Research Note: Validation of a new differentiation approach using the commercial ASAPTM media to detect the Salmonella 441/014 vaccine strain.

Vaccination is one of the most important control tools to reduce Salmonella in poultry production. In order for a live vaccine to be licensed for field use it should be provided with the detection methods to differentiate it from field strains. This paper aims to describe the validation of an alternative method for the differentiation of the Salmonella 441/014 vaccine strain from field strains, using a chromogenic Media, ASAP from bioMérieux. The ASAP-based differentiation method was compared with already authorized methods, namely the Anicon SE Kylt PCR DIVA 1 assay and Ceva S-Check Salmonella differentiation kit, following the ISO 16140-6:2019 validation method guidelines. A Generalised Linear Model was fitted to the data to determine the inclusivity and exclusivity of differentiation methods (PCR Kylt vs. S-Check vs. ASAPTM). Statistical differences were based on a P-value level of < 0.05 (SPSS Inc., Chicago, IL). In this study, we show that the ASAP media was able to differentiate Salmonella Enteritidis vaccine strains from field strains, obtaining 100% agreement between the three differentiation assays. This differentiation approach is quicker, easier to deploy and cheaper as compared to alternative methods.

New vascular structure in broiler breast: breast-vessel.

Chickens intensively selected for accelerating growth rate and enhanced muscle mass has resulted in the occurrence of breast alterations with a negative consumers perception. Together with the already known breast abnormalities (White Striping, Wooden Breast and Spaghetti Meat) a new one has been described by the consumers as a "long and thin worm". For this reason, the aim of this work was to macroscopically and microscopically describe this structure in broiler breast. To this end, 2 different experiments were performed. In the first one, 4 broiler breasts from a supermarket were analysed. Thereafter, a second experiment was carried out on an experimental farm simulating field management conditions in order to confirm the results of the first experiment. To this end, 120 chickens were reared in an experimental farm up to 42 d. Breast samples were collected and analysed macroscopically and microscopically to assess the abnormality on the broiler breast. In both experiments, the results of the analysis showed the finding of tubular structures located on the surface of pectoralis major muscle compatible with arteries. So far, currently, this occurrence has not been described in the literature and we propose calling this new broiler breast feature as breast-vessel.

Monitoring serologic response to single in ovo vaccination with an immune complex vaccine against infectious bursal disease in broilers.

The infectious bursal disease (IBD) virus is one of the most resistant and prevalent virus worldwide in the poultry industry, being vaccination the main tool to control the disease. For this reason, consistent and uniform immunization of broiler flocks against IBD is necessary to avoid the disease spreading. The aim of this study was to apply and assess an epidemiologic mapping tool focused on the immunization by in ovo single broiler vaccination using an immune complex IBD vaccine. With this regard, 7,576 serum samples were collected from 603 broiler flocks raised in 354 Spanish farms. To do so, blood samples were randomly collected from birds with ages between 35 to 51 d, and the serum was analyzed by ELISA. The results obtained from this study suggested a high uniform immunization against IBDV and a protective immunization between 35 and 51 d of age, with mean titer values ranging between 6,331 and 7,426. In addition, seroprevalence titer data of this large-scale monitoring study fitted a polynomial equation with a R2 value of 0.77, helping to explain and predict the humoral response to IBD vaccination. This seroprevalence map was applied to broiler production and was based on business intelligence tool that incorporates newly developed mapping tool to cover the need of having real-time information of humoral response to IBD vaccination and could be an effective tool for veterinary services to control and prevent IBD.

Genotyping and molecular characterization of antimicrobial resistance in thermophilic Campylobacter isolated from poultry breeders and their progeny in Eastern Spain.

Thermophilic Campylobacter spp. are recognized as a major cause of acute bacterial diarrhea in humans, with broiler meat being the most common source of human infection. Antibiotic therapy is usually necessary for severe or prolonged infections, especially in immunocompromised populations such as young or elderly individuals. However, different studies have demonstrated a close association between antibiotic use in animal production and antimicrobial resistance (AMR) in humans. In this sense, there is social pressure to reduce antibiotic administration and find adequate alternatives to control the presence of bacterial infections in farms. However, there is a lack of information related to Campylobacter AMR dynamics through the entire production system from breeders to their progeny. It is unknown if resistance genes are a result of adaptation through chromosomal mutation or through horizontal gene transfer, instead of vertical transmission of DNA from the parent to their progeny. Thus, the main objectives of this study were to assess the main AMR rates present in a poultry production system, to study the relationship between Campylobacter AMR profiles from breeders and their progeny, and to study the presence and distribution of antibiotic resistance genes in poultry production. Regarding AMR rates, ciprofloxacin was classified as extremely high, followed by nalidixic acid and tetracyclines that were classified as very high. Moreover, this study demonstrated a relationship between the AMR patterns and genes found from Campylobacter strains isolated in breeders and those present in their progeny.

The dynamic of antibiotic resistance in commensal Escherichia coli throughout the growing period in broiler chickens: fast-growing vs. slow-growing breeds.

Antimicrobial resistance (AMR) is an important threat to public health worldwide. Furthermore, different studies have demonstrated a close association between antibiotic use in animal production and AMR in humans. It is well known that it is necessary to reduce antibiotic administration in farms by finding effective alternative treatments, using more resistant breeds and improving animal welfare. However, to be able to assess the alternatives proposed, it is essential to study the epidemiology of AMR under production conditions. Hence, the aim of this study was to investigate the AMR dynamic in 2 genetic poultry breeds during the growing period. The study was performed in 2 experimental poultry houses to simulate real production conditions, and no antibiotics were administered during the growing period. In addition, 2 poultry breeds were used, fast-growing and slow-growing. To evaluate AMR evolution, Escherichia coli was selected as indicator bacterium. To this end, animals from each experimental group were sampled at different times: on day of arrival, at mid-period, and at slaughter day. In the laboratory, cecal content was removed and inoculated in selective media. Then, biochemical tests were performed to confirm E. coli. Finally, antibiotic susceptibility was assessed according to Decision 2013/653. At the onset of the cycle, significant differences were observed between breeds, as the E. coli strains isolated from fast-growing 1-day-old-chicks showed higher AMR rates. However, at the end of the period, no significant differences were found between breeds and their presence of resistant bacteria (above 95%). Therefore, although no antibiotics were administered during the growing period, a high level of AMR at slaughter day was demonstrated. Further studies are necessary to determine the main risk factors that increase the level of AMR throughout the productive cycle in broiler chickens. In conclusion, it is important to highlight that although it is crucial to control both antibiotic use and animal welfare during the growing period, measures should be taken at all levels of the production chain.

Salmonella Infantis and Salmonella Enteritidis specific bacteriophages isolated form poultry faeces as a complementary tool for cleaning and disinfection against Salmonella.

Salmonellosis represents an important public health concern. Several authors point out the inefficiency of the cleaning and disinfection protocols to remove the bacteria from the field. For this reason, innovative techniques, as bacteriophages, could be implemented to control the bacteria. The main objectives of this study were to assess the effect of bacteriophages against Salmonella Infantis and Salmonella Enteritidis on farm surfaces, and to evaluate bacteriophage procedure application as sanitiser against Salmonella in field conditions. Thus, most prevalent serovars in poultry production were selected (Salmonella Infantis and Salmonella Enteritidis) to contaminate farm facilities. Then, two specific bacteriophages isolated from poultry faeces were applied against them. Results showed Salmonella Infantis and Salmonella Enteritidis decreased of 4.55 log10CFU/mL and 3.85 log10CFU/mL, respectively; the maximum reduction in Salmonella was the 5th day, after 108 PFU/mL and 103 PFU/mL bacteriophage application. These results highlight bacteriophages as a promising tool together with cleaning and disinfection.

Autophage as a control measure for Salmonella in laying hens.

Notwithstanding the National Salmonella Control Programs, the latest data published by the European Food Safety Authority show an increase in Salmonella Enteritidis prevalence in laying hen flocks. For this reason, the implementation of innovative techniques such as phage therapy is needed to control Salmonella at farm level. Most common bacteriophage applications are a cocktail of 2 or more bacteriophages, as it has been described that cocktails could remove different Salmonella serotypes, thus providing cross efficacy. Nevertheless, resistance to the bacteriophage cocktail has been reported, resulting in a decrease in their effectiveness. Along these lines, some authors have reported the possibility of using autophage when commercial bacteriophage cocktails are not active against field strains. To our best knowledge, no autophage (bacteriophage isolated from the same environment where the pathogen is isolated) has been found to control Salmonella in laying hens. In this context, the aim of this study was to assess the application of autophage in reducing Salmonella Enteritidis in environmental and fecal samples in a layer farm. To this end, the bacteriophage was isolated from the same farm where the bacteria was present and was applied onto the facility installations and the animals, at 2 different times. After bacteriophage challenges, swab cloths from facility surfaces and feces samples were collected at 3 times according to the time spent after the bacteriophage challenge. The results obtained in our study showed that all the surface samples collected from the farm facilities after phage therapy were negative for Salmonella. Concerning faces samples, statistical differences were found in Salmonella counts, with the strongest decrease (1.78log10) occurring after the second challenge. Otherwise, depending of the moment of sampling, the results obtained were 2.34log10, 1.39log10, 0.56log10, and 0.97log10 cfu/g for T0, T1, T2, and T3 respectively. The study highlights the use of autophage therapy not only for Salmonella Enteritidis control in animals, but as a sanitizer in cleaning and disinfection.

The use of dynamic prevalence maps to assess the prevalence of Salmonella in broiler flocks in the Valencian Community, Spain.

The aim of this study was to design and implement a dynamic prevalence map for managing the data collected within the framework of the Salmonella spp. National control plan in broiler flocks, using a data set from the Valencian Community, Spain. Such a map would be a useful tool for veterinarians seeking to define control options. The development of the mapping tool for this study involved three main phases: data collection, data analysis and data representation. The data used were obtained from the results of the National Control Programme (based on European Commission [EC] Regulation No. 2160/2003). The Salmonella prevalence data were represented on a map of the Valencian Community, which included geographical information on flock locations to facilitate the interpretation of the results from monitoring. The average prevalence of Salmonella spp. was 2.74% from 2009 to 2013. Moreover, the proportion of broiler flocks that remained positive for the target serovars according to EC Regulation No. 2160/2003 (S. Enteritidis and/orS. Typhimurium [including monophasic S. Typhimurium]) was less than 1%. Over the five-year period of the study (2009-2013), the area of Bajo Maestrazgo had the highest level of Salmonella spp. prevalence every year, thereby validating the usefulness of this mapping tool. The aim of the study is to detect geographical 'highpressure' Salmonella zones over time and make suggestions on where efforts should be focused in this region to reduce future Salmonella spp. prevalence. Local Veterinary Services could, using the map, identify the infection pressure of Salmonella in the geographical areas where farms are located. Proximity to higher prevalences may imply an increased risk of flock contamination. Although annual data are represented, data can be presented in real time with regular frequency (daily, weekly, monthly or quarterly) or even over periods of several years. Therefore, the dynamic prevalence map provides extremely useful epidemiological information to veterinarians on the prevention of Salmonella.

L'étude présentée par les auteurs visait à concevoir et mettre en application une carte dynamique de la prévalence afin de gérer les données collectées dans le cadre du plan national de contrôle des Salmonella spp. dans les cheptels de poulets de chair à partir des informations transmises par la Communauté valencienne (Espagne). L'objectif attendu était que cette carte puisse aider les vétérinaires à définir la meilleure stratégie de lutte. La conception du logiciel de cartographie s'est faite en trois étapes, portant respectivement sur la collecte, l'analyse et la représentation des données. Les données utilisées provenaient des résultats du Programme national de lutte (basé sur le Règlement [CE] n° 2160/2003 du Parlement européen et du Conseil). Les taux de prévalence de Salmonella ont été reportés sur une carte de la Communauté valencienne, qui contenait également des informations géographiques relatives à la localisation des élevages afin de faciliter l'interprétation des résultats du suivi. De 2009 à 2013, la prévalence moyenne de Salmonella spp. était de 2,74 %. En outre, le pourcentage de cheptels de poulets de chair restant positifs au regard des sérovars visés par le Règlement CE n° 2160/2003 (S. Enteritidis et/ou S. Typhimurium [y compris la souche monophasique de S. Typhimurium]) était inférieur à 1 %. Sur les cinq années de l'étude (2009­2013), la prévalence annuelle la plus élevée de Salmonella spp. restait concentrée dans la région de Bajo Maestrazgo, ce qui confirme l'utilité de cet outil cartographique. Le but de l'étude est de pouvoir détecter les zones géographiques à « forte pression ¼ de Salmonella dans le temps afin d'orienter en conséquence les efforts de lutte dans cette région et de réduire ainsi la prévalence des Salmonella spp. à l'avenir. Grâce à cette carte, les Services vétérinaires locaux pourront connaître la pression exercée par Salmonella dans les zones géographiques où se situent les élevages. La proximité des zones à forte prévalence peut représenter un risque accru de contamination des cheptels. L'outil propose par défaut une représentation annuelle des données mais celles-ci peuvent également être présentées en temps réel à intervalles réguliers (fréquence quotidienne, hebdomadaire, mensuelle ou trimestrielle), voire même sur des périodes couvrant plusieurs années. Ainsi, la carte dynamique des prévalences fournit aux vétérinaires des informations épidémiologiques extrêmement utiles pour la prévention de Salmonella.

Los autores describen un estudio encaminado a concebir y realizar un mapa dinámico de prevalencia para explotar los datos obtenidos como parte del plan nacional de lucha contra las salmonelas en bandadas de pollos asaderos, utilizando para ello un conjunto de datos correspondientes a la Comunidad Valenciana (España). Semejante mapa ofrecería a los veterinarios una herramienta útil para definir las fórmulas idóneas de lucha. La elaboración del dispositivo de cartografía para este estudio entrañaba tres grandes fases: la de obtención, la de análisis y la de representación de los datos. Los datos utilizados derivan de los resultados obtenidos con el Programa Nacional de Control (basado en el Reglamento de la Comisión Europea [CE] Nº 2160/2003). Para facilitar la interpretación de los resultados obtenidos con la labor de vigilancia, los datos de prevalencia de salmonelas fueron representados en un mapa de la Comunidad Valenciana que contenía información geográfica sobre la localización de las bandadas. De 2009 a 2013, la prevalencia media de Salmonella spp. fue del 2,74%. Por otra parte, la proporción de bandadas de pollos asaderos que seguían siendo positivas para las serovariantes estudiadas con arreglo al Reglamento (CE)Nº 2160/2003 (S. Enteritidis y/o S. Typhimurium [incluida S. Typhimurium monofásica]) era inferior al 1%. En el quinquenio cubierto por el estudio (2009­2013), la zona del Bajo Maestrazgo registró cada año la mayor prevalencia de salmonelas, hecho que confirmó la utilidad de esta herramienta cartográfica. El estudio tiene por objetivo determinar a lo largo del tiempo las zonas geográficas de 'alta presión' de salmonelas y hacer propuestas sobre las áreas de esa región en las que convendría centrar el trabajo para reducir en el futuro la prevalencia de salmonelas. Empleando el mapa, los Servicios Veterinarios podrían determinar la presión infecciosa de salmonelas en las zonas geográficas en que se encuentran las explotaciones. Cuanto más cerca esté una explotación de zonas con niveles altos de prevalencia, mayor puede ser el riesgo de contaminación de las bandadas. Aunque en el mapa se representan datos anuales, también es posible presentarlos en tiempo real con determinada periodicidad (diaria, semanal, mensual o trimestral) o incluso cubrir periodos de varios años. El mapa dinámico de prevalencia proporciona así a los veterinarios información epidemiológica de gran utilidad para la prevención de las salmonelosis.