2. Universidad Cardenal Herrera-CEU

Campylobacter is recognised as one of the most important foodborne bacteria, with a worldwide health and socioeconomic impact. This bacterium is one of the most important zoonotic players in poultry, where efficient and fast detection methods are required. Current official culture methods for Campylobacter enumeration in poultry usually include >44 h of culture and >72 h for identification, thus requiring at least five working shifts (ISO/TS 10272-2:2017). Here, we have assembled a portable sequencing kit composed of the Bento Lab and the MinION and developed a workflow for on-site farm use that is able to detect and report the presence of Campylobacter from caecal samples in less than five hours from sampling time, as well as the relationship of Campylobacter with other caecal microbes. Beyond that, our workflow may offer a cost-effective and practical method of microbiologically monitoring poultry at the farm. These results would demonstrate the possibility of carrying out rapid on-site screening to monitor the health status of the poultry farm/flock during the production chain.

Background: Campylobacter is the main pathogen involved in zoonotic gastrointestinal diseases. Last year, the European regulation 2017/1495 on Campylobacter in broiler carcasses came into force. In this context, the aim of this study was to assess the potential risk factors associated with exceeding the 1,000 CFU/g criterion set by the European Commission in several slaughterhouses in Spain. Methods: Information relating to 12 factors were collected using questionnaires. Samples were collected from 12 Spanish abattoirs during June, July and August 2017 (n=1,725) and were analysed following ISO/TS 10272-2:2006 method. Results: The proportion of Campylobacter-positive samples was 23.7% (n=409). Analysis of the flock age (41-50 days) revealed a significantly increased OR in Campylobacter enumeration (OR=7.41). Moreover, scalding temperature (51.9.54 ºC) was positively associated with an increase in OR (OR=2.75). Time in transit to slaughter (1-1.5h), showed a significant OR decrease (OR=0.25). However, when processed for more than 2 hours, presented an increase in OR (OR=4.44). Regarding carcass weight, the range from 3.21-3.58 presented a decrease in OR (OR=0.01). Conclusion: The outcomes of this study suggest that although most chickens are contaminated by the bacterium, the prevalence that exceeds the limit of 1,000 CFU/ is not so high as we thought.

Different studies have reported the prevalence and antibiotic resistance of Salmonella in dromedaries’ camels and its role in camelid-associated salmonellosis in humans, but little is known about the epidemiology of Campylobacter in dromedaries. Here we investigate the prevalence, genetic diversity and antibiotic resistance of Campylobacter and Salmonella in dromedary camels (Camelus dromedarius). A total of 54 individuals were sampled from two unique dromedary farms located in Tenerife (Canary Islands, Spain). Whilst all the samples were Campylobacter-negative, Salmonella prevalence was 5.5% (3/54) and the only serovar isolated was S. Frintrop. The pulsed field gel electrophoresis analysis revealed a low genetic diversity, with all isolates showing a nearly identical pulsotype (similarity > 95%). Our results indicate that dromedaries’ camels could not be a risk factor for Campylobacter human infection, but seems to be a reservoir for Salmonella transmission. Since camel ride has become one of the main touristic attractions in several countries and its popularity has considerably risen in the last years, a mandatory control, especially for zoonotic pathogens, such as Campylobacter and Salmonella should be implemented.

In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the e ects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae–early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures.

The exploration of novel nonantibiotic interventions in the field, such as the use of bacteriophages, is necessary to avoid the presence of Salmonella. Bacteriophages are a group of viruses widely distributed in nature, strictly associated with the prokaryotic cell. Researchers have demonstrated the success of phage therapy in reducing Salmonella counts in poultry products. However, the impact that phage concentration in the environment may have against certain Salmonella serovars is not well understood. Therefore, the aim of this study was to assess Salmonella phage prevalence in commercial poultry farms in terms of the production type: layers or broilers. The most prevalent Salmonella serovars isolated in poultry production were used for phage isolation. Salmonella specific phages were isolated from 141 layer and broiler farms located in the Valencia region during 2019. Analysis of the samples revealed that 100% presented Salmonella phages, the most prevalent being against serovar S. Enteritidis (93%), followed by S. Virchow (59%), S. Typhimurium (55%), S. Infantis (52%) and S. Ohio (51%). These results indicate that poultry farms could represent an important source of Salmonella phages. Nevertheless, further studies are needed to assess the epidemiology of phages against other serovars present in other countries and their diversity from the point of view of molecular studies.