Effects of Eimeria acervulina infection on the luminal and mucosal microbiota of the cecum and ileum in broiler chickens.

Coccidiosis, an intestinal disease caused by Eimeria parasites, is responsible for major losses in the poultry industry by impacting chicken health. The gut microbiota is associated with health factors, such as nutrient exchange and immune system modulation, requiring understanding on the effects of Eimeria infection on the gut microbiota. This study aimed to determine the effects of Eimeria acervulina infection on the luminal and mucosal microbiota of the cecum (CeL and CeM) and ileum (IlL and IlM) at multiple time points (days 3, 5, 7, 10, and 14) post-infection. E. acervulina infection decreased evenness in CeL microbiota at day 10, increased richness in CeM microbiota at day 3 before decreasing richness at day 14, and decreased richness in IlL microbiota from day 3 to 10. CeL, CeM, and IlL microbiota differed between infected and control birds based on beta diversity at varying time points. Infection reduced relative abundance of bacterial taxa and some predicted metabolic pathways known for short-chain fatty acid production in CeL, CeM, and IlL microbiota, but further understanding of metabolic function is required. Despite E. acervulina primarily targeting the duodenum, our findings demonstrate the infection can impact bacterial diversity and abundance in the cecal and ileal microbiota.

Expression of Toll-like receptors and host defence peptides in the cecum of chicken challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria protozoa, affects poultry farming. Toll-like receptors (TLRs) and host defence peptides (HDPs) help host innate immune responses to eliminate invading pathogens, but their roles in Eimeria tenella infection remain poorly understood. Herein, 14-day-old chickens were treated orally with 50,000 E. tenella oocysts and the cecum was dissected at different timepoints. mRNA expression of 10 chicken TLRs (chTLRs) and five HDPs was measured by quantitative real-time PCR. chTLR7 and chTLR15 were upregulated significantly at 3 h post-infection while other chTLRs were downregulated (p < .05). chTLR1a, chTLR1b, chTLR2b and chTLR4 peaked at 36 h post-infection, chTLR3, chTLR5 and chTLR15 peaked at 72 h post-infection and chTLR21 expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For HDPs, cathelicidin (CATH) 1 to 3 and B1 peaked at 48 h post-infection, liver-expressed antimicrobial peptide 2 peaked at 96 h post-infection, and CATH 2 expression was highest among HDPs. CATH2 and CATH3 were markedly upregulated at 3 h post-infection (p < .05). The results provide insight into innate immune molecules during E. tenella infection in chicken, and indicate that innate immune responses may mediate resistance to chicken coccidiosis.

The interaction between free Ca2+ in host cells and invasion of E. tenella.

Eimeria tenella is the most pathogenic and common coccidia that causes chicken coccidiosis. The intracellular free Ca2+ of the host cell is closely related to the invasion, development, and proliferation of intracellular parasites. To determine the dynamic changes of intracellular free Ca2+ and its function in the process of E. tenella invading host cells, we established a chick embryo cecal epithelial cells model of E. tenella infection. Chick embryo cecal epithelial cells were treated with different Ca2+ signal inhibitor, respectively, and then infected with E. tenella. The results showed that extracellular Ca2+, Ca2+ channels on the cell membrane, IP3R ion channels on the endoplasmic reticulum membrane, and RyR ion channels regulated the free Ca2+ in cecal epithelial cells. Through fluorescence labeling and invasion rate detection, we found that the intracellular Ca2+ did not change significantly during the invasion of E. tenella, but its stability was critical to the invasion of parasites.

Excretion of Histomonas meleagridis following experimental co-infection of distinct chicken lines with Heterakis gallinarum and Ascaridia galli.

BACKGROUND: Histomonosis is a severe re-emerging disease of poultry caused by Histomonas meleagridis, a protozoan parasite which survives in the environment via the cecal worm Heterakis gallinarum. Following infection, the parasites reside in the ceca and are excreted via host feces. In the present work, male birds of conventional broiler (Ross 308, R), layer (Lohmann Brown Plus, LB) and a dual-purpose (Lohmann Dual, LD) chicken line were infected with 250 embryonated eggs of Ascaridia galli and Heterakis gallinarum, respectively, with the latter nematode harboring Histomonas meleagridis, to investigate a co-infection of nematodes with the protozoan parasite in different host lines. METHODS: In weekly intervals, from 2 to 9 weeks post infection (wpi), individual fecal samples (n = 234) from the chickens were collected to quantify the excretion of H. meleagridis by real-time PCR and to determine the number of nematode eggs per gram (EPG) in order to elucidate excretion dynamics of the flagellate and the nematodes. This was further investigated by indirect detection using plasma samples of the birds to detect antibodies specific for H. meleagridis and worms by ELISA. The infection with H. meleagridis was confirmed by histopathology and immunohistochemistry to detect the flagellate in the cecum of representing birds. RESULTS: The excretion of H. meleagridis could already be observed from the 2nd wpi in some birds and increased to 100% in the last week of the experiment in all groups independent of the genetic line. This increase could be confirmed by ELISA, even though the number of excreted H. meleagridis per bird was generally low. Overall, histomonads were detected in 60% to 78% of birds with temporary differences between the different genetic lines, which also showed variations in the EPG and worm burden of both nematodes. CONCLUSIONS: The infection with H. gallinarum eggs contaminated with H. meleagridis led to a permanent excretion of the flagellate in host feces. Differences in the excretion of H. meleagridis in the feces of genetically different host lines occurred intermittently. The excretion of the protozoan or its vector H. gallinarum was mostly exclusive, showing a negative interaction between the two parasites in the same host.

Rare cause of right iliac fossa pain in a UK patient.

A 62-year-old Asian man presented with a 3-month history of right iliac fossa pain which had progressively worsened over the last 3 weeks. All blood parameters were found to be unremarkable except for mildly elevated erythrocyte sedimentation rate. CT imaging demonstrated thickening of the ascending colon and caecum. Colonoscopic biopsies showed submucosal granulomas with features suggestive of schistosomiasis and parasite serology was positive for Schistosoma antibodies. He was treated with praziquantel and showed subsequent symptomatic and radiological improvement. However, he represented nearly 2 years later and underwent a right hemicolectomy for small bowel obstruction. The resected bowel showed an inflammatory caecal mass and a terminal ileal adenocarcinoma.

Molecular characterization of 60S ribosomal protein L12 of E. tenella.

This study analyzed the large-subunit (60S) ribosomal protein L12 of Eimeria tenella (Et60s-RPL12). A full-length cDNA was cloned, and the recombinant protein was expressed in E. coli BL21 and inoculated in rabbits to produce the polyclonal antibody. Quantitative real-time polymerase chain reaction and western blotting were used to analyze the transcription levels of Et60s-RPL12 and translation levels in different developmental stages of E. tenella. The results showed that the mRNA transcription level of Et60s-RPL12 was highest in second-generation merozoites, whereas the translation level was highest in unsporulated oocysts. Indirect immunofluorescence showed that Et60s-RPL12 was localized to the anterior region and surface of sporozoites, except for the two refractile bodies. As the invasion of DF-1 cells progressed, fluorescence intensity was increased, and Et60s-RPL12 was localized to the parasitophorous vacuole membrane (PVM). The secretion assay results using staurosporine indicated that this protein was secreted, but not from micronemes. The role of Et60s-RPL12 in invasion was evaluated in vitro. The results of the invasion assay showed that polyclonal antibody inhibited host cell invasion by the parasite, which reached about 12%. However, the rate of invasion was not correlated with the concentration of IgG.

Immunological evaluation of invasive Lactobacillus plantarum co-expressing EtMIC2 and chicken interleukin-18 against Eimeria tenella.

Chicken coccidiosis is a protozoan parasitic disease that leads to considerable economic losses in the poultry industry. In this study, we used invasive Lactobacillus plantarum (L.P) expressing the FnBPA protein as a novel bacterial carrier for DNA delivery into epithelial cells to develop a live oral DNA vaccine. A fusion DNA vaccine co-expressing EtMIC2 and chicken IL-18 (chIL-18) was constructed and then delivered to the host by invasive L.P. Its efficacy against Eimeria tenella challenge was evaluated in chickens by examining the relative weight gain rate; caecal lesion score; OPG; anti-coccidial index (ACI); levels of EtMIC2 antibody, FnBPA, IL-4, IL-18, IFN-γ and SIgA; and proliferation ability and percentages of CD4+ and CD8+ splenocytes. The experimental results showed that chickens immunized with invasive L.P carrying the eukaryotic expression vector pValac-EtMIC2 (pValac-EtMIC2/pSIP409-FnBPA) had markedly improved immune protection against challenge compared with that of chickens immunized with non-invasive L.P (pValac-EtMIC2/pSIP409). However, invasive L.P co-expressing EtMIC2 with the chIL-18 vector exhibited the highest protection efficiency against E. tenella. These results indicate that invasive Lactobacillus-expressing FnBPA improved humoural and cellular immunity and enhanced resistance to E. tenella. The DNA vaccine delivered by invasive Lactobacillus provides a new concept and method for the prevention of E. tenella.

Development of caecaloids to study host-pathogen interactions: new insights into immunoregulatory functions of Trichuris muris extracellular vesicles in the caecum.

The caecum, an intestinal appendage in the junction of the small and large intestines, displays a unique epithelium that serves as an exclusive niche for a range of pathogens including whipworms (Trichuris spp.). While protocols to grow organoids from small intestine (enteroids) and colon (colonoids) exist, the conditions to culture organoids from the caecum have yet to be described. Here, we report methods to grow, differentiate and characterise mouse adult stem cell-derived caecal organoids, termed caecaloids. We compare the cellular composition of caecaloids with that of enteroids, identifying differences in intestinal epithelial cell populations that mimic those found in the caecum and small intestine. The remarkable similarity in the intestinal epithelial cell composition and spatial conformation of caecaloids and their tissue of origin enables their use as an in vitro model to study host interactions with important caecal pathogens. Thus, exploiting this system, we investigated the responses of caecal intestinal epithelial cells to extracellular vesicles secreted/excreted by the intracellular helminth Trichuris muris. Our findings reveal novel immunoregulatory effects of whipworm extracellular vesicles on the caecal epithelium, including the downregulation of responses to nucleic acid recognition and type-I interferon signalling.

Effects of the PI3K/Akt signaling pathway on the apoptosis of early host cells infected with Eimeria tenella.

This study investigated the role of PI3K/Akt signaling pathway on host cell apoptosis in the early infection of Eimeria tenella. Chicken cecal epithelial cells were treated with apoptosis-inducer Actinomycin D (Act D) or PI3K/Akt signaling pathway inhibitor LY294002 and then infected with E. tenella. Results demonstrated that the E. tenella-infected group had less apoptosis 4-8 h after the infection and more apoptosis 12-20 h after the infection than the control group. At 4-20 h after the infection, the apoptotic/necrotic rate and the Caspase-3 activity in the Act D + E. tenella group were lower (P < 0.01) than those in the Act D-treated group. The p-Akt and NF-κB contents in the E. tenella-infected group were higher (P < 0.01) than those in the control group 4-12 h after the infection. However, the bad content and the Caspase-9/3 activity were lower (P < 0.05) in the E. tenella-infected group than in the control group. Compared with the E. tenella-infected group, the LY294002 + E. tenella group showed decreased p-Akt content and increased apoptotic/necrotic rate, bad content, NF-κB expression, membrane permeability transition pore (MPTP) openness, and Caspase-9/3 activity. Thus, the early development of E. tenella could inhibit host cell apoptosis by downregulating the Caspase-3 activity. Upregulating this activity promoted apoptosis. In addition, activating the PI3K/Akt signaling pathway inhibited the apoptosis of E. tenella host cells in the early infection by reducing the expression of the bad content, limiting the MPTP opening, and decreasing the Caspase-9 and Caspase-3 activities.

A phylogenetic study of the cecal amphistome Zygocotyle lunata (Trematoda: Zygocotylidae), with notes on the molecular systematics of Paramphistomoidea.

Zygocotyle lunata inhabits the caecum of birds and mammals from the American continent. This amphistome parasite is easily maintained in the laboratory and serves as a model organism in life-cycle studies, but it has seldom been studied using molecular data. Neither the position of Z. lunata in the superfamily Paramphistomoidea nor the monophyly of the Zygocotylidae has been evaluated with molecular phylogenetic methods. In the present study, adult specimens of Z. lunata obtained experimentally in mice from Brazil were submitted to molecular studies. Partial sequences of nuclear (1261 bp of 28S and 418 bp of 5.8S-ITS-2) and mitochondrial (1410 bp of cytochrome c oxidase 1, cox1) markers were compared with published data. In the most well-resolved phylogeny, based on 28S sequences, Z. lunata clustered in a well-supported clade with Wardius zibethicus, the only other species currently included in the Zygocotylidae, thus confirming the validity of this family. Divergence of 28S sequences between these species was 2.2%, which falls in the range of intergeneric variation (0.9-5.6%) observed in the other two monophyletic groups in the 28S tree, i.e., representatives of Gastrodicidae and Neotropical cladorchiids (Cladorchiidae). Analysis of ITS-2 and two parts of the cox1 gene placed Z. lunata within poorly resolved clades or large polytomies composed of several paramphistomoid families, without clarifying higher-level phylogenetic relationships. The cox1 of a Brazilian isolate of Z. lunata is 99.6% similar to a Canadian isolate, confirming the pan-American distribution of the species. Finally, our phylogenetic reconstructions of Paramphistomoidea revealed a complex scenario in the taxonomic composition of some amphistome families, which highlights a need for further integrative studies that will likely result in rearrangements of traditional morphology-based classifications.

A potential vaccine candidate towards chicken coccidiosis mediated by recombinant Lactobacillus plantarum with surface displayed EtMIC2 protein.

Eimeria tenella (E. tenella) has caused severe economic loss in chicken production, especially after the forbidden use of antibiotics in feed. Considering the drug resistant problem caused by misuse of chemoprophylaxis and live oocyst vaccines can affect the productivity of chickens, also it has the risk to reversion of virulence, the development of efficacious, convenient and safe vaccines is still deeply needed. In this study, the EtMic2 protein of E. tenella was anchored on the surface of Lactobacillus plantarum (L. plantarum) NC8 strain. The newly constructed strain was then used to immunize chickens, followed by E. tenella challenge. The results demonstrated that the recombinant strain could provide efficient protection against E. tenella, shown by increased relative body weight gains, percentages of CD4+ and CD8+ T cells, humoral immune response and inflammatory cytokines. In addition, decreased cecum lesion scores and fecal oocyst shedding were also observed during the experiment. In conclusion, this study proves the possibility to use L. plantarum as a vessel to deliver protective antigen to protect chickens against coccidiosis.

Effects of Eimeria maxima and Clostridium perfringens infections on cecal microbial composition and the possible correlation with body weight gain in broiler chickens.

With the voluntary and regulatory withdrawal of antibiotic growth promoters from animal feed, coccidiosis and necrotic enteritis (NE) emerge as the top two enteric poultry infectious diseases responsible for major economic loss worldwide. The objective of this study was to investigate the correlation between the cecal microbiota compositions with the growth trait after coccidiosis and NE. In this study, the effects of Eimeria maxima and/or Clostridium perfringens infections on the microbial composition and potential correlation with the body weight gain were investigated in broiler chickens using 16S rRNA gene sequencing. E. maxima and C. perfringens coinfection successfully induced NE with its typical gut lesions and significant reductions in the percentage of relative body weight gain (RBWG%). The NE challenge model did not affect cecal microbial diversity, but influenced the cecal microbial composition. KEGG enzymes in microbiota were significantly altered in abundance following dual infections. Furthermore, significant correlations between cecal microbiota modules and RBWG% were identified in the sham control, E. maxima or C. perfringens infected groups. Understanding of host-microbiota interaction in NE would enhance the development of antibiotics-independent strategies to reduce the harmful effect of NE on the gut microbiota structure, and improve the gut health and poultry production.

Research Note: Correlation analysis of interleukin-6, interleukin-8, and C-C motif chemokine ligand 2 gene expression in chicken spleen and cecal tissues after Eimeria tenella infection in vivo.

IL-6, IL-8, and C-C motif chemokine ligand 2 (CCLi2) are important factors in inflammatory and immune responses. To investigate their relationships in the spleen and cecum and between coccidiosis-infected and uninfected states, we performed quantitative real-time PCR to compare the relative expression difference of IL-6, IL-8, and CCLi2 in the same tissues between the infection and control groups. In addition, the correlations of the relative expression levels of these 3 genes were determined in the same and different tissues within the same group. The results showed that the expression levels of IL-6, IL-8, and CCLi2 in the spleen and cecum of the infected group were all higher than those of the uninfected group (P < 0.05). The correlation coefficients among the IL-6, IL-8, and CCLi2 expression levels in the spleen or cecum were all positive in both the infection and control groups. In the spleen tissues, CCLi2 expression was strongly correlated with IL-6 and IL-8 in the uninfected group (P < 0.01), and the correlation coefficients reached 0.853 (R2 = 0.728) and 0.996 (R2 = 0.992), respectively. The expression of CCLi2 was also strongly correlated with IL-8 (R reached 0.890, R2 = 0.792) in the infected group. In the cecal tissues, the expression levels of the 3 genes were all extremely significantly correlated in the uninfected group (P < 0.01), and the correlation coefficients ranged from 0.498 to 0.765, indicating moderate correlations. The expression of IL-6 was extremely significantly positively correlated with IL-8 and CCLi2 in the infected group (P < 0.01), with moderate correlations (R ranged from 0.469-0.639). In addition, the expression levels of the 3 genes were not significantly correlated (P > 0.05) between the spleen and cecum tissues in either the infection group or the control group. These results indicate that IL-6, IL-8, and CCLi2 were correlated and play an important role in coccidiosis infection of Jinghai yellow chicken. Our data also provide a basis for further exploring the role of these 3 genes in genetic breeding for coccidiosis resistance.

Dissection of the cecal microbial community in chickens after Eimeria tenella infection.

BACKGROUND: Eimeria spp. are responsible for chicken coccidiosis which is the most important enteric protozoan disease resulting in tremendous economic losses in the poultry industry. Understanding the interaction between the avian cecal microbiota and coccidia is of interest in the development of alternative treatments that do not rely on chemotherapeutics and do not lead to drug resistance. METHODS: We utilized 16S rRNA gene sequencing to detect the dynamics of the cecal microbial community in AA broilers challenged with Eimeria tenella. Histopathological analysis of the cecum was also conducted. RESULTS: We found that microbial shifts occur during the infection. Lactobacillus, Faecalibacterium, Ruminococcaceae UCG-013, Romboutsia and Shuttleworthia decreased in abundance. However, the opportunistic pathogens Enterococcus and Streptococcus increased in abundance over time in response to the infection. CONCLUSIONS: Eimeria tenella disrupts the integrity of the cecal microbiota and could promote the establishment and growth of potentially pathogenic bacteria. Defining bacterial populations affected by coccidial infection might help identify bacterial markers for intestinal disease as well as populations or species that could be beneficial in maintaining and restoring gut homeostasis during and after infection with E. tenella.

Pathology caused by three species of Eimeria that infect the turkey with a description of a scoring system for intestinal lesions.

Three-week-old turkey poults were infected with pure lines of three species of Eimeria (E. adenoeides, E. gallopavonis, and E. meleagrimitis) recently isolated from commercial turkey farms. The lines had been propagated from a single oocyst and identified by species-specific PCR amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Five to six days after infection their intestines were removed and examined for the presence of intestinal lesions. A description and review of the pathology caused by these parasites is provided, and a scoring system developed by which the severity of the lesions can be evaluated. The system is similar to that described by Johnson, J. and Reid, W. M. [1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitology, 28, 30-36] for chickens in which a score of zero to four is assigned to lesions of increasing severity. The intestinal lesions observed here, and their assigned scores, are supported by representative illustrations. It is hoped that they may prove a useful tool for evaluating the pathology caused by E. adenoeides, E. gallopavonis, and E. meleagrimitis in the turkey.RESEARCH HIGHLIGHTSA scoring system has been developed for intestinal lesions caused by three species of Eimeria that infect the turkey.The lesions attributable to these species are illustrated.

Life Cycle of the Trout Cecal Nematode, Truttaedacnitis truttae (Nematoda: Cucullanidae): Experimental and Field Observations.

Truttaedacnitis truttae is a cucullanid nematode of primarily salmonine fishes. Brown trout (Salmo trutta) in Europe reportedly become parasitized by ingesting lampreys (Lampetra planeri) carrying infective larvae. However, our field and laboratory observations suggested that North American specimens of T. truttae have an alternative life cycle. High abundances and potential impact of T. truttae in rainbow trout, Oncorhynchus mykiss, in the Colorado River drainage in Grand Canyon, where there are no lampreys, prompted a study on the transmission dynamics of this nematode. Eggs of T. truttae, collected from live gravid females, were incubated in the laboratory. Snails, Physa gyrina and Lymnaea sp., were exposed to T. truttae larvae 3-4 wk later. Active larvae of T. truttae were observed penetrating the intestinal wall of exposed snails, and worm larvae were found in the visceral tissues when examined 1 wk after exposure. Larvae in snails showed little growth and development 2 wk later and corresponded to L3 larvae. Infected snails were fed to hatchery-reared juvenile rainbow trout. Developing stages were subsequently found in the mucosal lining and lumen of trout intestines. Adult male and female (gravid) worms were found in the ceca of trout examined 5-6 mo after consuming infected snails. Larvae found in pepsin/trypsin digests and mucosal scrapings from wild, naturally infected, trout corroborate laboratory findings. Screening of Physa sp. and gammarids collected from Colorado River, Grand Canyon, for natural infections with T. truttae using the ITS1 rDNA marker gave positive results. Truttaedacnitis truttae is the second species, after Truttaedacnitis clitellarius of lake sturgeon, capable of using a snail first intermediate/paratenic host and is similar to several other cucullanids in having a histotropic phase of development in the definitive fish host.

Protective potential of diclazuril-treated oocysts against coccidiosis in layer chicks.

Diclazuril, which is widely used for the prevention of coccidiosis in chickens, has a lethal effect on asexual and sexual stages of Eimeria spp. However, little is known about its effect on the exogenous stages of Eimeria spp. In this study, we evaluated the effect of in vitro treatment with 0.2% diclazuril on unsporulated and sporulated oocysts of Eimeria spp. For this purpose, a total of 180 male layer chicks aged one day were randomly divided into 5 experimental groups. Each group was divided into 3 replicates of 12 chicks each. Group 1 (G1) and Group 2 (G2) were negative (non-immunized and non-challenged) and positive (non-immunized and challenged) controls, respectively. Group 3 (G3) was immunized per os with 1.0 × 104 non-diclazuril treated-sporulated oocysts. Groups 4 (G4) was immunized per os with 0.2% diclazuril treated-unsporulated oocysts (1.0 × 104) in which diclazzuril didn't affect sporulation. Group 5 (G5) was immunized per os with 0.2% diclazuril treated-sporulated oocysts (1.0 × 104). Chicks of G2, G3, G4, and G5 were challenged with 7.5 × 104 untreated sporulated oocysts at the age of 21 days, while the group 1 chicks remained unchallenged. G4 and G5 animals immunized with 0.2% diclazuril-treated oocysts showed a significant decrease in bloody diarrhea severity, lesion scores, and oocyst counts in comparison to those immunized with untreated oocysts. Furthermore, histopathologic findings showed a low number of parasitic stages in cecal tissues in G4 and G5. A significant increased body weight gain was observed in Gs 4 and 5 in comparison to G2. In addition, expression levels of IL-2, IL-12, and IFN-γ were significantly increased in G4 and G5. In conclusion, diclazuril is effective in attenuating Eimeria oocysts and thus provides an alternative approach for using diclazuril-treated oocysts to protect chicks against Eimeria challenge.

Molecular Identification and Phylogenetic Analysis of Heterakis dispar Isolated from Geese.

PURPOSE: Heterakidosis is a common parasitic infection caused in domestic birds by Heterakis species: Heterakis gallinarum, H. isolonche, and H. dispar. Among them, the best described species is H. gallinarum, noted mainly in gallinaceous birds. In waterfowl, H. dispar is the predominant species. The variations in morphology and host specificity qualify H. dispar as a different species, but the phylogenetic relationships between heterakids were unclear for a long time, because of a lack of H. dispar sequences. METHODS: The authors provided the molecular data for H. dispar and analyzed the obtained sequences of the partial 18S rRNA gene and region ITS1-5.8SrRNA-ITS2 with the homological sequences. RESULTS: The 18S rRNA PCR product of H. dispar was about 800 bp, and the ITS-5.8S-ITS2 PCR product was about 920 bp, noticeably smaller size compared to H. gallinarum product. The BLAST analysis of H. dispar 18S sequence showed a 99% similarity with the sequences of Heterakis gallinarum and Ascaridia galli, 98% with A. nymphii, but only 94% with the sequence of Heterakis sp. Our ITS sequence of H. dispar was almost identical to the H. isolonche isolate, there is only one nucleotide of difference among the 943 sites analyzed. It also showed a lower similarity to the ITS sequences of H. gallinarum (88%), H. spumosa (87%), and H. dahomensis (87%). CONCLUSIONS: In our phylogenetic analysis, it is the first attempt at the reconstruction of relationships within this superfamily Heterakoidea based on 18S rDNA and ITS region.

A new genus and species of pinworm (Nematoda, Oxyuridae) from the gray mouse opossum, Tlacuatzin canescens.

Tlacuatzoxyuris simpsoni n. gen. n. sp. is described from the cecum of the gray opossum, Tlacuatzin canescens, a species endemic to the deciduous dry forest of Mexico. The digestive tracts of four specimens were examined for parasites; three of these were archived in the American Museum of Natural History and one was a live capture. Relative to the other four monotypic genera of pinworms known to infect opossums, the new genus is diagnosed on the basis of a round cephalic plate with a semicircular stoma surrounded by a rim. In addition, males feature a prominent cephalic vesicle not fully developed in females, accounting for sexual dimorphism. The new species includes small worms that feature a conspicuous, not reticulated cephalic vesicle and semicircular stoma and lateral alae with two crests. In addition, the postcloacal cuticle of males features a small area with ornamentation between cloaca and submedial papillae. Finally, both spicule and gubernaculum are relatively short. Although the eggs of Tlacuatzoxyuris n. gen. are unknown, the conspicuous differences in traits used in the diagnosis of genera prompted us to propose a new genus for the new species. This is the first species of Oxyuridae reported in mouse opossums outside South America, and the fifth species of the family occurring in didelphimorph marsupials. This is an example of the usefulness of documenting the diversity of parasites associated with this unique clade of mammals through the examination of preserved tissues.