Centralized industrialization of pork in Europe and America contributes to the global spread of Salmonella enterica.

Salmonella enterica causes severe food-borne infections through contamination of the food supply chain. Its evolution has been associated with human activities, especially animal husbandry. Advances in intensive farming and global transportation have substantially reshaped the pig industry, but their impact on the evolution of associated zoonotic pathogens such as S. enterica remains unresolved. Here we investigated the population fluctuation, accumulation of antimicrobial resistance genes and international serovar Choleraesuis transmission of nine pig-enriched S. enterica populations comprising more than 9,000 genomes. Most changes were found to be attributable to the developments of the modern pig industry. All pig-enriched salmonellae experienced host transfers in pigs and/or population expansions over the past century, with pigs and pork having become the main sources of S. enterica transmissions to other hosts. Overall, our analysis revealed strong associations between the transmission of pig-enriched salmonellae and the global pork trade.

[Influence of breed and herd size on the intra-herd prevalence of Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) in sheep]. / Einfluss von Rasse und Herdengröße auf die Intraherdenprävalenz von Salmonella enterica subspecies diarizonae Serovar 61: k: 1, 5, (7) bei Schafen.

SUBJECT AND AIM: At present, only little information is available on the within-flock prevalence of Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) (SASd) in sheep flocks in Germany as well as their possible influencing factors. The aim of the study was to investigate relationships between flock size, breed and within-flock prevalence. MATERIAL AND METHODS: A total of 1610 clinically healthy ewes from 14 sheep flocks of 9 different breeds aged 2 to 12 years were microbiologically tested for SASd by nasal swab and fecal samples. Linear multivariable models were used to analyse the associations between within-flock prevalence and farm factors (flock size, breed) or detection frequencies in fecal or nasal swabs. RESULTS: SASd was detected in all sheep flocks examined, with 75% of adults having at least one positive nasal or fecal result. In comparison to the 11 flocks in which commercial breeds were kept, the 3 flocks of landraces had a lower apparent within-flock prevalence (p=0.01). No association with herd size was evident. With respect to the age of the ewes, there was a negative relationship (p=0.05) with the frequency of detection of SASd in the nasal swab but not in the fecal swab. The health status and fertility performance of the flocks were in line with a normal range for commercial sheep flocks, with lambing losses of 5% to 10% and lambing scores of 130% to 158%. CONCLUSIONS: Despite the high prevalence of SASd infections particularly within commercial breeds, there was no evidence of a relevant risk to sheep health. Compared with commercial breed flocks, a lower spread of SASd within flocks keeping landraces was evident. CLINICAL RELEVANCE: Despite a high prevalence, infections with SASd are very unlikely to lead to clinical symptoms or disease. Regulation and monitoring of SASd in sheep are of low priority for animal health authorities.

[Longitudinal study on the time of infection of sheep lambs with Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7)]. / Longitudinale Studie zum Zeitpunkt einer Infektion von Schaflämmern mit Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7).

OBJECTIVE: Infections with Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) (SASd) are widespread in sheep. SASd is considered as host associated with low virulence. The aim of the study was to determine the time of infection of lambs after birth with consideration of the Salmonella status of the mother. MATERIAL AND METHODS: Fecal and nasal swabs from 88 ewes of a flock with known SASd status but unknown intraherd prevalence were examined for SASd in the period of 6 weeks before the beginning of the lambing period. Fecal and nasal swabs from one female lamb from each of these ewes were also examined for SASd at 4-6 time points between the 4th and 50th day of life and at 7, 30 and 40 months of age. RESULTS: In the first two weeks after birth, significantly more lambs were positive in the nasal swab than in the fecal swab. SASd positive nasal swabs were mainly found in lambs originating from ewes also positive in nasal swabs. 43 lambs remained SASd-negative until day 50 of age. There was no correlation between the SASd status of ewes and their off-spring. At 7 months of age, the number of animals with a positive detection of SASd increased significantly. 32% of the nasal swabs and 62% of the fecal samples were positive for SASd. CONCLUSIONS: There is no rapid and intensive transmission of SASd from ewes to their offspring. Most likely, the infection begins by colonising the nasal mucosa of the lambs and does not cause any clinical symptoms. CLINICAL RELEVANCE: Even with a high intraherd prevalence, infections with SASd are very unlikely to cause clinical symptoms.

Oxford nanopore technologies-a valuable tool to generate whole-genome sequencing data for in silico serotyping and the detection of genetic markers in Salmonella.

Bacteria of the genus Salmonella pose a major risk to livestock, the food economy, and public health. Salmonella infections are one of the leading causes of food poisoning. The identification of serovars of Salmonella achieved by their diverse surface antigens is essential to gain information on their epidemiological context. Traditionally, slide agglutination has been used for serotyping. In recent years, whole-genome sequencing (WGS) followed by in silico serotyping has been established as an alternative method for serotyping and the detection of genetic markers for Salmonella. Until now, WGS data generated with Illumina sequencing are used to validate in silico serotyping methods. Oxford Nanopore Technologies (ONT) opens the possibility to sequence ultra-long reads and has frequently been used for bacterial sequencing. In this study, ONT sequencing data of 28 Salmonella strains of different serovars with epidemiological relevance in humans, food, and animals were taken to investigate the performance of the in silico serotyping tools SISTR and SeqSero2 compared to traditional slide agglutination tests. Moreover, the detection of genetic markers for resistance against antimicrobial agents, virulence, and plasmids was studied by comparing WGS data based on ONT with WGS data based on Illumina. Based on the ONT data from flow cell version R9.4.1, in silico serotyping achieved an accuracy of 96.4 and 92% for the tools SISTR and SeqSero2, respectively. Highly similar sets of genetic markers comparing both sequencing technologies were identified. Taking the ongoing improvement of basecalling and flow cells into account, ONT data can be used for Salmonella in silico serotyping and genetic marker detection.

Clonal relation between Salmonella enterica subspecies enterica serovar Dublin strains of bovine and food origin in Germany.

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) is a host-adapted serovar causing enteritis and/or systemic diseases in cattle. As the serovar is not host-restricted, it may cause infections in other animals, including humans with severe illness and higher mortality rates than other non-typhoidal serovars. As human infections are mainly caused by contaminated milk, milk products and beef, information on the genetic relationship of S. Dublin strains from cattle and food should be evaluated. Whole-genome sequencing (WGS) of 144 S. Dublin strains from cattle and 30 strains from food origin was performed. Multilocus sequence typing (MLST) revealed mostly sequence type ST-10 from both, cattle and food isolates. In total, 14 of 30 strains from food origin were clonally related to at least one strain from cattle, as detected by core-genome single nucleotide polymorphisms typing as well as core-genome MLST. The remaining 16 foodborne strains fit into the genome structure of S. Dublin in Germany without outliers. WGS proved to be a powerful tool not only to gain information on the epidemiology of Salmonella strains but also to detect clonal relations between organisms isolated from different stages of production. This study has shown a high genetic correlation between S. Dublin strains from cattle and food and, therefore, the potential to cause human infections. S. Dublin strains of both origins share an almost identical set of virulence factors, emphasizing their potential to cause severe clinical manifestations in animals, but also in humans and thus the need for effective control of S. Dublin in a farm-to-fork strategy.

Whole genome sequencing of Salmonella enterica serovars isolated from humans, animals, and the environment in Lagos, Nigeria.

BACKGROUND: Salmonella infections remain an important public health issue worldwide. Some serovars of non-typhoidal Salmonella (NTS) have been associated with bloodstream infections and gastroenteritis, especially in children in Sub-Saharan Africa with circulating S. enterica serovars with drug resistance and virulence genes. This study identified and verified the clonal relationship of Nigerian NTS strains isolated from humans, animals, and the environment. METHODS: In total, 2,522 samples were collected from patients, animals (cattle and poultry), and environmental sources between December 2017 and May 2019. The samples were subjected to a standard microbiological investigation. All the isolates were identified using Microbact 24E, and MALDI-TOF MS. The isolates were serotyped using the Kauffmann-White scheme. Antibiotic susceptibility testing was conducted using the disc diffusion method and the Vitek 2 compact system. Virulence and antimicrobial resistance genes, sequence type, and cluster analysis were investigated using WGS data. RESULTS: Forty-eight (48) NTS isolates (1.9%) were obtained. The prevalence of NTS from clinical sources was 0.9%, while 4% was recorded for animal sources. The serovars identified were S. Cotham (n = 17), S. Give (n = 16), S. Mokola (n = 6), S. Abony (n = 4), S. Typhimurium (n = 4), and S. Senftenberg (n = 1). All 48 Salmonella isolates carried intrinsic and acquired resistant genes such as aac.6…Iaa, mdf(A), qnrB, qnrB19 genes and golT, golS, pcoA, and silP, mediated by plasmid Col440I_1, incFIB.B and incFII. Between 100 and 118 virulence gene markers distributed across several Salmonella pathogenicity islands (SPIs), clusters, prophages, and plasmid operons were found in each isolate. WGS revealed that strains of each Salmonella serovar could be assigned to a single 7-gene MLST cluster, and strains within the clusters were identical strains and closely related as defined by the 0 and 10 cgSNPs and likely shared a common ancestor. The dominant sequence types were S. Give ST516 and S. Cotham ST617. CONCLUSION: We found identical Salmonella sequence types in human, animal, and environmental samples in the same locality, which demonstrates the great potential of the applied tools to trace back outbreak strains. Strategies to control and prevent the spread of NTS in the context of one's health are essential to prevent possible outbreaks.

Epidemiological Analysis on the Occurrence of Salmonella enterica Subspecies enterica Serovar Dublin in the German Federal State Schleswig-Holstein Using Whole-Genome Sequencing.

The cattle-adapted serovar Salmonella Dublin (S. Dublin) causes enteritis and systemic diseases in animals. In the German federal state Schleswig-Holstein, S. Dublin is the most important serovar in cattle indicating an endemic character of the infection. To gain information on dissemination and routes of infection, whole-genome sequencing (WGS) was used to explore the genetic traits of 78 S. Dublin strains collected over a period of six years. The phylogeny was analysed using core-genome single nucleotide polymorphisms (cgSNPs). Genomic clusters at 100, 15 and 1 cgSNPs were selected for molecular analysis. Important specific virulence determinants were detected in all strains but multidrug resistance in S. Dublin organisms was not found. Using 15 cgSNPs epidemiological links between herds were identified, clusters at 1 cgSNPs provided clear evidence on both persistence of S. Dublin at single farms in consecutive years and transmission of the organisms between herds in different distances. A possible risk factor for the repeated occurrence of S. Dublin in certain districts of Schleswig-Holstein might be the spreading of manure on pastures and grassland. Effective control of S. Dublin requires farm-specific analysis of the management supplemented by WGS of outbreak causing S. Dublin strains to clearly identify routes of infection.

[Assessment of available and currently applied typing methods including genome-based methods for zoonotic pathogens with a focus on Salmonella enterica]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden einschließlich genombasierter Verfahren von Zoonoseerregern am Beispiel von Salmonella enterica.

BACKGROUND: In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS: An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION: The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.

Interaction of Salmonella Gallinarum and Salmonella Enteritidis with peripheral leucocytes of hens with different laying performance.

Salmonella enterica ssp. enterica serovars Enteritidis (SE) and Gallinarum (SG) cause different diseases in chickens. However, both are able to reach the blood stream where heterophils and monocytes are potentially able to phagocytose and kill the pathogens. Using an ex vivo chicken whole blood infection model, we compared the complex interactions of the differentially host-adapted SE and SG with immune cells in blood samples of two White Leghorn chicken lines showing different laying performance (WLA: high producer; R11: low producer). In order to examine the dynamic interaction between peripheral blood leucocytes and the Salmonella serovars, we performed flow cytometric analyses and survival assays measuring (i) leucocyte numbers, (ii) pathogen association with immune cells, (iii) Salmonella viability and (iv) immune gene transcription in infected whole blood over a four-hour co-culture period. Inoculation of blood from the two chicken lines with Salmonella led primarily to an interaction of the bacteria with monocytes, followed by heterophils and thrombocytes. We found higher proportions of monocytes associated with SE than with SG. In blood samples of high producing chickens, a decrease in the numbers of both heterophils and Salmonella was observed. The Salmonella challenge induced transcription of interleukin-8 (IL-8) which was more pronounced in SG- than SE-inoculated blood of R11. In conclusion, the stronger interaction of monocytes with SE than SG and the better survivability of Salmonella in blood of low-producer chickens shows that the host-pathogen interaction and the strength of the immune defence depend on both the Salmonella serovar and the chicken line.

Epidemiological Analysis of Salmonella enterica subsp. enterica Serovar Dublin in German Cattle Herds Using Whole-Genome Sequencing.

Salmonella enterica subsp. enterica serovar Dublin is a cattle-adapted serovar that causes enteritis and systemic diseases in animals. In Germany, S. Dublin is not detected or is very rarely detected in some federal states but is endemic in certain regions. Information on detailed genetic characteristics of S. Dublin is not available. An understanding of the paths and spreading of S. Dublin within and between regions and over time is essential to establish effective control strategies. Whole-genome sequencing (WGS) and bioinformatic analysis were used to explore the genetic traits of S. Dublin and to determine their epidemiological context. Seventy-four S. Dublin strains collected in 2005 to 2018 from 10 federal states were studied. The phylogeny was analyzed using core-genome single-nucleotide polymorphisms (cgSNPs) and core-genome multilocus sequence typing. Genomic clusters at 100 cgSNPs, 40 cgSNPs, and 15 cgSNPs were selected for molecular epidemiology. WGS-based genoserotyping confirmed serotyping. Important specific virulence determinants were detected in all strains, but multidrug resistance in German S. Dublin organisms is uncommon. Use of different thresholds for cgSNP analysis enabled a broad view and also a detailed view of the occurrence of S. Dublin in Germany. Genomic clusters could be allocated nationwide, to a limited number of federal states, or to special regions only. Results indicate both persistence and spread of S. Dublin within and between federal states in short and longer time periods. However, to detect possible routes of infection or persistence of S. Dublin indicated by genomic analysis, information on the management of the cattle farms and contacts with corresponding farms are essential. IMPORTANCE Salmonella enterica subsp. enterica serovar Dublin is a bovine host-adapted serovar that causes up to 50% of all registered outbreaks of salmonellosis in cattle in Germany. S. Dublin is not detected or is only rarely detected in some federal states but has been endemic in certain regions of the country for a long time. Information on genetic traits of the causative strains is essential to determine routes of infection. WGS and bioinformatic analysis should be used to explore the genetic characteristics of S. Dublin. Combining the genomic features of S. Dublin strains with information on the management of the cattle farms concerned should enable the detection of possible routes of infection or persistence of S. Dublin. This approach is regarded as a prerequisite to developing effective intervention strategies.

Toward an Integrated Genome-Based Surveillance of Salmonella enterica in Germany.

Despite extensive monitoring programs and preventative measures, Salmonella spp. continue to cause tens of thousands human infections per year, as well as many regional and international food-borne outbreaks, that are of great importance for public health and cause significant socio-economic costs. In Germany, salmonellosis is the second most common cause of bacterial diarrhea in humans and is associated with high hospitalization rates. Whole-genome sequencing (WGS) combined with data analysis is a high throughput technology with an unprecedented discriminatory power, which is particularly well suited for targeted pathogen monitoring, rapid cluster detection and assignment of possible infection sources. However, an effective implementation of WGS methods for large-scale microbial pathogen detection and surveillance has been hampered by the lack of standardized methods, uniform quality criteria and strategies for data sharing, all of which are essential for a successful interpretation of sequencing data from different sources. To overcome these challenges, the national GenoSalmSurv project aims to establish a working model for an integrated genome-based surveillance system of Salmonella spp. in Germany, based on a decentralized data analysis. Backbone of the model is the harmonization of laboratory procedures and sequencing protocols, the implementation of open-source bioinformatics tools for data analysis at each institution and the establishment of routine practices for cross-sectoral data sharing for a uniform result interpretation. With this model, we present a working solution for cross-sector interpretation of sequencing data from different sources (such as human, veterinarian, food, feed and environmental) and outline how a decentralized data analysis can contribute to a uniform cluster detection and facilitate outbreak investigations.

Emergence of Multidrug-Resistant Salmonella enterica Subspecies enterica Serovar Infantis of Multilocus Sequence Type 2283 in German Broiler Farms.

During the last decade, Salmonella enterica subspecies enterica serovar Infantis (S. Infantis) has become more prevalent across Europe with an increased capability to persist in broiler farms. In this study, we aimed to identify potential genetic causes for the increased emergence and longer persistence of S. Infantis in German poultry farms by high-throughput-sequencing. Broiler derived S. Infantis strains from two decades, the 1990s (n = 12) and the 2010s (n = 18), were examined phenotypically and genotypically to detect potential differences responsible for increased prevalence and persistence. S. Infantis organisms were characterized by serotyping and determining antimicrobial susceptibility using the microdilution method. Genotypic characteristics were analyzed by whole genome sequencing (WGS) to detect antimicrobial resistance and virulence genes as well as plasmids. To detect possible clonal relatedness within S. Infantis organisms, 17 accessible genomes from previous studies about emergent S. Infantis were downloaded and analyzed using complete genome sequence of SI119944 from Israel as reference. In contrast to the broiler derived antibiotic-sensitive S. Infantis strains from the 1990s, the majority of strains from the 2010s (15 out of 18) revealed a multidrug-resistance (MDR) phenotype that encodes for at least three antimicrobials families: aminoglycosides [ant(3")-Ia], sulfonamides (sul1), and tetracyclines [tet(A)]. Moreover, these MDR strains carry a virulence gene pattern missing in strains from the 1990s. It includes genes encoding for fimbriae clusters, the yersiniabactin siderophore, mercury and disinfectants resistance and toxin/antitoxin complexes. In depth genomic analysis confirmed that the 15 MDR strains from the 2010s carry a pESI-like megaplasmid with resistance and virulence gene patterns detected in the emerged S. Infantis strain SI119944 from Israel and clones inside and outside Europe. Genotyping analysis revealed two sequence types (STs) among the resistant strains from the 2010s, ST2283 (n = 13) and ST32 (n = 2). The sensitive strains from the 1990s, belong to sequence type ST32 (n = 10) and ST1032 (n = 2). Therefore, this study confirms the emergence of a MDR S. Infantis pESI-like clone of ST2283 in German broiler farms with presumably high tendency of dissemination. Further studies on the epidemiology and control of S. Infantis in broilers are needed to prevent the transfer from poultry into the human food chain.

Complete genome and plasmid sequences of a multidrug-resistant Salmonella enterica subsp. enterica serovar Panama isolate from a German cattle farm.

We describe a rare isolate of Salmonella enterica subsp. enterica serovar Panama with an extended-spectrum ß-lactamase (ESBL) profile from a German cattle-fattening farm. Applying two next-generation sequencing methods we generated sequences of the genome as well as the plasmids; assembled the draft genome sequence of Salmonella enterica subsp. enterica serovar Panama isolate 18PM0209. Antimicrobial resistance genes, virulence-associated genes and plasmids were analyzed using bioinformatics. Occurrence of multidrug-resistant Salmonella serovars at cattle-fattening farms indicate the need of enhanced surveillance to prevent further spread of these organisms.

Efficacy of a competitive exclusion culture against extended-spectrum ß-lactamase-producing Escherichia coli strains in broilers using a seeder bird model.

BACKGROUND: Administration of a competitive exclusion culture (CE culture) has the potential to induce protective effects in very young chicks against caecal colonisation by EEC (= extended-spectrum ß-lactamases [ESBL] and AmpC-type [AmpC] beta-lactamases producing Escherichia coli). The study aimed to verify the protective capacity of a CE culture in broilers using the seeder bird model against EEC exposure of the chicks. RESULTS: Introduction of infected seeder birds resulted in rapid and strong caecal colonisation of four different EEC challenge strains tested in untreated contact broilers. Compared to controls the broilers pre-treated with the CE culture showed a considerable decrease in caecal load of different EEC challenge strains from about 3.0-3.5 log10 units (P < 0.05) on day 9 of life to 2.5-3.0 log10 units (P < 0.05) on day 37. A slightly higher protective level of the CE culture in layer birds than in broilers raises the question on reasons for possible differences in the efficacy of CE culture in broiler and layer breeds. Whether the diet's protein content has an impact on both normal intestinal flora composition and the efficacy of CE cultures against EEC or other pathogens remains open and needs further elucidation. CONCLUSIONS: Our findings suggest that CE cultures of undefined composition can be valuable to reduce the intestinal colonisation by EEC in newly hatched broilers.

Whole-Genome Sequence of Salmonella enterica subsp. diarizonae Serovar 61:k:1,5,(7) Strain 1569 (14PM0011), Isolated from German Sheep.

Here, we report the draft genome sequence of Salmonella enterica subsp. diarizonae serovar 61:k:1,5,(7) strain 1569, alternatively named 14PM0011, which is a common serovar in German sheep that is unrepresented in the databases and considered and described as being host adapted with low virulence.

Occurrence and characterisation of Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) in sheep in the federal state of Thuringia, Germany.

BACKGROUND: The occurrence of Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) (SASd) and other Salmonella organisms in sheep in the German federal state of Thuringia was examined for the first time. Pooled faecal samples from 90 flocks located in this state were monitored. RESULTS: Only SASd was detected in 74 (82.2%) out of the 90 sheep herds, other Salmonella serovars were not identified. A positive correlation was found between the flock size and the detection probability of SASd. Despite the agent's high prevalence, clinical symptoms of a disease exclusively due to SASd have not been observed. The SASd strains were characterised by macrorestriction analysis, antimicrobial testing and the biochemical profile. All strains were sensitive to 13 out of 14 antimicrobial substances and resistant to only sulfamethoxazole. The high number of macrorestriction groups of SASd strains indicated a low clonality of the serovar. CONCLUSIONS: Data from sheep derived foods and public health data in Germany strongly suggest that the significance of SASd for public health is considerably lower than that of serovars belonging to Salmonella enterica subspecies enterica. For this reason and because of the low disease-causing potential of SASd in sheep, it is worthwile to consider a reduction in ongoing activities from combating to monitoring serovar 61: k: 1, 5, (7) in the sheep population.

Salmonella enterica subspecies enterica serovar Choleraesuis in a German wild boar population: occurrence and characterisation.

BACKGROUND: The swine-adapted serovar Choleraesuis of Salmonella enterica subspecies enterica is found rarely in domestic pigs in Germany. However, a considerable and increasing number of S. Choleraesuis organisms have been isolated from wild boars in Germany in recent years. To investigate a possible epidemiological context, S. Choleraesuis strains from a regional German wild boar population and other hosts were characterised by genotyping methods. RESULTS: Macrorestriction analysis, biochemical differentiation and antimicrobial susceptibility typing enabled the identification of several clusters of S. Choleraesuis. Some clusters occurred almost permanently in a certain district, whereas other groups circulated among different wild boar herds in larger regions. Non-porcine hosts were infected with the same cluster as the wild boars. CONCLUSIONS: The emergence of S. Choleraesuis in wild boars might be caused by a higher prevalence in the wild boar population, but also the higher awareness to infections with African swine fever may have resulted in a higher number of examined animals. Separation of wild boar populations and, as a result, also the diverse S. Choleraesuis organisms might be due to natural barriers and artificial barriers like arterial roads. The findings of S. Choleraesuis in domestic pigs emphasize the importance of strict biosecurity measures to prevent transmission from wild boars of this but also other pathogens. To avoid risks for humans by zoonotic pathogens regular inspections of meat from wildlife need to be conducted.

In vitro and in vivo generation of heterophil extracellular traps after Salmonella exposure.

The release of extracellular traps (ETs) by granulocytes is a unique strategy to stop the dissemination of microbial pathogens. This study was undertaken to elucidate the potential of avian granulocytes (heterophils) to form ETs that can arrest and kill Salmonella organisms. After in vitro exposure of isolated heterophils and in vivo infection of day-old chicks with Salmonella enterica subsp. enterica serovars Infantis (SI) or Enteritidis (SE), the generation of ETs as well as the trapping and survivability of Salmonella organisms in the ET meshwork were determined by means of microscopy and spectrophotometry. In vitro, heterophils were able to form ETs within 15min after SE and SI inoculation. At 120min and with a multiplicity of infection of 1 and 5, SI induced significantly more ETs and DNA release than SE. Both SE and SI were found to be associated with the ET structures. Live-dead staining showed most of the microorganisms within the extracellular scaffold alive. In vivo, heterophils were detected in cecal lumen of SE-, but not SI-infected chicks. In cecum of the SE-exposed chicks, ET formations were scarcely detected whereas intact heterophils with phagocytosed bacteria were frequently found. The results evidence the capability of heterophils to generate ETs after SE and SI exposure in vitro. However, an infection of chicks with Salmonella did not significantly induce the formation of ET structures in cecum. Thus, the process to form ETs (ETosis) seems not to be of special relevance for Salmonella defense within the cecal lumen of young chicks.

Avian CD25(+) gamma/delta (γδ) T cells after Salmonella exposure.

Although an expansion of distinct γδ T-lymphocyte subpopulations has been shown after Salmonella infection of chicks, functions and activation states of these cells are still largely unknown. To characterize the activation status of avian γδ T lymphocytes after Salmonella exposure, an avian whole-blood Salmonella Enteritidis live-vaccine-strain (SE-LV) stimulation assay and flow cytometry were used, and the bacteria-provoked CD25-antigen expression of CD8-characterized γδ T-cell subsets determined in comparison to αß T cells. In non-stimulated controls, low numbers of CD25-positive cells were observed in all T-cell populations. The SE-LV stimulation resulted in a significantly enhanced percentage of CD25-expressing CD8α(-), CD8α(lo+) and CD8αα(hi+) γδ T cells accompanied by an increased intensity of the CD25 expression of these cells. Highest values were always seen within the CD8αα(hi+) γδ T-lymphocyte subset. The αß T cells showed no changed CD25 expression. In conclusion, the increased CD25 expression on discrete peripheral T-lymphocyte populations after ex vivo stimulation demonstrates a Salmonella-induced activation of γδ T-cell subsets and underlines the unique, probably activated character, especially of the avian CD8αα(hi+) γδ T cells.

Immune reaction and survivability of salmonella typhimurium and salmonella infantis after infection of primary avian macrophages.

Salmonella serovars are differentially able to infect chickens. The underlying causes are not yet fully understood. Aim of the present study was to elucidate the importance of Salmonella Pathogenicity Island 1 and 2 (SPI-1 and -2) for the virulence of two non-host-specific, but in-vivo differently invasive, Salmonella serovars in conjunction with the immune reaction of the host. Primary avian splenic macrophages were inoculated with Salmonella enterica sub-species enterica serovar (S.) Typhimurium and S. Infantis. The number and viability of intracellular bacteria and transcription of SPI-1 and -2 genes by the pathogens, as well as transcription of immune-related proteins, surface antigen expression and nitric oxide production by the macrophages, were compared at different times post inoculation. After infection, both of the Salmonella serovars were found inside the primary macrophages. Invasion-associated SPI-1 genes were significantly higher transcribed in S. Infantis- than S. Typhimurium-infected macrophages. The macrophages counteracted the S. Infantis and S. Typhimurium infection with elevated mRNA expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-12, IL-18 and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) as well as with an increased synthesis of nitric oxide. Despite these host cell attacks, S. Typhimurium was better able than S. Infantis to survive within the macrophages and transcribed higher rates of the SPI-2 genes spiC, ssaV, sifA, and sseA. The results showed similar immune reactions of primary macrophages after infection with both of the Salmonella strains. The more rapid and stronger transcription of SPI-2-related genes by intracellular S. Typhimurium compared to S. Infantis might be responsible for its better survival in avian primary macrophages.