Th17 Cells Provide Mucosal Protection against Gastric Trypanosoma cruzi Infection.

Trypanosoma cruzi is the intracellular parasite of Chagas disease, a chronic condition characterized by cardiac and gastrointestinal morbidity. Protective immunity requires CD4+ T cells, and Th1 cells and gamma interferon (IFN-γ) are important players in host defense. More recently, Th17 cells and interleukin 17A (IL-17A) have been shown to exert protective functions in systemic T. cruzi infection. However, it remains unclear whether Th17 cells and IL-17A protect in the mucosa, the initial site of parasite invasion in many human cases. We found that IL-17RA knockout (KO) mice are highly susceptible to orogastric infection, indicating an important function for this cytokine in mucosal immunity to T. cruzi. To investigate the specific role of Th17 cells for mucosal immunity, we reconstituted RAG1 KO mice with T. cruzi-specific T cell receptor transgenic Th17 cells prior to orogastric T. cruzi challenges. We found that Th17 cells provided protection against gastric mucosal T. cruzi infection, indicated by significantly lower stomach parasite burdens. In vitro macrophage infection assays revealed that protection by Th17 cells is reduced with IL-17A neutralization or reversed by loss of macrophage NADPH oxidase activity. Consistently with this, mice lacking functional NADPH oxidase were not protected by Th17 cell transfer. These data are the first report that Th17 cells protect against mucosal T. cruzi infection and identify a novel protective mechanism involving the induction of NADPH oxidase activity by IL-17A. These studies provide important insights for Chagas vaccine development and, more broadly, increase our understanding of the diverse roles of Th17 cells in host defense.

Dynamic transcriptomic changes of goat abomasal mucosa in response to Haemonchus contortus infection.

Gastrointestinal nematode (GIN) infections are one of the major constraints for grazing sheep and goat production worldwide. Genetic selection for resistant animals is a promising control strategy. Whole-transcriptome analysis via RNA-sequencing (RNA-seq) provides knowledge of the mechanisms responsible for complex traits such as resistance to GIN infections. In this study, we used RNA-seq to monitor the dynamics of the response of the abomasal mucosa of Creole goat kids infected with Haemonchus contortus by comparing resistant and susceptible genotypes. A total of 8 cannulated kids, 4 susceptible and 4 resistant to GIN, were infected twice with 10 000 L3 H. contortus. During the second infection, abomasal mucosal biopsies were collected at 0, 8, 15 and 35 days post-infection (dpi) from all kids for RNA-seq analysis. The resistant animals showed early activation of biological processes related to the immune response. The top 20 canonical pathways of differentially expressed genes for different comparison showed activation of the immune response through many relevant pathways including the Th1 response. Interestingly, our results showed a simultaneous time series activation of Th2 related genes in resistant compared to susceptible kids.

Gastric coccidiosis in Thoracocharax stellatus caused by Goussia guamaensis n. sp. (Apicomplexa: Eimeriidae) from the Amazon region.

Ninety-seven specimens of spotfin hatchetfish, Thoracocharax stellatus, an ornamental freshwater species from the Amazon basin, were captured in the basin of the Guamá River in the municipality of Belém, in northern Brazil, and analysed for coccidiosis infection. Overall, 26 of the specimens were infected by apicomplexan parasites of the genus Goussia, with unsporulated forms being found in the gastric epithelium and sporulated forms in the intestinal lumen. The spheroid oocysts (mean diameter: 13.2 ± 1.7 µm) have four elliptical sporocysts. A partial sequence of the SSU rDNA of the new species was obtained, which contained 1,121 base pairs, with 43.8% guanine + cytosine (G + C), and the bases distributed as follows: A = 28.1%, C = 18.3%, G = 25.5% and T = 28.1%. The combined analysis of the morphometric and phylogenetic evidence confirmed that the specimens represented the genus Goussia and were allocated to a new species, Goussia guamaensis n. sp., which is described here.

Severe strongyloidiasis and systemic vasculitis: comorbidity, association or both? Case-based review.

A possible association between strongyloidiasis and systemic vasculitis is rarely reported in the literature. We report the case of a patient with severe strongyloidiasis and an angiographic finding consistent with polyarteritis nodosa. Diagnosis of strongyloidiasis was made by finding of larvae and adult parasites in samples of the upper gastrointestinal tract mucosa and stool. The patient was treated with albendazole, ivermectin and corticosteroid withdrawal. This therapy led to the resolution of symptoms, with repeated stool samples negative for S. stercoralis. However, the clinical course was complicated with pulmonary tuberculosis. Despite tuberculostatic therapy and supportive measures, a lethal outcome occurred. The report is followed by a focused review of the available literature on the association of strongyloidiasis and systemic vasculitis.

Anisakiasis: The risks of seafood consumption.

CONTEXT AND AIMS: Japanese cuisine is now popular worldwide, and consumption of raw fish has thus increased at sushi bars and Japanese restaurants outside Japan. Anisakiasis, also known as herring-worm disease, is caused by ingesting larval nematodes in raw seafood and is a common illness in Japan. However, due to the rising popularity of Japanese food, gastroenterologists outside Japan need to be familiar with this disease. SUBJECTS AND METHODS AND RESULTS: We treated 158 patients presenting with acute gastrointestinal manifestations caused by anisakiasis from April 1991 to April 2000. One or more nematodes were removed endoscopically within 48 h of presentation in 44% of these patients, which resulted in prompt resolution of symptoms. Major endoscopic findings were gastric ulcer accompanied by hemorrhage, erosion, redness, and edema of the gastric mucosa in areas penetrated by larvae and other areas. CONCLUSIONS: Endoscopy was valuable for the diagnosis and treatment of anisakiasis. We recommend endoscopy in suspected cases of anisakiasis. Moreover, it is desirable to combine complementary tests such as immunological tests/IgE measurement. As the popularity of Japanese cuisine increases, reports of anisakiasis are likely to be more frequent in countries other than Japan.

Characterization of Ostertagia ostertagi annexin-like proteins at different developmental stages.

Ostertagiosis remains an economically important parasitic disease in cattle in the temperate regions of the world. Repeated exposures to Ostertagia ostertagi in calves cause significant pathology in the abomasum but elicit little protective immunity. The larvae use the host's gastric glands as a niche for development, where the parasite completes its parasitic stages, while in the gastric glands, the larvae must down-regulate the host inflammatory immune responses. Annexin (ANX) A1, commonly found in most eukaryotes, is heavily involved in controlling anti-inflammatory responses by binding receptors on leukocytes. We hypothesized, therefore, that parasite proteins of the ANX family may be involved in host-parasite interactions during ostertagiosis. BLASTN search with the bovine ANXA1 identified two families of Oos-ANX like proteins (Oos-ANXL), each of which was highly conserved at the genetic level and identical at the amino acid sequence level. Oos-ANXL-1 is encoded by two transcripts and Oos-ANXL-2 by 20 transcripts. The present study characterized one Oos-ANXL, representing the most abundant Oos-ANXL, which was further defined as Oost-ANXL-2.1. Oos-ANXL-2.1 with a coding sequence of 519 bp was PCR-amplified, cloned, and expressed. Oos-ANXL-2.1 was immunolocalized to both L3 and adult, but not L4. The staining appeared to be associated with the gut and hypodermis in L3, but it was specifically localized to the hypodermis in adult worms. Western blots detected three protein bands in parasite lysates using anti-recombinant Oos-ANXL-2.1 antibody. Integrated optical density for each of the 3 Oos-ANXL-2s or the total Oos-ANXL-2s detected by Western blots (P < 0.05) was higher in adult worms than in L3 or L4. The results indicate that the production of Oos-ANXL-2s is developmentally regulated and most abundant in the adult worm. This rather large family of proteins could be a potential vaccine target against O. ostertagi infection and warrants further investigation.

Experimental infection of calves with Haemonchus placei or Haemonchus contortus: Assessment of clinical, hematological and biochemical parameters and histopathological characteristics of abomasums.

The present study had the primary objective of evaluating clinical, hematological and biochemical parameters, as well as observing anatomical and histopathological characteristics of abomasums, from calves prime-infected with Haemonchus contortus or H. placei. Ten male Holstein newborns were subdivided in three groups (GI placebo; GII infected with H. contortus; GIII inoculated with H. placei). Eye mucosa staining was evaluated. Hematological and biochemical tests were performed on animals. The euthanasia of all ten experimental calves was performed on the 42nd day post-inoculation. Fragments were collected from each of all 10 abomasums for histopathological analysis. Discrete submandibular edema was diagnosed in animals from both infected groups (H. contortus or H. placei). However, there were no significant changes (P > 0.05) in the color of the ocular mucosa of calves from all three experimental groups across the entire experimental period. Hematological and biochemical changes diagnosed on animals could not be linked to infections by species of Haemonchus spp. Regarding histopathological exams, it was possible to diagnose hypertrophy, hyperplasia, binucleated cells, inflammatory infiltrate, multifocal hemorrhage and edema in abomasums from calves of both groups infected with H. placei and H. contortus. It can, thus, be concluded that not only are calves susceptible to infections by both Haemonchus species, but they can also present clinical changes and similar anatomic histopathological lesions independent of being infected by Haemonchus placei or Haemonchus contortus. These results reflect a negative effect on helminth control by mixed grazing between sheep and cattle, especially when using calves.

Third-stage nematode larvae of Contracaecum osculatum from Baltic cod (Gadus morhua) elicit eosinophilic granulomatous reactions when penetrating the stomach mucosa of pigs.

Third-stage larvae of the anisakid nematode Contracaecum osculatum were recovered from livers of Atlantic cod (Gadus morhua) caught in the Baltic Sea (June 2014) and used for experimental infection of two pigs (one male and one female). Each pig received 215 larvae by oral infection (feeding with minced cod liver containing live nematode larvae). Pigs were euthanized after 5 days, necropsied, and subjected to parasitological investigation. A total of 12 larvae were found penetrating the mucosa of the ventricle (7 in the female pig and 5 in the male pig) eliciting a granulomatous reaction at the penetration site. Four non-attached larvae were found in the female pig stomach and one in the male pig. Petechial bleeding was observed at several locations in the ventricular mucosa where larvae were located. Histological examination of the stomach mucosa revealed a massive cellular infiltration (giant cells, lymphocytes, macrophages, granulocytes, and fibroblast like cells) around the penetrating larva. Mononuclear and polymorphonuclear cells containing eosinophilic granulae were particularly prominent in the granulomas. Reactions correspond to reactions in pigs following experimental infection with the human pathogenic anisakid larvae Anisakis sp. and Pseudoterranova sp. which suggests that C. osculatum might have a zoonotic potential as well.

An anisakis larva attached to early gastric cancer: report of a case.

Gastrointestinal anisakidosis is a nematode infection caused by the ingestion of larvae-infected raw or undercooked fish. The Japanese like to eat raw or undercooked fish, so gastric anisakiasis is a common disease in Japan. However, reports of anisakiasis with gastrointestinal cancer are rare. A 63-year-old Japanese male was diagnosed with a small early gastric cancerous lesion associated with gastric anisakiasis. From our experience and based on a review of the literature, the attachment of an anisakis larva to early gastric cancer is not considered accidental.

Anisakids (Nematoda: Anisakidae) from stomachs of northern fur seals (Callorhinus ursinus) on St. Paul Island, Alaska: parasitological and pathological analysis.

Anisakid nematodes parasitize the alimentary tracts of aquatic vertebrates, including northern fur seals (Callorhinus ursinus) (NFS). The main purpose of this study was to estimate the prevalence, intensity, and species composition of anisakids in NFSs on St. Paul Island, Alaska, and to analyze changes in NFS infection with these nematodes during the last decades. The study was carried out on St. Paul Island, Alaska, in July-August 2011-2013. Stomachs of 443 humanely harvested NFS males were collected from five separate haul-out areas. A total of 4,460 anisakid specimens were collected and identified by morphological criteria. Gross and histological examinations of stomachs were also performed. The overall prevalence of NFS infection was 91.2%; overall mean intensity 10.9, and overall median intensity 6. Five species, Anisakis simplex s. l., Contracaecum osculatum s. l., Pseudoterranova decipiens s. l., P. azarazi and Phocascaris cystophorae, were found. The prevalence of Anisakis was 46.5%, its intensity 7.5. The prevalence and intensity of Contracaecum, Pseudoterranova, and Phocascaris were 33.6% and 3.5, 81.9% and 6.5, and 5.2% and 1.7, respectively. Significant differences in the prevalence of NFS infection were observed between separate haul-outs. Comparison of the present data with the results of studies performed in the North Pacific in the 1960s, showed a significant decrease in the intensity of NFS infection with anisakids during the last decades. The prevalence of mucosal lesions in NFS stomachs caused by anisakids also decreased from 92 to 20.9%. Possible reasons for the changes in NFS infection with anisakids are discussed.

Molecular and morphological comparison of two different types of Habronema muscae (Nematoda: Habronematidae) in horse.

Habronema muscae is a spirurid nematode that undergoes developmental stages in the stomach of equids, causing chronic catarrhal gastritis. Despite preceding investigations have developed polymerase chain reaction (PCR)-based assays for molecular diagnosis, we aimed to assess the applicability of cytochrome c oxidase subunit 1 (cox1) sequences to identify the H. muscae infection and to assess the level of intraspecific variations in this parasite obtained from affected horses in Southern Iran. According to the morphological characterizations, two different isolates of H. muscae were identified. Although the majority of the recovered specimens had normal characterizations of H. muscae, a number of parasites showed an abnormal feature as large, asymmetrical, and thick cuticular extensions was observed at their anterior end (head region) in gross and histologic examinations. Unexpectedly, molecular assay disclosed that both morphologically distinct samples were completely identical to each other based on cox1 sequence. Multiple alignment of the cox1 amino acid sequences showed that all polymorphism sites were silent. Also, phylogenetic analysis provided strong support that H. muscae form a sister group to Spirocerca lupi and Thelazia callipaeda.

Experimental comparison of pathogenic potential of two sibling species Anisakis simplex s.s. and Anisakis pegreffii in Wistar rat.

OBJECTIVES: There are little data available on the pathology caused by the sibling species Anisakis simplex s.s. and Anisakis pegreffii. The differences shown in their ability to penetrate the muscle of fish may also be manifested in humans. The purpose of this study is to confirm possible differences in pathogenicity between A. simplex s.s. and A. pegreffii using an experimental model which simulates infection in humans. METHODS: Female Wistar rats were infected with 190 Anisakis type I L3 larvae from the Iberian coastline. After the animal was sacrificed, these L3 larvae were then recovered and identified via PCR-RFLP of the ITS1-5.8S-ITS2. A logistic regression analysis was performed searching for association between experimental pathogenic potential and species. RESULTS: The distribution of A. simplex s.s. and A. pegreffii between Atlantic and Mediterranean waters of the Iberian Peninsula showed statistically significant differences (P < 0.001) which were not observed in the hybrid genotypes (P > 0.3). 21.6% showed pathogenic potential, interpreted as the capacity of the larvae to cause lesions, stick to the gastrointestinal wall or penetrate it. The species variable showed association with the pathogenic role of the larva (P = 0.008). Taking A. simplex s.s. as our reference, the OR for A. pegreffii is 0.351 (P = 0.028). CONCLUSIONS: Despite this difference, A. pegreffii is also capable of causing anisakiasis, being responsible for 14.3% of the penetrations of the gastric mucosa found in rats, which justifies both species being considered aetiologic agents of this parasitic disorder.

Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection.

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm's Excretory/secretory antigens.

Importance of the CCR5-CCL5 axis for mucosal Trypanosoma cruzi protection and B cell activation.

Trypanosoma cruzi is an intracellular parasite and the causative agent of Chagas disease. Previous work has shown that the chemokine receptor CCR5 plays a role in systemic T. cruzi protection. We evaluated the importance of CCR5 and CCL5 for mucosal protection against natural oral and conjunctival T. cruzi challenges. T. cruzi-immune CCR5(-/-) and wild-type C57BL/6 mice were generated by repeated infectious challenges with T. cruzi. CCR5(-/-) and wild-type mice developed equivalent levels of cellular, humoral, and protective mucosal responses. However, CCR5(-/-)-immune mice produced increased levels of CCL5 in protected gastric tissues, suggesting compensatory signaling through additional receptors. Neutralization of CCL5 in CCR5(-/-)-immune mice resulted in decreased mucosal inflammatory responses, reduced T. cruzi-specific Ab-secreting cells, and significantly less mucosal T. cruzi protection, confirming an important role for CCL5 in optimal immune control of T. cruzi replication at the point of initial mucosal invasion. To investigate further the mechanism responsible for mucosal protection mediated by CCL5-CCR5 signaling, we evaluated the effects of CCL5 on B cells. CCL5 enhanced proliferation and IgM secretion in highly purified B cells triggered by suboptimal doses of LPS. In addition, neutralization of endogenous CCL5 inhibited B cell proliferation and IgM secretion during stimulation of highly purified B cells, indicating that B cell production of CCL5 has important autocrine effects. These findings demonstrate direct effects of CCL5 on B cells, with significant implications for the development of mucosal adjuvants, and further suggest that CCL5 may be important as a general B cell coactivator.

The Lesser Egyptian Gerbil (Gerbillus gerbillus) is a suitable host for the long-term propagation of Cryptosporidium andersoni.

We describe the course of infection of Cryptosporidium andersoni LI03, originally isolated from cattle, in outbred Gerbillus gerbillus (Lesser Egyptian Gerbil), Meriones unguiculatus (Mongolian gerbil), and Meriones tristrami (Tristram's jird). While both Meriones spp. partially cleared the infection and shed a low number of oocysts (less than 15,000 oocysts per gram (OPG)), chronic infection with a mean infection intensity reaching 200,000 OPG was observed in G. gerbillus. These data suggest that G. gerbillus can be used as a laboratory model for the long-term maintenance and study of C. andersoni without the need for host immunosuppression.

Pathological features of Cryptosporidium andersoni-induced lesions in SCID mice.

To assess the infectivity and the istopathological features of Cryptosporidium andersoni (C. andersoni) in laboratory animals, SCID mice were orally inoculated with oocysts of C. andersoni. Starting one week after inoculation, the SCID mice began shedding oocysts, and this continued for ten weeks. Histopathologically, myriads of C. andersoni were observed on the apical surface of the epithelium in the gastric pit of the glandular stomach. There were few lesions in the gastric epithelium except C. andersoni adhesion. In the lamina propria of the affected mucosa, minimum infiltration of inflammatory cells was observed. Immunohistochemically, C. andersoni demonstrated a positive reaction to a number of primary antibodies of Cryptosporidium parvum. In the experiment described here, few increases were seen in apoptotic epithelial cells in the affected mucosas of the SCID mice, and the nuclear augmentation was not enhanced. It was hypothesized that the absence of apoptosis and cell division were due to a lack of inflammatory cell reaction in the lamina propria.

Activity of chitosan-encapsulated Eucalyptus staigeriana essential oil on Haemonchus contortus.

Phytotherapy has been described as an alternative method for the control of gastrointestinal nematodes in small ruminants. Goal of the encapsulation of essential oils in biopolymer matrices is to optimize the biological effects of these oils. The aim of the present study was to evaluate the in vitro and in vivo anthelmintic activity of encapsulated Eucalyptus staigeriana essential oil (EncEs) on the eggs and larvae of Haemonchus contortus. Therefore, the egg hatching test (EHT), larval development test (LDT) and worm load evaluation were performed to evaluate Meriones unguiculatus experimentally infected with H. contortus. The chemical constituents of E. staigeriana essential oil (EsEO) and the in vitro oil release profile from the chitosan matrix at a pH of 1.2 and 7.0 were also characterized. EncEs and EsEO inhibited larval hatching by 97.19% and 99.96% at doses of 1.5 and 1.0 mg ml(-1), respectively. In the LDT, EncEs and EsEO induced a larvicidal effect greater than 95% at concentrations of 5.8 and 8 mg ml(-1), respectively. EncEs and EsEO decreased H. contortus load in M. unguiculatus by 40.51% and 46.44%, respectively. The major chemical constituents of EsEO were (+)-Limonene (72.9%), 1,8-Cineole (9.5%) and o-Cimene (4.6%). The release profile of EsEO was 30% in acid and 25% at neutral pH. The similar efficacy of EncEs and EsEO demonstrates that there was no optimization of anthelmintic action following the encapsulation process. Therefore, the use of new encapsulation matrices with controlled release in the pH of the abomasum should be investigated.

Perkinsus sp. infecting oyster Crassostrea rhizophorae (Guilding, 1828) on the coast of Bahia, Brazil.

This study investigated the occurrence of the protozoan Perkinsus in the oyster Crassostrea rhizophorae on the coast of Bahia State, Brazil. The oysters (n = 900) were collected in February-March and July-August 2010. The Ray's fluid thioglycollate medium (RFTM) analysis of gills and rectum revealed hypnospores of Perkinsus sp. with a high mean prevalence (63%). The infection intensity varied from very light to advanced. The polymerase chain reaction confirmed Perkinsus in 87.2% of the RFTM-positive oysters. Histological analysis showed trophozoites and schizonts phagocytized by hemocytes, mainly in the intestine and the stomach epithelium.

Local immune response of Canarian Majorera goats infected with Teladorsagia circumcincta.

BACKGROUND: Due to increased anthelmintic resistance, alternative methods to drugs are necessary to control gastrointestinal nematodes (GINs). Some of the most promising alternatives are based on the immune response of the host, such as the selection of genetically resistant breeds or the use of vaccines against these parasites. Given the limited information available on the immune response against GINs in goats, this study investigated the local immune response of goat kids of an indigenous Canary Islands breed (Majorera breed) experimentally infected with Teladorsagia circumcincta, one of the most pathogenic and prevalent GIN species. METHODS: For this purpose, the relationship between different parasitological (number of mature and immature worms, worm length, and number of intrauterine eggs) and immunological parameters at the local level (related to both the humoral and cellular immune response) was analyzed at early (1 week post-infection [wpi]) and late (8 wpi) stages of infection. RESULTS: Primary infection of goat kids with T. circumcincta infective larvae (L3) generated a complex immune response that could be defined as Th2 type, characterized by increased infiltration in abomasal tissues of several effector cells as well as a progressive presence of specific antibodies against parasitic antigens in the gastric mucus. Cellular responses were evidenced from 1 wpi onward, showing an increase in antigen-presenting cells and various lymphocyte subsets in the gastric mucosa. CONCLUSIONS: The complexity of the host response was evidenced by statistically significant changes in the number of all these subpopulations (MHCII+, CD4+, CD8+, γδ+, CD45R+, IgA+, and IgG+), as well as in the evolution of the relative cytokine gene expression. From a functional point of view, negative associations were observed between the number of most of the immune cells (CD4, IgA, IgG, and CD45R cells) and parameters that could be related to the fecundity of worms, a phenomenon that was especially evident when the number of IgG and CD45R cells or the specific IgA levels of the gastric mucus were compared with parasitological parameters such as the female worm length or fecal egg counts at 8 wpi.