engleski

Levamisole stimulates intestinal T cell-mediated immune responses of weaned pigs to vaccination against colibacillosis

Apart from its anthelmintic activity in domestic food animals, levamisole may also be used as an adjuvant for preventive vaccines (5, 9). But information on its potential adjuvant activity in hosts vaccinated against gastrointestinal pathogens is generally lacking. Moreover, it is the systemic immune system that has been the focus of most levamisole research. We have recently found that levamisole appears to prime the T cells and macrophages in the draining mesenteric lymph node for interaction with the vaccinal Escherichia coli-derived F4ac antigen and the potentiating action of the drug and vaccine abrogated the inefficacy of vaccination against porcine post-weaning colibacillosis (PWC) induced by the vaccine alone (2, 4). Priming by levamisole of pigs vaccinated against PWC also was found to induce CD25 expression by both jejunal lamina propria (LP) and ileal Peyer&#8217; s patch (PP) cells (3). This can mean that levamisole acts by stimulating jejunal LP and ileal PP T cell-mediated immune response in the vaccinated weaned pigs, but this has never been proven directly. Therefore, the aim of the present study was to gain insight into this issue. Fifteen commercial crossbred pigs were randomly divided into 3 groups of 5 pigs each, immediately after weaning at 4 weeks of age. One group of 5 pigs (experimental group) was intramuscularly primed with levamisole (Nilverm&#174; Pliva, Zagreb, Croatia) at an immunostimulatory dose of 2.5 mg/kg given daily, over 3 consecutive days (5). The control group (N = 5) received saline according to the same schedule. Following the last levamisole or saline dose was given, both groups were orally vaccinated with the vaccinal F4ac+ E. coli strain (serotype O9: K36: H19: F4ac: LT- STb-). Seven days later, all pigs were challenge-inoculated with the virulent F4ac+ E. coli strain (serotype O149: K91: F4ac: 987P: Hly+ LT+ STb+), isolated from diarrhoeic pigs reared on swine farms in Croatia, and killed with T-61&#174; (Hoechst, München, Germany) on post-challenge day 6. After pig&#8217; s euthanasia, the small intestines were excised for isolation of jejunal LP and ileal PP lymphocytes, as previously described (2). Single cell suspensions were prepared and incubated with monoclonal antibody (mAb) BB23-8E6 that reacts with an epitope on porcine CD3e designated CD3a (7). Flow cytometric analysis of the positively stained cells expressing CD3 antigen was performed for each animal and the data presented as arithmetic mean &#177; SD. Levels of significance between primed vaccinated and un-primed vaccinated challenge-infected groups were determined by two-tailed Student's t-test and a value of P<0.05 was considered significant. In order to confirm the possibility that the drug exerts its potentiating activity only in the vaccinated, but not non-vaccinated, weaned pigs (3), 5 remaining pigs were primed with levamisole according to the same schedule as experimental group, but they were sham-vaccinated with Trypticase soy broth (positive control group). This latter group was housed separately. Results. and discussion. The data of the quantitative phenotypic analysis of isolated cells show that both the experimental and control groups contained a comparable numbers of CD3+ jejunal LP T cells, there being no difference in cells from primed vaccinated, or un-primed vaccinated, challenge-infected weaned pigs (Fig. 1). However, the number of ileal PP cells expressing the CD3 molecule increased almost twice in primed vaccinated vs. un-primed vaccinated challenge-infected weaned pigs (P < 0.001). These findings were confirmed by immunohistochemical in situ staining, showing a numerous CD3+ T cells within the follicules of the ileal PP in the primed vaccinated but not un-primed vaccinated challenge-infected pigs (data not shown). These results, together with our recently published data (3), suggest enhanced recruitment and activation of ileal PP T cells and probably physical interaction between the T and B cells in the ileal PP of the primed vaccinated weaned pigs. As the pig ileal PP is a primary lymphoid organ generating B lymphocites and since the antigen-specific interaction between the T and B cells represents a potent regulator of the T cell-dependent antibody response, it is likely that levamisole may contribute to immune protection from challenge-induced porcine PWC by influencing antibody secretion from plasma cells, antibody isotype switching and memory B cell formation (1, 6, 8). The present study also shows that there was no significant difference in the proportion of the CD3+ T cells between primed sham-vaccinated (positive control group) and un-primed vaccinated weaned pigs (17.9 &#177; 6.3 vs. 17.1 &#177; 4.5 for jejunal LP cells) or (4.9 &#177; 1.4 vs. 5.5 &#177; 2.3 for ileal PP cells), implying that levamisole exerts its potentiating activity only in the vaccinated but not non-vaccinated weaned pigs (3). In normal pigs, the ileal PP is populated with up to 5% of CD3+ T cells (1). 1. Levamisole synergizes experimental F4ac+ Escherichia coli oral vaccine in recruiting ileal PP T cells in weaned pigs upon challenge infection. 2. Levamisole may probably contribute to immune protection from challenge-induced porcine PWC by influencing interaction between the T and B cells in the ileal PP. Acknowledgments. This project was supported financially by grant No. 053079 founded by the Ministry of Science and Technology of Croatia. The authors would like to thank Drs T.A. Casey for providing vaccinal non-ETEC strain, V. Bilić for providing ETEC strain, M.D. Pesovitz for providing mAb, D. Žubčić for help in vaccination and infection procedures and Mrs Z. Miletić for flow cytometry support.

levamisole; T cell-mediated immune responses; vaccination against colibacillosis

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