1. Investigación

Free-living cats usually live in colonies in urban areas, especially close to parks and neighbourhoods where people feed them without any sanitary control. This can pose a human, animal and environmental health concern due to the close contact between uncontrolled colonies, the population and other domestic and/or wild animals. Thus, this study aimed to assess the genetic diversity and antimicrobial resistance (AMR) among Salmonella enterica subsp. enterica strains isolated from feral cats in a previous epidemiological study in the Gran Canaria island (Spain). A total of nineteen Salmonella isolates were obtained from November 2018 to January 2019 in a Salmonella epidemiological study in feral cats. All isolates obtained were genotyped by pulsed-field gel electrophoresis (PGFE) and were tested for antimicrobial susceptibility, in accordance with Decision 2013/652/EU. PFGE analysis revealed isolates clustering by serovar, with identical clones for serovars Bredeney and Grancanaria, while differing pulsotypes were observed for serovars Florida (88.89 % similarity) and Nima (83.23 % similarity). All but two isolates were resistant to at least one antimicrobial. The results obtained demonstrate that feral cats in the region investigated are a reservoir of Salmonella strains resistant to gentamicin (94.1 %) and of the critically important antimicrobial tigecycline (23.5 %). Hence, they could excrete AMR strains through their faeces and contaminate the environment, favoring the spread of such bacteria to cohabiting pets. Moreover, this widespread presence of AMR Salmonella clones across various serovars highlights the urgent need to implement efficient antimicrobial stewardship and control programs by the local governments due to the ongoing need to protect human and animal health under a One Health concept.

Vultures are nature's most successful scavengers, feeding on the carcasses of dead animals present in the field. Availability of domestic carrion has been unstable due to rapidly changing agro-grazing economies and increasing sanitary regulations that may require burial or burning of livestock carcasses. Thus, several griffon vulture (Gyps fulvus) recoveries are based on European legislation that guarantees the animals' welfare, avoids intense persecution of the vultures and allows the feeding of threatened wildlife in supplementary feeding stations (SFS). However, in recent years, many studies have speculated on the likelihood that avian scavengers may be infected by feeding on pig carcasses at SFS from intensive livestock. In this context, the present study evaluated whether free-living griffon vultures and pig farms share zoonotic Salmonella strains to test the hypothesis that vulture are infected during consumption of carcasses provided at SFS. Here, the occurrence, serotypes and genomic DNA fingerprinting (phage typing and pulsed-field gel electrophoresis) of isolated strains were carried out in griffon vultures and pig farms authorised to provided carcasses at SFS in Castellón province (eastern Spain). The bacteriological analyses revealed that 21.1% of vultures and 14.5% for pig farms samples tested were Salmonella-positive. Monophasic S. typhimurium 1,4,[5],12:i:- was the most frequently isolated serovar. Comparison of Salmonella strains isolated from vultures and pig farms revealed that monophasic S. typhimurium 1,4,[5],12:i:-, S. Derby and S. Rissen strains were highly genetically homogeneous (similar DNA fingerprint). In conclusion, the current study indicates that free-living griffon vultures and pig farms that provide the carcasses at SFS share several zoonotic Salmonella strains. On this basis, and although transmission could be bidirectional, our result seems to corroborate the pig carcasses-to-vulture transmission and cross-infection at SFS. As an immediate Salmonella control strategy in wild avian scavengers, we suggest the implementation of a programme to guarantee that solely pig carcasses from Salmonella-free farms arrive at SFS.

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.

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.