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Modulation by levamisole of microfold (M) cells in the ileal Peyer’ s patch of weaned pigs experimentally vaccinated against colibacillosis

Levamisole is anthelmintic agent that can also be used as an adjuvant for preventive bacterial and viral vaccines (1, 2). It could also be coplidered as a candidate mucosal adjuvant for experimental F4ac+ Escherichia coli oral vaccine in weaned pigs as it was found to synergize this vaccine in stimulating intestinal humoral and cell-mediated immune responses (2-4). Although it is not clear how levamisole interacts with experimental F4ac+ E. coli oral vaccine against post-weaning colibacillosis (PWC), enhanced translocations of F4ac antigen to the mesenteric lymph nodes and modulation of T cell maturation in the MLN and ileal Peyer’ s patch (IPP) are potential mechanisms (2-4). In an attempt to better understand the mechanisms by which levamisole induces protective mucosal immune responses of pigs to vaccination against PWC, immunohistology was used to investigate whether levamisole synergizes experimental F4ac+ E. coli oral vaccine in recruiting microfold (M) cells in the IPP of weaned pigs. Ten commercial crossbred pigs weaned at 4 weeks were i.m. primed with levamisole (Nilverm® Pliva, Zagreb, Croatia) at an immunostimulatory dose of 2.5 mg/kg given daily, over 3 consecutive days (5). Following the last levamisole dose was given, experimental group (N = 5) was orally vaccinated with the vaccinal F4ac+ E. coli strain, and control group (N = 5) were sham-vaccinated with Trypticase soy broth. Both groups were challenge-inoculated with the virulent F4ac+ E. coli strain 7 days later and killed on post-challenge day 6. The small intestines were excised for immunohistology (6) and murine anti-cytokeratin 18 mAb was used (7) to distinguish the M cells in the follicule-associated epithelium (FAE). Digital image processing system was used for the quantification of M cells in immunohistochemically stained tissue sections. The quantification of M cells was performed by computerized image analysis based on Adobe Photoshop software using commercial imaging Lucia G program as described previously (8). In every tissue section sample, count of M cells were in 12 randomly chosen fields (with the area of 700480 µ m2) using a magnification of 200 . Quantification of cytokeratin 18+ M cells within ileal FAE showed significant difference (p  0.05 ; Student’ s t-test) in the proportion of these cells between primed vaccinated (6.53  10-5  1.34) and primed sham-vaccinated pigs (1.08  10-4  0.87) implying that levamisole exerts its potentiating activity in the vaccinated but not non-vaccinated pigs. Since M cells are considered as antigen/microorganisms transporting cells (9), it is likely that levamisole may contribute to immune protection from challenge-induced porcine PWC not only by influencing of intestinal T and B cell maturation (2-4), but also by enhancing transportation of the vaccinal antigen by M cells. This study was supported by the grants no. 053-0532265-2255 and 053-0532265-2248 from the Ministry of Science, Education and sport of Croatia. References 1. Jin et al. Vaccine 2004 ; 22: 2925-2935. 2. Janjatović et al. J Vet Pharmacol Therap. 2008 ; 31: 328-333. 3. Božić et al. J Vet Pharmacol Therap 2003 ; 26: 225-231. 4. Božić et al. J Vet Pharmacol Therap 2006 ; 29: 199-204. 5. Brunner & Muscoplat JAVMA 1980 ; 176 ; 1159-1162. 6. Lacković et al. Eur J Histochem 1999 ; 43: 39-46. 7. Lugering et al. Clin Exp Immunol 2004 ; 136: 232-238. 8. Oberholzer et al. Histochem Cell Biol 1996 ; 105: 333-355. 9. Kerneis et al. Science 1997 ; 277: 949-952.

Levamisole; Weaned pigs; Vaccination; Colibacillosis; M cells

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