Immunostimulatory and Antibacterial Effects of Cannabis sativa L. Leaves on Broilers.

The aim of this study was to evaluate the effect of dried Cannabis sativa L. leaves as a phytogenic mixture added to broiler feed on CD4+ and CD8+ T lymphocyte subpopulations, Newcastle disease virus (NDV) antibody titres, and the presence of E. coli in faecal samples. The study was conducted on 100 male Ross 308 broilers, divided into four groups of 25 broilers, for a 42-day research period. The groups were housed separately in boxes on a litter of softwood shavings and were fed starter mixture from day 1 to day 21 and finisher mixture from day 22 to day 42. Industrial hemp (C. sativa) was grown in the Crkvina area, Croatia (latitude: 45°18'46.8″ N; longitude: 15°31'30″ E). The hemp leaves were manually separated, sun-dried, and ground to a powder. The mixture offered to the control group did not contain cannabis leaves, whereas the three experimental groups received mixtures containing mixed cannabis leaves in a quantity of 10 g/kg, 20 g/kg, or 30 g/kg (E_10, E_20, and E_30, respectively). The mean NDV antibody level was uniform in all study groups until post-vaccination day 14 and increased comparably with time. The percentage of CD4+ and CD8+ lymphocytes in the peripheral blood subpopulation showed statistically significant differences (p < 0.001) in the E_20 group as compared with the control group and both the E_10 and E_30 groups throughout the study period. As the broiler age increased, the CD4+-to-CD8+ ratios also increased and were statistically significant (p < 0.0001) on day 42 in all experimental groups as compared to the control group. Comparing the control group with the experimental groups indicated that the bacterial count was lower in broiler groups having received feed with the addition of 20 g/kg and 30 g/kg C. sativa leaves. In conclusion, the C. sativa leaves were found to elicit a favourable immunomodulatory effect on cell-mediated and humoral immune responses in broilers via increased CD4+ and CD8+ lymphocyte subpopulations and higher CD4+:CD8+ cell ratios, thus indicating enhanced immune function capacity. In addition, C. sativa leaves may have complementary effects on the broiler post-vaccination immune response, increase broilers' resistance to infectious diseases, reduce the effect of stress associated with vaccination, and improve broiler health and welfare.

IVO HORVAT, BOTANIST (1897 - 1963), WORK AT THE DEPARTMENT OF BOTANY OF THE FACULTY OF VETERINARY MEDICINE AT THE UNIVERSITY OF ZAGREB / BOTANICAR IVO HORVAT (1897. ­ 1963.), RAD NA BOTANICKOM ZAVODU VETERINARSKOG FAKULTETA SVEUCILISTA U ZAGREBU

Professor Ivo Horvat, a world-renowned Croatian scientist, botanist, and university teacher, was born in Cazma on October 7, 1897. After finishing the Classical Gymnasium in Zagreb in 1916, he graduated from the University of Zagreb, Faculty of Philosophy in 1920 and obtained his Ph.D. in botany on July 31, 1920. From his invaluable scientific and educational heritage, we have selected out for this occasion only a small part dedicated to the period from June 11, 1947 to April 23, 1963, in which Prof. Horvat worked at the Faculty of Veterinary Medicine, University of Zagreb. He worked in a wide range of both natural and biomedical veterinary sciences: from systematic botany, ecology, and environmental protection to the study of phylogeny, floristic, and vegetation research, including vegetation mapping and scientific-organizational work. Following the bibliographic and archival sources of the original documents, the given data represent a brief overview of Prof. Horvat's contribution to phytocoenological work and an overview of scientific and educational heritage with an emphasis on the years spent at the Faculty of Veterinary Medicine, University of Zagreb.

A New Method of Assessing Sheep Red Blood Cell Types from Their Morphology.

Data concerning the morphometric parameters of sheep red blood cells (RBCs) obtained using computer-assisted image analysis have not yet been investigated, and there are no data on any analyses of ovine RBC subpopulations based on their morphometric parameters. The aims of this study are to determine the values of RBC haematological and morphometric size and shape parameters, to form groups according to the obtained values of haematological parameters; to determine the differences in RBC morphometric parameters between the formed groups, and to determine RBC subpopulations and their respective proportions in the formed groups. Thirty-six blood samples were collected from the jugular vein of clinically healthy Lika pramenka sheep, aged between 2 and 5 years. Haematological parameters including haemoglobin (HGB), haematocrit (HTC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and RBC distribution width were analysed using a haematology analyser. Haematological parameters were categorized into two groups: those with lower values or values below the physiological range (Groups 1) and groups with higher values or values above the physiological range (Groups 2). Morphometric parameters of RBCs were determined from stained blood smears using SFORM, a computer-assisted program. Significantly higher values of RBC area, outline, convex, minimal and maximal radius, as well as length and breadth were established in Groups 2 compared to Groups 1 of HGB, HCT, MCV, MCH, and MCHC, respectively. Based on the morphometric parameters of RBCs, three RBC subpopulations were obtained using principal component and cluster analysis: ES 1-the smallest and most elongated RBCs, ES 2-the biggest and most rounded RBCs, and ES 3-average size and shape RBCs. Significantly higher proportions of ES 2 and ES 3 subpopulations, as well as a significantly lower proportion of ES 1 subpopulation, were established in Groups 2 compared to Groups 1 of HGB, HTC, MCV, and MCH, respectively. It can be concluded that ovine RBC subpopulations, based on their morphometric parameters, can be obtained by using computer-assisted image analysis of RBC morphometry and multivariate statistical methods, including principal component and cluster analysis. RBC morphometry, including classification into subpopulations, could serve as a basis for future possibilities in the diagnostic interpretation of anaemic syndromes in veterinary medicine, especially in normocytic, macrocytic, and microcytic anaemias in sheep.

Analysis of 12 X-chromosomal markers in the population of central Croatia.

Investigator® Argus X-12 Kit is a commercially available set that allows simultaneous PCR amplification of 12 X-STR markers belonging to four linkage groups (LG). To assess the forensic efficiency of these markers for the population of central Croatia and consequent applicability in routine forensic casework, DNA from 200 blood samples of unrelated donors (100 female and 100 male) was amplified by Investigator® Argus X-12 Kit and analyzed by capillary electrophoresis. Statistical computations based on allele and haplotype frequencies for LG1 - LG4 were performed using Arlequin 3.5 software and on-line tool available at ChrX-STR.org. In female samples, all X-STR markers were in Hardy-Weinberg equilibrium (HWE). The most informative marker for central Croatia population was DXS10135 with polymorphism information content (PIC) 0.9296. The least polymorphic locus was DXS8378 (PIC=0.6363). Power of discrimination (PD) varied from 0.6968 to 0.9336 in male and from 0.8476 to 0.9916 in female samples. Combined PD exceeded 0.999999999 in both men and women. In male samples, linkage disequilibrium (LD) test revealed significant association (P=0.0000) of one marker pair in LG4 and two marker pairs in LG3. Portion of observed haplotypes in the number of possible haplotypes varied from 2.86% to 7.47% across all LGs. LG1 was the most informative with haplotype diversity (H) 0.9972. High PD of all analyzed markers exhibited for central Croatia population confirms suitability of Investigator® Argus X-12 for forensic pertinence. Moreover, results of this study will be included in establishing a national reference X-STR database based on 12 X-STR loci, which is necessary for the correct interpretation of the forensic casework results.

Synthetic vs natural scaffolds for human limbal stem cells.

AIM: To investigate the impact of synthetic electrospun polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, before and after hydrolytic surface modification, on viability and differentiation of cultured human eye epithelial cells, in comparison with natural scaffolds: fibrin and human amniotic membrane. METHODS: Human placenta was taken at elective cesarean delivery. Fibrin scaffolds were prepared from commercial fibrin glue kits. Nanoscaffolds were fabricated by electrospinning. Limbal cells were isolated from surpluses of human cadaveric cornea and seeded on feeder 3T3 cells. The scaffolds used for viability testing and immunofluorescence analysis were amniotic membrane, fibrin, PU, and PCL nanoscaffolds, with or without prior NaOH treatment. RESULTS: Scanning electron microscope photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. There was a significant difference in viability performance between cells with highest viability cultured on tissue culture plastic and cells cultured on all other scaffolds. On the other hand, electrospun PU, PCL, and electrospun PCL treated with NaOH had more than 80% of limbal cells positive for stem cell marker p63 compared to only 27%of p63 positive cells on fibrin. CONCLUSION: Natural scaffolds, fibrin and amniotic membrane, showed better cell viability than electrospun scaffolds. On the contrary, high percentages of p63 positive cells obtained on these scaffolds still makes them good candidates for efficient delivery systems for therapeutic purposes.

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

BACKGROUND: Because 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. METHODS: The 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. RESULTS: Pigs 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. CONCLUSION: This 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.