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In mammals, enteric salmonellas can use tetrathionate (ttr), formed as a by-product from the inflammatory process in the intestine, as electron acceptor in anaerobic respiration, and it can fuel its energy metabolism by degrading the microbial fermentation product 1,2-propanediol. However, recent studies have shown that this mechanism is not important for Salmonella infection in the intestine of poultry, while it prolongs the persistence of Salmonella at systemic sites in this species. In the current study, we show that ΔttrApduA strains of Salmonella enterica have lower net survival within chicken-derived HD-11 macrophages, as CFU was only 2.3% (S. Enteritidis ΔttrApduA), 2.3% (S. Heidelberg ΔttrApduA), and 3.0% (S. Typhimurium ΔttrApduA) compared to wild-type strains after 24 h inside HD-11 macrophage cells. The difference was not related to increased lysis of macrophages, and deletion of ttrA and pduA did not impair the ability of the strains to grow anaerobically. Further studies are indicated to determine the reason why Salmonella ΔttrApduA strains survive less well inside macrophage cell lines.

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Intestinal infections caused by non-typhoidal Salmonella spp., along with antimicrobial resistance spread are a major food safety concern worldwide. Here, we evaluate the potential of competitive exclusion products developed by anaerobic or aerobic conditions to control systemic infection, cecal colonization, fecal excretion, and improve the intestinal health in broilers challenged by Salmonella Heidelberg (SH). A total of 105 day-old chickens were randomly distributed into three experimental groups: A (untreated control), B (treated with anaerobic culture), and C (treated with aerobic culture). During 21 days, morphometric parameters of the small intestine were analyzed using microscopy, fecal excretions by cloacal swabs, systemic infection, and cecal colonization by colony-forming unit counts (CFU/g). The results indicated the lowest number of positive swabs (45.33%) recovered from Group C, followed by Group B (71.8%) and Group A (85.33%). The bacterial enumeration revealed the lowest amounts in Group C at the necropsy realized in 5-, 7-, and 14-days post-infection (DPI) (P = 0.0010, P = 0.0048, and P = 0.0094, respectively). Statistical differences between intestinal morphometrics were observed in the Group C at 21 DPI. Our results suggest that the product developed under aerobic conditions can improve intestinal health, protecting birds against SH.

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In recent years, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has been increasingly isolated from laying hens and shell eggs around the world. Moreover, this serovar has been identified as the causative agent of several salmonellosis outbreaks in humans. Surprisingly, little is known about the characteristics of this emerging serovar, and therefore, we investigated antimicrobial resistance, virulence, and prophage genes of six selected Brazilian strains of Salmonella Mbandaka using Whole Genome Sequencing (WGS). Multi-locus sequence typing revealed that the tested strains belong to Sequence Type 413 (ST413), which has been linked to recent multi-country salmonellosis outbreaks in Europe. A total of nine resistance genes were detected, and the most frequent ones were aac(6')-Iaa, sul1, qacE, blaOXA-129, tet(B), and aadA1. A point mutation in ParC at the 57th position (threonine → serine) associated with quinolone resistance was present in all investigated genomes. A 112,960 bp IncHI2A plasmid was mapped in 4/6 strains. This plasmid harboured tetracycline (tetACDR) and mercury (mer) resistance genes, genes contributing to conjugative transfer, and genes involved in plasmid maintenance. Most strains (four/six) carried Salmonella genomic island 1 (SGI1). All S. Mbandaka genomes carried seven pathogenicity islands (SPIs) involved in intracellular survival and virulence: SPIs 1-5, 9, and C63PI. The virulence genes csgC, fimY, tcfA, sscA, (two/six), and ssaS (one/six) were absent in some of the genomes; conversely, fimA, prgH, and mgtC were present in all of them. Five Salmonella bacteriophage sequences (with homology to Escherichia phage phiV10, Enterobacteria phage Fels-2, Enterobacteria phage HK542, Enterobacteria phage ST64T, Salmonella phage SW9) were identified, with protein counts between 31 and 54, genome lengths of 24.7 bp and 47.7 bp, and average GC content of 51.25%. In the phylogenetic analysis, the genomes of strains isolated from poultry in Brazil clustered into well-supported clades with a heterogeneous distribution, primarily associated with strains isolated from humans and food. The phylogenetic relationship of Brazilian S. Mbandaka suggests the presence of strains with high epidemiological significance and the potential to be linked to foodborne outbreaks. Overall, our results show that isolated strains of S. Mbandaka are multidrug-resistant and encode a rather conserved virulence machinery, which is an epidemiological hallmark of Salmonella strains that have successfully disseminated both regionally and globally.

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Conjugation plays an important role in the dissemination of antimicrobial resistance genes. Besides, this process is influenced by many biotic and abiotic factors, especially temperature. This study aimed to investigate the effect of different conditions of temperature and storage (time and recipient) of poultry meat, intended for the final consumer, affect the plasmid transfer between pathogenic (harboring the IncB/O-plasmid) and non-pathogenic Escherichia coli organisms. The determination of minimal inhibitory concentrations (MIC) of ampicillin, cephalexin, cefotaxime, and ceftazidime was performed before and after the conjugation assay. It was possible to recover transconjugants in the poultry meat at all the treatments, also these bacteria showed a significant increase of the MIC for all antimicrobials tested. Our results show that a non-pathogenic E. coli can acquire an IncB/O-plasmid through a conjugation process in poultry meat, even stored at low temperatures. Once acquired, the resistance genes endanger public health especially when it is about critically and highly important antimicrobials to human medicine.

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Salmonella spp. is one of the major foodborne pathogens responsible for causing economic losses to the poultry industry and bringing consequences for public health as well. Both the pathogen survival ability in the intestinal environment during inflammation as well as their relationship with the host immune system, play a key role during infections in poultry. The objective of this study was to quantify the presence of the macrophages and CD4+/CD8+ cells populations using the immunohistochemistry technique, in commercial lineages of chickens experimentally infected by wild-type and mutant strains of Salmonella Enteritidis and Salmonella Typhimurium lacking ttrA and pduA genes. Salmonella Enteritidis ∆ttrA∆pduA triggered a higher percentage of the stained area than the wild-type, with exception of light laying hens. Salmonella Typhimurium wild-type strain and Salmonella Typhimurium ∆ttrA∆pduA infections lead to a similar pattern in which, at 1 and 14 dpi, the caecal tonsils and ileum of birds showed a more expressive stained area compared to 3 and 7 dpi. In all lineages studied, prominent infiltration of macrophages in comparison with CD4+ and CD8+ cells was observed. Overall, animals infected by the mutant strain displayed a positively stained area higher than the wild-type. Deletions in both ttrA and pduA genes resulted in a more intense infiltration of macrophages and CD4+ and CD8+ cells in the host birds, suggesting no pathogen attenuation, even in different strains of Salmonella.

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Genetic profiles of Salmonella Minnesota isolates were analyzed using pulsed-field gel electrophoresis (PFGE). In total, 13 isolates obtained from the broiler industry collected in the states of Minas Gerais (11) and São Paulo (2), as well as five recovered from cases of foodborne infections in humans in the states of Minas Gerais (2), Santa Catarina (1), and Rio Grande do Sul (2), were submitted to PFGE. These 18 S. Minnesota isolates together with other 12 of poultry origin were also subjected to antimicrobial susceptibility testing. The PFGE analysis of 18 strains of S. Minnesota generated a dendrogram that grouped the isolates with 83-90% similarity into four main clusters. Among them, cluster "A" grouped the majority of isolates (13), including two of human origin that showed 90% similarity with a broiler isolate, both recovered in Minas Gerais. The S. Minnesota isolates showed resistance to tetracycline (80%), cefoxitin (80%), ceftazidime (46.7%), nalidixic acid (23.3%), ciprofloxacin (13.3%), and streptomycin (10%). No resistance to gentamicin, chloramphenicol, meropenem, nitrofurantoin, and sulfamethoxazole-trimethoprim was found. Moreover, 23.3% of the evaluated isolates presented multi-resistance profile, all from Minas Gerais. The results highlight the importance of further studies involving S. Minnesota, which is prevalent in the Brazilian broiler flocks and could provoke foodborne infection in humans.

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European wild boars (Sus scrofa) are considered exotic invasive species worldwide. Invasions of wild boars are a growing public health concern, as wild boars may represent an important reservoir of zoonotic pathogens, including bacteria of the genus Salmonella. The aim of this study was to determine the prevalence and serovars of Salmonella spp. in free-ranging wild boars legally hunted in the state of São Paulo, Brazil, and the susceptibility of those Salmonella spp. to antimicrobials. Fecal samples and mesenteric lymph nodes were acquired from 63 wild boars. The prevalence of Salmonella spp. in free-ranging wild boars was 9.5 % (6/63; confidence interval: 4.4 % - 19.2 %). Six serovars were isolated: S. enterica subsp. enterica ser. 4,5,12:-:1,2, S. enterica ser. Cerro, S. enterica ser. Madelia, S. enterica ser. Typhimurium, S. enterica ser. I (4,5,12:i:-) and S. enterica ser. Muenster. Analysis of antimicrobial resistance of Salmonella spp. showed that the majority of serovars were fully susceptible to the tested antimicrobials. Only S. enterica ser. Typhimurium and S. enterica ser. Muenster showed a resistance pattern to at least one antimicrobial analyzed. To our knowledge, this study is the first report the prevalence and serovars of Salmonella spp. in free-ranging wild boars in the State of São Paulo, Brazil. Results indicate a low prevalence with variability of Salmonella serovars, with some pattern of antimicrobial resistance. This study highlights the potential role of wild boars as carriers of Salmonella and could pose a risk to wild and domestic animals as well as humans.

Os javalis europeus (Sus scrofa) são considerados uma espécie exótica invasora em todo o mundo. As invasões de javalis são uma preocupação crescente de saúde pública, pois os javalis podem representar um importante reservatório de patógenos zoonóticos, incluindo bactérias do gênero Salmonella. O objetivo deste estudo foi determinar a prevalência e os sorovares de Salmonella spp. em javalis de vida livre caçados legalmente no Estado de São Paulo, Brasil, e a suscetibilidade dessa Salmonella spp. aos antimicrobianos. Amostras fecais e linfonodos mesentéricos foram adquiridos de 63 javalis. A prevalência de Salmonella spp. em javalis selvagens foi de 9,5% (6/63; intervalo de confiança: 4,4% - 19,2%). Seis sorovares foram isolados: S. enterica subsp. enterica ser. 4,5,12:-:1,2, S. enterica ser. Cerro, S. enterica ser. Madelia, S. enterica ser. Typhimurium, S. enterica ser. I (4,5,12:i:-) e S. enterica ser. Muenster. As análises de resistência antimicrobiana de Salmonella spp. evidenciaram que a maioria dos sorovares era pansensível aos antimicrobianos testados. Apenas S. enterica ser. Typhimurium e S. enterica ser. Muenster mostraram um padrão de resistência a pelo menos um antimicrobiano analisado. A saber, este estudo é o primeiro relato da prevalência e de sorovares de Salmonella spp. em javalis de vida livre no Estado de São Paulo, Brasil. Os resultados indicaram baixa prevalência com variabilidade de sorovares de Salmonella, com algum padrão de resistência antimicrobiana. Este estudo destaca o papel potencial dos javalis como portadores de Salmonella spp. e pode representar um risco para os animais domésticos e selvagens, bem como para os humanos.

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Salmonella Enteritidis (SE) is a major cause of foodborne diseases in humans being frequently related to the consumption of poultry products. Therefore, guaranteeing early immunity to chicks is an important tool to prevent the colonization and infection by this pathogen. The present study evaluated the effectiveness of a candidate recombinant vaccine against SE. Thirty female and five male broiler breeders that were ten weeks-old were divided into 3 groups: unvaccinated (UV), vaccinated with recombinant vaccine candidate (VAC) and vaccinated with commercial bacterin (BAC). Samples of serum and embryonated egg were collected at seven and twelve weeks after the booster dose to quantify the transfer rate of IgY to egg yolks and offspring. Subsequently, forty day-old offspring were divided into two groups (UV and VAC) and challenged on the following day with 107 CFU/chick of SE. Samples of serum, intestine, liver, and cecal content were harvested. Throughout the experiment period, significantly higher levels of IgY were observed in the egg yolk and also in the serum of broiler breeders and offspring of the VAC group in comparison to the UV group. In addition, increased transfer rates of IgY were observed in the VAC group when compared to the BAC group. Furthermore, higher villus-crypt ratios were found out in duodenum, jejunum and ileum at four days post-infection in the offspring from the VAC group. A high challenge dose of SE (107 CFU per chick) was used and despite the stronger humoral immune response provoked by the candidate vaccine, there were no statistical differences in the recovery of viable SE cells from the offspring cecal contents. Therefore, the effect of vaccination to improve intestinal quality may affect the development of the chickens and consequently increase the resistance to lower SE challenge doses.

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Salmonella Gallinarum (SG) is an avian-restricted pathogen that causes fowl typhoid in poultry. Although it has been reported frequently over many decades in poultry flocks worldwide, the microorganism is more commonly associated with poultry in developing countries, particularly those with high ambient temperatures, where the acute form of the disease results in considerable economic losses. A more detailed investigation of environmental factors that affect the course of disease may assist in identifying effective prevention and control measures. Heat stress is known to impair the immunological response to a variety of pathogens and clearly may be an important contributory factor in the prevalence of disease in countries with warm or hot climates. Thus, the objective of the present study was to evaluate the effects of heat stress on chickens infected with SG. For this, light and semi-heavy commercial laying hens were distributed randomly within four groups as follows: infected and non-infected groups in rooms held at ambient temperature, and infected and non-infected groups under heat stress. Clinical signs, egg production, and mortality were recorded daily. Bacteriological counts in liver and spleen samples were estimated at 2, 5, 7, and 14 days post-infection. The results showed that both SG infection and heat stress had similar effects on egg production and a synergistic effect of the two stressors was observed. The data show an interaction between disease and heat stress which could point towards environmental and biosecurity approaches to resolving the possible 30% fall in production observed in such countries.

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The number of foodborne gastroenteritis caused by nontyphoidal Salmonella (NTS) worldwide is estimated to be 80.3 million each year. Currently, antimicrobial-resistant NTS disseminated in the animal environment increases the risk of aggravated foodborne outbreaks. Poultry are important source of foodborne NTS infections. This study was conducted to evaluate the phenotypic and genotypic characteristics of 83 NTS isolates from poultry, classified within 36 different serovars. The most prevalent serovar was S. Schwarzengrund (10/83), from which 8/10 were multidrug resistant (MDR). The antimicrobial susceptibility testing showed a total of 18 MDR isolates, from which 8/18 coharbored blaCTX-M-2 and qnrB5. The genes qnrB5, blaCTX-M-2, qnrB2, or blaCMY-2 were also found alone in other MDR isolates. All resistance genes were harbored in large plasmids, ranging from 30 to 270 kb. The pColE replicon was present in 8 MDR isolates; however it was not associated with resistance. ISCR1 and class I integron structures were always associated with blaCTX-M-2.

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