Immunogenicity of a live bivalent non-enterotoxigenic Escherichia coli (non-ETEC) vaccine and dietary clinoptilolite efficacy against postweaning diarrheal disease of pigs due to F4+ and F18+ ETEC strains
Authors:   Clinic for Obstetrics and Reproduction, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia, Vince Silvijo , Večkovec Ana Marija , Valpotić Hrvoje , Špoljarić Daniel , Žura Žaja Ivona , Đuričić Dražen , Leiner Denis , Šavorić Juraj , Butković Ivan , Habrun Boris , Njari Bela , Kovšca Janjatović Ana , Efendić Maša , Samardžija Marko , Popović Maja , Valpotić Ivica , Špoljarić Branimira 
Published: April 2023

No safe and effective vaccine exists against porcine enterotoxigenic Escherichia coli (ETEC) strains, which are the etiological agents of post-weaning diarrhoea (PWD), economically one of the most significant diseases of swine, which encountered for major productive losses in swine industry worldwide. The current study was designed to evaluate: (1) efficacy of an oral bivalent F4ac+/F18ac+ non-ETEC live vaccine candidate (VACCINE) in stimulating systemic and intestinal cellular immunity in 4-week-old weaned pigs, (2) the onset and duration of protective immunity of weaned pigs against naturally occurring PWD during the period of 6 weeks following weaning, and (3) the dietary supplement potential of zeolite clinoptilolite (CPL), an antimicrobial mineral and/or immunomodulator/ vaccine adjuvant (VACCINE + CPL). The pigs immunized either with the VACCINE or its combination with dietary CPL had significantly increased body weight gain from Day 7 to Day 42 (P<0.05) of the experiment, as compared to the control pigs (CONTROLS). Conversely, the pigs that were only supplemented with CPL had mostly significantly lower (P<0.05) body weight. The pigs that received VACCINE + CPL were neither diarrheic nor were there any mortalities during the entire period of the experiment. On Day 42 the total bacterial load in the jejunum was much lower in the pigs from all three principal groups than in the CONTROLS (30 x 108 vs. 18 x 107 vs. 14 x 107 vs. 13 x 107 CFU/mL, respectively). Regarding CD4+ and CD8+ T cells, specific immunization with either VACCINE or with VACCINE + CPL stimulated a significantly higher proportion of these cells (P<0.05) from Day 7 to Day 42 of the experiment. Quite similar findings were obtained for CD21+ B cells, as their proportion was significantly elevated in the pigs treated with either VACCINE or VACCINE + CPL (P<0.05 and P<0.05, respectively). The pigs from the VACCINE + CPL group had a significantly higher proportion of CD45+ lymphoid cells than the pigs from the CONTROL group (P<0.05). Quite sparsely distributed CD45RA+ naïve T lymphocytes were observed in the jejunal lamina propria of the intestinal villi, within the Lieberkühn crypts (LC) and in the submucosa of the CONTROLS and CPL supplemented pigs. The treatment with the VACCINE induced moderate recruitment of CD45 RA+ cells within both the IFA and FA of the jejunal PP of the pigs 6 weeks following the vaccination. In the CONTROLS, CD45 RC+ memory T cells were not abundant and were mostly dispersed in the middle of the villous lamina propria, but only rarely adjacent to the basal membrane of the intestinal enterocytes. These cells were more frequent within the lamina propria of the villi in the CPL group of pigs than in the CONTROLS. Even more numerous CD45 RC+ memory T cells were observed in the villous lamina propria of the jejunum of the pigs from the VACCINE group. These cells were predominantly found in the villous lamina propria and in the IFA, but were less frequent in the FA of the jejunal PP of the pigs that received the VACCINE and CPL. Due to the nature of the disease, challenges in PWD vaccine development will continue to exist. Although our research approach was at least partially successful, developing a safe and immunogenically effective live oral vaccine against PWD that will provide solid protection and sustained cellular and humoral immune responses remains a significant challenge.