simultaneous identification of the most important diarrhea-causing protozoa that infect humans. Additionally, this molecular assay presents several advantages compared to conventional tools, such as the standardization and near-total automation of the process. Although critical issues related to the employment of molecular assays are still evident, the system is suitable for clinical parasitological diagnosis as long as it is used in association with conventional tools.

The main etiologic agent of human toxocariasis, a zoonotic disease, is the helminth Toxocara canis. Among the diagnostics used for human toxocariasis, ELISA using T. canis excretion and secretion antigen (TES) is considered as a standard technique. TES antigen requires the cultivation of T. canis larvae, which makes its production difficult. Besides this, the use of TES antigen does not eliminate the cross-reactions with other similar proteins that are produced by other intestinal worms. In this context, recombinant antigens are being tested to improve the diagnosis of human toxocariasis. Herein, we describe the production of polyclonal antibodies against recombinant protein TES30 (pAb-rTES30) and evaluate its use in a blocking ELISA (b-ELISA) using human sera. The b-ELISA showed 95.6% sensitivity and 94.4% specificity. Thus, the b-ELISA using pAb-rTES30 offers a viable option for toxocariasis diagnosis owing to its configuration, which prevents cross-reactivity with non-species-specific antibodies.

Malaria is caused by Plasmodium spp., a parasitic protist that infects erythrocytes. A method that can detect the parasite with high sensitivity and that can identify the parasite species is urgently required for the control of malaria. The cell microarray chip was made using polystyrene with 200 cone-shaped frustum microchambers (800-µm top diameter, 636-µm bottom diameter, and 225 µm deep). Approximately 3,000 erythrocytes could be accommodated in each microchamber with monolayer formation, there being 60,000 erythrocytes in total microchambers on a cell microarray. Plasmodium could be quantitatively detected with high sensitivity with the use of cell microarray chips. Plasmodium parasitizing in erythrocytes was labeled with a cell-permeant fluorescent nucleic acid stain (SYTO 21), which could be detected in erythrocytes in the microchambers. Next, we used loop-mediated isothermal amplification (LAMP) in the microchambers (on-chip LAMP) to identify the parasite species detected in the microchambers. LAMP was performed in the microchambers (in a reaction volume of 0.09 µl) using Plasmodium falciparum–infected erythrocytes as the template and specific primers targeting 18S rRNA. To avoid evaporation of the reaction buffer during heat treatment, mineral oil was overlaid on each microchamber and the cell microarray chips were heated at 63 C for 1 hr. The results of on-chip LAMP were assessed using a portable ultraviolet transilluminator. We showed that this method has the potential for detection of parasites in 600,000 erythrocytes and for identification of the parasite species on a cell microarray chip. In conclusion, the parasites can be detected quantitatively with high sensitivity, and the species can be identified with the use of cell microarray chips.

Calodium hepaticum (Trichinellida: Capillaridae) is a parasitic nematode of mammals distributed worldwide. Although this parasite can infect the liver of a wide diversity of mammals (including humans), it is mostly associated with Muroidea hosts. Sigmodontinae rodents were recently recognized as important hosts of this parasite in Argentina, but the impact of this parasitism on these hosts has not been established. Here we report results of histopathological analyses of 40 livers of Akodon azarae infected with C. hepaticum. Lesions were classified into 4 categories: level 0, absence of lesions; level 1, with focal granulomatous hepatitis; level 2, presence of multifocal granulomatous hepatitis, fibrosis and focal necrosis with neutrophils, and level 3, absence of intact adult parasites, diffuse distribution, necrosis, and fibrosis. Most samples presented lesions of level 2 (55%), but all categories of lesions were found. This is the first study to describe the lesions caused by C. hepaticum in the liver of Sigmodontinae rodents, and the results suggest that infection by this parasite is costly to A. azarae populations.

A new species of the recently erected genus of proteocephalid cestodes, Synbranchiella Arredondo, Alves and Gil de Pertierra, 2017, is described based on specimens found in the mountain mullet, Dajaus monticola (Bancroft, 1834), from Costa Rica. The new species is placed in Synbranchiella because of the cortical position of the genital organs (typical of the former subfamily Monticelliinae); a robust scolex (lacking a metascolex) having a dome-shaped apex and biloculate suckers (lacking free posterior margins); vitelline follicles in 2 narrow lateral bands; a vagina always anterior to the cirrus-sac; and a genital pore that is markedly pre-equatorial, i.e., close to the anterior margin of proglottids. The new species, Synbranchiella megacirrus, differs from the type and only other species of the genus, Synbranchiella mabelae Arredondo, Alves and Gil de Pertierra, 2017, that infects the marbled swamp eel, Synbranchus marmoratus Bloch, 1795, in Argentina by the absence of prep oral vitelline follicles (present in S. mabelae); a well-developed, large, elongate vaginal sphincter (small and ring-like in S. mabelae); and a large cirrus-sac relative to the proglottid size (cirrus-sac length is on average 48% of the proglottid width in S. megacirrus vs. 31% in the latter species). This is the first adult tapeworm reported from mountain mullet and the first record of the genus outside of South America, thereby greatly extending the range of distribution of species of Synbranchiella.

A new species of chigger mites, Morelacarus uazi n. sp., parasitizing lizards of the species Sceloporusgrammicus from Mazapil, Zacatecas, Mexico, is described and illustrated. This new taxon differs from other species of the genus by having a palpal claw with 4 subequal paired prongs, and it can be further distinguished by the combination of the following characters: presence of onychotriches and mastitarsala III, absence of mastitibiala III, subterminala I, and parasubterminala I, and the palpal setal formula B/B/BBb. Additionally, an identification key for the species of the genus Morelacarus is presented. The new species represents the seventh species assigned to the genus and the first species described from a Mexican lizard.

Ticks are important vectors and reservoirs of several zoonotic pathogens. Recently, both known and unknown tick-borne pathogens have emerged and re-emerged, causing unpredictable epidemics. In this study, 211 soft tick samples were collected from Tongliao and Alxa in Inner Mongolia, China. Tick species were identified by morphological and molecular biological analyses. Morphological analysis showed that there was no significant difference in surface features between ticks from the 2 areas. Cloning by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene showed that all ticks belonged to the species Argas persicus. Analysis using Genetyx software indicated that there was a limited degree of diversity between ticks from the 2 areas. Three base changes were detected in the 16S rRNA gene. We constructed phylogenetic trees using MEGA 6.0 software and showed that the ticks from the 2 areas might have evolved independently from species in other geographical areas. To assess the presence of Rickettsia, Streptococcus suis, and Staphylococcus aureus pathogens in tick samples, over 100 16S rRNA sequences belonging to these 3 pathogens were obtained from GenBank. We used CLC Sequence Viewer 7.0 to determine conserved sequences for the design of degenerate primers. Using standard PCR, we detected Rickettsia-specific genes, including 16S rRNA, 17KD, and omp B, in gDNA samples of ticks from Alxa. This study has laid a foundation for future studies on the biodiversity of ticks and for a new pathogen information database of ticks in local areas.

Adults of the genus Austrodiplostomum are parasites in cormorants of the New World, whereas metacercariae are parasites from eye globe and brain of freshwater and brackish water fishes. In this study, specimens of Austrodiplostomum mordax from South America (type-species) were analyzed together with other specimens of Austrodiplostomum spp. collected from several locations across Middle America and North America. Partial DNA sequences of the mitochondrial gene cytochrome c oxidase subunit I (COI), the internal transcribed spacers (ITS1, ITS2, and 5.8S gene), and the D2–D3, domains of the large subunit (LSU) of nuclear ribosomal DNA, were generated for both developmental stages and compared with available sequences of Austrodiplostomum spp. Phylogenetic analyses inferred with each molecular marker using maximum likelihood and Bayesian inference revealed the existence of 4 lineages representing 2 described species, A. mordax and Austrodiplostomum compactum (syn. Austrodiplostomum ostrowskiae) and 2 undescribed species of Austrodiplostomum recognized in previous studies. The COI haplotype network inferred with 172 sequences detected 28 haplotypes divided into 4 clusters, separating each other by 33 and 40 substitutions and with a genetic divergence ranging from 9 to 12%. The largest group included specimens identified as A. compactum plus those identified as A. ostrowskiae, supporting the synonymy of both species. As a result, we conclude that A. compactum is widely distributed across the Americas, in locations of the United States, Mexico, El Salvador, Honduras, Costa Rica, Venezuela, Peru, and Brazil. The other 2 undescribed species of the genus Austrodiplostomum were previously recorded in the United States and now are reported in Mexico. These 2 species cannot be described because adult forms have not been found in their definitive hosts. Finally, the species A. mordax has been found only in some lakes from Argentina, and it was validated in this study through molecular analyses.

Legless lizards (Amphisbaenia) belong to a group of mostly legless squamates that include about 196 species. One genus (Bipes) retains a pair of forelimbs, but all other 19 genera in the clade are limbless. They are widely distributed, occurring in the Middle East and the Caribbean and nearly all of the major continents (except Australia). Only 2/6 (33%) families, 3/20 (15%) genera, and 4/195 (2%) species in the Amphisbaenia clade of the Sauria have been examined for coccidia and 8 coccidia species are now known. Here, we summarize information on the 8 species of coccidia (3 Choleoeimeria, 1 Eimeria, 4 Isospora) reported from legless lizards of the world. In addition, Eimeria amphisbaeniarum Huntington, Cisper, Smith, Powell, Parmerlee Jr., and Lathrop, 1996, is placed in the genus Choleoeimeria. We speculate that another 380 intestinal coccidia infecting this unique reptilian lineage wait to be discovered.

Oarfish are rarely seen and seldom studied, which makes their parasite fauna even more enigmatic. Necropsy of 12 oarfish, Regalecus russelii (Regalecidae) (Cuvier, 1816), from Japan yielded 2 species of acanthocephalans. One species was found in 2 oarfish and a total of 76 specimens was collected, but only a single, immature specimen of the second species was found. The former represents an undescribed species from the order Echinorhynchida and is described here. Morphological and phylogenetic analyses of the small subunit (SSU) rDNA place this species in the family Gymnorhadinorhynchidae, and genus Gymnorhadinorhynchus which is characterized by a cylindrical proboscis with longitudinal rows of hooks, basal circle of enlarged hooks, asymmetry of hook shape, 4 cement glands, and a spineless trunk. Diagnostic characters of this species within the genus are the number of longitudinal rows of hooks (14), smaller body size (males: 4.8–6.6 mm and females: 5.3–6.3 mm) and a number of molecular autapomorphies including a number of long insertions in both the SSU and large subunit rDNA (LSU). A single immature female of Bolbosoma sp. (Palaeacanthocephala: Plagiorhynchidae) was also found with its anterior end embedded in the mucosa of the pyloric ceca. The characters of this specimen are not consistent with any other known species of Bolbosoma; however, because only 1 immature specimen with a partially invaginated proboscis was recovered, it was not designated as a new species.

The study of arthropods is still scarce in paleoparasitology, especially their molecular identification. In this experimental study, we amplify DNA using a polymerase chain reaction from 2 ectoparasite species, Rhipicephalus sanguineus and Pediculus humanus capitis, in experimentally desiccated feces. This study shows perspectives for the study of the identification of arthropods in coprolites when taxonomic identification is not possible.

Bluegill sunfish (Lepomis macrochirus) are an important North American sport fish distributed across the United States and Canada. These fish are sexually dimorphic, with males being larger and more brightly colored than females. Additionally, there are 2 male morphotypes, dominant, brightly colored α-males, and β-males, which resemble females in both appearance and behavior. The 2 male morphotypes differ significantly in terms of mating behavior, territoriality, and diet. These behavioral and feeding differences may result in α-males harboring greater parasite diversity and parasite loads compared to β-males. This was tested by collecting, necropsying, and identifying parasites from 636 L. macrochirus sampled from 9 ponds in northwest Virginia and comparing parasite species richness and parasite load in the male morphotypes. The parasite infracommunities infecting the male morphotypes differed significantly between them at 7 of the 9 sample sites. When compared to β-males, α-males consistently had greater parasite species richness as well as greater abundance for a majority of both trophically and non-trophically transmitted parasite species sampled in this study. The separation of male morphotypes must be accounted for in studies of L. macrochirus parasites due to sex bias differences between males and females being masked when male morphotypes are combined.

Here, we report confirmation of sarcocysts of Sarcocystis jamaicensis in an experimental intermediate host, IFN-γgene knockout (KO) mice orally inoculated sporocysts from its natural definitive host, a red-tailed hawk (Buteo jamaicensis) (RTH). A RTH submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because it could not be rehabilitated and released. Fully sporulated sporocysts from intestinal scrapings of the RTH were orally fed to 2 laboratory-reared outbred Swiss Webster mice (SW; Mus musculus) and to 2 KO mice. The sporocysts were infective for KO mice but not to SW mice. Both SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in their tissues when euthanized on day 54 postinoculation (PI). The KO mice developed neurological signs and were necropsied 38–54 days PI. Schizonts/merozoites were found in both KO mice euthanized and they were confined to the brain. The predominant lesion was meningoencephalitis. Microscopic sarcocysts were found in muscles of both KO mice. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 µm thick) and smooth. Ultrastructural details of sarcocysts are described.

To evaluate the effect of mitogen-activated protein kinase (MAPK) signal transduction pathway inhibitors against alveolar echinococcosis in vitro and in vivo, Echinococcus multilocularis metacestode cysts and protoscolices were obtained from infected mice. Protein chip technology was utilized to screen for key highly expressed target proteins in the MAPK pathway in this parasite and their corresponding inhibitors. Four-week-old Balb/c female mice used for the in vivo experiment underwent inoculation of E. multilocularis by intraperitoneal injection, as well as intragastric administration of MAPK inhibitors for 6 wk. We included 6 groups of mice: a phosphate-buffered saline (PBS) group (negative control); an albendazole-treated group (positive group); and 4 experimental groups treated with TRx0237 mesylate, GDC-0994, pifithrin-β hydrobromide, or Selonsertib. Echinococcus multilocularis protoscolices were collected and cultured in 1066 medium with penicillin/streptomycin and 10% fetal bovine serum. The in vitro experiment included a PBS group (negative control), a dimethyl sulfoxide–treated group (solvent group), and 4 inhibitor-treated groups as in the in vivo experiment (experimental groups). Each inhibitor group received 4 drug concentrations (5, 30, 55, and 80 µM), and the experiment was performed in triplicate per sample. Fluorescence microscopy was used to evaluate the survival rate of the protoscolices every 48 hr beginning from the first 24 hr. The same grouping was used to evaluate cytotoxicity on E. multilocularis germinal cells and L02 cells. The average weights of E. multilocularis metacestode cyst tissue from each group of the in vivo experiment were 873 mg (PBS), 335 mg (albendazole), 323 mg (TRx0237 mesylate), 420 mg (GDC-0994), 340 mg (pifithrin-β hydrobromide), and 642 mg (Selonsertib). Results showed albendazole, TRx0237 mesylate, and pifithrin-β hydrobromide had significant inhibitory effects on inhibition of E. multilocularis. We found a positive correlation between drug concentrations and the inhibitory effects seen in the in vitro experiment, with the differences in contrast with the control group becoming statistically significant after 72 hr of treatment (P < 0.05). The inhibition rates of TRx0237 mesylate to germinal cells by drug concentration were 23.73, 46.59, 74.71, and 77.44%. Other drugs had no effect on germinal cells. All the inhibitors had low toxicity on L02 cells. Inhibitors of the MAPK signal transduction pathway showed significant inhibitory effects on E. multilocularis, suggesting these may be potential candidates for the treatment of alveolar echinococcosis.

The control of emerging parasites requires a fundamental knowledge of where and when rates of transmission are high. Data on spatiotemporal patterns of infection are challenging to obtain, particularly for complex life cycle parasites that involve transmission into multiple obligate hosts. The lancet liver fluke, Dicrocoelium dendriticum, has a long history of colonization outside its native host and geographical range in continental Europe. Infection patterns involving adult and metacercarial stages have been characterized for this trematode in a region of emergence in western Canada within co-grazing herbivores and ants, but infection patterns in snail intermediate hosts in this region are unknown. We combined spatiotemporal prevalence surveys with sequence analyses of the cytochrome c oxidase subunit 1 (COI) barcoding gene from samples of sporocyst tissue in infected snails to confirm that D. dendriticum utilizes 3 sympatric species of Oreohelid land snail (Oreohelix subrudis, Oreohelix sp., and Oreohelix cooperi) as first intermediate host. Mean prevalence within a total sample of 900 adult snails collected over 1 field season from 6 sites was 9.9 ± 2.4%. For each species of snail, prevalence ranged between 5–30% within monthly samples, with peaks in midsummer followed by declines in fall. Between-site variation in prevalence was low and nonsignificant, implying that rates of transmission of D. dendriticum miracidia from domestic stock and wildlife into snails are similar within localized sites, despite high variation in local habitat characteristics and in the structure of the definitive host community.

In this study, 542 individual fish from 20 species from the Patagonian continental shelf of Argentina were examined for acanthocephalans. A total of 1,547 acanthocephalans belonging to 5 species were collected from 18 species of fish. Adult forms were represented by 2 species: Aspersentis johni (Baylis, 1929) (Heteracanthocephalidae) from longtail southern cod, Patagonotothen ramsayi (Regan) (new host record), and Breizacanthus aznari Hernández-Orts, Alama-Bermejo, Crespo, García, Raga and Montero, 2012 (Arhythmacanthidae) from raneya, Raneya brasiliensis (Kaup). Immature worms of B. aznari were also collected from the intestine of pink cusk-eel, Genypterus blacodes (Forster) (new host record). Cystacanths of 3 species of Corynosoma Lühe, 1904 (Polymorphidae) were found encapsulated in the mesenteries of fish. Corynosoma australe Johnston, 1937 was the most abundant acanthocephalan in our study, infecting 18 species of fish and accounting for >89.9% of all specimens collected. A cystacanth of Corynosoma bullosum (Linstow, 1892) was found in “castan˜ eta”, Nemadactylus bergi (Norman) (new host record), and cystacanths of Corynosoma cetaceum Johnston and Best, 1942 were collected from red searobin, Prionotus nudigula Ginsburg, and flounders Paralichthys isosceles Jordan (new host record) and Xystreurys rasile (Jordan). The Patagonian shelf of Argentina represents a new locality record for A. johni and C. bullosum. This survey is a starting point for understanding the diversity of marine acanthocephalans in Patagonian waters.

This study reports for the first time the molecular characterization of Cryptosporidium spp. in Salmo trutta. A total number of 613 brown trout was captured by local anglers in 44 Galician rivers within 10 river basins (NW Spain) during the 2015 fishing season (March–August) and classified into groups according to their size. The gastrointestinal tracts were dissected and differentiated in pyloric ceca and intestine, which were homogenized and concentrated in phosphate-buffered saline 0.04 M pH 7.2/diethyl ether (2:1). Cryptosporidium oocysts were observed by immunofluorescence microscopy in 103 of 613 specimens (16.8%), with a mean intensity of 326.7 oocysts/trout. The highest prevalence rate was detected in specimens <2 yr (23.1%). Considering the anatomical location, Cryptosporidium oocysts were observed in pyloric ceca (72 trout, 69.9%), intestine (15 trout, 14.6%), or in both locations (16 trout, 15.5%), showing statistically significant differences between the 2 locations (P < 0.01). The prevalence rate in the pyloric ceca increased with the age/size of the fish (62.2% vs. 70.8% vs. 83.3% for trout <2, 2–3, and >3 yr, respectively). By contrast, the prevalence rate in the intestinal location decreased with the age/size of specimens (21.6% vs. 12.5% vs. 7.7% for trout <2, 2–3, and >3 yr, respectively), but statistically significant differences were not determined. The microscopic observation of clusters of 4–20 oocysts in the pyloric ceca from 5 specimens of 20–28-cm body length is remarkable. By polymerase chain reaction amplification and sequencing of fragments of small-subunit ribosomal DNA (SSU-rDNA), GP60, hsp70, and actin loci, Cryptosporidium molnari-like genotype was identified in 1 trout and Cryptosporidium parvum (subtypes IIaA15G2R1 and IIaA18G3R1) in 47 fish, including those specimens in which oocyst clusters were observed. This finding may indicate a true infection by C. parvum, as the homogenization process would break the epithelial cells, releasing oocysts, free or in clusters. Cryptosporidium oocysts were detected in wild trout captured from 27 of 44 rivers sampled in Galicia (61.4%), belonging to 9 of the 10 river basins considered, confirming the presence of this protozoan parasite in Galician rivers and proving their wide dispersion in aquatic freshwater environments. The identification of the zoonotic species C. parvum in brown trout may indicate a risk to public health as trout may be a potential source of infection to humans. Thus, edible wild fish extend the range of foodstuffs involved in the transmission of cryptosporidiosis.

Pallisentis (Brevitritospinus) indica Mittal and Lal, 1976 was found infecting the spotted snakehead Channa punctatus Bloch and Schneider (Channidae) from Meerut, Uttar Pradesh (U.P.), India. The species was identified on the basis of proboscis hooks, trunk spines, and other structures that separate it from all described species. Molecular analysis based on 18S rDNA placed the P. indica isolates within a clade of Pallisentis spp. but distinct from other representatives of the same genus. This study documents the first molecular characterization of P. indica from India.

Equine piroplasmosis stands out among the diseases that affect Equidae in Brazil and the world. It is caused by the protozoa Theileria equi and Babesia caballi. The objective of the present study was to carry out the molecular characterization of T. equi using equine blood samples collected in the 5 geographic regions of Brazil. Samples from all over the country were tested for the presence of T. equi by real-time PCR. The 18S rRNA sequences (∼1,600 bp) obtained from 23 samples taken from naturally infected horses were characterized by sequencing and analyzed to identify the genotypes and the possible sites of genetic variability. Thirteen different T. equi 18S rRNA sequences were identified, and 2 different genotypes were demonstrated to be in circulation in Brazil. Alignment entropy analysis demonstrated the existence of three hypervariable regions (V2, V4, and V8) within the 18S rRNA sequence of T. equi. The V2 region is located between nucleotides 63 and 75, V4 is located between nucleotides 524 and 586, and V8 is located between nucleotides 1,208 and 1,226. The hypervariable region V4 demonstrated the greatest variation within the 18S rRNA sequence of T. equi. Phylogenetic analysis based on the 18S rRNA sequences revealed the formation of 3 distinct clades (A, B, and C). The Brazilian samples belonged to 2 clades (A and C). The present study describes the characterization and heterogeneity of the circulating T. equi 18S rRNA sequences in Brazil. The results confirm that the country is an endemic area for the disease, and they indicate that at least 2 distinct T. equi genotypes are naturally infecting equines in Brazil.