Granule-exocytosis of granulysin and granzyme B as a potential key mechanism in vaccine-induced immunity in cattle against the nematode Ostertagia ostertagi

Ostertagia ostertagi is considered one of the most economically important bovine parasites. As an alternative for anthelmintic treatment, an experimental host-protective vaccine was previously developed based on ASP-proteins derived from the adult worms. Intramuscular injection of this vaccine, combined with QuilA as adjuvant, significantly reduced the faecal egg counts by 59 %. 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 micro-array, combined with histological analysis. Sixty nine genes were significantly 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 three 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 Overall design: 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.