Fighting Salmonella Infantis: bacteriophage-driven cleaning and disinfection strategies for broiler farms.

Introduction: Salmonella is a bacterium that can cause food-borne infections and is responsible for the most common gastrointestinal illnesses. The emergence of multi-drug resistant (MDR) strains worldwide is a major threat, representing a major challenge in public health. To reduce its incidence, the One Health approach is required, and the development of new biocontrol protocols will help prevent or eliminate the spread of Salmonella. Prevention measures, such as on-farm cleaning and disinfection protocols, are a crucial step in reducing infection to new flocks and eliminating bacteria that remain in the facilities. However, MDR Salmonella species, such as S. Infantis, are highly resistant to conventional cleaning and disinfection protocols, with an increased ability to persist in the broiler farm environment. The need for alternative biocontrol methods has led to the use of bacteriophages or phages, viruses that target bacteria, as promising tools. Thus, the aim of this study was to evaluate the efficacy of phages as a biocide against S. Infantis isolates in combination with cleaning and disinfection protocols in 10 commercial poultry farms. Methods: All commercial farms selected in this study had persistent Salmonella, even after the routinely used cleaning and disinfection procedures. In addition, Salmonella isolated before treatment were phenotypically characterized by antimicrobial resistance patterns. Results: The results showed that 100% of S. Infantis were resistant to at least one antibiotic, and > 70% were MDR. Phages were then isolated against the in-farm bacteria, purified, and multiplied for each poultry farm. The cleaning and disinfection protocols included the application of the lytic phages (vB_Si_CECAV_FGS009; vB_Si_CECAV_FGS017; vB_Si_CECAV_FGS029 and vB_Si_CECAV _FGS030) twice at 24-h intervals between cleaning and disinfection. Following the cleaning and disinfection procedures, Salmonella detection was reduced from 100% after cleaning to 36% after applying the phages and dropped to 0% after the final step of disinfection, thus eliminating Salmonella from the farm facilities. Discussion: This study demonstrates that bacteriophage application after cleaning and before disinfection enhances the removal of MDR Salmonella Infantis in commercial broiler farms, suggesting their use as biocontrol agents to reduce Salmonella, a major public health concern.

Simultaneous Salmonella and bacteriophage isolation on Modified Semisolid Rappaport Vassiliadis media.

Salmonella represents a food safety concern worldwide. Despite the application of National Control Programs (NCP) against Salmonella, regulated by the European Union, every year the European Food Safety Authority reports new cases. On the look for new alternatives to antibiotics, bacteriophages, or phages, rise as a promising alternative to treat multidrug resistance infections. Although they are known to be ubiquitous in the environment, their high specificity to host cells hinders their isolation and usage for phage therapy. The ISO 6579-1:2017 is performed as a reference method in the NCP and uses an unspecific media to enrich the sample the same way most phage isolation protocols do. Later, the protocol uses a more selective media to isolate the Salmonella, Modified Semisolid Rappaport Vassiliadis (MSRV). This paper aims to find out whether, due to the similarity between phage isolation protocols and the ISO 6579-1:2017, this last one could be used as a protocol to also isolate phages against the same bacterium that is being simultaneously isolated. To do so, 2 experiments were performed to assess phage isolation from MSRV media in in-vivo conditions. The results from experiments 1 and 2 proved that the MSRV media was usable for simultaneous phage and pathogen isolation through a single procedure. Additionally, there is a correlation between the antigenic formulae from the bacteria and the phage's host range, seeming to be effective against bacteria with similar antigenic formulae.

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.

Research Note: Campylobacter spp. control at field level two years after the implementation of European Regulation (EU) 2017/1495.

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.

Antimicrobial Resistance Trends of Escherichia coli Isolates: A Three-Year Prospective Study of Poultry Production in Spain.

Antimicrobial resistance (AMR) poses a major threat to health worldwide. Poultry products are one of the main threats, due to the transmission of antimicrobial resistance genes throughout the food chain. Escherichia coli is the main cause of mortality in the poultry industry, mainly mitigated with antibiotics, but due to the high genetic strain variability, recurrent outbreaks of multidrug resistant E. coli take place. The major challenge to tackling AMR is understanding the burden of resistance. For this reason, one of the main strategies is monitoring AMR by phenotypic characterisation. Our study aimed to monitor the resistance of E. coli strains isolated from the poultry sector over a period of three years (2019-2021) to provide information on the resistance magnitude and trends. Promising results have been found concerning the low frequency of resistance to cephalosporins, polymyxin, and fluoroquinolones. However, levels of resistance found to antimicrobials such as erythromycin (100%), tylosin (98%), or penicillin (97%) suggest the need to continue working on the limitation of use of antimicrobials in poultry to achieve the demise of MDR.

Monitoring antimicrobial resistance trends in Salmonella spp. from poultry in Eastern Spain.

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.

Seroprevalence and prevalence of Infectious Bronchitis Virus in broilers, laying hens and broiler breeders in Spain.

Infectious Bronchitis Virus (IBV) is one of the most important viral diseases which causes important economic losses in poultry industry. This study aimed to assess the seroprevalence, prevalence, and variants of IBV in broilers, layers, and broiler breeders´ farms of Gallus gallus species in Eastern Spain. Thus, 29, 16, and 14 flocks of broilers, layers and broiler breeders, respectively were analyzed. To assess seroprevalence, sera samples were analyzed by ELISA. Tracheal swabs and tissue samples were tested by PCR to know the prevalence and detect specific variants. An IBV seroprevalence of 100% was detected in the 3 productive orientations. According to PCR results, a prevalence of 38% in broilers, 44% in layers and 43% in broiler breeders was obtained. The variant-specific RT-PCR analysis showed that 4/91, Massachusetts, QX, Italy-02 and D274 strains were present in commercial flocks in eastern Spain, being 4/91 the most prevalent in all the productive orientations. In layers 100% of QX prevalence, 14% of Italy 02 and 14% of D274 was detected. Regarding broilers, a prevalence of 18% of Massachusetts strain was also detected. In contrast, in broiler breeders´ farms only 4/91 strain was found. In conclusion, our findings showed the presence of IBV in eastern Spain and the changing situation of the IBV variants´ prevalence, being different according to the productive orientation. The continuous emergence of new variants emphasizes the importance of continuous IBV monitoring in order to optimize vaccination strategies.

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.

Dynamic paramyxovirus type 1 seroprevalence maps in broilers in the Valencian Community (eastern Spain) during a five-year period (2008-2012).

Newcastle disease is a devasting disease in poultry production worldwide, thus it is important to implement control measures to avoid entrance of the disease and its spread in the field. In this context, the aim of this study was to design and implement a seroprevalence map based on business intelligence for avian paramyxovirus type 1 (APMV-1) in broilers in the Valencia Community (eastern Spain). This tool consists in software mapping based on data collection, data analysis and data representation. In order to obtain the serological data, 12 495 sera from 131 broiler farms over 5 years were analysed (2008-2012). The data were represented on a map of the Valencian Community including geographical information of flock locations to facilitate disease monitoring. No clinical signs of APMV-1 were reported in the studied flocks. The data from this study showed no evidence contact with APMV-1 in broiler flocks and the novel software mapping tool as a valuable method for easily monitoring the serological response to avian paramyxovirus type 1 (APMV-1) including geographical information.

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.

Seroprevalence and prevalence of Mycoplasma synoviae in laying hens and broiler breeders in Spain.

Mycoplasma species are worldwide recognized poultry pathogens, with Mycoplasma synoviae being the second most important species from the clinical point of view that causes considerable economic losses in the poultry industry. The aim of this study was to assess the seroprevalence, prevalence, and phylogenetic variants of M. synoviae present in layers and broiler breeders' farms of Gallus gallus species located in eastern Spain. Thus, 19 and 23 flocks of layers and broiler breeders, respectively, were analyzed at 3 different ages. To assess seroprevalence, sera samples were analyzed by ELISA. Tracheal swabs were tested by PCR to assess the prevalence. A M. synoviae seroprevalence of 95 and 74% was detected in layers and broiler breeders, respectively. Regarding age-wise analysis, the positive rates obtained seemed to be higher as the age of sampling increased. As per PCR results, a prevalence of 95% in layers and 35% in broiler breeders was obtained. The genetic analysis showed that the strains present in broilers breeders were vaccine strains (MS H strain). In contrast, 6 different field strains were detected in layer hens. In conclusion, this study carried out in eastern Spain showed a higher seroprevalence and prevalence of M. synoviae field strains in layer flocks regarding broiler breeders, highlighting the usefulness of monitoring flocks to control this poultry pathogen. Moreover, our findings suggest M. synoviae vaccination in broiler breeders could be an effective prevention strategy.

Salmonella Bacteriophage Diversity According to Most Prevalent Salmonella Serovars in Layer and Broiler Poultry Farms from Eastern Spain.

The exploration of novel nonantibiotic interventions in the field, such as the use of bacteriophages, is necessary to avoid the presence of Salmonella. Bacteriophages are a group of viruses widely distributed in nature, strictly associated with the prokaryotic cell. Researchers have demonstrated the success of phage therapy in reducing Salmonella counts in poultry products. However, the impact that phage concentration in the environment may have against certain Salmonella serovars is not well understood. Therefore, the aim of this study was to assess Salmonella phage prevalence in commercial poultry farms in terms of the production type: layers or broilers. The most prevalent Salmonella serovars isolated in poultry production were used for phage isolation. Salmonella specific phages were isolated from 141 layer and broiler farms located in the Valencia region during 2019. Analysis of the samples revealed that 100% presented Salmonella phages, the most prevalent being against serovar S. Enteritidis (93%), followed by S. Virchow (59%), S. Typhimurium (55%), S. Infantis (52%) and S. Ohio (51%). These results indicate that poultry farms could represent an important source of Salmonella phages. Nevertheless, further studies are needed to assess the epidemiology of phages against other serovars present in other countries and their diversity from the point of view of molecular studies.

Campylobacter prevalence and risk factors associated with exceeding allowable limits in poultry slaughterhouses in Spain.

BACKGROUND: Campylobacter is the main pathogen involved in zoonotic gastrointestinal diseases. In 2018, European Regulation 2017/1495 on Campylobacter in broiler carcases came into force. In this context, the aim of the study was to assess the potential risk factors associated with exceeding the 1000 cfu/g (colony-forming units per gram) limit set by the EC in several slaughterhouses in Spain. METHODS: Data relating to 12 factors were collected using questionnaires. Samples were collected from 12 Spanish abattoirs in June, July and August 2017 (n=1725) and were analysed following the ISO 10272-2:2006 method. RESULTS: The proportion of Campylobacter-positive samples was 23.7 per cent (n=409). Analysis of flock age (41-50 days) revealed a significantly increased odds ratio (OR) in Campylobacter enumeration (OR=7.41). Moreover, scalding temperature (51.9°C-54°C) was positively associated with an increase in OR (OR=2.75). Time in transit to slaughter for 1-1.5 hours showed a significant decrease in OR (OR=0.25), while time in transit for more than two hours showed an increase in OR (OR=4.44). With regard to carcase weight, a weight of 3.21-3.58 kg showed a decrease in OR (OR=0.01). CONCLUSION: The outcomes of this study suggest that although most chickens are contaminated by the bacterium, the prevalence of those exceeding the 1000 cfu/g limit is not so high as thought.

[Use of bacteriphages against Salmonella Enteritidis: a prevention tool]. / EMPLEO DE BACTERIÓFAGOS FRENTE A SALMONELLA ENTERITIDIS COMO HERRAMIENTA DE PREVENCIÓN.

INTRODUCTION: Salmonellosis is a highly prevalent disease still searching for preventive tools to avoid contamination level priority public health. OBJECTIVE: The in vitro effect of bacteriophages against Salmonella enteritidis was evaluated as a prevention tool. METHOD: Two tests with three concentrations of bacteriophages were conducted against two strains of Salmonella Enteritidis inoculated in fresh faeces of laying hens. Each test had a positive control. Thus, four groups in each test were evaluated. Each experimental group included two replicates, and three plates were incubated per replicate. The concentrations tested were three: commercial solution (5 × 10(7) pfu/mL), and two dilutions (1/10 and 1/30). One of the strains tested was CECT 4300, a certified strain of Colección Española de Cultivo Tipo and the other a field isolated strain in a sacrificed hen farm. Both strains were inoculated at 1.3 × 10(5) cfu/g of faeces in each of the four groups. Isolation and identification of bacteria by ISO6579 was done at various times after inoculation: 1 minute, 24 hours and 7 days. RESULTS: In the first test, with certified strain, Salmonella was isolated in all groups at time 1 minute. After 24 hours, Salmonella was isolated in all groups except in one of the replicas treated with 1/10 dilution of bacteriophages, one of the other replica plate treated with 1/10 dilution, and two plates of the two replicas treated with the commercial solution. After 7 days, the bacteria were not isolated from any of the experimental groups. In the second test, with the field strain, Salmonella was isolated in all groups at time 1 minute. After 24 hours, Salmonella was isolated in all groups except in one of the replicas treated with 1/10 dilution of bacteriophages and the two replicas treated with the commercial solution. Salmonella was not isolated in any of the experimental groups at 7 days. CONCLUSIONS: The use of bacteriophages reduced Salmonella enteritidis isolates in faeces at 24 hours after the application, so it could be considered as a prevention tool. At 7 days after inoculation of bacteria, no one was isolated in any of the experimental groups.

Introducción: la salmonelosis es una enfermedad de alta prevalencia, siendo la búsqueda de herramientas preventivas para evitar la contaminación una prioridad a nivel de salud pública. Objetivo: en el presente trabajo se evaluó el efecto in vitro de bacteriófagos frente a Salmonella enteritidis como una herramienta de prevención. Método: se realizaron dos pruebas con tres concentraciones de bacteriófagos frente a dos cepas de Salmonella enteritidis inoculadas en muestras de heces frescas de gallinas ponedoras, y el correspondiente control positivo. Así, se testaron cuatro grupos en cada una de las dos pruebas. Cada grupo experimental contó con dos réplicas, y en cada réplica se incubaron tres placas. Las concentraciones ensayadas fueron tres: solución comercial (5 × 107 pfu/mL), y dos diluciones de la misma (1/10 y 1/30). Una de las cepas testada fue la cepa CECT 4300, cepa certificada de la Colección Española de Cultivo Tipo, y la otra una cepa de campo aislada en una explotación de ponedoras sacrificadas. Ambas cepas se inocularon en muestras de heces a la dosis de 1,3 × 105 ufc/g de heces en cada uno de los cuatro grupos. Se procedió con el aislamiento e identificación de la bacteria por ISO 6579 a varios tiempos desde la inoculación: 1 minuto, 24 h y 7 días. Resultados: en la primera prueba, con la cepa certificada, se aisló Salmonella en todos los grupos a tiempo 1 minuto. A las 24 h se aisló Salmonella en todos los grupos excepto en una de las réplicas tratada con la dilución 1/10 de bacteriófagos, en una de las placas de la otra réplica tratada con la dilución 1/10, y en dos placas de cada una de las dos réplicas tratadas con la solución comercial. A partir de los 7 días ya no se aisló la bacteria de ninguno de los grupos experimentales. En la segunda prueba, con la cepa de campo, se aisló Salmonella en todos los grupos a tiempo 1 minuto. A las 24 h se aisló Salmonella en todos los grupos excepto en una de las réplicas tratada con la dilución 1/10 de bacteriófagos y en las dos réplicas tratadas con la solución comercial. Al igual que en la prueba primera, a los 7 días tampoco se aisló Salmonella en ninguno de los grupos experimentales. Conclusiones: el empleo de bacteriófagos contribuyó a reducir los aislamientos de Salmonella enteritidis en las muestras de heces a las 24 h de su aplicación, por lo que podría ser considerado una herramienta de prevención. A los 7 días tras la inoculación de la bacteria no se aisló la misma en ninguno de los grupos experimentales.