Project description:The gastrointestinal nematode Ostertagia ostertagi is one of major causal agents that contribute to production inefficiency in cattle industry in the temperate region of the world. One of pathophysiological factors that lead to reduced weight gain and milk yield is altered gastrointestinal functions, resulting from considerable tissue damage in the abomasal mucosa during infections. Protective immunity to Ostertagia ostertagi infections in cattle develops very slowly. Resistance to reinfection becomes manifest only after a prolonged period of exposure. Mechanisms underlying the development of protective immunity remain largely unexplored. Immune animals, with significantly reduced worm burdens, were developed after multiple drug-attenuated experimental infections and were compared to the primary infected group and their respective uninfected controls. In this study, transcriptomic analysis identified 3 signaling pathways, the complement system, leukocyte extravasation and acute phase responses, significantly impacted during both primary and repeat infections. The markedly increased mRNA levels of complement components C3, factor B (CFB), and factor I (CFI) in the abomasal mucosa of the infected cattle were confirmed using quantitative PCR. Western blot analysis established the presence of elevated levels of activated C3 proteins in the mucosa. One of the iniators of local complement activation could be related with secretory IgA and IgM because infections significantly upregulated expression of J chain (IGJ) as well as polymeric Ig receptor (PIGR) and an IgM-specific receptor (FAIM3), suggesting sustained increase in both synthesis and transepithelial transport of IgA and IgM during the infection. The elevated levels of pro-inflammatory cytokines, such as IL-4 and IL-1β, during the infection may be involved in gene regulation of complement components. Our data suggested enhanced tissue repair and mucin secretion in immune animals may also contribute to protective immunity. Our results presented the first piece of evidence that local complement activation may be involved in the development of long term protective immunity and provided a novel mechanistic insight into resistance against Ostertagia ostertagi in cattle.
Project description:The gastrointestinal nematode Ostertagia ostertagi is one of major causal agents that contribute to production inefficiency in cattle industry in the temperate region of the world. One of pathophysiological factors that lead to reduced weight gain and milk yield is altered gastrointestinal functions, resulting from considerable tissue damage in the abomasal mucosa during infections. Protective immunity to Ostertagia ostertagi infections in cattle develops very slowly. Resistance to reinfection becomes manifest only after a prolonged period of exposure. Mechanisms underlying the development of protective immunity remain largely unexplored. Immune animals, with significantly reduced worm burdens, were developed after multiple drug-attenuated experimental infections and were compared to the primary infected group and their respective uninfected controls. In this study, transcriptomic analysis identified 3 signaling pathways, the complement system, leukocyte extravasation and acute phase responses, significantly impacted during both primary and repeat infections. The markedly increased mRNA levels of complement components C3, factor B (CFB), and factor I (CFI) in the abomasal mucosa of the infected cattle were confirmed using quantitative PCR. Western blot analysis established the presence of elevated levels of activated C3 proteins in the mucosa. One of the iniators of local complement activation could be related with secretory IgA and IgM because infections significantly upregulated expression of J chain (IGJ) as well as polymeric Ig receptor (PIGR) and an IgM-specific receptor (FAIM3), suggesting sustained increase in both synthesis and transepithelial transport of IgA and IgM during the infection. The elevated levels of pro-inflammatory cytokines, such as IL-4 and IL-1β, during the infection may be involved in gene regulation of complement components. Our data suggested enhanced tissue repair and mucin secretion in immune animals may also contribute to protective immunity. Our results presented the first piece of evidence that local complement activation may be involved in the development of long term protective immunity and provided a novel mechanistic insight into resistance against Ostertagia ostertagi in cattle. There were four treatment groups: naive control (never infected), primary infection, drug-attenuated control, and drug-attenuated 5th reinfection. Each group had 4 biolgical replicates. A total of 16 arrays were used for this experiment. The 2 major contrast were 1). The primary infection vs naive control; and 2). The drug-attenuated 5th reinfection vs the drug-attenuated control.
Project description:IL-10 is a master regulator of immune responses, but its cellular source and function in cattle during the initial phase of immune priming have not been well established. Despite a massive B cell response in the abomasal draining lymph nodes in Ostertagia ostertagi (OO)-infected cattle, protective immunity is slow to develop, and partial protection requires years of repeated exposure. In addressing this problem, our initial hypothesis was that B cells produce IL-10 that downregulates the host protective immune response. However, our results showed that neutrophils made up the majority of IL-10-producing cells in circulation and in secondary lymphoid tissues, particularly the spleen (80%). Conversely, IL-10-producing B cells were rare. In addition, approximately 10% to 20% of the neutrophils in the blood and spleen expressed MHC II and were IL-10 negative, suggesting that neutrophils could also participate in antigen presentation. In vitro investigation of bovine neutrophils revealed that exposure thereof to OO extract increased IL-10 and MHC II expression in these cells in a dose-dependent manner, consistent with IL-10+/MHC II+ neutrophils detected in cattle shortly after experimental OO infection. Co-culture of untreated neutrophils with anti-CD3 antibody (Ab)-stimulated CD4+ T cells led to enhanced T cell activation; also, IL-10 depletion with neutralizing Ab enhanced the stimulatory function of neutrophils. OO extract depressed neutrophil stimulation of CD4+ T cells in the presence of IL-10-neutralizing Ab, suggesting that OO utilizes both IL-10-dependent and independent mechanisms to manipulate the bovine immune response. Finally, contact and viability were required for T cell-stimulatory neutrophil function. This report, to the best of our knowledge, is the first to demonstrate that neutrophil-derived IL-10 is directly involved in T cell regulation in cattle. Our data suggest that neutrophils and neutrophil-derived IL-10 are co-opted by nematode parasites and other pathogens to attenuate host immune responses and facilitate pathogen survival.
Project description:High levels of L3 overwinter survival for multiple cattle gastrointestinal nematode species on western Canadian pastures as revealed by ITS-2 rDNA metabarcoding
Project description:As an alternative to antihelminthic drugs, we are exploiting vaccination to control infections with the abomasal nematode Ostertagia ostertagi in cattle. Our focus for vaccine targets is excretory-secretory (ES) products of this parasite. One of the most abundant antigens in larval and adult Ostertagia ES products is a protein homologous to nematode polyprotein allergens. We found that the Ostertagia polyprotein allergen (OPA) is encoded by a single-copy gene. OPA comprises three or more repeated units, and only the 15-kDa subunits are found in ES products. The native antigen is localized in the intestinal cells of third-stage larvae and in the hypodermis and cuticle of fourth-stage larvae and adult parasites. Vaccination of cattle with native OPA (nOPA) in combination with QuilA resulted in protection against Ostertagia challenge infections. The geometric mean cumulative fecal egg counts in the nOPA-vaccinated animals were reduced by 60% compared to the counts in the control group during the 2-month course of the experiment. Both male and female adult worms in nOPA-vaccinated animals were significantly shorter than the worms in the control animals. In the abomasal mucus of vaccinated animals the nOPA-specific immunoglobulin G1 (IgG1) and IgG2 levels were significantly elevated compared to the levels in the control animals. Reductions in the Ostertagia egg output and the length of the adult parasites were significantly correlated with IgG1 levels. IgG2 titers were only negatively associated with adult worm length. Protected animals showed no accumulation of effector cells (mast cells, globular leukocytes, and eosinophils) in the mucosa. In contrast to the native antigen, recombinant OPA expressed in Escherichia coli did not stimulate any protection.
Project description:Ostertagia ostertagi (OO) is a widespread parasite that causes chronic infection in cattle and leads to annual losses of billions of dollars in the cattle industry. It remains unclear why cattle are unable to mount an effective immune response despite a large influx of immune cells to the infected abomasal mucosa and draining lymph nodes. Neutrophils, the immune system's first responders, have the capacity to release neutrophil extracellular traps (NETs) to contain various pathogens, including some parasites. In the present study, the mechanisms by which O. ostertagi influences bovine NET formation were investigated. O. ostertagi larval soluble extract (OO extract) was able to induce typical NETs by purified neutrophils in vitro, confirmed by co-localization of extracellular DNA with typical NET-associated proteins histone and neutrophil elastase (NE). Consistent with existing literature, inhibition assays demonstrated that these OO extract-induced NETs were dependent upon the enzymes NADPH oxidase and myeloperoxidase (MPO). Live OO stage 4 larvae (L4) stimulated neutrophils to form NETs similar to those induced by OO extract. Bovine neutrophils also released NETs in response to Caenorhabditis elegans, a free-living soil nematode, suggesting that bovine NET production may be a conserved mechanism against a broad range of nematodes. This is the first report demonstrating O. ostertagi-induced NET formation by bovine neutrophils, a potentially underappreciated mechanism in the early immune response against nematode infections.
Project description:A dynamic, deterministic model was developed to investigate the consequences of parasitism with Ostertagia ostertagi, the most prevalent and economically important gastrointestinal parasite of cattle in temperate regions. Interactions between host and parasite were considered to predict the level of parasitism and performance of an infected calf. Key model inputs included calf intrinsic growth rate, feed quality and mode and level of infection. The effects of these varied inputs were simulated on a daily basis for key parasitological (worm burden, total egg output and faecal egg count) and performance outputs (feed intake and bodyweight) over a 6 month grazing period. Data from published literature were used to parameterise the model and its sensitivity was tested for uncertain parameters by a Latin hypercube sensitivity design. For the latter each parameter tested was subject to a 20% coefficient of variation. The model parasitological outputs were most sensitive to the immune rate parameters that affected overall worm burdens. The model predicted the expected larger worm burdens along with disproportionately greater body weight losses with increasing daily infection levels. The model was validated against published literature using graphical and statistical comparisons. Its predictions were quantitatively consistent with the parasitological outputs of published experiments in which calves were subjected to different infection levels. The consequences of model weaknesses are discussed and point towards model improvements. Future work should focus on developing a stochastic model to account for calf variation in performance and immune response; this will ultimately be used to test the effectiveness of different parasite control strategies in naturally infected calf populations.