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.

Genetic Diversity and Resistome Analysis of Campylobacter lari Isolated from Gulls in Croatia.

Campylobacter lari is a thermotolerant bacterium that sporadically causes gastrointestinal diseases in humans and can be found in wildlife and the environment. C. lari is an understudied species, especially in wild birds such as gulls. Gulls are potentially good carriers of pathogens due to their opportunistic behavior and tendency to gather in large flocks. During winter and their breeding period, 1753 gulls were captured, and cloacal swabs were taken to be tested for the presence of C. lari. From isolated bacteria, the DNA was sequenced, and sequence types (ST) were determined. Sixty-four swabs were positive for C. lari, and from those, forty-three different STs were determined, of which thirty-one were newly described. The whole genome was sequenced for 43 random isolates, and the same isolates were tested for antimicrobial susceptibility using the broth microdilution method to compare them to WGS-derived antimicrobial-resistant isolates. All the tested strains were susceptible to erythromycin, gentamicin, and chloramphenicol, and all were resistant to ciprofloxacin. Resistance to ciprofloxacin was attributed to a gyrA_2 T86V mutation. Genes connected to possible beta-lactam resistance (blaOXA genes) were also detected.

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.

First Data on Campylobacter spp. Presence in Shellfish in Croatia.

This study aimed to assess the presence of thermotolerant Campylobacter spp., as one of the most important foodborne zoonotic pathogens, in three shellfish species: mussels (Mytilus galloprovincialis), oysters (Ostrea edulis) and queen scallops (Aequipecten opercularis). The samples were collected from nine locations in the Istrian aquatory, Croatia. Isolation of Campylobacter was done according to standard ISO method, and species were identified using multiplex PCR. Isolates identified as C. jejuni and C. lari were genotyped using multilocus sequence typing (MLST) to determine the potential source of contamination. Among 108 examined samples of bivalve molluscs, mussels dominated and were the only ones found positive for the presence of Campylobacter (25.6%). In total, 19 C. lari and 1 C. jejuni strains were isolated. C. lari isolates found in this study belong to 13 sequence types (STs), and 9 of them are newly described in this paper. Two out of the four previously described C. lari STs that were found in this study were previously found in human stool. The only C. jejuni isolate was found to be sequence type 1268, which belongs to ST-1275 clonal complex that is almost exclusively found in seabirds and can sporadically cause infection in humans. Regarding the obtained results, introducing surveillance of thermotolerant Campylobacter in shellfish in the Republic of Croatia is advised as an improvement for public health safety.

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.

Occurrence of Campylobacter jejuni in Gulls Feeding on Zagreb Rubbish Tip, Croatia; Their Diversity and Antimicrobial Susceptibility in Perspective with Human and Broiler Isolates.

Campylobacteriosis is the most common gastrointestinal bacterial disease in the European Union (EU). Wild birds are one of the natural reservoirs of these pathogens. In this study we tested cloacal swabs of 643 gulls captured on rubbish tip in Zagreb, Croatia for the presence of Campylobacter spp. and found 168 Campylobacter positive samples. We used multilocus sequence typing (MLST) to genotype 62 random C. jejuni isolates from gulls, 24 isolates from broiler caeca, 27 isolates from broiler neck skins and 23 human isolates. Altogether, we identified 44 different STs, from which 19 were newly described. Most of the new STs (14) originate from gulls. Although humans and broilers share the majority of STs and isolates from gulls are separated from these, there was one ST present in all three hosts: 45. Additionally antimicrobial susceptibility to six antimicrobials was performed on 123 C. jejuni strains isolated from broiler caeca (n = 22), neck skins of broilers (n = 20), gulls cloacal swabs (n = 50) and human faeces (n = 31) by the broth microdilution method. Results show lower resistance of gull isolates to NAL and CIP, while resistance to TET was as high as in human and broiler isolates.

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.

First finding of libyostrongylosis in farm-reared ostriches (Struthio camelus) in Croatia: unusual histopathological finding in the brain of two ostriches, naturally infected with Libyostrongylus douglasi.

In the present work, the very first finding of Libyostrongylus douglasi in farm-reared ostriches in Croatia, was described, not only as the main cause of the death, but also as the cause of persistent infection. The results of histopathological examination of almost all parenchymatous organs in two routinely necropsied ostriches were presented, including atypical histopathological finding in the brain and the result of the L. douglasi scanning electron microscopical (SEM) examination. In order to determine the parasite species to which the larval form found in the brain belonged, polymerase chain reaction (PCR) was performed. Total DNA was isolated from fresh L. douglasi, and from archival formalin-fixed and paraffin-embedded brain section. Additionally, the smears of the proventricular mucosal lining were cytologically examined. Virological examination for newcastle disease virus (NDV) was also performed. As there was very limited information concerned ostrich's health status in Croatian farms, a preliminary evaluation of the parasite infestation level in the Croatian ostrich population over the period 2001-2002 was also done, and an attempt at characterising individual parasite species, was made.

Stress-induced Cushing's syndrome in fur-chewing chinchillas.

One of the most serious problems in the chinchilla industry is 'fur-chewing', when the chinchilla bites off areas of its own or some other animal's fur. The condition generally develops in both genders at the age of 6-8 months. In chinchilla farms in Croatia an incidence of 15-20% has been observed. A pathomorphological, microbiological and parasitological investigation was conducted on eleven 6- to 11-month-old chinchillas of both sexes with clinical symptoms of 'fur-chewing' and three chinchillas without such signs. Histopathology of the adrenal glands and of the chewed skin revealed changes typical of Cushing's syndrome in 'fur-chewed' chinchillas, such as hyperkeratinisation of the epidermis, epidermal atrophy, pronounced follicular and sebaceous gland atrophy, hyperkeratinisation of the follicles with comedo formations and the presence of calcium salts in subcutis.