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.

Impact of Deoxynivalenol and Zearalenone as Single and Combined Treatment on DNA, Cell Cycle and Cell Proliferation in HepG2 Cells.

The study aimed to investigate toxicity and the mechanism of toxicity of two Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZEA). DON and ZEA were applied to HepG2 cells as single compounds and in combination at low environmentally relevant concentrations. HepG2 cells were exposed to DON (0.5, 1, and 2 µM), ZEA (5, 10, and 20 µM) or their combinations (1 µM DON + 5 µM ZEA, 1 µM DON + 10 µM ZEA and 1 µM DON + 20 µM ZEA) for 24 h and cell viability, DNA damage, cell cycle and proliferation were assessed. Both mycotoxins reduced cell viability, however, combined treatment with DON and ZEA resulted in higher reduction of cell viability. DON (1 µM) induced primary DNA damage, while DON (1 µM) in combination with higher ZEA concentrations showed antagonistic effects compared to DON alone at 1 µM. DON arrested HepG2 cells in G2 phase and significantly inhibited cell proliferation, while ZEA had no significant effect on cell cycle. The combined treatment with DON and ZEA arrested cells in G2 phase to a higher extend compared to treatment with single mycotoxins. Potentiating effect observed after DON and ZEA co-exposure at environmentally relevant concentrations indicates that in risk assessment and setting governments' regulations, mixtures of mycotoxins should be considered.

Prevalence and Characterization of Shiga Toxin Producing Escherichia coli Isolated from Animal Feed in Croatia.

A survey on prevalence and number of Shiga toxin-producing Escherichia (E.) coli (STEC) in animal feed was carried out over a period of nine years in the Republic of Croatia. A total of 1688 feed samples were collected from feed factories and poultry farms. Analysis included two standard procedures: sample enrichment and (a) immunomagnetic separation and plating on two selective media; or (b) plating on two selective media. Confirmation of STEC included morphological examination, biochemical tests, serotyping, and polymerase chain reaction. Morphological and biochemical characterization revealed 629 E. coli strains. Further serological screening method revealed 78 STEC and EPEC serotypes, while only 27 strains were confirmed as STEC with PCR. All positive samples (1.6%) originated from poultry farms and contained combination of virulence genes: eaeA, stx1, and/or stx2. Since the presence of stx (especially stx2) and eae are identified as risk factors for development of severe diseases in humans, results of this survey indicate that avian sources of STEC infections might be one of those "undefined sources" of human illnesses. Further research is necessary for evaluation of risks posed by contaminated feed, poultry, and environment.

Determination of Fumonisins in Grains and Poultry Feedstuffs in Croatia: A 16-Year Study.

Fumonisins are a group of closely related mycotoxins produced by Fusarium, Alternaria alternata and Aspergillus species. Their occurrence is correlated with various factors during growth, processing and storage. Fumonisins occurrence data in the literature mainly include the B group of fumonisins (FB1 & FB2) in raw materials, showing high frequency of positive samples in a wide range of concentrations. In this study, a total of 933 grains (63.7%) and poultry feed (36.3%) samples, collected in the 16-year period (2006-2021), were analysed with commercial enzyme-linked-immunosorbent assay for detection of three fumonisins (FB1, FB2 & FB3). All positive and suspect samples were confirmed with high-performance-liquid-chromatography method with fluorescence detection. Overall, we have determined high occurrence of FBs in grains and poultry feed in all tested years, while the lowest occurrence was determined in 2019, followed by 2009 and 2008. Although, contamination levels varied from year-to-year, majority of analyzed samples in all tested years were around 1 mg/kg, while the maximum values varied from 3 mg/kg to 22.23 mg/kg. This study highlights the importance of regular monitoring of raw materials and understanding of the fate of FBs in the food chain in order to avoid undesirable health effects in animals and accompanied economic losses.

Beneficial Effects of Laurel (Laurus nobilis L.) and Myrtle (Myrtus communis L.) Extract on Rat Health.

Polyphenols of Laurel and Myrtle exhibit structural diversity, which affects bioavailability, metabolism, and bioactivity. The gut microbiota plays a key role in modulating the production, bioavailability and, thus the biological activities of phenolic metabolites, particularly after the intake of food containing high-molecular-weight polyphenols. The aim of this study was to investigate whether the polyphenolic components of Laurel and Myrtle aqueous extract have beneficial effects on rat health. The growth of lactic acid bacteria (LAB), ß-glucuronidase, ß-glucosidase, ß-galactosidase activity, pH value, body weight change and food efficacy ratio after intragastric treatment of rats with Laurel and Myrtle extract at doses of 50 and 100 mg/kg for two weeks were investigated. The endogenous populations of colonic probiotic bacteria (Lactobacilli and Bifidobacteria) were counted on selective media. According to the obtained data, Laurel extract in the applied dose of 50 and 100 and Myrtle extract (100 mg/kg) positively affects the rats health by increasing the number of colonies of Lactobacilli and Bifidobacteria compared to the control group, causes changes in glycolytic enzymatic activity and minor change in antioxidative tissue activity. In addition, high doses of Laurel increase food efficiency ratio, while Myrtle has the same effect at a lower dose.

Correlation of deoxynivalenol and fumonisin concentration determined in maize by ELISA methods.

Maize (Zea mays) is one of the most common crops in Croatia that is often contaminated with mycotoxins. The enzyme linked immunosorbent assay (ELISA) method as a screening immunological method has become very popular in the determination of mycotoxins contamination of cereals, as it has many advantages such as a rapid and simple determination of their concentration. In this present study, 40 maize samples collected from animal feed factories were analyzed for the concentration of deoxynivalenol (DON) and fumonisin (FUM) using the validated ELISA method of two different kit manufacturers to investigate the correlation of the determined mycotoxins concentration. The correlation coefficients for the concentration of DON and FUM, obtained using different kit manufacturers, were r = 0.9698 and r = 0.9303, respectively, yielding a high correlation between the applied ELISA methods. Concerning the legislation, in one maize sample, the DON concentration higher than the maximum recommended value was determined (16.24 mg/kg), whereas the FUM concentration in all the analyzed samples, without exception, were in accordance with recommended values.

The natural occurrence of T-2 toxin and fumonisins in maize samples in Croatia.

Maize (Zea mays) considered to be one of the most frequent crops in Croatia, is often contaminated with Fusarium mycotoxins. The aim of this study was to investigate the possible contamination of maize with T-2 toxin and fumonisins on 46 samples from different regions of Croatia. The highest concentrations of T-2 toxin and fumonisins were 210 and 25,200 ng/g, with mean values of 110 and 4,509 ng/g, respectively, pointing to maize contamination with these mycotoxins after the period of the year with extremely high rainfall.

T-2 toxin: incidence and toxicity in poultry.

T-2 toxin is the most toxic type A trichothecene mycotoxin. It is the secondary metabolite of the Fusarium fungi, and is common in grain and animal feed. Toxic effects have been shown both in experimental animals and in livestock. It has been implicated in several outbreaks of human mycotoxicoses. Toxic effects in poultry include inhibition of protein, DNA, and RNA synthesis, cytotoxicity, immunomodulation, cell lesions in the digestive tract, organs and skin, neural disturbances and low performance in poultry production (decreased weight gain, egg production, and hatchability). Concentrations of T-2 toxin in feed are usually low, and its immunosuppressive effects and secondary infections often make diagnosis difficult. If at the onset of the disease, a change in diet leads to health and performance improvements in animals, this may point to mycotoxin poisoning. Regular control of grain and feed samples is a valuable preventive measure, and it is accurate only if representative samples are tested. This article reviews the incidence and toxic effects of T-2 toxin in poultry.

A pathological condition possibly caused by spontaneous trichotecene poisoning in Brahma poultry: first report.

Trichotecene poisoning in poultry can cause oral lesions, haemorrhages, depletion and necrosis in the lymphopoetic organs and death. Spontaneous poisonings with these toxins are rarely described. This paper describes the spontaneous poisoning of two Brahma chickens with T-2 toxin, diacetoxyscirpenol and deoxynivalenol. Two out of 10 chickens died under signs of depression and loss of appetite. Histopathological analysis revealed vacuolar dystrophy of the liver, necrosis and depletion of lymphocyte in the bursa of Fabricius as well as multiple necroses in the glandular stomach and gut. Even though quantities of 0.70 mg/kg T-2 in the food together with 0.50 mg/kg diacetoxyscirpenol significantly differ from the median lethal dose for chickens reported in literature (4.97 mg/kg), parasitological, virological and histopathological results indicate trichotecenes as the causative agents of this pathological condition.