1. Investigación

Salmonella spp. is one of the most important zoonotic pathogens with economic impact in public health worldwide. The relevance of Salmonella increases with the appearance of resistant strains. The aim of this study was to determine the level of antimicrobial resistance in 332 Salmonella isolates selected from 3 different poultry productive orientations in Eastern Spain during 3 yr (2015−2017). Antimicrobial susceptibly was evaluated by broth microdilution method using 14 antibiotics. Epidemiological cut-off values (ECOFF) were used to evaluate the microbiological resistance to antibiotics. The rates of Salmonella resistance at least to one antibiotic were 96, 98, and 56% in broilers, turkeys, and layers, respectively. Regarding multidrug resistance, all productive orientations seems to present a decreasing trend along the study, being the mean rates 80% in turkeys followed by broilers (40%) and layers (6%). Throughout the study, the highest percentage of resistance was found to sulfamethoxazole in all productive orientations. Strains from broilers showed the highest resistance rates to sulfamethoxazole (73%), gentamicin (57%), ciprofloxacin (50%), nalidixic acid (29%), and tetracycline (24%). Relative to turkeys the highest resistance rates were to sulfamethoxazole (76%), ciprofloxacin (69%), tetracycline (75%), nalidixic acid (63%), and ampicillin (63%). Layers presented the most elevated resistance rates to sulfamethoxazole (39%) and tetracycline (13%). Regarding serovars the most MDR common serovars to the 3 productive orientations were S. Kentucky and S. Hadar. In the other hand, high MDR rates were found in other serovars like S. Infantis and S. Typhimurium in broilers and turkeys. Results shown in the present study suggest that the reduction in the use of antibiotics begins to be reflected in the reduction of the number of MDRs, especially in layers, with no MDR Salmonella strains in the last period. However, the level of resistances found in this study suggests the necessity of continuing working on the limitation of the use of antimicrobials in poultry to achieve (as in layers) the control of MDRs.

Campylobacteriosis was the most frequently reported foodborne infection in humans in the European Union in the last years. Campylobacter spp. in broiler flocks from Spain was monitored at farm level during 12-month period (2020−2021). Feces samples were analyzed according to ISO (International Standard Organization) 10272-2:2018. From all samples collected, 54% were Campylobacter spp. positive. Regarding the age, Campylobacter spp. was isolated in 36% of the flocks during thinning and 64% in flocks at slaughter age. In addition, Campylobacter spp. counts increased with the age of the animals. On the other hand, the presence of Campylobacter showed statistical differences between the months of the year (P-value <0.05) in flocks at thinning age that exceeded the ≥1,000 CFU/g limit. The highest rates were found from June to December coinciding with the seasons of summer and autumn. In conclusion, our study shows the situation of Campylobacter spp. in broiler flocks in Spain considering age and season effects. This way, it was found higher rates and counts in broilers close to slaughter age and peaking during the summer to autumn period.

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.

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.