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.

Investigating the Presence of SARS CoV-2 in Free-Living and Captive Animals.

Due to SARS CoV-2 recombination rates, number of infected people and recent reports of environmental contamination, the possibility of SARS CoV-2 transmission to animals can be expected. We tested samples of dominant free-living and captive wildlife species in Croatia for the presence of anti-SARS CoV-2 antibodies and viral RNA. In total, from June 2020 until February 2021, we tested blood, muscle extract and fecal samples of 422 free-living wild boars (Sus scrofa), red foxes (Vulpes vulpes) and jackals (Canis aureus); blood and cloacal swabs of 111 yellow-legged gulls (Larus michahellis) and fecal samples of 32 zoo animals. A commercially available ELISA (ID.Vet, France) and as a confirmatory test, a surrogate virus neutralization test (sVNT; GenScript, Netherlands) were used. Fecal samples were tested for the presence of viral RNA by a real-time RT-PCR protocol. Fifteen out of 533 (2.8%) positive ELISA results were detected; in wild boars (3.9%), red foxes (2.9%) and jackals (4.6%). However, the positive findings were not confirmed by sVNT. No viral RNA was found. In conclusion, no spillover occurred within the investigated period (second COVID-19 wave). However, further investigation is needed, especially regarding wildlife sample features for serological tests.

Multiple introduction of Asian H5N1 avian influenza virus in Croatia by wild birds during 2005-2006 and isolation of the virus from apparently healthy black-headed gulls (Larus ridibundus).

This study describes the introduction and spread of avian influenza A (H5N1) subtype in Croatia. Seventeen isolates were identified during the period from October 2005 to March 2006, all originating from wild birds. The full-length nucleotide sequence analysis of the hemagglutinin (HA) gene of seven representative isolates revealed that three distinct genetic strains involved in the outbreaks, implicating at least three independent introductions of the virus into Croatia during a relatively short period of time. All three genetic strains belonged to clade 2.2 (Qinghai-like viruses) and each strain displayed significant similarity to concurrent H5N1 viruses from other European countries. The dominant strain of the virus was present in all four affected areas and in all three bird species (mute swan, mallard, and black-headed gull), indicating cross-species transmission of the virus. Two other genetic strains were found, together with the dominant strain, only in a marsh at the Adriatic coast during late February and early March 2006, which could be associated with frozen water surfaces in the continental part of Croatia as well as in Eastern Europe in early 2006 and the movement of birds toward warmer areas. This is also the first isolation of highly pathogenic avian influenza virus of H5N1 subtype from apparently healthy black-headed gulls.