Hrvoje Valpotić

Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac+ nonenterotoxigenic E. coli strain.

Colidiarrhea and colienterotoxemia caused by F4+ and/or F18+ enterotoxigenic E. coli (ETEC) strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac+ non-ETEC vaccine candidate strain against challenge infection with F4ac+ ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs.We also evaluated levamisole as an immune response modifier (IRM) and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0) or with levamisole and perorally given F18ac+ non-ETEC strain (at day 0), and challenged with F4ac+ ETEC strain 7 days later.At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3+, CD45RA+, CD45RC+, CD21+, IgA+ and myeloid SWC3+ cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer’s patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3+, CD45RC+ and SWC3+ cells (p<0.05) as compared to those recorded in nontreated control pigs.In the ileum of these pigs we have recorded that only CD21+ cells were significantly increased (p<0.01). The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac+ non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.

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.

Blood biochemical parameters of crossbred Istrian x East Friesian dairy ewes: relation to milking period

Abstract The research was conducted to investigate the serum biochemistry values for crossbred Istrian x East Friesian dairy ewes and additionally to test the effects of the milking period on biochemistry values. The values could be used for monitoring metabolic status and breeding program. Biochemical values for crossbred Istrian x East Friesian dairy ewes were derived from one hundred and twenty ewes raised in the Mediterranean region of Croatia. The milking period was divided into early, mid and late milking periods in order to examine the effects of the milking period on biochemical values. The concentrations of calcium, phosphorus, magnesium, blood urea nitrogen (BUN), creatinine, glucose, total protein, albumin, globulin, total lipids, triglycerides, cholesterol, beta-hydroxy-butirate as well as aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl-transferase (GGT), alkaline phosphatase (ALP), and creatine phosphokinase (CK) activities were examined.

Prophylaxis and therapeutic potential of ozone in buiatrics: Current knowledge.

Ozone therapy has been in use since 1896 in the USA. As a highly reactive molecule, ozone may inactivate bacteria, viruses, fungi, yeasts and protozoans, stimulate the oxygen metabolism of tissue, treat diseases, activate the immune system, and exhibit strong analgesic activity. More recently, ozone has been used in veterinary medicine, particularly in buiatrics, but still insufficiently. Medical ozone therapy has shown effectiveness as an alternative to the use of antibiotics, which are restricted to clinical use and have been withdrawn from non-clinical use as in-feed growth promoters in animal production. This review is an overview of current knowledge regarding the preventive and therapeutic effects of ozone in ruminants for the treatment of puerperal diseases and improvement in their fertility. In particular, ozone preparations have been tested in the treatment of reproductive tract lesions, urovagina and pneumomovagina, metritis, endometritis, fetal membrane retention and mastitis, as well as in the functional restoration of endometrium in dairy cows and goats. In addition, the preventive use of the intrauterine application of ozone has been assessed in order to evaluate its effectiveness in improving reproductive efficiency in dairy cows. No adverse effects were observed in cows and goats treated with ozone preparations. Moreover, there is a lot of evidence indicating the advantages of ozone preparation therapy in comparison to the application of antibiotics. However, there are certain limitations on ozone use in veterinary medicine and buiatrics, such as inactivity against intracellular microbes and selective activity against the same bacterial species, as well as the induction of tissue inflammation through inappropriate application of the preparation.

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.

The Intrauterine Treatment of the Retained Foetal Membrane in Dairy Goats by Ozone: Novel Alternative to Antibiotic Therapy

One of the major post-parturient complications in dairy goats is the retention of foetal membrane (RFM), which negatively influences their health, reproductive efficacy and welfare. The aim of this study was to compare the efficiency of intrauterine either ozone (OZ) or antibiotic (AB) treatments to establish the use of OZ as a novel and potential alternative to AB therapy in does with the RFM. The study was performed on 7 herds of dairy goats (n = 563) kept in the farms in Croatia. The conception rate was 563 of 641 total matings or 87.83%. The does from selected farms were observed during early puerperium and were divided into animals without the RFM (n = 522) and with the RFM (n = 41), treated either with foam spray OZ (n = 21) or with foaming AB oxytetracycline tablets (n = 20). The does with the RFM were mated successfully and became pregnant next kidding season, regardless of the treatment applied. Treatment with OZ attained similar results to the standard AB therapy, indicating that it could be novel potential alternative therapy of the RFM in dairy goats.

Influence of Body Condition on Serum Metabolic Indicators of Lipid Mobilization and Oxidative Stress in Dairy Cows During the Transition Period

The objectives of this study were to examine the influence of body condition of cows on metabolic and antioxidative status, as well as to investigate the relationship between metabolic indicators of lipid mobilization and oxidative stress during transition period. The study was conducted on 24 Holstein-Friesian dairy cows divided into 2 groups according to their body condition score (BCS) as optimal (n = 12; BCS from 3.25 to 3.75) or adipose (n = 12; BCS ≥4). Metabolic status (glucose, triglycerides, total cholesterol, HDL cholesterol, NEFA and BHB), paraoxonase-1 (PON1) and apolipoprotein A-I (ApoA-I) were analysed in sera taken on days -30, -10, -2, 0, 5, 12, 19, 26 and 60 relative to parturition. Adipose cows had significantly higher glucose concentration at parturition being significantly decreased after parturition on days 12 and 19. Total cholesterol and HDL-C concentrations were the lowest at parturition and significantly higher on days 26 and 60 after parturition in both groups of cows. Both investigated groups had significantly higher NEFA concentration from parturition until day 19 after parturition, indicating energy deficit and an increased lipid mobilization after calving. There were no significant differences in BHB concentration during transition period in both groups. No significant differences were found in PON1 activity and ApoA-I concentration during transition period in both groups of cows. However, in adipose cows, although not significantly different, PON1 was decreased from calving until day 19 after parturition indicating a disturbance in antioxidative status in adipose cows. PON1 significantly positively correlated with total cholesterol and HDL-C concentrations and negatively with NEFA indicating a strong relationship of PON1 with lipid metabolism. Significant positive correlation between NEFA and BHB in both groups of cows points out on energy deficit during transition period that cows tend to overcome by lipid mobilization providing alternative source of energy needed for parturition and lactation.

Stimulation by levamisole of cell-mediated immunity in weaned pigs

Abstract Commercial crossbred pigs weaned at four weeks were allocated into three equal groups. The experimental group 1 was primed intramuscularly, and the experimental group 2 was primed per os, with levamisole at an immunostimulatory dose of 2.5mg/kg once daily, for three consecutive days, and controls received saline. Venous blood samples from all pigs were collected after the last levamisole or saline dose was given (day 0), and 35 days later (day 35) for flow cytometry analysis. The results obtained by immunophenotyping of isolated circulating lymphocytes on day 35 indicate that priming by levamisole of weaned pigs selectively recruited CD4+, CD8+ and CD4+CD8+, but not CD21+ cells in the systemic circulation.

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.

Age-related differences of semen quality, seminal plasma, and spermatozoa antioxidative and oxidative stress variables in bulls during cold and warm periods of the year

The aims of this study were to determine the presence and quantities of antioxidative status and oxidative stress (OS) variables in the seminal plasma and spermatozoa of bulls of varying age during cold and warm periods of the year, and to establish the correlation of these variables with semen quality parameters. The study was conducted on two groups each comprising nine Simmental bulls: one group contained younger animals (aged 2 to 4 years) and the second older animals (aged 5 to 10 years). Semen samples were collected using an artificial vagina for biochemical analysis. Seminal plasma and spermatozoa activities of total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), catalase (CAT), selenium-dependent glutathione peroxidase, reduced glutathione and concentrations of total protein (TP), thiobarbituric acid reactive substances (TBARS) and protein carbonyl content (PCC) were determined. Several antioxidants in seminal plasma were also determined: total glutathione peroxidase (TGSH-Px), selenium-independent glutathione peroxidase (Non-SeGSH-Px), uric acid, albumins (ALB) and alkaline phosphatase (ALP). Significantly higher spermatozoa motility was observed during the cold v. warm period, and a significantly higher volume and total number of spermatozoa per ejaculate was observed in older than in younger bulls. Significantly higher values of ALP, TP and ALB were found in seminal plasma of older bulls than in younger bulls during the warm period. The seminal plasma of younger bulls showed significantly higher activities of TSOD, MnSOD, CuZnSOD, TGSH-Px and Non-SeGSH-Px. Younger bulls had significantly higher PCC concentration and activity of CAT in seminal plasma than older bulls during the cold period. Significantly higher concentrations of PCC and TBARS, and activities of TSOD, MnSOD and CuZnSOD were established in spermatozoa of the younger than in older bulls during the warm period. It could be concluded that antioxidative and OS variables differ significantly depending on bull age and time of year. Younger bulls were more sensitive to elevated ambient temperatures during the warm period, when the higher enzymatic antioxidative protection in seminal plasma and spermatozoa were insufficient to counteract the intensive oxidative processes in spermatozoa, which eventually resulted in decreased spermatozoa motility. The estimation of antioxidative and OS variables in seminal plasma and spermatozoa may have practical value for the assessment of bull semen quality.