Increased number of intestinal villous M cells in levamisole -pretreated weaned pigs experimentally infected with F4ac+ enterotoxigenic Escherichia coli strain

Immunoprophylaxis of porcine postweaning colibacillosis (PWC) caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae is an unsolved problem. Just as ETEC strains can exploit intestinal microfold (M) cells as the entry portal for infection, their high transcytotic ability make them an attractive target for mucosally delivered vaccines, adjuvants and therapeutics. We have developed a model of parenteral/oral immunization of 4-weeks-old pigs with either levamisole or vaccine candidate F4ac+ non-ETEC strain to study their effects on de novo differentiation of antigen-sampling M cells. Identification, localization and morphometric quantification of cytokeratin 18 positive M cells in the ileal mucosa of 6-weeks-old pigs revealed that they were: 1) exclusively located within villous epithelial layer, 2) significantly numerous (P< 0.01) in levamisole pretreated/challenged pigs, and 3) only slightly, but not significantly numerous in vaccinated/challenged pigs compared with non-pretreated/challenged control pigs. The fact that levamisole may affect the M cells frequency by increasing their numbers, makes it an interesting adjuvant to study development of an effective M cell-targeted vaccine against porcine PWC

Effect of levamisole on the number of intestinal goblet cells in weaned pigs experimentally vaccinated against colibacillosis

VALPOTIĆ: Effect of levamisole on the number of intestinal goblet cells in weaned pigs experimentally vaccinated against colibacillosis. Vet. arhiv 79, 543-553, 2009. Postweaning colibacillosis (PWC) is an etiologically complex disease commonly induced by porcine F4ac + enterotoxigenic Escherichia coli (ETEC) for which no effective vaccine is available. The objective of this study was to determine the nonspecific immunomodulatory effect of levamisole in combination with specific intragastric immunization of weaned pigs with a candidate F4ac + non-ETEC oral vaccine on the population of intestinal goblet cells (GC). The pigs were immunized with F4ac + non-ETEC strain, in combination with or without levamisole. Seven days after immunization the pigs were challenged with F4ac + ETEC strain and 14 days following immunization they were euthanatized for sampling of specimens of the small intestine for immunohistochemistry and morphometric analyses. Samples of the ileum were tested for the presence of acidic and neutral carbohydrates, components of mucus produced and secreted by the intestinal goblet cells (GC). The volume density (V) of the PAS V + and AB +/PAS+ GC was determined using the stereological point-counting method. The Vv of the ileal PAS + GC was lowest (0.130 ± 0.075 mm3) in the pigs that were immunized wit

Identifikacija, lokalizacija i kvantifikacija subpopulacija crijevnih imunosnih stanica prasadi tijekom prvih sedam tjedana postnatalnog razvitka.

By using immunohistology and digital image analysis for histomorphometry, we investigated the influence of porcine postnatal development on distribution and quantitative patterns of naïve/memory lymphoid cell subsets (CD45RA+, CD45RC+, respectively), helper/cytotoxic T cell subsets (CD4+, CD8+, respectively) as well as of IgA+ plasma cells in the gut-associated lymphoid tissues (GALT) of pigs reared under intensive conditions during the first 7 weeks of life. Current research on the postnatal ontogenesis/maturation of porcine small intestinal mucosal immune system have shown that newborn pigs are immunologically rather immature and, thus, totally dependent on passively acquired immunity. Their jejunal mucosa almost lacks immune cells, with the exception of a low number of CD45RA+ lymphoid cells (1.25×10-5) at Day 0 post partum. A very few CD45RC+ lymphoid cells (8.79×10-6 ) appeared at Day 7, followed by a similar number of IgA+ plasma cells (7.65×10-6) which were observed at Day 14. Such first signs of the postnatal development of both innate and adaptive immunity by identification/localization of tested cell subsets residing porcine GALT, i e. jejunal mucosal sites are proceeded by a lag period of three weeks until the appearance of low quantities of CD4+ (3.62×10-5) and CD8+ (3.13×10-5) T cells at Day 35 after birth. After that phase of early postnatal development of the crucial cell subsets participating in the antigen-specific immunity on the gut mucosal surfaces of young pigs, further progress was visible through their different distribution patterns within the jejunal mucosa compartments, and a gradual increase in their numbers, particularly between Day 35 and Day 49 of age. A significant increase was recorded within that period in the numbers of naïve, memory and plasma cells, whereas the number of helper and cytotoxic T cells only slightly increased between Day 42 and Day 49 of life.Pomoću imunohistoloških i histomorfometrijskih analiza računalnom obradom slike istraživali smo utjecaj postnatalnog razvitka u svinje na distribucijske i kvantitativne pokazatelje subpopulacija djevičanskih/ memorijskih limfoidnih stanica (CD45RA+ odnosno CD45RC+), pomoćničkih/citotoksičnih T-stanica (CD4+ odnosno CD8+), kao i za IgA+ plazma stanice u limfatičkim tkivima probavnog sustava (LTPS) prasadi uzgajane u intenzivnim uvjetima tijekom prvih 7 tjedana života. Istraživanje postnatalnog razvitka/sazrijevanja imunosnog sustava sluznice tankoga crijeva svinje pokazalo je da je neonatalna prasad imunološki prilično nezrela, pa je stoga u potpunosti ovisna o pasivno stečenoj imunosti. U sluznici njihovog jejunuma gotovo potpuno nedostaju imunosne stanice, s izuzetkom malobrojnih CD45 RA+ limfoidnih stanica (1,2510–5) 0. dana nakon prasenja. Sedmog dana post partum pojavljuje se nešto malo CD45RC+ limfoidnih stanica (8,7910-6), a potom se 14. dana mogu identificirati malobrojne IgA+ plazma stanice (7,650-6). Ovi prvi znakovi postnatalnog razvitka urođene i stečene imunosti, temeljem identifi kacije/lokalizacije subpopulacija istraživanih stanica koje naseljavaju LTPS svinje, odnosno sluznicu jejunuma, nastavljaju se nakon razdoblja od tri tjedna do pojavljivanja malobrojnih CD4+ (3,6210-5) i CD8+ (3,13 10-5) T-stanica 35. dana nakon prasenja. Nakon ove faze ranog postnatalnog razvitka ključnih subpopulacija stanica koje sudjeluju u antigenski specifičnoj imunosti na sluzničkim površinama crijeva mlade prasadi, daljnji je napredak vidljiv u različitosti obrazaca njihove raspodjele u odjeljcima sluznice jejunuma i postupnom porastu njihove brojnosti, napose između 35. i 49. dana starosti. Tijekom tog razdoblja zabilježen je značajan porast brojnosti djevičanskih, memorijskih i plazma stanica, dok je brojnost pomoćničkih i citotoksičnih T-stanica samo blago porasla između 42. i 49. dana života

Učinak levamisola na brojnost crijevnih vrčastih stanica odbijene prasadi pokusno cijepljene protiv kolibaciloze

Postweaning colibacillosis (PWC) is an etiologically complex disease commonly induced by porcine F4ac+ enterotoxigenic Escherichia coli (ETEC) for which no effective vaccine is available. The objective of this study was to determine the nonspecific immunomodulatory effect of levamisole in combination with specific intragastric immunization of weaned pigs with a candidate F4ac+ non-ETEC oral vaccine on the population of intestinal goblet cells (GC). The pigs were immunized with F4ac+ non-ETEC strain, in combination with or without levamisole. Seven days after immunization the pigs were challenged with F4ac+ ETEC strain and 14 days following immunization they were euthanatized for sampling of specimens of the small intestine for immunohistochemistry and morphometric analyses. Samples of the ileum were tested for the presence of acidic and neutral carbohydrates, components of mucus produced and secreted by the intestinal goblet cells (GC). The volume density (VV) of the PAS+ and AB+/PAS+ GC was determined using the stereological point-counting method. The Vv of the ileal PAS+ GC was lowest (0.130 ± 0.075 mm3) in the pigs that were immunized with the vaccine candidate F4ac+ non-ETEC strain. Interestingly, AB+/PAS+ GC Vv were found to be highest (0.262 ± 0.091 mm3) in this group of pigs. The Vv of PAS+ GC was the largest (0.201 ± 0.064 mm3) in the negative control group of pigs. Pigs from the levamisole pretreated group were shown to have the lowest Vv (0.166 ± 0.051 mm3) of AB+/PAS+ GC. According to the data presented, there was no significant influence of nonspecific/specific immunization on the nonimmune defence mechanism of the intestinal mucosa, as measured by GC Vv in weaned pigs. Hence, it is likely that the production of mucus was not affected and that nonspecific protection of porcine intestines provided by mucus layer was not compromised following peroral immunization against PWC. Also, presumably the anti-inflammatory effect of levamisole was observed as the population of ileal mast cells was not considerably affected by non-specific/specific immunization and challenge infection.Kolibaciloza odbijene prasadi (KOP) etiološki je složena bolest, najčešće izazvana svinjskim F4ac+ enterotoksigenim sojevima bakterije Escherichia coli (ETEC), za koju nema djelotvorne vakcine. Namjera ovoga rada bila je da se utvrdi imunomodulacijski učinak levamisola, u kombinaciji sa specifičnom intragastričnom imunizacijom odbijene prasadi pokusnim vakcinalnim F4ac+ ne-ETEC sojem, na populaciju crijevnih vrčastih stanica (VS). Prasad je bila imunizirana F4ac+ ne-ETEC sojem u kombinaciji s levamisolom ili bez njega, sedam dana nakon toga bila je izazivački inficirana F4ac+ ETEC sojem, a 14 dana nakon imunizacije bila je usmrćena radi uzimanja uzoraka crijeva za imunohistokemijske i morfometrijske analize. Uzorci ileuma testirani su na prisutnost kiselih i neutralnih ugljikohidrata, sastavnica sluzi koju proizvode i izlučuju VS. Volumna gustoća (Vg) VS koje se boje kombinacijom alcijanskoga modrila (AM) i periodičnoga kiseloga Schiffovoga (PKS) reagensa određivana je pomoću stereologijske metode brojanja točaka. Vg ilealnih PKS+ VS bila je najniža (0,130 ± 0,075 mm3) u prasadi imunizirane pokusnim vakcinalnim F4ac+ ne-ETEC sojem. Međutim, zanimljivo je da je utvrđena najveća Vg AM+/PKS+ VS (0,262 ± 0,091 mm3) u prasadi iz te skupine. U prasadi iz negativne kontrolne skupine utvrđena je najveća Vg PKS+ VS (0,201 ± 0,064 mm3), dok je prasad iz skupine obrađene levamisolom i vakcinalnim ne-ETEC sojem imala najnižu Vv ukupnih (AM+/PKS+) VS (0,159 ± 0,023 mm3). Primjereno dobivenim rezultatima, nije utvrđen značajan utjecaj nespecifične/specifične imunizacije na neimunosne obrambene mehanizme crijevne sluznice vrednovane temeljem Vg i distribucije VS u odbijene prasadi. Stoga je vrlo vjerojatno da proizvodnja sluzi nije umanjena i da nespecifična zaštita crijeva prasadi slojem sluzi ne bi bila slabija nakon peroralne imunizacije protiv KOP. Također je zapažen potencijalni protuupalni učinak levamisola s obzirom na činjenicu da populacija ilealnih mastocita nije bila znatnije stimulirana nespecifičnom/specifičnom imunizacijom i izazivačkom infekcijom

Promjene humoralnih i staničnih imunosnih pokazatelja povezane s dobi svinje.

The sequence of development of porcine systemic and local humoral and cellular immunity was analyzed by the age-dependent patterns of total serum immunoglobulin (Ig) levels, responsiveness of peripheral blood lymphocytes (PBL) to common mitogens (phytohemagglutinin, PHA; concanavalin A, ConA; poke-weed mitogen, PWM), or allogeneic PBL in a mixed lymphocyte culture (MLC), and the expression/distribution patterns of intestinal mucosal immune cell subsets, in order to establish the basic immune parameters for an assessment of immunocompetence in clinically normal pigs. Thus, we surveyed the species-related physiological values of these parameters in different age/technological categories, comprising infant (suckling and weaned pigs) and adult (gilts, sows, and boars) swine, kept in intensive rearing conditions. The highest level of total Igs was determined in neonatal pigs (43.5 ± 4.6 g/L), and this value was significantly higher (P<0.001) than that in weaned pigs (12.1 ± 1.5 g/L). Among adult swine, the highest level of total Igs was recorded in sows (32.0 ± 3.1 g/L), and this was much higher (P<0.001) than that found in gilts (25.1 ± 3.5 g/L) or boars (21.2 ± 5.0 g/L). The reactivity of PBL in neonatal pigs decreased by 60-70% compared to weaned pigs, regardless ofthe stimulator applied. In adult swine, the strongest reactivity of PBL, except to PHA, was determined in sows. Histomorphometric analyses showed that CD45RA+, CD45RC+ and IgA+ cells in sections of the jejunum and ileum were significantly higher in weaned pigs (P<0.05) compared to neonatal pigs. The immune parameters obtained for a sample of the swine population in Croatia may serve as a basis for further research on porcine systemic and local (intestinal) immune responsiveness regarding (i) differences among breeds, (ii) the influence of paragenetic factors, (iii) the impact of microbial etiology diseases, particularly those accompanied by immunosuppression, and (iv)the validation of specific/nonspecific immunomodulation.Slijed razvitka sustavne i lokalne humoralne i stanične imunosti u svinje analiziran je s pomoću dobno ovisnih promjena u razinama serumskih imunoglobulina (Ig), odgovorima limfocita periferne krvi (LPK) na uobičajene mitogene (fitohemaglutin, PHA; konkanavalin A, ConA; poke-weed mitogen, PWM) ili specifični antigen (alogenske LPK u miješanoj kulturi limfocita, MKL), te u obrascima ekspresije/distribucije subpopulacija imunosnih stanica u sluznici crijeva radi utvrđivanja temeljnih imunosnih pokazatelja za procjenu imunosne kompetencije u klinički normalnih svinja. Stoga smo istražili za vrstu specifične fiziološke vrijednostitih pokazatelja u različitih dobnih, odnosno tehnoloških kategorija, koje su uključivale mladu prasad (sisančad i odbijenike) i odrasle svinje (nazimice, krmače i nerastove) držane u uvjetima intenzivnog uzgoja. U neonatalne prasadi utvrđema je najviša razina ukupnih Ig (43,5 ± 4,6 g/L), i ta je vrijednost bila značajno viša (P<0,001) od one u odbijene prasadi (12,1 ± 1,5 g/L). U odraslih svinja, najviša razina ukupnih Ig zabilježena je u krmača (32,0 ± 3,1 g/L) i bila je mnogo viša (P<0,001) od onih utvrđenih u nazimica (25,1 ± 3,5 g/L) i nerastova (21,2 ± 5,0 g/L). Reaktivnost LPK u neonatalne prasadi bila je snižena za 60 - 70% u odnosu na onu zabilježenu u odbijene prasadi bez obzira na primijenjeni stimulator. U odraslih svinja, najjača je reaktivnost LPK, osim na PHA, utvrđena u krmača. Histomorfometrijske analize pokazale su da su CD45RA+, CD45RC+ i IgA+ stanice u dijelovima jejunuma i ileuma značajno brojnije u odbijene prasadi (P<0,05) u usporedbi s vrijednostima u neonatalne prasadi. Dobiveni imunosni pokazatelji na uzorku populacije svinja u Hrvatskoj mogu poslužiti kao temelj za dalja istraživanja njihovog sustavnog i lokalnog (crijevnog) imunosnog odgovora s obzirom na: (I) različitosti među pasminama, (II) utjecaje paragenetičkih činitelja, kao što su uvjeti uzgoja/držanja, način prehrane, te ambijentalni i okolišni stresori, (III) učinke bolesti mikrobne etiologije, posebice onih povezanih s imunosupresijom, kao i na (IV) vrednovanje specifične/nespecifične imunomodulacije

De novo diferencijacija M stanica crijevnih resica u odbijene prasadi imunizirane pokusnim cjepnim F4ac+ ili F18ac+ neenterotoksigenim sojevima bakterije Escherichia coli s levamisolom kao adjuvansom.

Active immunization against porcine postweaning colidiarrhea (CD) and/or colienterotoxemia (CE) caused by F4+ and/or F18+ enterotoxigenic Escherichia coli (ETEC) is still an unsolved problem. The intestinal microfold (M) cells play a role in the entry/invasion of intraluminal pathogens (such as ETEC strains), in antigen sampling, and in facilitating the induction of immunity to gut infections. Just as ETEC strains can exploit M cells as the portal of entry for infections, such as CD and/or CE, their high transcytotic ability makes them an attractive target for mucosally delivered vaccines, adjuvants and therapeutics. The objective of our study was to evaluate the effects of levamisole-adjuvanted vaccine candidate F4ac+ and F18ac+non-ETEC strains on incidence/frequency of ileal M cells and up-regulation of antigen delivery by de novo formation of these cells in weaned pigs. Conventionally reared 4-week-old pigs were divided into three groups, of which two were parenterally and orally immunized with levamisole (at days -2, -1 and 0) in combination with either vaccine candidate non-ETEC strain (at day 0), respectively. The third group of pigs received saline as a placebo. Challenge was performed (at day 7) with the F4ac+ ETEC strain, and the pigs were euthanatized (at day 13) and sampled for immunohistology. Distribution patterns of cytokeratin 18 positive M cells revealed that they are interspersed between enterocytes than as small clusters, and most of them were found to be located at the apex of the villi in the ileum of 6-weeks-old-pigs. Morphometric quantififi cation of M cells in the ileal mucosa showed that levamisole-pretreated F18ac+non-ETEC-immunized and challenged pigs had significantly increased numbers (P<0.01) of ileal M cells as compared to the values obtained in the control non-primed and challenged pigs. The proportion of these cells in this group of pigs was increased by 145%. In the levamisole-pretreated F4ac+ non-ETEC-immunized and challenged pigs only a slightly increased (for 7%) proportion of M cells was recorded. However, this increase was not significantly different from the numerical values obtained for control pigs. Our finding that levamisole-adjuvanted F18ac+non-ETEC vaccine may affect de novo differentiation of antigen-sampling M cells by increasing their number in the ileum, indicated that the vaccine probably utilizes these cells as a target for entry/delivery to the nearby lymphocytes and induces protective immunity against CE. On the other hand, the failure of levamisole-adjuvanted F4ac+non-ETEC vaccine to produce a similar effect on M cells remains to be elucidated.Aktivna imunizacija odbijene prasadi protiv kolidijareje (KD) i/ili kolienterotoksemije (KE) uzrokovane F4ac+ i/ili F18ac+ enterotoksigenim sojevima bakterije Escherichia coli (ETEC) još je uvijek neriješen problem. Crijevne mikronaborane (M) stanice imaju funkciju pri ulasku/invaziji intraluminalnih patogena (kao što su ETEC sojevi), unosu uzoraka antigena i pogodovanju tvorbi imunosti na probavne infekcije. Činjenica da ETEC sojevi rabe M stanice kao ulazna vrata za uzročnike infekcija, kao što su KD i/ili KE, a njihova velika sposobnost transcitoze čini ih ujedno pogodnim stanicama za unos mukoznih cjepiva, adjuvanata i lijekova. Cilj ovoga rada bilo je vrednovanje učinaka pokusnih cjepnih sojeva F4ac+ i F18ac+ ne-ETEC, s levamisolom kao adjuvansom, na pojavnost i brojnost M stanica u ileumu, te na poticanje unosa antigena nakon de novo tvorbe tih stanica u odbijene prasadi. Prasad iz uobičajenoga uzgoja, u dobi od 4 tjedna, bila je razvrstana u tri skupine od kojih su dvije parenteralno/oralno imunizirane levamisolom (-2., -1. i 0. dana pokusa) u kombinaciji s jednim od dva pokusna vakcinalna ne-ETEC soja (0. dana pokusa). Treća je skupina primila fiziološku otopinu kao placebo. Izazivačka je infekcija provedena s F4ac+ ETEC sojem (7. dana pokusa), a prasad je usmrćena (13. dana pokusa) radi uzimanja uzoraka za imunohistologiju. Distribucijski obrasci M stanica, pozitivnih na biljeg za citokeratin 18, pokazuju da su pretežito raspršene između enterocita, a manje ih je u malim nakupinama, te da se većina tih stanica nalazi pri vrhu resica ileuma prasadi u dobi od 6 tjedana. Morfometrijska kvantifikacija M stanica u sluznici ileuma pokazuje da prasad koja je prethodno dobivala levamisol i imunizirana F18ac+ ne- ETEC sojem ima značajno veći broj (P<0,01) M stanica u usporedbi s vrijednostima dobivenima u kontrolne neimunizirane prasadi. Udjel M stanica u te prasadi bio je povećan za 145%. U skupini prasadi prethodno obrađene levamisolom i imuniziranih F4ac+ ne-ETEC sojem zabilježen je samo blagi porast (za 7%) udjela M stanica. Međutim, taj porast nije bio statistički značajno različit od vrijednosti dobivenih u kontrolne prasadi. Naš nalaz da F18ac+ ne-ETEC vakcina s levamisolom kao adjuvansom može pospješiti de novo diferencijaciju M stanica time što povećava njihovu brojnost u ileumu, upućuje na povećanu sposobnost tih stanica da unose pokusni vakcinalni soj i tako dostavljaju imunogen do obližnjih limfocita što potiče zaštitnu imunost protiv KE. Međutim, trebalo bi objasniti izostanak sličnog učinka na M stanice u prasadi koja je primila F4ac+ ne-ETEC vakcinu s levamisolom kao adjuvansom

Identifikacija, lokalizacija i kvantifikacija subpopulacija crijevnih imunosnih stanica prasadi tijekom prvih sedam tjedana postnatalnog razvitka.

By using immunohistology and digital image analysis for histomorphometry, we investigated the influence of porcine postnatal development on distribution and quantitative patterns of naïve/memory lymphoid cell subsets (CD45RA+, CD45RC+, respectively), helper/cytotoxic T cell subsets (CD4+, CD8+, respectively) as well as of IgA+ plasma cells in the gut-associated lymphoid tissues (GALT) of pigs reared under intensive conditions during the first 7 weeks of life. Current research on the postnatal ontogenesis/maturation of porcine small intestinal mucosal immune system have shown that newborn pigs are immunologically rather immature and, thus, totally dependent on passively acquired immunity. Their jejunal mucosa almost lacks immune cells, with the exception of a low number of CD45RA+ lymphoid cells (1.25×10-5) at Day 0 post partum. A very few CD45RC+ lymphoid cells (8.79×10-6 ) appeared at Day 7, followed by a similar number of IgA+ plasma cells (7.65×10-6) which were observed at Day 14. Such first signs of the postnatal development of both innate and adaptive immunity by identification/localization of tested cell subsets residing porcine GALT, i e. jejunal mucosal sites are proceeded by a lag period of three weeks until the appearance of low quantities of CD4+ (3.62×10-5) and CD8+ (3.13×10-5) T cells at Day 35 after birth. After that phase of early postnatal development of the crucial cell subsets participating in the antigen-specific immunity on the gut mucosal surfaces of young pigs, further progress was visible through their different distribution patterns within the jejunal mucosa compartments, and a gradual increase in their numbers, particularly between Day 35 and Day 49 of age. A significant increase was recorded within that period in the numbers of naïve, memory and plasma cells, whereas the number of helper and cytotoxic T cells only slightly increased between Day 42 and Day 49 of life.Pomoću imunohistoloških i histomorfometrijskih analiza računalnom obradom slike istraživali smo utjecaj postnatalnog razvitka u svinje na distribucijske i kvantitativne pokazatelje subpopulacija djevičanskih/ memorijskih limfoidnih stanica (CD45RA+ odnosno CD45RC+), pomoćničkih/citotoksičnih T-stanica (CD4+ odnosno CD8+), kao i za IgA+ plazma stanice u limfatičkim tkivima probavnog sustava (LTPS) prasadi uzgajane u intenzivnim uvjetima tijekom prvih 7 tjedana života. Istraživanje postnatalnog razvitka/sazrijevanja imunosnog sustava sluznice tankoga crijeva svinje pokazalo je da je neonatalna prasad imunološki prilično nezrela, pa je stoga u potpunosti ovisna o pasivno stečenoj imunosti. U sluznici njihovog jejunuma gotovo potpuno nedostaju imunosne stanice, s izuzetkom malobrojnih CD45 RA+ limfoidnih stanica (1,2510–5) 0. dana nakon prasenja. Sedmog dana post partum pojavljuje se nešto malo CD45RC+ limfoidnih stanica (8,7910-6), a potom se 14. dana mogu identificirati malobrojne IgA+ plazma stanice (7,650-6). Ovi prvi znakovi postnatalnog razvitka urođene i stečene imunosti, temeljem identifi kacije/lokalizacije subpopulacija istraživanih stanica koje naseljavaju LTPS svinje, odnosno sluznicu jejunuma, nastavljaju se nakon razdoblja od tri tjedna do pojavljivanja malobrojnih CD4+ (3,6210-5) i CD8+ (3,13 10-5) T-stanica 35. dana nakon prasenja. Nakon ove faze ranog postnatalnog razvitka ključnih subpopulacija stanica koje sudjeluju u antigenski specifičnoj imunosti na sluzničkim površinama crijeva mlade prasadi, daljnji je napredak vidljiv u različitosti obrazaca njihove raspodjele u odjeljcima sluznice jejunuma i postupnom porastu njihove brojnosti, napose između 35. i 49. dana starosti. Tijekom tog razdoblja zabilježen je značajan porast brojnosti djevičanskih, memorijskih i plazma stanica, dok je brojnost pomoćničkih i citotoksičnih T-stanica samo blago porasla između 42. i 49. dana života

Imunofenotipizacija dendritičnih stanica u organiziranim limfnim tkivima probavnog sustava svinja.

The aim of the study was to analyze the distribution patterns of porcine dendritic cells (DCs) of the residing mucosa of the ileum, caecum and colon, as well as mesenteric/colonic lymph nodes (MLN/CLN) of adult pigs. Three-color immunofluorescence (IF) was applied using monoclonal antibodies specific for porcine cell surface molecules (CD172, CD11R1 and CD16) on DCs. DCs were strategically localized within the lamina propria (LP) of the ileal villi, adjacent to the epithelial layer and around the crypts of the caecum/colon. Within the ileal LP, CD16+ and CD11R1+ cells were prevalent, whereas CD172+ cells were much rarer. Conversely, in the caecum the latter cells were more prominent, the CD16+ cells were also numerous, while CD11R1+ cells were extremely rare. In the colonic mucosa the most frequent were CD16+.CD11R1+ cells were very rare, whereas CD172+ cells were almost absent. In the MLN, CD172+ cells were prevalent within the follicular area, CD16+ cells were rare and none of the CD11R1+ cells were examined. Within the perifollicular area the most numerous were CD16+ cells, CD11R1+ cells were less prevalent, while CD172+ cells were sporadic. In the CLN, CD16+ cells were the most frequent in the perifollicular area, CD11R1+ cells were less frequent and none of the CD172+ cells were observed. Within the follicular area the most numerous were CD11R1+ cells, CD172+ cells were very rare and CD16+ cells were absent.Cilj istraživanja bio je odrediti razmještaj svinjskih dendritičnih stanica (DS) u sluznici ileuma, cekuma i kolona te u mezenterijskim limfnim čvorovima (MLČ) i limfnim čvorovima kolona (LČK) odraslih svinja. Trobojna imunofluorescencijska (IF) pretraga korištena je uz upotrebu monoklonskih protutijela specifičnih za svinjske površinske molekule (CD172, CD11R1, MHC II i CD16) na dendritične stanice. DS su strateški lokalizirane unutar lamine proprije (LP) crijevnih resica, neposredno uz epitelni sloj te uokolo kripti cekuma i kolona. Unutar LP ileuma prevladavale su CD16+ i CD11R1+ stanice, dok su CD172+ stanice bile mnogo rijeđe. Nasuprot tome, u cekumu je bila izraženija prisutnost CD172+ stanica, dok su CD16+ stanice bile također brojne, a CD11R1+ stanice izrazito rijetke. U mukozi kolona najčešće su bile CD16+ stanice, CD11R1+ stanice su bile vrlo rijetke, dok su CD172+ stanice bile gotovo odsutne. U MLČ su CD172+ stanice prevladavale unutar područja folikula, CD16+ stanice su bile rijetke, a nije zapažena niti jedna CD11R1+ stanica. Unutar perifolikularnog područja najbrojnije su bile CD16+ stanice, CD11R1+ stanice bile su mnogo manje zastupljene, dok su CD172+ stanice bile sporadične. U LČK, CD16+ stanice su bile najčešće u perifolikularnom području, CD11R1+ stanice su bile mnogo manje zastupljene, a nije primjećena niti jedna CD172+ stanica. Unutar područja folikula najbrojnije su bile CD11R1+ stanice, CD172+ stanice su bile vrlo rijetke, a CD16+ stanice nisu primijećene

Supkonjunktivalna primjena alogeničnih limbalnih stanica u pasa s poremećajima rožnice.

Limbal stem cells play a crucial role in maintaining the integrity of the corneal surface in a healthy condition or after injury through corneal renewal and repair. In pathologic conditions these cells could be replenished with cultivated allogenic limbal cells. In this investigation, limbal stem cells from small fragments of donor tissue were cultivated in vitro for treatment of corneal lesions in dogs. Fourteen dogs were divided into two groups of seven animals each: a group of dogs with corneal lesions treated using the classical veterinary ophthalmology approach, and a group of dogs with corneal lesions treated with the application of cultivated allogenic limbal cells. Regardless of the size and location of the corneal lesions, after 30 days in the group of dogs with corneal lesions treated with the application of cultivated allogenic limbal cells ophthalmological examination showed no signs of any eye disorder except corneal edema (14.29 %). In the group of dogs with corneal lesions cured using the classical veterinary ophthalmology approach, 30 days after the beginning of treatment ophthalmological examination showed 42.86 % mild/incisional blepharospasm, 85.71 % secretion, 100 % observable conjuctival congestion, 14.29 % eye liquor dimming, 85.71 % corneal edema and 85.71 % irritation. After application of cultivated allogenic limbal cells, corneal lesions were completely healed 15 days earlier than the lesions treated with conventional ophthalmological therapy. Allogenic limbal cells were capable of restoring corneal clarity with no development of ocular complications.Limbalne matične stanice igraju ključnu ulogu u održavanju integriteta površine rožnice u zdravom stanju ili nakon ozljede, kroz obnavljanje i popravak rožnice. U patološkim stanjima te se stanice mogu nadomijestiti s uzgojenim alogeničnim limbalnim stanicama. U ovom istraživanju limbalne matične stanice iz malih fragmenata tkiva donora uzgojene su in vitro radi liječenja ozljeda rožnice u pasa. Četrnaest pasa bilo je podijeljeno u dvije skupine od sedam životinja: skupina pasa s ozljedama rožnice liječenim klasičnim veterinarskim oftalmološkim pristupom i skupina pasa s ozljedama rožnice liječenim primjenom in vitro uzgojnih alogeničnih limbalnih stanica. Neovisno o veličini i smještaju lezije rožnice, deset dana nakon primjene stanica, oftalmološki pregled je pokazao 80% manje izražene simptome blefarospazma, 85% smanjenu sekreciju, 43% manje uočljivu konjunktivalnu kongestiju, 31% manji edem rožnice i 55% smanjenu iritaciju u usporedbi s psima liječenim klasičnim oftalmološkim pristupom, bez znakova zamućenja očne vodice ili priraslica. Nakon primjene uzgojeni alogeničnih limbalnih stanica, lezije rožnice u potpunosti su bile izliječene 15 dana ranije nego lezije liječene konvencionalnim oftalmološkim načinom. Supkonjunktivalna primejna limbalnih stanica omogućuje potpunu regeneraciju ozljeda rožnice nakon 30 dana bez obzira na veličinu i opseg defekta. Alogenične limbalne stanice sposobne su obnoviti prozirnost rožnice bez razvoja komplikacija. Iako su postignuti rezultati bili ohrabrujući, potrebna su daljnja istraživanja za optimizaciju protokola liječenja

Vrednovanje profilaktičkog potencijala imunizacije cjepivom neenterotoksigenog soja bakterije Escherichia coli (ne-ETEC) i pogodnosti kompetitivne ekskluzije dodatkom hrani manan-oligosaharida protiv infekcija izazvanih sojevima ETEC u odbijene prasadi

Enterotoxigenic Escherichia coli (ETEC) strains expressing F4 and F18 fimbriae are the most common causative agents of post-weaning diarrhoea (PWD) in pigs. The growing global restriction on the use of antibiotics in food animals has encouraged research into the development of nutritional and feeding strategies as well as vaccination against PWD. The aim of this study was to evaluate the efficacy of a live oral F4ac+ F18ac+ non-ETEC vaccine candidate (VAC) to stimulate gut and systemic cellular immunity in 4-week old pigs over 5 weeks following immunization. The onset and duration of protective immunity against on-farm occurring PWD, growth performance, diarrhoea scoring and mortality, as well as the phenotypic proportions of immune cells, were determined. Faecal and ileal samples were taken for determining the microbial composition or phenotyping of naïve/memory T cells. Also, the effect of prebiotic supplement mannan oligosaccharide (MOS) in the prevention of small intestinal colonization by ETEC, and its potential adjuvanticity in combination with the vaccine (VAC+MOS) were assessed. The pigs supplemented with MOS or that received VAC had significantly higher body weight (BW) (P<0.05) on Day 14, whereas the VAC+MOS treated pigs had significantly lower BW on Day 35. Treatment with VAC+MOS resulted in considerably reduced clinical PWD, in particular the incidence and severity of diarrhoea and mortality. The total bacterial load in the ileum was much lower in the pigs from all 3 principal groups (MOS, VAC, and VAC+MOS) than in the control (CON) group (19 x 107, 17 x 107 and 12 x 107 vs. 23 x 108 CFU/mL, respectively) on Day 35. The pigs from the principal groups had significantly higher proportions of tested immune cells (P<0.05) on Days 28 and 35. The localization and frequency of naive CD45RA+ and memory CD45RC+ T lymphocytes indicated their different distribution patterns within particular tissue structures, such as the villi, crypts, epithelium, lamina propria and areas (interfollicular follicular and Peyer’s patches) of ileal mucosa. This may indicate their different functions in intestinal immune responses to intraluminal microbes and their products, vaccinal immunogens and/or immunomodulators/adjuvants. To conclude, active mucosal immunity is needed to protect pigs against PWD. Hence, oral vaccination of pigs against both F4 and F18 ETEC, in combination with prebiotic supplementation represents a sustainable, practical and effective approach in PWD control.Enterotoksigeni sojevi bakterije Escherichia coli (ETEC), koji proizvode F4 i F18 fimbrije, najuobičajeniji su uzročnici dijareje nakon odbića (DNO) u prasadi. Rastuće globalno ograničavanje uporabe antibiotika u farmskih životinja usmjeruje istraživanja prema razvijanju nutritivnih i prehrambenih strategija, kao i prema cijepljenju protiv DNO-a. Cilj ovog istraživanja bio je vrednovanje učinkovitosti živog, oralnog F4ac+ F18ac+ ne-ETEC cjepiva-kandidata (VAK) u poticanju crijevne i sistemske stanične imunosti u prasadi u dobi od 4 tjedna, tijekom 5 tjedana nakon imunizacije. U pokusu su određivani početak i trajanje zaštitne imunosti od pojavnosti farmskog DNO-a, proizvodni rezultati, ocjenjivanje učestalosti i jačine dijareje te mortalitet i fenotipski udjeli imunosnih stanica. Uzimani su uzorci fecesa i ileuma za određivanje sastava mikrobiota ili za fenotipiziranje naivnih/memorijskih T-limfocita. Procijenjena je i učinkovitost prebiotika manan-oligosaharida (MOS), kao dodatka hrani, koji bi mogao uspostaviti kompetitivnu ekskluziju naseljavanja tankog crijeva sojevima ETEC, a mogao bi pokazati i adjuvantnost u testiranoj kombinaciji (VAK + MOS). Prasad hranjena dodatkom MOS-a u hrani, ili koja je primila VAK, imala je znakovito povećanu (P < 0,05) tjelesnu masu 14. dan pokusa, dok je prasad tretirana kombinacijom VAK-a i MOS-a imala znakovito nižu tjelesnu masu 35. dan pokusa. Tretman kombinacijom VAK-a i MOS-a rezultirao je znatno blažom kliničkom slikom DNO-a, napose u odnosu na pojavnost i jačinu dijareje te na mortalitet. Ukupno bakterijsko opterećenje u ileumu bilo je mnogo niže u prasadi iz sve tri pokusne skupine (MOS, VAK i VAK + MOS) od onog u kontrolnoj (KON) skupini (19 x 107, 17 x 107 i 12 x 107 prema 23 x 108 CFU/mL) 35. dan pokusa. Prasad iz pokusnih skupina imala je znakovito veće udjele testiranih imunosnih stanica (P < 0,05) 28. i 35. dan pokusa. Lokalizacija i učestalost naivnih CD45RA+ te memorijskih CD45RC+ T-limfocita pokazuju njihove različite obrasce smještanja u posebne tkivne strukture, kao što su crijevne resice, kripte, epitelij i lamina propria te područja u sluznici ileuma, što može specificirati njihove različite funkcije u crijevnim imunosnim odgovorima na intraluminalne mikrobe i njihove proizvode, vakcinalne imunogene i/ili imunomodulatore/adjuvanse. Zaključujemo da je radi zaštite prasadi od DNO-a nužno uspostaviti aktivnu mukoznu imunost. Stoga je oralno cijepljenje prasadi protiv F4 i F18 ETEC-a održiv, praktičan i učinkovit pristup u pronalaženju odgovarajućega bivalentnog, izrazito imunogenog i sigurnog cjepiva