First Report of Campylobacter jejuni Strains Belonging to ST-21 Clonal Complex Isolated from Human, Poultry and Wild Birds in Croatia: Antimicrobial Resistance and Genetic Distance.

In the era of growing antimicrobial resistance, a threat affecting humans, endangering animals, as well as livelihoods and food security worldwide, we wanted to find possible explanations for its continuous spread from a new perspective. The ubiquity of resistance genes requires a One Health approach to finding the explanations for continuous AMR spread. The natural transformability of Campylobacter jejuni, its high incidence of infections, and emerging resistance worldwide inspired us to choose C. jejuni ST-21CC to be our pathogen for analyzing its contribution and connection to the cycle of AMR dissemination. ST-21CC is known as a generalist among humans and broilers, the most prevalent lineage worldwide, but it is rarely found in wild birds. Emerging in wild birds, genetic relatedness and similar resistance profiles were expected. We analyzed 23 Croatian C. jejuni strains belonging specifically to ST-21CC from humans, broilers, and wild birds. The genomic data obtained through whole genome sequencing and phenotypic susceptibility data of strains were compared. Our findings suggest high fluoroquinolone resistance in ST-21CC strains, with more diverse genetic backgrounds in wild birds. Intriguing were three isolates of ST-822 (from human and storks), sharing a similar genetic fingerprint.

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.

Heavy metal(loid) effect on multi-biomarker responses in apex predator: Novel assays in the monitoring of white stork nestlings.

The goal of the present study was to investigate differences in biomarker responses related to metal(loid)s in white stork (Ciconia ciconia) nestling's blood from continental Croatia. To achieve this, a battery of biomarkers that can be affected by environmental pollutants, including metal(loid)s, was assessed (esterase activity, fluorescence-based oxidative stress biomarkers, metallothionein levels, glutathione-dependent enzyme activity). The research was conducted during the white stork breeding season in diverse areas (a landfill, industrial and agricultural sites, and an unpolluted area). White storks' nestlings near the landfill exhibited reduced carboxylesterase (CES) activity, elevated glutathione (GSH) concentration, as well as high Pb content in the blood. Increased As and Hg concentrations in blood were attributable to environmental contamination in agricultural area and an assumed unpolluted area, respectively. Furthermore, agricultural practices appeared to affect CES activity, as well as elevate Se levels. In addition to the successful implementation of biomarkers, present research showed that agricultural areas and a landfill are areas with increased metal(loid) levels possibly causing adverse effects on the white storks. This first-time heavy metal and metalloid analyses in the white stork nestlings from Croatia point to the necessary monitoring and future assessments of pollution impact to prevent irreversible adverse effects.

Active breeding seabirds prospect alternative breeding colonies.

Compared to other animal movements, prospecting by adult individuals for a future breeding site is commonly overlooked. Prospecting influences the decision of where to breed and has consequences on fitness and lifetime reproductive success. By analysing movements of 31 satellite- and GPS-tracked gull and tern populations belonging to 14 species in Europe and North America, we examined the occurrence and factors explaining prospecting by actively breeding birds. Prospecting in active breeders occurred in 85.7% of studied species, across 61.3% of sampled populations. Prospecting was more common in populations with frequent inter-annual changes of breeding sites and among females. These results contradict theoretical models which predict that prospecting is expected to evolve in relatively predictable and stable environments. More long-term tracking studies are needed to identify factors affecting patterns of prospecting in different environments and understand the consequences of prospecting on fitness at the individual and population level.

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.

Resistome Analysis of Campylobacter jejuni Strains Isolated from Human Stool and Primary Sterile Samples in Croatia.

Campylobacteriosis represents a global health challenge due to continuously increasing trends of antimicrobial resistance in Campylobacter jejuni. C. jejuni can sometimes cause life-threatening and severe systematic infections (bacteremia, meningitis, and other extraintestinal infections) with very few antibiotics left as treatment options. Bearing in mind that C. jejuni is the predominant species in humans, in this paper, we present a study of the C. jejuni differences in antimicrobial resistance and genotype distribution between strains isolated from stool and primary sterile sites. We compared the genomic data obtained through whole genome sequencing (WGS) and phenotypic susceptibility data of C. jejuni strains. Once antimicrobial susceptibility testing of C. jejuni strains was carried out by the broth microdilution method for six of interest, results were compared to the identified genotypic determinants derived from WGS. The high rate of resistance to fluoroquinolones presented in this study is in accordance with national surveillance data. The proportion of strains with acquired resistance was 71% for ciprofloxacin and 20% for tetracycline. When invasive isolates were analysed separately, 40% exhibited MIC values of ciprofloxacin higher than the ECOFFs, suggesting a lower flouroquinolone resistance rate in invasive isolates. All isolates demonstrated wilde-type phenotype for chloramphenicol, erythromycin, gentamicin, and ertapenem. A special focus and review in this study was performed on a group of C.jejuni strains found in primary sterile samples. Apart from demonstrating a lower resistance rate, these isolates seem genetically more uniform, showing epidemiologically more homogenous patterns, which cluster to several clonal complexes, with CC49 being the most represented clonal complex.

Blood biomarkers in white stork (Ciconia ciconia) nestlings show different responses in several areas of Croatia.

White stork nestlings can provide quantitative data on the quality of the environment, as they are dependent on their parents that provide locally foraged food. Blood was sampled from the brachial vein (n = 109) and the sampling was performed in parallel with ringing during breeding season 2020 from five areas in eastern Croatia: Lonjsko polje, Jelas polje, Slavonski Brod-east, Podunavlje, and Donje Podravlje. In the present study, for the first time in Croatia, the following enzymatic biomarkers were assessed in white stork nestlings: activities of acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as nonenzymatic biomarkers: levels of glutathione (GSH) and reactive oxygen species (ROS). All endpoints were measured in two blood fractions: plasma and a postmitochondrial fraction (S9). Nestlings from Podunavlje and Donje Podravlje, areas known for intensive agriculture, showed lower AChE and CES activity when compared to the other investigated areas, indicating the presence of inhibitory xenobiotics. Higher oxidative stress was observed in Slavonski Brod-east, an area surrounded by metal and engineering industry, and Podunavlje compared to the other sampling areas. Hence, this study shows the impact of pollutants from the surrounding metal, petroleum, and agricultural industry might have on the biomarkers in white stork nestlings, which are often seen as early-warning signals.

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.

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.

Abundance of IFN-α and IFN-γ gene transcripts and absence of IL-2 transcripts in the blood of chickens vaccinated with live or inactivated NDV.

As immune responses to live and inactivated vaccines might differ, temporal responses of broiler chickens to vaccination were examined on the basis of the abundance in the circulating blood of gene transcripts of IFN-α, IFN-γ and IL-2, critical cytokines for immune responses. Blood samples were collected 6, 12 and 24 hours, and 7 and 14 days following vaccination with either live or inactivated Newcastle disease virus, La Sota strain, at 14 days of age, and the abundance of transcripts for each cytokine was assayed by real-time RT-PCR. Physiological saline and vaccine emulsion without viral antigen were administered to control groups for live and inactivated vaccine groups, respectively. The abundance of IFN-γ transcripts was elevated during the early times following vaccination and had reached baseline by the seventh day but the abundance of IFN-α transcripts remained elevated. Transcripts for neither IFN gene were detected in the control birds. The abundance of transcripts for each IFN was not different between the two vaccinated groups at any time. Transcripts for IL-2 were detected only in spleens from chicken embryos that had been inoculated with the live virus. The results show that cells stimulated to produce IFN-α and IFN-γ enter the circulating blood but those stimulated to produce IL-2 do not, or in very low number, and the IFN responses to both vaccines are the same.

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.