STUDY OF THE IMMUNE SYSTEM IN TWO LIVESTOCK SPECIES OF VETERINARY INTEREST

Abstract Pathogenic microorganisms employ various strategies to replicate, spread, and pose threats to host functions. Meanwhile, the immune system's task involves eliminating these harmful microbes and toxins while avoiding excessive damage to self-tissues and beneficial microbiota. This complex task necessitates a diverse array of protective mechanisms. In mammals, the immune system comprises both innate and adaptive components, working together to respond to microbial infections. A comprehensive understanding of the host immune system is crucial for advancing vaccine and prevention strategies, as well as diagnostic markers. In light of these considerations, my PhD thesis investigates different aspects of the immune system with an emphasis on my latest discoveries in two pivotal projects. The virulence factors of pathogens play a crucial role in determining the adaptation of bacteria to specific host niches. Enteric colibacillosis is a commonly occurring illness and cause of death globally in pigs that are being nursed or weaned. F4 (K88) or F18 adhesive fimbriae are involved in enterotoxigenic (ETEC) infections, leading to post-weaning diarrhoea (PWD). They mediate bacterial binding on enterocytes of the small intestine. After colonisation, F4 and F18 E. coli strains produce enterotoxins that can cause local or systemic effects. These enterotoxins induce secretory diarrhoea, leading to severe clinical signs, reducing production performances and increasing use of drugs. Vaccination of pregnant sows with inactivated E. coli stimulates the production of specific antibodies passed into colostrum and milk for the protection of suckling piglets against ETEC diarrhoea. However, passive protection is rapidly lost after weaning, and newborns can rapidly become susceptible to disease. At weaning, triggering gut mucosal immunity with local production of IgA antibodies by secreting B cells (SIgA) could represent a valid approach to prevent PWD. The present study aims to clarify the immunogenicity of a new oral inactivated F4/F18 vaccine for active immunisation of piglets against PWD caused by F4-ETEC or F18-ETEC by ELISA and ELISPOT assay. Furthermore, some pigs can be genetically resistant to ETEC due to the lack of specific receptors in the gut for fimbriae. It is important to consider the piglet's genotype when interpreting the immunogenicity of the vaccine. This could affect the outcome of the main objective of the project, which is to evaluate the effectiveness of the oral vaccine. To determine whether a pig is susceptible or resistant to F4/F18, I applied a genotyping protocol based on PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Two heat-inactivated ETEC strains along with human-interferon-alpha (IFNα) as adjuvant were administrated to piglets born from unvaccinated sows. Piglets were divided into vaccinated and control groups, and various samples were analysed using ELISA for anti-F4/F18 IgA and IgG levels. Specific F4/F18 immunoglobulins (IgG and IgA) were found in sow colostrum regardless of vaccination, suggesting E. coli circulation in the farm. Results revealed elevated anti-F4/F18 IgA levels in vaccinated piglets' faecal and saliva samples post-vaccination and significant differences in serum IgA levels between vaccinated and control groups and within the control group. The control group had higher levels of maternal-derived anti-F4 IgG, indicating potential pathogen circulation. The pregnant sows, each at day 70 of gestation, were obtained from a conventional herd in which there had been no clinical outbreaks of E. coli enteritis in post-weaning piglets. The selection of sows (from a conventional farm instead of from an SPF facility) was in line with our goal to create realistic field conditions, ensuring the vaccine's effectiveness in a typical farm environment where E. coli exposure is common. Once the animals arrived at the IZSLER animal facility, in order to implement the 3Rs, the project provided for the search of ETEC F4+/F18+ strains in faecal samples, instead to check for antibodies (in order to avoid bleeding of the animals). During this phase, faecal samples were collected from the sows at weekly intervals to ascertain whether they were carriers of F4 and F18 fimbriae-producing E. coli. IgA-secreting B cell responses were observed in vaccinated piglets' mesenteric lymph nodes by ELISPOT assay. The genotyping of piglets was not initially planned and was only conducted after observing the high variability in the immunoassay results. However, genotyping indicated different resistance polymorphisms in vaccinated and control groups, suggesting further investigation is needed. Overall, the study improves my understanding of vaccine immunogenicity and the impact of environmental E. coli on vaccination efficacy and paves the way for developing more effective immunisation strategies against specific E. coli strains during the early post-weaning period. The second project addressed the capacity of bacterial killing of acellular skim milk of dairy cattle against different bacteria. Mastitis is a prevalent disease in dairy cattle farming and is the primary reason for the use of antibiotics, which has significant public health implications. Recent studies have focused on mastitis resistance; however, the mechanisms of susceptibility to intramammary infections need to be clarified. Gram-positive and gram-negative pathogens trigger different immune responses in the mammary glands of dairy cows. Therefore, I defined a new protocol, based on fluorocytometric analysis, to discriminate the bacterial killing activity of bovine colostrum and milk. The experiment involved incubating E. coli and S. aureus strains, that were freshly cultured from clinical cases of bovine mastitis, with milk and colostrum samples at different lactation time points from cows belonging to two cattle breeds: the autochthonous Modenese breed and the common Holstein Friesian breed. Flow cytometry was used to analyse the vitality of the bacteria by using vital dyes after incubation with colostrum and milk samples. I analysed the milk and colostrum samples using traditional bacteriology and assessed NAGase (N-acetyl-β-D-glucosaminidase) activity. The results revealed some important findings. The bacterial killing activity was found to be associated with milk NAGase levels. Additionally, both S. aureus killing and NAGase activity decreased as lactation progressed. Samples of milk from Modenese cows (hypothesised to be a more resistant breed) displayed greater levels of bacterial killing and NAGase activity compared to samples from Holstein Friesian cows. My study demonstrated a more specific killing activity of colostrum and milk against S. aureus, compared to E. coli and suggested that this capacity is mainly mediated by NAGase activity. My data contributes to a better understanding of the mechanisms of pathogenesis and immunoregulation during mastitis. By combining NAGase activity with other diagnostic markers like somatic cell counts (SCC) and bacteriological analysis, a more comprehensive and accurate diagnosis can be achieved. NAGase activity is closely linked to the inflammatory response in the mammary gland. Even in samples with low SCC, a correlation between bacterial killing and NAGase activity is observed. This correlation is notably weaker in culture-negative samples, suggesting that NAGase activity can help differentiate between pathogenic and non-pathogenic bacteria, as non-pathogenic bacteria can also modulate NAGase levels in mammary epithelial cells of healthy quarters. Additionally, NAGase's synergistic activity with lysozyme against peptidoglycan enhances its diagnostic value. The combined activity can provide insights into the effectiveness of the immune response, especially in subclinical or early-stage infections. This could help in the future definition of new diagnostic markers that can contribute to the accurate classification of ambiguous cases of mastitis.