2. Universidad Cardenal Herrera-CEU

Stray cat populations can represent a significant threat of the transmission of zoonotic diseases such as salmonellosis. The objective of this study was to assess Salmonella carriage by free-living cats in Gran Canaria island and the Salmonella serovars involved, in order to inform to those responsible for the colonies about the possible risk factors. One hundred rectal swabs of feral cats were taken. Salmonella strains were serotyped in accordance with Kauffman-White-Le-Minor technique. Of a total of 100 animals under study, 19% were found to be positive to Salmonella spp. This is the first report that described the zoonotic serovars S. Nima, S. Bredeney, S. Grancanaria and S. Kottbus in cats. The present study demonstrates that feral cats may represent a source of risk for the spread of different Salmonella zoonotic serovars. It has been reported that there is a certain correlation between Salmonella isolates from pets and wild animals. Further studies are needed from other animal species and environmental sources to make this correlation.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA. Case presentation: In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a twotube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, nextgeneration sequencing (NGS) covered > 90% SARS-CoV-2 genome sequence, allowing to classify it as variant B.1.177. Remarkably, the sequence revealed the Ile402Val substitution in the spike protein (S), of potential concern because it mapped in the receptor binding domain (RBD) that mediates virus interaction with the cell. NGS reads mapping to bacterial genomes showed that the dog fecal microbiome fitted best the characteristic microbiome of dog’s acute hemorrhagic diarrhea. Conclusion: Our findings exemplify dog infection stemming from the human SARS-CoV-2 pandemic, providing nearly complete-genome sequencing of the virus, which is recognized as belonging to the B.1.177 variant, adding knowledge on variant circulation in a geographic region and period for which there was little viral variant characterization. A single amino acid substitution found in the S protein that could have been of concern is excluded to belong to this category given its rarity and intrinsic nature. The dog’s pathology suggests that SARS-CoV-2 could affect the gastrointestinal tract of the dog.

Salmonellosis remains one of the main foodborne zoonoses in Europe, with poultry products as the main source of human infections. The slaughterhouse has been identified as a potential source for Salmonella contamination of poultry meat. Despite the mandatory programme of the EU, there are companies with persistent Salmonella that are unable to remove the bacteria from their processing environment, compromising the entire production line. In this context, an intensive sampling study was conducted to investigate a slaughterhouse with persistent Salmonella problems, establishing the genetic relationship among Salmonella strains isolated during the slaughter process. A total of 36 broiler flocks were sampled during processing at the slaughterhouse. Salmonella was identified based on ISO 6579-1:2017 (Annex D), serotyped by Kauffman-White-Le-Minor technique, and the genetic relationship was assessed with ERIC-PCR followed by PFGE. The outcomes showed that 69.4% of the batches sampled carried Salmonella upon arrival at the slaughterhouse and that 46.3% of the different samples from carcasses were contaminated with Salmonella. The two serovars isolated at the different steps in the slaughterhouse were Enteritidis (98.2%) and Kentucky (1.8%). Pulsed-field gel electrophoresis analysis revealed a low genetic diversity, with all S. Enteritidis isolates showing a nearly identical pulsotype (similarity >85%) and S. Kentucky strains showed the same XbaI PFGE profile (95.0% genetic similarity). The results of this study showed a high genetic relationship among isolates recovered from carcasses and environmental samples in the slaughterhouse from both Salmonella-positive and Salmonella- free flocks. Salmonella strains re-circulated across to poultry flocks and re-entered the slaughterhouse to survive on the processing line. Thus, it is necessary to implement molecular diagnosis methods in time at the field level to determine the Salmonella epidemiology of the flock, to make rapid decisions for the control of Salmonella and prevent entry into the slaughterhouse environment.

Bacteriophages selectively infect and kill their target bacterial host, being a promising approach to controlling zoonotic bacteria in poultry production. To ensure confidence in its use, fundamental questions of safety and toxicity monitoring of phage therapy should be raised. Due to its high specificity, a minimal impact on the gut ecology is expected; however, more in-depth research into key parameters that influence the success of phage interventions has been needed to reach a consensus on the impact of bacteriophage therapy in the gut. In this context, this study aimed to investigate the interaction of phages with animals; more specifically, we compared the caecum microbiome and metabolome after a Salmonella phage challenge in Salmonella-free broilers, evaluating the role of the phage administration route. To this end, we employed 45 caecum content samples from a previous study where Salmonella phages were administered via drinking water or feed for 24 h from 4, 5 to 6-weeks-old broilers. High-throughput 16S rRNA gene sequencing showed a high level of similarity (beta diversity) but revealed a significant change in alpha diversity between broilers with Salmonella-phage administered in the drinking water and control. Our results showed that the phages affected only a few genera of the microbiota’s structure, regardless of the administration route. Among these, we found a significant increase in Streptococcus and Sellimonas in the drinking water and Lactobacillus, Anaeroplasma and Clostridia_vadinBB60_group in the feed. Nevertheless, the LC-HRMS-based metabolomics analyses revealed that despite few genera were significantly affected, a substantial number of metabolites, especially in the phage administered in the drinking water were significantly altered (64 and 14 in the drinking water and feed groups, respectively). Overall, our study shows that preventive therapy with bacteriophages minimally alters the caecal microbiota but significantly impacts their metabolites, regardless of the route of administration.

Antimicrobial resistance (AMR) and the increase in multi-resistant bacteria are among the most important threats to public health worldwide, according to the World Health Organisation (WHO). Moreover, this issue is underpinned by the One Health perspective, due to the ability of AMR to be transmitted between animals and humans living in the same environment. Therefore, since 2014 different surveillance and control programmes have been established to control AMR in commensal and zoonotic bacteria in production animals. However, public health authorities’ reports on AMR leave out companion animals, due to the lack of national programmes and data collection by countries. This missing information constitutes a serious public health concern due to the close contact between companion animals, humans and their surrounding environment. This absence of control and harmonisation between programmes in European countries leads to the ineffectiveness of antibiotics against common diseases. Thus, there is a pressing need to establish adequate surveillance and monitoring programmes for AMR in companion animals and further develop alternatives to antibiotic use in this sector, considering the impact this could have on the gut microbiota. In this context, the aim of this review is to evaluate the current control and epidemiological situations of AMR in companion animals in the European Union (EU), as well as the proposed alternatives to antibiotics.

Salmonella is mostly noted as a food-borne pathogen, but contact with chelonians has also been reported as a source of infection. Moreover, high levels of antimicrobial resistance (AMR) have been reported in Salmonella isolated from wild and captive reptiles. The aim of this study was to assess the occurrence of Salmonella AMR carriage by chelonians admitted to two zoological institutions in Spain, characterizing the isolates to assess the Salmonella AMR epidemiology in wildlife. To this end, 152 chelonians from nine species were sampled upon their arrival at the zoological nuclei. Salmonella identification was based on ISO 6579-1:2017 (Annex D), isolates were serotyped and their AMR analysed according to the EU Decision 2013/652. Moreover, the genetic relationship of the isolates was assessed by pulsed-field gel electrophoresis (PFGE). Results showed 19% (29/152) of the chelonians positive to Salmonella, all of them tortoises. For all isolates, 69% (20/29) were resistant and 34% (10/29) multidrug-resistant (MDR) strains. PFGE clustered isolates according to the serovar, confirming a low genetic diversity. In conclusion, this study shows a high presence of MDR Salmonella strains in tortoises at their entry into zoological nuclei. This condition highlights the need to establish Salmonella detection protocols for the entry of animals into these centres.

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.

Pork is considered a major source of Salmonella Typhimurium infection in humans in the EU, including monophasic strains. Widespread distribution of virulent serotypes such as monophasic variants of S. Typhimurium have emerged as a public health threat. Despite the current situation, within the EU there is no mandatory programme for the control of Salmonella at pork production level. In this context, the aims of this study were: to examine the presence of Salmonella in the swine production system from arrival at the slaughterhouse until the end of processing, and investigate the genetic relationship among the Salmonella serovars isolated. During the study, a total of 21 pig herds were intensively sampled during processing at the slaughterhouse. ERIC-PCR was performed among isolates recovered at the different steps in the slaughterhouse to assess the genetic relationship. Then, PFGE was done to study the pulsotypes among the different Salmonella serovars isolated. The results showed a high level of Salmonella pork batch contamination upon arrival at the slaughterhouse (71.4%) and at the end of the slaughtering process (66.7%), with mST the main serovar isolated from both origins (53.1% and 38.2%, respectively). The slaughter environment poses a potential risk for carcass contamination and it is considered an important source of Salmonella spp. Similarly, this study shows that 14.3% of the strains isolated from carcasses have the same Xbal-PFGE profile as those previously recovered in the slaughterhouse environment, but not in the live animals from that same batch. In conclusion, there is a high level of Salmonella swine batch contamination upon arrival at the slaughterhouse and at the end of the slaughtering process, mST being the most frequently isolated serovar. Moreover, a strong genetic relationship has been observed between Salmonella strains isolated from the batch on arrival at the slaughterhouse, the processing environment and pork carcass contamination. In this sense, it would be necessary to implement a control programme to reduce the bacterium from pork farms and raise the awareness of biosecurity measures.

Background: Salmonella enterica serovar Infantis is a zoonotic pathogen isolated in broilers causing great economic losses in the European poultry sector. It is demonstrated that an investment in management measures at farm level could directly affect the control of food chain microorganisms. The aim of this study was to investigate the development of S. Infantis antimicrobial resistance (AMR) patterns during the growing period, according to flock density and ventilationmanagement, without antibiotic administration. Methods: The experiment was performed in two identical poultry houses, evaluating commercial and optimal farm conditions. At 24 h of rearing, 20% of the animals were orally infected with a S. Infantis strain susceptible to all the antibiotics tested. To study Salmonella shedding, faeces samples from each experimental groupwere takenweekly and analysed as per ISO/TS 6579- 2:2017. Antibiotic susceptibilitywas assessed according toDecision 2013/653. Results: Salmonella shedding showed that the lowest counts were observed in the first week post-infection and highest at slaughter day for both groups. Moreover, 100% of the isolates were multi-resistant. Conclusion: The acquisition of AMR by S. Infantis starts at the onset of the production cycle and is maintained until the end, demonstrating the importance of transmission of AMR in zoonotic bacteria at farmlevel.