Dubravko Kezić

Histomorphometric evaluation of intestinal cellular immune responses in pigs immunized with live oral F4ac+ non-enterotoxigenic E. coli vaccine against postweaning colibacillosis

Enterotoxigenic Escherichia coli (ETEC) infection is the most common type of porcine postweaning colibacillosis (PWC). Among fimbriae of porcine ETEC strains the best studied family of fimbriae are the members of F4 adhesins, existing in at least three variants: ab, ac, ad. Active immunization against porcine PWC is difficult due to: i) ETEC strains are only one of the essential predisposing factors, ii) the success of vaccinal antigen uptake depends on the presence of enterocyte receptors for F4 adhesins, iii) the intestinal immune system may react with tolerance or hypersensitivity to the same antigens depending on the dose and form of the vaccinal immunogen, and iv) kinetics of the specific immune responses may be different in the case of F4 (earlier) and the other ETEC adhesins, particularly F18 (later). The aim of this study was to test the effectiveness of a live attenuated F4ac+ non-ETEC vaccine against porcine PWC by analyzing quantitative differences in the small intestinal lymphoid and myeloid cell subsets of immunized (with or without levamisole given as an adjuvant) vs control non-immunized pigs. Four week-old pigs were intragastrically immunized with a vaccine candidate F4ac + non-ETEC strain 2407 at day 0, challenged 7 days later with a virulent F4ac+ strain ETEC 11-800/1/94, euthanatized at day 13 and sampled for immunohistology. Non-immunized pigs received saline at day 0 and were processed as the principals. Immunophenotypes of lymphoid and myeloid cell subsets were demonstrated within jejunal and ileal mucosa by immunohistochemical avidinbiotin complex method and corresponding morphometric data were analyzed using software program Lucia G for digital image analyses. Monoclonal antibodies reactive with surface molecules on porcine immune cells such as CD3, CD45RA, CD45RC, CD21 and SWC3 enabled clear insight into distribution patterns and amount of these cells within the gut-associated lymphoid tissues (GALT) examined. The numbers of jejunal and ileal cell subsets tested were significantly increased (at P<0.5 or lower) in both principal groups (vaccinated or levamisole primed-vaccinated) of pigs, compared to those recorded in the control non-vaccinated pigs. Based on the histomorphometric quantification of porcine intestinal immune cells from the GALT compartments tested, it is possible to differentiate the responses of pigs immunized by an experimental mucosal vaccine from those of non-immunized pigs.

Colony development of two Carniolan genotypes (Apis mellifera carnica) in relation to environment

Summary The objective of this study was to compare the colony development cycle (unsealed and sealed worker brood, drone brood, pollen and colony strength) of two Apis mellifera carnica subpopulations in two distinct environments (alpine and continental). At each test location were two sub groups of 12 colonies headed by naturally mated sister queens from either the Institute of Apiculture Lunz am See, Austria (AT) or from the Faculty of Agriculture University of Zagreb, Croatia (HR). Colony development was monitored every 14 days. The HR genotype, adapted to a continental climate, had faster spring brood development in both environments. During spring and early summer the AT genotype maintained the number of sealed brood cells at a constant level in the more favourable conditions, although the amount of unsealed brood reached its maximum in early June. The environment influenced colony development, food stores and colony strength. Interaction between genotype and environment did not affect the number of unsealed brood cells, but the difference was statistically significant for the number of sealed brood cells. The study indicated the presence of a number of genotype and environment interactions between the two honey bee genotypes and their colony traits.

In vitro cultivation of porcine limbal stem cells

Abstract Similarly as in other organ structures stem cells are present in cornea residing basal epithelial layer termed palisade of Voight. A growing interest in allografts and xenografts implies a thorough study of regenerative potentials of these cells, as well as a clear description of their patterns in in vitro tissue cultures to be grafted. Recently we have developed a simple method for cultivation of porcine corneal epithelial stem cells obtained by biopsy from the limbal region. Eight enucleated eyes were obtained from four slaughtered pigs. 5 mm2 samples of limbal epithelium were taken by keratotomy method. The primary cultures of these cells showed phenotypic and morphometric characteristics of porcine corneal epithelial cells following May-Grunwald-Giemsa staining. After 5d of sowing they reached 80% of confluence. With the «Night & Day» lenses a total confluence was achieved 5d earlier in comparison to the cells that were grown in the secondary cultures. Accordingly, the use of porcine limbal stem cells has potentials in veterinary medicine (as novel approach in reparative/regenerative medicine of pets, horses and selected breeds), but also are in accordance with the extensive studies on the potential use of xenografts, mainly swine tissues/organs, in humans.

Effect of polyoxyethylene and polyoxypropylene nonionic block copolymers on performance and recruitment of immune cell subsets in weaned pigs.

BackgroundBecause European-wide directives are restricting the non-clinical use of antibiotics as in-feed growth promotors in swine production, there is an intensive search for alternative strategies for control and prevention of losses among young pigs. With the growing knowledge of the porcine immune system and its endogenous modulation, it has been clearly established that exogenous immunomodulation using adjuvants and immune response modifiers (IRMs) represents an important prophylactic/therapeutic approach in the prevention/treatment of both stress- and microbial-induced disorders that accompaning weaning. However, it is essential to select a fully evaluated agent which may act either as a nonspecific IRM or synergistically as an adjuvant with vaccines. The synthetic macromolecules with a long history as adjuvant and IRM are nonionic block copolymers which consist of polyoxyethylene (POE) and polyoxypropylene (POP) molecules.MethodsThe aim of this work was to evaluate the effectiveness of POE-POP given as a single peroral dose on productivity parameters such as body weight gain, feed intake and feed conversion ratio, and systemic and intestinal immune parameters by assessing the proportions of CD45+ lymphoid cells, CD4+ and CD8+ T cells, and CD21+ B cells in the peripheral blood as well as the number of CD45RA+ naive lymphoid cells residing in the ileal mucosa in weaned pigs during a follow-up study 5 weeks after the treatment.ResultsPigs treated with POE-POP had better feed intake (+ 14.57%), higher average body mass at the end of the experiment (20.91 kg vs. 17.61 kg), and higher body weight gain in relation to Day 0 (191.63% vs. 144.58%) as well as in relation to nontreated pigs (+ 18.74%), with a lower feed conversion ratio (− 30.26%) in comparison to the control pigs. A much lower diarrhea severity score (5 vs. 54) was recorded in pigs treated with POE-POP (− 90.74%) than in the control pigs. A higher average diarrhea severity (ADS) was recorded in the control pigs (1.54 vs. 0.14), whereas the treatmant group had much a lower ADS ratio (− 90.91%) after 35 days of the experiment. The pigs that were treated with POE-POP had an increased proportion of CD45+, CD4+ and CD8+ cells at Day 21 (at p < 0.05, p < 0.05 or p < 0.01, respectively), Day 28 (at p < 0.01, respectively) and Day 35 (at p < 0.01, p < 0.05 or p < 0.01, respectively) as well as of CD21+ cells at Day 28 (p < 0.05) and Day 35 of the experiment (p < 0.01). Also, these pigs had more numerous CD45RA+ cells in interfollicular (p < 0.05) and follicular areas (p < 0.01) of the ileal Peyer’s patches than did control pigs.ConclusionThis property of POE-POP to induce recruitment of circulating and intestinal immune cell subsets in weaned pigs may allow the use of IRM-active block copolymers as adjuvants for vaccines, particularly those orally delivered and targeted to the gut-associated lymphoid tissues that are well known to promote rather tolerogenic than protective immune responses.