Proteomic Insights into Seminal Plasma and Spermatozoa Proteins of Small-Spotted Catsharks, Scyliorhinus canicula: Implications for Reproductive Conservation in Aquariums.

In the ex situ conservation of chondrichthyan species, successful reproduction in aquaria is essential. However, these species often exhibit reduced reproductive success under human care. A key aspect is that conventional sperm analyses do not provide insights into the functional competence of sperm. However, proteomics analysis enables a better understanding of male physiology, gaining relevance as a powerful tool for discovering protein biomarkers related to fertility. The present work aims to build the first proteome database for shark semen and to investigate the proteomic profiles of seminal plasma and spermatozoa from small-spotted catsharks (Scyliorhinus canicula) related to the underlying adaptations to both natural and aquarium environments, thereby identifying the reproductive impact in aquarium specimens. A total of 305 seminal plasma and 535 spermatozoa proteins were identified. Among these, 89 proteins (29.2% of the seminal plasma set) were common to both spermatozoa and seminal plasma. In the seminal plasma, only adenosylhomocysteinase protein showed differential abundance (DAP) between wild and aquarium animals. With respect to the spermatozoa proteins, a total of 107 DAPs were found between groups. Gene Ontology enrichment analysis highlighted the primary functional roles of these DAPs involved in oxidoreductase activity. Additionally, KEGG analysis indicated that these DAPs were primarily associated with metabolic pathways and carbon metabolism. In conclusion, we have successfully generated an initial proteome database for S. canicula seminal plasma and spermatozoa. Furthermore, we have identified protein variations, predominantly within spermatozoa, between aquarium and wild populations of S. canicula. These findings provide a foundation for future biomarker discovery in shark reproduction studies. However, additional research is required to determine whether these protein variations correlate with reproductive declines in captive sharks.

Phage-Based Biosanitation Strategies for Minimizing Persistent Salmonella and Campylobacter Bacteria in Poultry.

Control strategies to minimize pathogenic bacteria in food animal production are one of the key components in ensuring safer food for consumers. The most significant challenges confronting the food industry, particularly in the major poultry and swine sectors, are antibiotic resistance and resistance to cleaning and disinfection in zoonotic bacteria. In this context, bacteriophages have emerged as a promising tool for zoonotic bacteria control in the food industry, from animals and farm facilities to the final product. Phages are viruses that infect bacteria, with several advantages as a biocontrol agent such as high specificity, self-replication, self-limitation, continuous adaptation, low inherent toxicity and easy isolation. Their development as a biocontrol agent is of particular interest, as it would allow the application of a promising and even necessary "green" technology to combat pathogenic bacteria in the environment. However, bacteriophage applications have limitations, including selecting appropriate phages, legal restrictions, purification, dosage determination and bacterial resistance. Overcoming these limitations is crucial to enhance phage therapy's effectiveness against zoonotic bacteria in poultry. Thus, this review aims to provide a comprehensive view of the phage-biosanitation strategies for minimizing persistent Salmonella and Campylobacter bacteria in poultry.

Multidrug-Resistant Escherichia coli Strains to Last Resort Human Antibiotics Isolated from Healthy Companion Animals in Valencia Region.

Failure in antibiotic therapies due to the increase in antimicrobial-resistant (AMR) bacteria is one of the main threats to public and animal health. In recent decades, the perception of companion animals has changed, from being considered as a work tool to a household member, creating a family bond and sharing spaces in their daily routine. Hence, the aim of this study is to assess the current epidemiological situation regarding the presence of AMR and multidrug resistance (MDR) in companion animals in the Valencia Region, using the indicator bacteria Escherichia coli as a sentinel. For this purpose, 244 samples of dogs and cats were collected from veterinary centres to assess antimicrobial susceptibility against a panel of 22 antibiotics with public health relevance. A total of 197 E. coli strains were isolated from asymptomatic dogs and cats. The results showed AMR against all the 22 antibiotics studied, including those critically important to human medicine. Moreover, almost 50% of the strains presented MDR. The present study revealed the importance of monitoring AMR and MDR trends in companion animals, as they could pose a risk due to the spread of AMR and its resistance genes to humans, other animals and the environment they cohabit.

Modulation of Caecal Microbiota and Metabolome Profile in Salmonella-Infected Broilers by Phage Therapy.

Bacteriophage therapy is considered one of the most promising tools to control zoonotic bacteria, such as Salmonella, in broiler production. Phages exhibit high specificity for their targeted bacterial hosts, causing minimal disruption to the niche microbiota. However, data on the gut environment's response to phage therapy in poultry are limited. This study investigated the influence of Salmonella phage on host physiology through caecal microbiota and metabolome modulation using high-throughput 16S rRNA gene sequencing and an untargeted metabolomics approach. We employed 24 caecum content samples and 24 blood serum samples from 4-, 5- and 6-week-old broilers from a previous study where Salmonella phages were administered via feed in Salmonella-infected broilers, which were individually weighed weekly. Phage therapy did not affect the alpha or beta diversity of the microbiota. Specifically, we observed changes in the relative abundance of 14 out of the 110 genera using the PLS-DA and Bayes approaches. On the other hand, we noted changes in the caecal metabolites (63 up-accumulated and 37 down-accumulated out of the 1113 caecal metabolites). Nevertheless, the minimal changes in blood serum suggest a non-significant physiological response. The application of Salmonella phages under production conditions modulates the caecal microbiome and metabolome profiles in broilers without impacting the host physiology in terms of growth performance.

Holistic Strategies to Control Salmonella Infantis: An Emerging Challenge in the European Broiler Sector.

Salmonella spp. has been globally recognized as one of the leading causes of acute human bacterial gastroenteritis resulting from the consumption of animal-derived products. Salmonella Enteritidis, S. Typhimurium, and its monophasic variant are the main serovars responsible for human disease. However, a serovar known as S. Infantis has emerged as the fourth most prevalent serovar associated with human disease. A total of 95% of isolated S. Infantis serovars originate from broilers and their derived products. This serovar is strongly associated with an elevated antimicrobial (AMR) and multidrug resistance, a resistance to disinfectants, an increased tolerance to environmental mercury, a heightened virulence, and an enhanced ability to form biofilms and attach to host cells. Furthermore, this serovar harbors genes that confer resistance to colistin, a last-resort antibiotic in human medicine, and it has the potential to acquire additional transferable AMR against other critically important antimicrobials, posing a new and significant challenge to global public health. This review provides an overview of the current status of the S. Infantis serovar in the poultry sector, focusing on its key virulence factors, including its virulence genes, antimicrobial resistance, and biofilm formation. Additionally, novel holistic strategies for controlling S. Infantis along the entire food chain are presented in this review.

Zoonotic Parasites in Playgrounds in Southern Spain: A One Health Approach.

Zoonotic parasitic diseases are considered a global threat to public health. In this sense, canines and felines may be infected by different cosmopolitan parasites, with playgrounds serving as an important focus of infection for humans, as well as domestic or wild animals. Knowledge of the epidemiological situation of parasites in animal reservoirs integrated into the environment, identifying the spread pathways, is a key element for an effective response to this threat. Thus, the aim of this study was to assess the frequency of intestinal parasites with zoonotic potential in 120 playgrounds in the Malaga province (Spain). Samples were processed and analysed following standard parasitological procedures. Some 36.7% of playgrounds were parasite-positive with one or more zoonotic parasites. The most common parasites recovered were nematodes (60.0%), followed by protozoan species (33.3%) and cestodes (6.7%). In the parasite-positive playgrounds, Toxocara spp. (17.0 ± 3.5%) and Giardia duodenalis (17.0 ± 3.4%) were the most predominant parasites. In addition, 34.1% of playgrounds were infected with multiple parasites. Our results show a high presence of parasitic forms with zoonotic potential in playgrounds in Malaga, Spain. Due to the close contact between pets and humans in playgrounds, the potential zoonotic risk may increase if prevention and control measures are not designed.

Liver Steatosis and Steatohepatitis Alter Bile Acid Receptors in Brain and Induce Neuroinflammation: A Contribution of Circulating Bile Acids and Blood-Brain Barrier.

A tight relationship between gut-liver diseases and brain functions has recently emerged. Bile acid (BA) receptors, bacterial-derived molecules and the blood-brain barrier (BBB) play key roles in this association. This study was aimed to evaluate how non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) impact the BA receptors Farnesoid X receptor (FXR) and Takeda G-protein coupled receptor 5 (TGR5) expression in the brain and to correlate these effects with circulating BAs composition, BBB integrity and neuroinflammation. A mouse model of NAFLD was set up by a high-fat and sugar diet, and NASH was induced with the supplementation of dextran-sulfate-sodium (DSS) in drinking water. FXR, TGR5 and ionized calcium-binding adaptor molecule 1 (Iba-1) expression in the brain was detected by immunohistochemistry, while Zonula occludens (ZO)-1, Occludin and Plasmalemmal Vesicle Associated Protein-1 (PV-1) were analyzed by immunofluorescence. Biochemical analyses investigated serum BA composition, lipopolysaccharide-binding protein (LBP) and S100ß protein (S100ß) levels. Results showed a down-regulation of FXR in NASH and an up-regulation of TGR5 and Iba-1 in the cortex and hippocampus in both treated groups as compared to the control group. The BA composition was altered in the serum of both treated groups, and LBP and S100ß were significantly augmented in NASH. ZO-1 and Occludin were attenuated in the brain capillary endothelial cells of both treated groups versus the control group. We demonstrated that NAFLD and NASH provoke different grades of brain dysfunction, which are characterized by the altered expression of BA receptors, FXR and TGR5, and activation of microglia. These effects are somewhat promoted by a modification of circulating BAs composition and by an increase in LBP that concur to damage BBB, thus favoring neuroinflammation.

Examining the effects of Salmonella phage on the caecal microbiota and metabolome features in Salmonella-free broilers.

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.

Antibiotic removal does not affect cecal microbiota balance and productive parameters in LP robust rabbit line.

Antimicrobial resistance is an important threat to public health worldwide, being one of the main death causes in 2050. Moreover, global health is currently underpinned by the "One Health" concept, whereby livestock is strictly related to human and environmental health. However, in the case of the meat rabbit industry, antibiotic additives are still added to prevent gastrointestinal diseases. Current food and consumer awareness require the implementation of sustainable production systems, where robustness and resilience are increasingly important. Hence, the aim of this study was to evaluate the effect of antibiotic feed supplementation on microbiota, and productive performance during the rabbit growing period in a robust genetic line. For this purpose, a total of 432 weaned rabbits were randomly housed, cecum samples were taken on the weaning day and at the end of the growing period (28 and 61 days of age, respectively), and 16S rRNA sequencing analysis was performed. Results showed a higher microbiota complexity at the end of growing in both experimental groups. Firmicutes represented the dominant phylum of the cecal community, followed by Bacteroidota in both groups. Moreover, Victivallis and Escherichia-Shigella genera were only identified in the experimental group without antibiotic supplementation at the end of the growing period. In conclusion, antibiotic feed supplementation had no effect on microbiota composition and productive performance in the robust genetic line reared. These results evidence the importance of the development of rabbit robust genetic lines as an alternative tool to antibiotic administration in epizootic enteropathy control.

Microencapsulated bacteriophages incorporated in feed for Salmonella control in broilers.

Bacteriophage inclusion as a feed additive could offer the prospect of its en masse application and reduce the intestinal carriage of Salmonella by broiler chickens. The objective of this study was to evaluate the application of microencapsulated phages as a strategy to control Salmonella and assess the impact of their use during the broiler rearing period. One-hundred one-day-old chicks were randomly divided in two identical poultry houses according to the two experimental groups (control vs Φ-treated group) simulating field production conditions. Half of the animals in each experimental group, were challenged with 105 CFU/bird of Salmonella Enteritidis per os. In the Φ-treated group, microencapsulated Salmonella-phage encapsulated in Eudragit®L100 (a pH-responsive formulation) were incorporated in the starter diet feed. To assess Salmonella colonization, excretion and diffusion, cecum samples, cloacal swabs and boot swabs were taken weekly. Salmonella detection was based on ISO 6579-1:2017 (Annex D). Salmonella colonization was significantly reduced in most of the rearing period, meanwhile the excretion was significantly reduced on the 2nd, 4th and 5th week of rearing. Moreover, Salmonella contamination of the farm environment was eliminated at the end of the cycle. This study provides important insights into the potential use of phages as a preventative and biocontrol strategy against Salmonella infection from farm-to-table.

Gut-Brain Axis: Insights from Hippocampal Neurogenesis and Brain Tumor Development in a Mouse Model of Experimental Colitis Induced by Dextran Sodium Sulfate.

Chronic inflammatory bowel disorders (IBD) are idiopathic diseases associated with altered intestinal permeability, which in turn causes an exaggerated immune response to enteric antigens in a genetically susceptible host. A rise in psych cognitive disorders, such as anxiety and depression, has been observed in IBD patients. We here report investigations on a model of chemically induced experimental colitis by oral administration of sodium dextran sulfate (DSS) in C57BL/6 mice. We investigate, in vivo, the crosstalk between the intestine and the brain, evaluating the consequences of intestinal inflammation on neuroinflammation and hippocampal adult neurogenesis. By using different DSS administration strategies, we are able to induce acute or chronic colitis, simulating clinical characteristics observed in IBD patients. Body weight loss, colon shortening, alterations of the intestinal mucosa and fecal metabolic changes in amino acids-, lipid- and thiamine-related pathways are observed in colitis. The activation of inflammatory processes in the colon is confirmed by macrophage infiltration and increased expression of the proinflammatory cytokine and oxidative stress marker (Il-6 and iNOS). Interestingly, in the hippocampus of acutely DSS-treated mice, we report the upregulation of inflammatory-related genes (Il-6, Il-1ß, S-100, Tgf-ß and Smad-3), together with microgliosis. Chronic DSS treatment also resulted in neuroinflammation in the hippocampus, indicated by astrocyte activation. Evaluation of stage-specific neurogenesis markers reveals deficits in the dentate gyrus after acute and chronic DSS treatments, indicative of defective adult hippocampal neurogenesis. Finally, based on a possible causal relationship between gut-related inflammation and brain cancer, we investigate the impact of DSS-induced colitis on oncogenesis, using the Ptch1+/-/C57BL/6 mice, a well-established medulloblastoma (MB) mouse model, finding no differences in MB development between untreated and DSS-treated mice. In conclusion, in our experimental model, the intestinal inflammation associated with acute and chronic colitis markedly influences brain homeostasis, impairing hippocampal neurogenesis but not MB oncogenesis.

Rapid Oxford Nanopore Technologies MinION Sequencing Workflow for Campylobacter jejuni Identification in Broilers on Site­A Proof-of-Concept Study

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.

Antimicrobial Resistant Salmonella in Chelonians: Assessing Its Potential Risk in Zoological Institutions in Spain.

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.

Antimicrobial Resistance in Companion Animals: A New Challenge for the One Health Approach in the European Union.

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.

Gastrointestinal Dynamics of Non-Encapsulated and Microencapsulated Salmonella Bacteriophages in Broiler Production.

Bacteriophage therapy is being considered as a promising tool to control Salmonella in poultry. Nevertheless, changes in gastrointestinal tract environmental conditions throughout the production cycle could compromise the efficacy of phages administered orally. The main objectives of this study were to assess the optimal timing of the phage administration over a 42-day production cycle and to compare microencapsulated and non-encapsulated phages and the spatial and temporal dynamics of the phage delivery along the gastrointestinal tract. Phage FGS011 was encapsulated in the pH-responsive polymer Eudragit® L100 using the process of spray drying. At different weeks of the chicken rearing period, 15 broilers were divided into three groups. Over a period of 24 h, group 1 received non-encapsulated phages (delivered through drinking water), group 2 received microencapsulated phages (incorporated in animal feed), and group 3 did not receive any phages. Microencapsulation was shown to enable efficient delivery of the bacteriophages to the animal gut and cecum throughout the animal rearing period. During the six weeks of application, the crop displayed the highest phage concentration for both phage delivery methods. The L100 based encapsulation offered significant protection to the phages from the harsh environmental conditions in the PV-Gizzard (not seen with phages administered in drinking water) which may help in the delivery of high phage doses to the cecum. Future Salmonella challenge studies are necessary to demonstrate the benefits of microencapsulation of phages using L100 formulation on phage therapy in field studies during the rearing period.

In Vitro and In Vivo Gastrointestinal Survival of Non-Encapsulated and Microencapsulated Salmonella Bacteriophages: Implications for Bacteriophage Therapy in Poultry.

The therapeutic use of bacteriophages is recognized as a viable method to control Salmonella. Microencapsulation of phages in oral dosage forms may protect phages from inherent challenges of the gastrointestinal tract in chickens. Therefore, the main objective of this study was to assess the survival of Salmonella BP FGS011 (non-encapsulated and microencapsulated) through the gastrointestinal tract under in vitro as well as in vivo conditions after oral administration to 1-day-old chicks. To this end, the phage FGS011 was encapsulated in two different pH-responsive formulations with polymers Eudragit® L100, and Eudragit® S100 using the process of spray drying. Phages encapsulated in either of the two formulations were able to survive exposure to the proventriculus-gizzard in vitro conditions whereas free phages did not. Moreover, phages formulated in polymer Eudragit® S100 would be better suited to deliver phage to the caeca in chickens. In the in vivo assay, no statistically significant differences were observed in the phage concentrations across the gastrointestinal tract for either the free phage or the encapsulated phage given to chicks. This suggested that the pH of the proventriculus/gizzard in young chicks is not sufficiently acidic to cause differential phage titre reductions, thereby allowing free phage survival in vivo.

Multidrug-Resistant Campylobacer jejuni on Swine Processing at a Slaughterhouse in Eastern Spain.

Campylobacteriosis is the most commonly reported gastrointestinal disease in humans in the EU, mainly from poultry meat consumption. C. jejuni is the main species involved in the human disease. However, little is known about the role of swine meat in its epidemiology. Thus, the aim of this study was to assess the epidemiology and antimicrobial resistance of C. jejuni on swine processing at the slaughterhouse. To this end, a total of 21 pig herds were intensively sampled at the slaughterhouse. Campylobacter isolation was based on official method ISO 10272-1:2018, speciation was determined by the hippurate hydrolysis test, and antibiotic susceptibility was performed according to standard disc diffusion assay. The results showed that all batches shed Campylobacter in faeces upon arrival at the slaughterhouse and remained positive at the end of the slaughtering process (42.8%). Moreover, 41.5% of Campylobacter strains isolated were C. jejuni and all of them were resistant to at least one antibiotic, and 96.3% were multidrug-resistant strains. In conclusion, the high level of multidrug-resistant C. jejuni swine batch contamination at the slaughterhouse makes it necessary to include the swine sector in national control programmes to reduce the bacterium and its resistance.

Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks.

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 effects 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.

Tackling the Threat of Rabies Reintroduction in Europe.

Rabies is one of the oldest, most important zoonoses worldwide due to its extreme and inevitably lethal nature, causing one death every 9 min worldwide. Recent reports have demonstrated that the Lyssavirus continues more alive than ever, despite the control carried out against the virus throughout Europe. In this context, this work reviews the main immunological implications, transmission risk factors and current prevention measures for virus control in Europe, and especially in Spain.

Pet Reptiles: A Potential Source of Transmission of Multidrug-Resistant Salmonella.

Salmonella spp. is widely considered one of the most important zoonotic pathogens worldwide. The close contact between reptiles and their owners provides favourable conditions for the transmission of zoonotic pathogen infections, and ~6% of human salmonellosis cases are acquired after direct or indirect contact with reptiles. Moreover, antimicrobial resistance is one of the most important health threats of the twenty-first century and has been reported in Salmonella strains isolated from pet reptiles, which could entail therapeutic consequences for their owners and breeders. The aim of this study was to assess Salmonella carriage by pet reptiles in pet shops and households, and their role in the transmission of antimicrobial resistance, to inform the owners about the possible risks factors. During the period between January 2019 and December 2019, 54 reptiles from pet shops and 69 reptiles from households were sampled in the Valencian Region (Eastern Spain). Three different sample types were collected from each reptile: oral cavity, skin, and cloacal swabs. Salmonella identification was based on ISO 6579-1:2017 (Annex D), serotyped in accordance with Kauffman-White-Le-Minor technique, and antibiotic susceptibility was assessed according to Decision 2013/652. The results of this study showed that 48% of the pet reptiles examined from households and pet shops carry Salmonella spp. All the strains isolated presented resistance to at least one antibiotic, and 72% were multidrug-resistant strains, the most frequently observed resistance patterns being gentamicin-colistin and gentamicin-colistin-ampicillin. The present study demonstrates that pet reptiles could be a source of human multidrug-resistant Salmonella infection. In this context, the most optimal prevention of multidrug-resistant Salmonella infections necessarily involves strict control of the sanitary status of reptile pet shops and hygienic handling by the individual owners at home.