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:Ostertagia ostertagi is considered one of the most economically important bovine parasites. As an alternative for anthelmintic treatment, an experimental host-protective was previously developed based on ASP-proteins derived from the adult worms. Intramuscular injection of this vaccine, combined with QuilA as adjuvant, significantly reduces the faecal egg counts by 60-85%. However, the immunological mechanisms triggered by the vaccine are still unclear. Therefore, in this study, the differences in immune responses at the site of infection, i.e., the abomasal mucosa, between ASP/QuilA-vaccinated animals and QuilA-vaccinated control animals were investigated on a transcriptomic level, using a whole genome bovine microarray, combined with histological analysis. Sixty-nine genes were signicantly impacted in animals protected by the vaccine, 48 of which were upregulated. A correlation study between the parasitological parameters and gene transcription levels showed that the transcription levels of two of the upregulated genes, granulysin (GNLY) and granzyme B (GZMB), negatively correlated to cumulative faecal egg counts and total worm counts, respectively. Both genes also positively correlated to each other, and to another upregulated gene, the IgE receptor subunit FCER1A. Surprisingly, these 3 genes also correlated significantly to CMA1, a mast cell marker, and to cell counts for mast cells and cells previously described as globule leukocytes. Furthermore, immunohistochemical data showed that GNLY was present in the granules of globule leukocytes and that it was secreted in the mucus. Overall, the results suggest a potential role of granule exocytosis by globule leucocytes, potentially IgE-mediated, in the vaccine induced protection against O. ostertagi. 12 helminth‐free Holstein crossbred calves at 7‐10 months of age were randomly divided in 2 groups of 6 animals. All animals were immunized three times intramuscularly in the neck with a three‐week interval. One group received 750 μg of QuilA with Tris‐buffer instead of antigen (negative control). The other group received ~30 μg of the native ASP fraction per immunization in combination with 750 μg of QuilA adjuvant. After the final immunization, the animals were challenged with a trickle infection of 30,000 infective L3 larvae (1,000 L3/day for 30 days, 5 days a week during a period of 6 weeks). 3 weeks after the last challenge infection, all animals were sacrified and total RNA from fundic abomasa was extracted. A total of 12 microarrays were used. Only biological replicates were used.

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-1Î², 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.

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