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. Graphical Abstract

Trichostrongyloid nematodes in ruminants of northern Iran: prevalence and molecular analysis

Background This study was carried out to investigate the prevalence and analyze the molecular characteristics based on the internal transcribed spacer (ITS) 2 region of the ribosomal RNA (RNA) gene of trichostrongylid nematodes in different ruminants from Guilan province, northern of Iran. Methods The gastrointestinal tracts of 144 ruminants including 72 cattle, 59 sheep, and 13 goats were collected from an abattoir in Guilan province during July to September 2018. After isolation the helminths, male specimens were identified based on morphological parameters. PCR and partial sequencing of the ITS2 fragment were conducted. After phylogenetic analysis, the intraspecific and interspecific differences were calculated. Results The prevalence of total infections with the nematodes was 38.9, 74.6 and 84.6% among cattle, sheep and goats, respectively. Eleven species of trichostrongylid nematodes including Haemonchus contortus, Marshallagia marshalli, Trichostrongylus axei, T. colubriformis, T. vitrinus, Ostertagia trifurcata, Teladorsagia circumcincta, Marshallagia occidentalis, O. lyrata, O. ostertagi, and Cooperia punctate were recovered from the ruminants. The most prevalent trichostrongyloid nematodes in cattle, sheep and goats were O. ostertagi (26.4%), M. marshalli (64.4%) and T. circumcincta (69.2%), respectively. Phylogenetic tree was discriminative for Trichostrongylidae family, while phylogenetic analysis of the ITS2 gene represented low variations and no species identification of Haemonchidae and Cooperiidae families. Conclusions This study suggests the high prevalence and species diversity of trichostrongyloid nematodes in different ruminants, indicating the importance of implement antiparasitic strategies in north regions of Iran. As well, this study showed that the ITS2 fragment is not a discriminative marker for Haemonchidae and Cooperiidae families, and investigation of other genetic markers such as mitochondrial genes would be more valuable for better understanding of their phylogenetic relationships.

Development and evaluation of an indirect ELISA for detection of Teladorsagia circumcincta infection in sheep

Background The gastrointestinal helminth, Teladorsagia circumcincta, is one of the major health risks and production-limiting diseases in small ruminant populations, particularly in temperate regions. With the increasing importance of disease management and recruited anthelmintic resistant types, accurate approaches are needed for the diagnosis of the infection in the host. Due to uncertain results using faecal examinations, the ELISA method was indicated for the detection of nematode antigenic materials. Despite some promising results, problems were described in terms of test specificity and cross-reactions. Therefore, this study aimed to evaluate the IgG response to worm somatic and excretory/secretory (ES) products using western blot analysis and an indirect ELISA for the detection of T. circumcincta infection in sheep. Results Based on the immuno-reactivity analysis, immunogenic fractions with molecular weights (MWs) of approximately 60, 75 and 100 kDa were detected in somatic content and two antigens of about 63 and 75 kDa in ES material. Accordingly, a specific product at 75 kDa had the strongest reaction and appeared as the most common antigenic protein. In ELISA, all the sera from the infected sheep revealed the OD rates above the calculated cut-off value with about two-fold greater average. Negative control samples were also specifically recognized with the mean OD rate of about 1/3 of the estimated cut-off value. The cross-reaction test, using rabbit anti-T. circumcincta IgG, did not show reactivity with the ES antigens of other prevalent nematodes including Haemonchus contortus, Protostrongylus rufescens and Marshallagia marshalli. In contrast, a strong positive reaction was observed with the somatic antigens of M. marshalli. Conclusions The results of this study indicated that the indirect ELISA method using the ES content enables distinguishing the T. circumcincta infected sheep with high specificity. Those antigenic ES peptides with 63 and particularly 75 kDa MWs should be further investigated due to the potential for serological diagnostic methods and immunoprotective targets in the host.

61 Oral Dosing Loline Alkaloid and Its Effects Against Mixed Infection of L4 Teladorsagia Circumcincta, and Trichostrongylus Colubriformis, and Adult Haemonchus Contortus

Loline, an alkaloid produced by Epichloë endophytes in pastoral grass species, is considered non-toxic with suggested antimicrobial and anthelmintic properties. In vivo metabolism of loline suggests ability to reach the abomasum or intestine, and anthelmintic benefit is indicated in vitro when parasites are in either a developmental stage close to the gastric mucus layers or adult stage consuming blood. Therefore, the objective of this project was to investigate the validation of using a loline seed extract as a natural anthelmintic in parasitized lambs. Sixteen Coopworth lambs were individually penned, offered ad-libitum lucerne pellets and water and randomly assigned to 3 treatment groups; CON; no treatment, or treatment with a Festuca pratensis seed extract with (LOL) or without loline (NIL). Lambs were orally infected with 20,0000 L3 Haemonchus contortus on day 0, and 10,000 L3 Teladorsagia circumcincta, and Trichostrongylus colubriformis on day 14. Parasites then matured to determine lolines efficacy on blood feeding adult Haemonchus contortus and L4 mucosal browsers Teladorsagia circumcincta, and Trichostrongylus colubriformis. Loline was orally dosed at 52.5 mg/kg LW starting on day 13, and every other day until slaughter for worm enumeration on day 28. Feed intake and refusals were recorded daily, and fecal samples were taken prior to infection, on loline dose days and at slaughter. Worm and fecal egg counts were transformed to log10 and all data were analyzed in Genstat (18th edition). Significance was declared at P < 0.05. Worm burdens of T. circumcincta (P = 0.96), T. colubriformis (P = 0.43), and H. contortus (P = 0.15) were not different between treatments. No treatment difference (P = 0.39) was observed in fecal egg counts, weight gain (P = 0.51), or feed intake (P = 0.18). Average growth efficiency (kg LWG/ kg DM intake) was 0.18 in CON which was less (P = 0.01) than LOL (0.24) or NIL (0.23). In conclusion, limited evidence supported an in vivo anti-parasitic effect of loline.

Teladorsagia Circumcincta Galectin-Mucosal Interactome in Sheep

Teladorsagia circumcincta is the most important gastrointestinal parasite in the livestock industry in temperate regions around the world, causing great economic losses. The infective third-stage larvae (L3) of Teladorsagia circumcincta secrete a large number of excretory-secretory (E/S) molecules, some of which are likely to play critical roles in modulating the host immune response. One of the most abundant E/S molecules is a protein termed Tci-gal-1, which has similarity to mammalian galectins. Galectins are a family of carbohydrate-binding molecules, with characteristic domain organisation and affinity for β-galactosids that mediate a variety of important cellular functions including inflammation and immune responses. To understand the role of Tci-gal-1 at the host–parasite interface, we used a proteomics pull-down approach to identify Tc-gal-1 interacting proteins from sheep abomasal scrapes and whole tissue. A total of 135 unique proteins were identified from whole abomasal tissue samples, while 89 proteins were isolated from abomasal scrape samples. Of these proteins, 63 were present in both samples. Many of the host proteins identified, such as trefoil factors and mucin-like proteins, play critical roles in the host response. The identification of Tci-gal-1 binding partners provides new insights on host–parasite interactions and could lead to the development of new control strategies.

Local Immune Response of Canarian Majorera Goats Primary Infected With Teladorsagia Circumcincta

Due to increased anthelmintic resistance, alternative methods to drugs are necessary to control gastrointestinal nematodes (GIN). Some of the most promising alternatives for their sustainability 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 GIN in goats, this study was carried out on the local immune response of goat kids of a Canary Islands indigenous breed (Majorera breed) experimentally infected with Teladorsagia circumcincta, one of the most pathogenic and prevalent GIN species. For this purpose, the relationship between different parasitological and immunological parameters at local level (both related to the humoral and cellular immune responses) have been analyzed at early (1 week post-infection –wpi-) and late (8 wpi) stages of infection.Primoinfection of goat kids with T. circumcincta L3 generates a complex immune response that could be defined as Th2 type, characterized by an increase of infiltration in abomasal tissues of several effector cells as well as a progressive presence of specific antibodies against parasitic antigens in the gastric mucus. These cellular responses could be evidenced from 1 wpi onward, showing an increase in the gastric mucosa of antigen presenting cells and various lymphocyte subsets. The complexity of the responses developed is evidenced by the statistically significant changes in numbers of all these subpopulations, 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 these 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 or CD45R cells or the specific IgA levels of the mucus were compared with parasitological parameters such as the lenght of female worms or faecal egg counts at 8wpi.

Interactions between Teladorsagia circumcincta Infections and Microbial Composition of Sheep with or without Successful Monepantel Treatment—A Preliminary Study

Nematodes are one of the main impactors on the health, welfare and productivity of farmed animals. Teladorsagia circumcincta are endemic throughout many sheep-producing countries, particularly in the northern hemisphere, and contribute to the pathology and economic losses seen on many farms. Control of these nematode infections is essential and heavily reliant on chemotherapy (anthelmintics), but this has been compromised by the development of anthelmintic resistance. In mammals, the composition of the intestinal microbiota has been shown to have a significant effect on overall health. The interactions between host, microbiota and pathogens are complex and influenced by numerous factors. In this study, comparisons between intestinal and faecal microbiota of sheep infected with sensitive or resistant strains of T. circumcincta, with or without monepantel administration were assessed. The findings from both faecal samples and terminal ileum mucosal scrapings showed clear differences between successfully treated animals and those sheep that were left untreated and/or those carrying resistant nematodes. Specifically, the potentially beneficial genus Bifidobacterium was identified as elevated in successfully treated animals. The detection of these and other biomarkers will provide the basis for new therapeutic reagents particularly relevant to the problems of emerging multidrug anthelmintic resistance.

Cellular and humoral immune responses associated with protection in sheep vaccinated against Teladorsagia circumcincta

Due to increased anthelmintic resistance, complementary methods to drugs are necessary to control gastrointestinal nematodes (GIN). Vaccines are an environmentally-friendly and promising option. In a previous study, a Teladorsagia circumcincta recombinant sub-unit vaccine was administered to two sheep breeds with different levels of resistance against GIN. In the susceptible Canaria Sheep (CS) breed, vaccinates harboured smaller worms with fewer eggs in utero than the control group. Here, we extend this work, by investigating the cellular and humoral immune responses of these two sheep breeds following vaccination and experimental infection with T. circumcincta. In the vaccinated CS group, negative associations between antigen-specific IgA, IgG2 and Globule Leukocytes (GLs) with several parasitological parameters were established as well as a higher CD4+/CD8+ ratio than in control CS animals, suggesting a key role in the protection induced by the vaccine. In the more resistant Canaria Hair Breed (CHB) sheep the vaccine did not significantly impact on the parasitological parameters studied and none of these humoral associations were observed in vaccinated CHB lambs, although CHB had higher proportions of CD4+ and CD8+ T cells within the abomasal lymph nodes, suggesting higher mucosal T cell activation. Each of the component proteins in the vaccine induced an increase in immunoglobulin levels in vaccinated groups of each breed. However, levels of immunoglobulins to only three of the antigens (Tci-MEP-1, Tci-SAA-1, Tci-ASP-1) were negatively correlated with parasitological parameters in the CS breed and they may be, at least partially, responsible for the protective effect of the vaccine in this breed. These data could be useful for improving the current vaccine prototype.

Chemical Analysis and Anthelmintic Activity Against Teladorsagia Circumcincta of Nordic Bark Extracts In vitro

Helminth parasitic infections are common in small ruminants in Norway; infection is usually treated with anthelmintic drugs, but anthelmintic resistance is an increasing problem. It is necessary to identify strategies to reduce the use of anthelmintic drugs and mitigate the impact of anthelmintic resistance. Condensed tannin (CT)-rich forages have been shown to reduce the helminth burden in small ruminants, but these forages have limited cultivation potential in Scandinavia. A good source for CT in cold climatic regions may be the bark of several commercially utilized tree species. In the present study, we determined the content and characterized the type of CT in bark extracts of pine (Pinus sylvestris L.), spruce (Picea abies L.), and birch (Betula pubescens). Extracts of selected bark samples were tested for their anthelmintic efficacy against the ovine infectious nematode Teladorsagia circumcincta. Total CT content was higher in the bark from younger (10–40 years old) pine and spruce trees; it decreased with tree age in pine, whereas it remained relatively stable in the bark of spruce and birch. Pine trees consisted of 100% procyanidins, whereas prodelphinins were present in most spruce (4–17%) and all birch samples (5–34%). Our studies clearly showed that there is variation in the anthelmintic activity of water and acetone extracts of bark samples collected from various sites around Norway, as this was measured with two independent in vitro assays, the egg hatch and larvae motility assays. The anthelmintic activity of some extracts was consistent between the two assays; for example, extracts from the three samples with the highest CT content showed very high activity in both assays, whereas the extract from the sample with the lowest CT content showed the lowest activity in both assays. For other extracts, activity was not consistent across the assays, which could be attributed to the susceptibility of the different stages of the parasitic life cycle. We demonstrated that bark extracts from commercially used trees in Scandinavia have the potential to be used as alternatives to anthelmintics. Further work should focus on refining the associations between bark extracts and anthelmintic activity to identify the best strategies to reduce the input of anthelmintic drugs in livestock production systems.