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.
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.
Valpotić, H., Šperanda, M., Kovšca-Janjatović, A., Ðidara, M., Lacković, G., Božić, F., Habrun, B., Srečec, S., Mataušić-Pišl, M. and Valpotić, I. 2014. Levamisole stimulates proliferation of ...circulating and intestinal immune cell subsets, gut health and performance in weaned pigs. Can. J. Anim. Sci. 94: 43–53. With the growing knowledge of the porcine immune system and its endogenous modulation, it has been clearly stated that exogenous modulation through the use of substances able to modulate immune functions represents an important prophylactic/therapeutic approach in prevention/treatment of both stress- and F4
+
and F18
+
enterotoxigenic E. coli (ETEC)-induced infections accompanied weaning. The aim of this study was to evaluate the effectiveness of levamisole (LEVA; 2.5 mg kg
−1
BW in 10 mL) applied per os to weaned pigs in proliferation of circulating and intestinal immune cell subsets throughout a period of 5 wk. Changes in proportion or number of peripheral blood and ileal mucosal leukocytes tested were studied either weekly by flow cytometry or at the end of the experiment (day 35) by immunohistology/histomorphometry, respectively. Pigs treated with LEVA had increased proportions of peripheral blood CD45
+
lymphoid cells, CD4
+
and CD8
+
T cells, and CD21
+
B cells (P<0.01) between days 14 and 35 following the treatment. Also, LEVA stimulated the proliferation of CD45RA
+
naïve lymphoid cells in interfollicular (P<0.001) and follicular areas (P<0.05) of ileal Peyer’s patches at day 35 of the experiment. These pigs had a significantly higher (P<0.05) average body weight (19.7 vs. 17.1 kg) and weight gain at the end of experiment compared with the control pigs (for 15%). We conclude that LEVA stimulated the proliferation of circulating and intestinal lymphoid cell subsets tested and improved performance in weaned pigs, and thus, the drug may nonspecifically enhance their immunity/resistance to F4
+
and F18
+
ETEC strains.
Valpotić, H., šperanda, M., Kovšca-Janjatovic, A., đidara, M., Lackovic, G., Božic, F., Habrun, B., Srecec, S., Mataušic-Pišl, M. and Valpotic, I. 2014. Levamisole stimulates proliferation of ...circulating and intestinal immune cell subsets, gut health and performance in weaned pigs. Can. J. Anim. Sci. 94: 43-53. With the growing knowledge of the porcine immune system and its endogenous modulation, it has been clearly stated that exogenous modulation through the use of substances able to modulate immune functions represents an important prophylactic/therapeutic approach in prevention/treatment of both stress- and F4+ and F18+ enterotoxigenic E. coli (ETEC)-induced infections accompanied weaning. The aim of this study was to evaluate the effectiveness of levamisole (LEVA; 2.5 mg kg-1 BW in 10 mL) applied per os to weaned pigs in proliferation of circulating and intestinal immune cell subsets throughout a period of 5 wk. Changes in proportion or number of peripheral blood and ileal mucosal leukocytes tested were studied either weekly by flow cytometry or at the end of the experiment (day 35) by immunohistology/histomorphometry, respectively. Pigs treated with LEVA had increased proportions of peripheral blood CD45+ lymphoid cells, CD4+ and CD8+ T cells, and CD21+ B cells (P<0.01) between days 14 and 35 following the treatment. Also, LEVA stimulated the proliferation of CD45RA+ naïve lymphoid cells in interfollicular (P<0.001) and follicular areas (P<0.05) of ileal Peyer's patches at day 35 of the experiment. These pigs had a significantly higher (P<0.05) average body weight (19.7 vs. 17.1 kg) and weight gain at the end of experiment compared with the control pigs (for 15%). We conclude that LEVA stimulated the proliferation of circulating and intestinal lymphoid cell subsets tested and improved performance in weaned pigs, and thus, the drug may nonspecifically enhance their immunity/resistance to F4+ and F18+ ETEC strains.
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.sup.+/F18ac.sup.+ 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 * 10.sup.8 vs. 18 * 10.sup.7 vs. 14 * 10.sup.7 vs. 13 * 10.sup.7 CFU/mL, respectively). Regarding CD4.sup.+and CD8.sup.+ 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.sup.+ 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.sup.+ lymphoid cells than the pigs from the CONTROL group (P<0.05). Quite sparsely distributed CD45RA.sup.+naive T lymphocytes were observed in the jejunal lamina propria of the intestinal villi, within the Lieberkiihn crypts (LC) and in the submucosa of the CONTROLS and CPL supplemented pigs. The treatment with the VACCINE induced moderate recruitment of CD45 RA.sup.+ cells within both the IFA and FA of the jejunal PP of the pigs 6 weeks following the vaccination. In the CONTROLS, CD45 RC.sup.+ 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.sup.+ 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 Key words: immunogenicity; bivalent; live vaccine; non-enterotoxigenic Escherichia coli; clinoptilolite; post-weaning diarrhoea; pigs
Dijareja nakon odbića (DNO) prasadi uzrokuje glavninu proizvodnih gubitaka u svinjogojskoj industriji širom svijeta. Ne postoji sigurno i učinkovito cjepivo protiv svinjskih enterotoksigenih sojeva ...bakterije Escherichia coli (ETEC) koji se smatraju etiološkim uzročnicima DNO. Ovo je istraživanje planirano radi vrednovanja: (1) učinkovitosti oralnog bivalentnog F4ac+/F18ac+ ne-ETEC pokusnog živog cjepiva (cjepivo) u stimuliranju sistemske i crijevne stanične imunosti 4 tjedna stare odbijene prasadi, (2) pojave i trajanja zaštitne imunosti odbijene prasadi protiv prirodnog zaražavanja uzročnicima DNO tijekom razdoblja od 6 tjedana nakon odbića i (3) potencijala dodatka hrani zeolita klinoptilolita (KPL), antimikrobnog minerala i/ili imunomodulatora/adjuvansa cjepiva (CJEPIVO + KPL). Prasad imunizirana ili cjepivom ili njegovom kombinacijom s dodatkom hrani KPL-om imala je znakovito povećan prirast tjelesne mase od 7. do 42. dana (P<0,05) pokusa u usporedbi s kontrolnom skupinom prasadi (KONTROLA). Suprotno tome, prasad koja su primila samo dodatak hrani KPL, imala su pretežito znakovito nižu (P<0,05) tjelesnu masu. Prasad koja je primila CJEPIVO + KPL nije imala ni dijareju, niti je bilo uginuća tijekom razdoblja trajanja pokusa. Ukupni broj izdvojenih bakterija iz jejunuma bio je mnogo manji u prasadi iz sve tri pokusne skupine od onoga u kontrolne prasadi (30 x 108 vs. 18 x 107 vs. 14 x 107 vs. 13 x 107 CFU/mL). Specifična imunizacija s CJEPIVOM
ili CJEPIVOM + KPL stimulirala je znakovit porast udjela CD4+ i CD8+ T stanica (P<0,05) od 7. do 42. dana pokusa. Vrlo su slični nalazi dobiveni za CD21+ B stanice, s obzirom da je njihov udjel bio znakovito povišen u prasadi tretirane ili s CJEPIVOM ili s CJEPIVOM + KPL (P<0,05 ili P<0,05). Prasad iz skupine koja je primila CJEPIVO + KPL, imala je značajno viši udjel CD45+ lifoidnih stanica od prasadi iz KONTROLA skupine (P<0,05). Rijetko raspodjeljeni CD45RA+ naivni T limfociti opaženi su u lamini propriji crijevnih resica jejunuma, u Lieberkühnovim kriptama te u submukozi prasadi KONTROLE skupine i skupine hranjene s dodatkom KPL. Tretman CJEPIVOM izazvao je umjerenu proliferaciju CD45RA+ u interfolikularnim (IFP) i folikularnim područjima (FP) Peyerovih ploča (PP) jejunuma u prasadi 6 tjedana nakon cijepljenja. U KONTROLNE prasadi, CD45RC+ memorijske T stanice nisu bile brojne i uglavnom su bile dispergirane u sredini lamine proprije resica, a samo rijetko u blizini bazalne membrane crijevnih enterocita. Te su stanice brojnije u lamini propriji resica u prasadi iz KPL skupine u odnosu na KONTROLNU prasad. No, CD45RC+ memorijske T stanice još su brojnije, što je primijećeno u lamini propriji resica jejunuma prasadi koja je primila CJEPIVO. Te su stanice pretežito nađene u lamini propriji resica i u IFP-u, ali su manje brojne u FP-u jejunalnih Peyerovih ploča u prasadi koja je primila CJEPIVO i KPL. Zbog naravi bolesti, izazovi vezani za razvoj cjepiva protiv DPO će i dalje postojati. Premda je naš istraživački pristup bio samo djelomično uspješan, razvoj sigurnog i imunogeno učinkovitog živog oralnog cjepiva protiv DPO, koje će pružati solidnu zaštitu i održive stanične i humoralne imunosne odgovore, i nadalje je izvanredan izazov.
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.
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.sup.+ and CD11R1.sup.+ cells were prevalent, whereas CD172.sup.+ cells were much rarer. Conversely, in the caecum the latter cells were more prominent, the CD16.sup.+ cells were also numerous, while CD11R1.sup.+ cells were extremely rare. In the colonic mucosa the most frequent were CD16.sup.+.CD11R1.sup.+ cells were very rare, whereas CD172.sup.+ cells were almost absent. In the MLN, CD172.sup.+ cells were prevalent within the follicular area, CD16.sup.+ cells were rare and none of the CD11R1.sup.+ cells were examined. Within the perifollicular area the most numerous were CD16.sup.+ cells, CD11R1.sup.+ cells were less prevalent, while CD172.sup.+ cells were sporadic. In the CLN, CD16.sup.+ cells were the most frequent in the perifollicular area, CD11R1.sup.+ cells were less frequent and none of the CD172.sup.+ cells were observed. Within the follicular area the most numerous were CD11R1.sup.+ cells, CD172.sup.+ cells were very rare and CD16.sup.+ cells were absent. Key words: dendritic cells, gut-associated lymphoid tissues, pig Cilj istrazivanja bio je odrediti razmjestaj svinjskih dendriticnih stanica (DS) u sluznici ileuma, cekuma i kolona te u mezenterijskim limfnim cvorovima (MLC) i limfnim cvorovima kolona (LCK) odraslih svinja. Trobojna imunofl uorescencijska (IF) pretraga koristena je uz upotrebu monoklonskih protutijela specifi cnih za svinjske povrsinske molekule (CD172, CD11R1, MHC II i CD16) na dendriticne stanice. DS su strateski lokalizirane unutar lamine proprije (LP) crijevnih resica, neposredno uz epitelni sloj te uokolo kripti cekuma i kolona. Unutar LP ileuma prevladavale su CD16.sup.+ i CD11R1.sup.+ stanice, dok su CD172.sup.+ stanice bile mnogo rijede. Nasuprot tome, u cekumu je bila izrazenija prisutnost CD172.sup.+ stanica, dok su CD16.sup.+ stanice bile takoder brojne, a CD11R1.sup.+ stanice izrazito rijetke. U mukozi kolona najcesce su bile CD16.sup.+ stanice, CD11R1.sup.+ stanice su bile vrlo rijetke, dok su CD172.sup.+ stanice bile gotovo odsutne. U MLC su CD172.sup.+ stanice prevladavale unutar podrucja folikula, CD16.sup.+ stanice su bile rijetke, a nije zapazena niti jedna CD11R1.sup.+ stanica. Unutar perifolikularnog podrucja najbrojnije su bile CD16.sup.+ stanice, CD11R1.sup.+ stanice bile su mnogo manje zastupljene, dok su CD172.sup.+ stanice bile sporadicne. U LCK, CD16.sup.+ stanice su bile najcesce u perifolikularnom podrucju, CD11R1.sup.+ stanice su bile mnogo manje zastupljene, a nije primjecena niti jedna CD172.sup.+ stanica. Unutar podrucja folikula najbrojnije su bile CD11R1.sup.+ stanice, CD172.sup.+ stanice su bile vrlo rijetke, a CD16.sup.+ stanice nisu primijecene. Kljucne rijeci: dendriticne stanice, limfno tkivo, probavni sustav, svinja
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. Key words: corneal disorders, allogenic limbal cells, dogs Limbalne maticne stanice igraju kljucnu ulogu u odrzavanju integriteta povrsine roznice u zdravom stanju ili nakon ozljede, kroz obnavljanje i popravak roznice. U patoloskim stanjima te se stanice mogu nadomijestiti s uzgojenim alogenicnim limbalnim stanicama. U ovom istrazivanju limbalne maticne stanice iz malih fragmenata tkiva donora uzgojene su in vitro radi lijecenja ozljeda roznice u pasa. Cetrnaest pasa bilo je podijeljeno u dvije skupine od sedam zivotinja: skupina pasa s ozljedama roznice lijecenim klasicnim veterinarskim oftalmoloskim pristupom i skupina pasa s ozljedama roznice lijecenim primjenom in vitro uzgojnih alogenicnih limbalnih stanica. Neovisno o velicini i smjestaju lezije roznice, deset dana nakon primjene stanica, oftalmoloski pregled je pokazao 80% manje izrazene simptome blefarospazma, 85% smanjenu sekreciju, 43% manje uocljivu konjunktivalnu kongestiju, 31% manji edem roznice i 55% smanjenu iritaciju u usporedbi s psima lijecenim klasicnim oftalmoloskim pristupom, bez znakova zamucenja ocne vodice ili priraslica. Nakon primjene uzgojeni alogenicnih limbalnih stanica, lezije roznice u potpunosti su bile izlijecene 15 dana ranije nego lezije lijecene konvencionalnim oftalmoloskim nacinom. Supkonjunktivalna primejna limbalnih stanica omogucuje potpunu regeneraciju ozljeda roznice nakon 30 dana bez obzira na velicinu i opseg defekta. Alogenicne limbalne stanice sposobne su obnoviti prozirnost roznice bez razvoja komplikacija. Iako su postignuti rezultati bili ohrabrujuci, potrebna su daljnja istrazivanja za optimizaciju protokola lijecenja. Kljucne rijeci: poremecaji roznice, alogenicne limbalne stanice, psi
By using immunohistology and digital image analysis for histomorphometry, we investigated the influence of porcine postnatal development on distribution and quantitative patterns of naive/memory ...lymphoid cell subsets (CD45RA.sup.+, CD45RC.sup.+, respectively), helper/cytotoxic T cell subsets (CD4.sup.+, CD8.sup.+, respectively) as well as of IgA.sup.+ 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.sup.+ lymphoid cells (1.25 x 10.sup.-5) at Day 0 post partum. A very few CD45RC.sup.+ lymphoid cells (8.79x10.sup.-6) appeared at Day 7, followed by a similar number of IgA.sup.+ plasma cells (7.65x10.sup.-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.sup.+ (3.62x10.sup.-5) and CD8.sup.+ (3.13x10.sup.-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 naive, memory and plasma cells, whereas the number of helper and cytotoxic T cells only slightly increased between Day 42 and Day 49 of life. Key words: postnatal development, immune cells, gut mucosa, pigs Pomocu imunohistoloskih i histomorfometrijskih analiza racunalnom obradom slike istrazivali smo utjecaj postnatalnog razvitka u svinje na distribucijske i kvantitativne pokazatelje subpopulacija djevicanskih/ memorijskih limfoidnih stanica (CD45RA.sup.+ odnosno CD45RC.sup.+), pomocnickih/citotoksicnih T-stanica (CD4.sup.+ odnosno CD8.sup.+), kao i za IgA.sup.+ plazma stanice u limfatickim tkivima probavnog sustava (LTPS) prasadi uzgajane u intenzivnim uvjetima tijekom prvih 7 tjedana zivota. Istrazivanje postnatalnog razvitka/sazrijevanja imunosnog sustava sluznice tankoga crijeva svinje pokazalo je da je neonatalna prasad imunoloski prilicno nezrela, pa je stoga u potpunosti ovisna o pasivno stecenoj imunosti. U sluznici njihovog jejunuma gotovo potpuno nedostaju imunosne stanice, s izuzetkom malobrojnih CD45 RA.sup.+ limfoidnih stanica (1,25 x10.sup.-5) 0. dana nakon prasenja. Sedmog dana post partum pojavljuje se nesto malo CD45RC.sup.+ limfoidnih stanica (8,79x10.sup.6), a potom se 14. dana mogu identificirati malobrojne IgA.sup.+ plazma stanice (7,65 x0.sup.-6). Ovi prvi znakovi postnatalnog razvitka urodene i stecene imunosti, temeljem identifikacije/lokalizacije subpopulacija istrazivanih stanica koje naseljavaju LTPS svinje, odnosno sluznicu jejunuma, nastavljaju se nakon razdoblja od tri tjedna do pojavljivanja malobrojnih CD4.sup.+ (3,62x10.sup.-5) i CD8.sup.+ (3,13x10.sup.-5) T-stanica 35. dana nakon prasenja. Nakon ove faze ranog postnatalnog razvitka kljucnih subpopulacija stanica koje sudjeluju u antigenski specificnoj imunosti na sluznickim povrsinama crijeva mlade prasadi, daljnji je napredak vidljiv u razlicitosti obrazaca njihove raspodjele u odjeljcima sluznice jejunuma i postupnom porastu njihove brojnosti, napose izmedu 35. i 49. dana starosti. Tijekom tog razdoblja zabiljezen je znacajan porast brojnosti djevicanskih, memorijskih i plazma stanica, dok je brojnost pomocnickih i citotoksicnih T-stanica samo blago porasla izmedu 42. i 49. dana zivota. Kjucne rijeci: postnatalni razvitak, imunosne stanice, sluznica crijeva, prasad
