Single-cell RNA-seq mapping of chicken peripheral blood leukocytes.

BACKGROUND: Single-cell transcriptomics provides means to study cell populations at the level of individual cells. In leukocyte biology this approach could potentially aid the identification of subpopulations and functions without the need to develop species-specific reagents. The present study aimed to evaluate single-cell RNA-seq as a tool for identification of chicken peripheral blood leukocytes. For this purpose, purified and thrombocyte depleted leukocytes from 4 clinically healthy hens were subjected to single-cell 3' RNA-seq. Bioinformatic analysis of data comprised unsupervised clustering of the cells, and annotation of clusters based on expression profiles. Immunofluorescence phenotyping of the cell preparations used was also performed. RESULTS: Computational analysis identified 31 initial cell clusters and based on expression of defined marker genes 28 cluster were identified as comprising mainly B-cells, T-cells, monocytes, thrombocytes and red blood cells. Of the remaining clusters, two were putatively identified as basophils and eosinophils, and one as proliferating cells of mixed origin. In depth analysis on gene expression profiles within and between the initial cell clusters allowed further identification of cell identity and possible functions for some of them. For example, analysis of the group of monocyte clusters revealed subclusters comprising heterophils, as well as putative monocyte subtypes. Also, novel aspects of TCRγ/δ + T-cell subpopulations could be inferred such as evidence of at least two subtypes based on e.g., different expression of transcription factors MAF, SOX13 and GATA3. Moreover, a novel subpopulation of chicken peripheral B-cells with high SOX5 expression was identified. An overall good correlation between mRNA and cell surface phenotypic cell identification was shown. CONCLUSIONS: Taken together, we were able to identify and infer functional aspects of both previously well known as well as novel chicken leukocyte populations although some cell types. e.g., T-cell subtypes, proved more challenging to decipher. Although this methodology to some extent is limited by incomplete annotation of the chicken genome, it definitively has benefits in chicken immunology by expanding the options to distinguish identity and functions of immune cells also without access to species specific reagents.

Genome characteristics related to the virulence of Streptococcus suis in Swedish pigs.

The impact of S. suis on Swedish pig production has increased in recent years, and characterization of the strains present in the pig population is needed to aid in surveillance and prevention. Therefore, the aim of this study was to identify and characterize differences in the genomes between Swedish S. suis isolates associated with disease and isolates from healthy animals. Isolates categorized as being pathogenic (n = 100) or non-pathogenic (n = 117) were whole-genome sequenced, serotyped in silico, and sequence-typed using traditional MLST and core-genome MLST, and a genome-wide association study was performed to identify virulence-associated genes. In decreasing order, serotypes 2, 1, and 7 were the most common in the pathogenic group, and serotypes 15 and 12 were the most common in the non-pathogenic group. Among the commonly disease-associated sequence types, ST28 and ST25 were identified, whereas ST1 was scarcely found. The majority of isolates belonged to novel sequence types, revealing differences between Swedish isolates and those reported from other countries. The genomes of the pathogenic isolates were on average smaller and less heterogenic as compared to those of the non-pathogenic isolates. Although a majority of the previously published virulence-associated genes included in the study were found in the genomes of both pathogenic and non-pathogenic isolates, several new, significantly virulence-associated genes were identified.

An investigation of household dogs as the source in a case of human bacteraemia caused by Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is a commensal and an opportunistic pathogen in dogs, and is also an opportunistic pathogen in humans. Here we report about a case of bacteraemia with a fatal outcome in a 77-year-old co-morbid male likely caused by a S. pseudintermedius and the investigation into the possible transmission from the two dogs in the patient's household. The two dogs carried the same S. pseudintermedius strain, but this dog strain was unrelated to the strain from the patient. In contrast to the patient strain, the dog strain showed reduced susceptibility to several antibiotics and both dogs had received antibiotic treatment prior to sampling. So, it is conceivable that these treatments can have eliminated the patient's strain between the transmission event and the dog sampling. It is also worth noting that the patient strain was positive for the expA gene, which encodes an exfoliative toxin closely related to the S. aureus exfoliative toxin B. This toxin has been linked to canine pyoderma, but its effect on humans remains unknown. Transmission of S. pseudintermedius was confirmed in the household between the dogs. However, we could not verify that the dogs were the source for the S. pseudintermedius in the patient.

Shiga toxin-producing Escherichia coli (STEC) and atypical enteropathogenic E. coli (aEPEC) in Swedish retail wheat flour.

Wheat flour has been identified as the source of multiple outbreaks of gastrointestinal disease caused by shiga toxin-producing Escherichia coli (STEC). We have investigated the presence and genomic characteristics of STEC and related atypical enteropathogenic E. coli (aEPEC) in 200 bags of Swedish-produced retail wheat flour, representing 87 products and 25 brands. Samples were enriched in modified tryptone soya broth (mTSB) and screened with real-time PCR targeting stx1, stx2 and eae, and the serogroups O157, O121 and O26. Isolation was performed by immunomagnetic separation (IMS) for suspected STEC/aEPEC O157, O121 and O26, and by screening pools of colonies for other STEC. Real-time PCR after enrichment revealed 12 % of samples to be positive for shiga toxin genes (stx1 and/or stx2) and 11 % to be positive for intimin (eae). Organic production, small-scale production or whole grain did not significantly influence shiga toxin gene presence or absence in a generalized linear mixed model analysis. Eight isolates of STEC were recovered, all of which were intimin-negative. Multiple serotype/sequence type/shiga toxin subtype combinations that have also been found in flour samples in other European countries were recovered. Most STEC types recovered were associated with sporadic cases of STEC among humans in Sweden, but no types known to have caused outbreaks or severe cases of disease (i.e. haemolytic uraemic syndrome) were found. The most common finding was O187:H28 ST200 with stx2g, with possible links to cervid hosts. Wildlife associated with crop damage is a plausible explanation for at least some of the surprisingly high frequency of STEC in wheat flour.

Dissemination of carbapenemase-producing Enterobacterales through wastewater and gulls at a wastewater treatment plant in Sweden.

Here we report the detection of carbapenemase-producing Enterobacterales (CPE) isolated from Swedish wastewater and gull faeces. CPE have not been detected in samples from animals in Sweden preceding this report. Sampling of wastewater treatment plant (WWTP) inlet and outlet, sedimentation basins, surface seawater from key aquatic bird habitats and freshly deposited gull faeces was done on six separate occasions during May to September 2021. Following broth enrichment, selective screening of putative CPE was performed on mSuperCarba™ (CHROMagar). Species identification was done with MALDI-TOF. Antimicrobial susceptibility testing was performed according to EUCAST. In total, seventeen CPE were verified by genome sequencing carrying blaGES-5, blaIMI-3, blaOXA-181 or blaOXA-244. The blaGES-5 was carried on IncP plasmids in four different species; Escherichia coli ST10 isolated from WWTP outlet, Raoultella ornithinolytica isolated from WWTP inlet, outlet and sedimentation basins as well as gull faeces collected at the WWTP and Klebsiella spp. isolates from WWTP inlet and outlet. The genetic environment surrounding blaGES-5 was similar in two Citrobacter freundii causing human infections. The blaIMI-3 was carried on IncFII(Yp) plasmids in four Enterobacter ludwigii, isolated from WWTP outlet and gull faeces collected at a recreational city park 2 km from the WWTP. The blaOXA-181 was located on a COLKP3 plasmid found in an E. coli, while blaOXA-244 was chromosomally located in an E. coli ST10, both isolated from WWTP inlet. Phylogenetic analysis of R. ornithinolytica and E. ludwigii isolates indicate that the gulls carried strains related to those identified in the WWTP samples. The results thus add to the increasing evidence of WWTPs as anthropogenic reservoirs for mobile genetic elements with antibiotic-resistance functionality. Such environments could profoundly impact the dissemination and spread of such genetic elements via for example aquatic birds, thereby warranting further study and surveillance.

Core genome multilocus sequence typing (cgMLST) confirms systemic spread of avian pathogenic Escherichia coli (APEC) in broilers with cellulitis.

Broiler cellulitis has emerged as an important cause of economic losses for farmers and slaughter plants from carcass condemnation at processing. Avian pathogenic Escherichia coli (APEC) has been identified as the main causative agent. The aim was to characterize E. coli isolated from cellulitis and organs in broilers at slaughter by whole genome sequencing analysis to study if systemic spread could be confirmed. Isolates were collected post-mortem from 101 carcasses condemned due to dermatitis/cellulitis from five commercial farms and six flocks. Forty-six isolates were characterised to determine serotypes, sequence types and virulence-associated genes. Analysis by cgMLST was performed to study the genetic similarity between isolates from the same broiler, among birds from the same flock and between flocks. Escherichia coli was isolated from 90% of birds from subcutaneous samples. In 20 broilers, E. coli was isolated from organs in pure culture or mixed with sparse growth of other bacteria. In eight of these, there were post-mortem findings suggestive of systemic bacterial spread. The majority of the isolates from the same bird and flock belonged to the same serotype and sequence type and were genetically indistinguishable, but differed when compared between flocks. Common APEC virulence genes, i.e. chuA, fyuA, hlyF, iroN, irp2, iss, ompT, sitA, TerC, TraT, were present in > 87% of the isolates. We conclude that evidence of systemic spread of E. coli from cellulitis was present in some birds at time of slaughter but cannot be reliably detected at meat inspection.

Characterization of the bacterial microbiome of Swedish ticks through 16S rRNA amplicon sequencing of whole ticks and of individual tick organs.

BACKGROUND: The composition of the microbial flora associated with ixodid ticks has been studied in several species, revealing the importance of geographical origin, developmental stage(s) and feeding status of the tick, as well as substantial differences between tissues and organs. Studying the microbiome in the correct context and scale is therefore necessary for understanding the interactions between tick-borne pathogens and other microorganisms as well as other aspects of tick biology. METHODS: In the present study the microbial flora of whole Ixodes ricinus, I. persulcatus and I. trianguliceps ticks were analyzed with 16S rRNA amplicon sequencing. Additionally, tick organs (midguts, Malpighian tubules, ovaries, salivary glands) from flat and engorged I. ricinus female ticks were examined with the same methodology. RESULTS: The most abundant bacteria belonged to the group of Proteobacteria (Cand. Midichloria mitochondrii and Cand. Lariskella). 16S amplicon sequencing of dissected tick organs provided more information on the diversity of I. ricinus-associated microbial flora, especially when organs were collected from engorged ticks. Bacterial genera significantly associated with tick feeding status as well as genera associated with the presence of tick-borne pathogens were identified. CONCLUSIONS: These results contribute to the knowledge of microbial flora associated with ixodid ticks in their northernmost distribution limit in Europe and opens new perspectives for other investigations on the function of these bacteria, including those using other approaches like in vitro cultivation and in vitro models.

Erysipelothrix rhusiopathiae-specific T-cell responses after experimental infection of chickens selectively bred for high and low serum levels of mannose-binding lectin.

Erysipelas, caused by infection with Erysipelothrix rhusiopathiae (ER) is an important emerging disease in laying hens. We have earlier observed prominent mannose-binding lectin (MBL) acute phase responses in experimentally ER infected chickens. The present study aimed to further examine immune responses to ER by using chickens selectively bred for high (L10H) and low (L10L) serum MBL levels. Chickens were infected with ER at 3 weeks of age and immune parameters and bacterial load were monitored in blood until day 18 after infection. Blood and spleen leukocytes collected on day 18 were stimulated in vitro with ER antigens and blast transformation of different T-cell populations was assessed. The ER infection gave a very varied outcome and no clear differences were observed between L10H and L10L chickens with respect to leukocyte counts, bacterial load or clinical outcome. Nonetheless, rapid innate responses, e.g., heterophilia and increased serum MBL levels were noted in bacteraemic chickens. All ER infected chickens also showed transient increased expression of mannose receptor MRC1L-B and decreased expression of major histocompatibility complex II on monocytes day 1 after infection indicating monocyte activation or relocation. In vitro ER stimulation showed antigen specific blast transformation of CD4+, TCRγ/δ-CD8αß+ and TCRγ/δ+CD8αß+ spleen cells from all infected chickens. For CD4+ and TCRγ/δ-CD8αß+ cells the proportions of blast transformed cells were significantly higher for samples from L10L chickens than those for samples from L10H chickens. This is the first observation of ER-specific T-cells in chickens and interestingly a Th1-type response comprising cytotoxic T-cells was indicated.

Genomic signatures of host adaptation in group B Salmonella enterica ST416/ST417 from harbour porpoises.

A type of monophasic group B Salmonella enterica with the antigenic formula 4,12:a:- ("Fulica-like") has been described as associated with harbour porpoises (Phocoena phocoena), most frequently recovered from lung samples. In the present study, lung tissue samples from 47 porpoises found along the Swedish coast or as bycatch in fishing nets were analysed, two of which were positive for S. enterica. Pneumonia due to the infection was considered the likely cause of death for one of the two animals. The recovered isolates were whole genome sequenced and found to belong to sequence type (ST) 416 and to be closely related to ST416/ST417 porpoise isolates from UK waters as determined by core-genome MLST. Serovars Bispebjerg, Fulica and Abortusequi were identified as distantly related to the porpoise isolates, but no close relatives from other host species were found. All ST416/417 isolates had extensive loss of function mutations in key Salmonella pathogenicity islands, but carried accessory genetic elements associated with extraintestinal infection such as iron uptake systems. Gene ontology and pathway analysis revealed reduced secondary metabolic capabilities and loss of function in terms of signalling and response to environmental cues, consistent with adaptation for the extraintestinal niche. A classification system based on machine learning identified ST416/417 as more invasive than classical gastrointestinal serovars. Genome analysis results are thus consistent with ST416/417 as a host-adapted and extraintestinal clonal population of S. enterica, which while found in porpoises without associated pathology can also cause severe opportunistic infections.

Dual RNA-seq transcriptome analysis of caecal tissue during primary Eimeria tenella infection in chickens.

BACKGROUND: Coccidiosis is an infectious disease with large negative impact on the poultry industry worldwide. It is an enteric infection caused by unicellular Apicomplexan parasites of the genus Eimeria. The present study aimed to gain more knowledge about interactions between parasites and the host immune system during the early asexual replication phase of E. tenella in chicken caeca. For this purpose, chickens were experimentally infected with E. tenella oocysts, sacrificed on days 1-4 and 10 after infection and mRNA from caecal tissues was extracted and sequenced. RESULTS: Dual RNA-seq analysis revealed time-dependent changes in both host and parasite gene expression during the course of the infection. Chicken immune activation was detected from day 3 and onwards with the highest number of differentially expressed immune genes recorded on day 10. Among early (days 3-4) responses up-regulation of genes for matrix metalloproteinases, several chemokines, interferon (IFN)-γ along with IFN-stimulated genes GBP, IRF1 and RSAD2 were noted. Increased expression of genes with immune suppressive/regulatory effects, e.g. IL10, SOCS1, SOCS3, was also observed among early responses. For E. tenella a general up-regulation of genes involved in protein expression and energy metabolism as well as a general down-regulation genes for DNA and RNA processing were observed during the infection. Specific E. tenella genes with altered expression during the experiment include those for proteins in rhoptry and microneme organelles. CONCLUSIONS: The present study provides novel information on both the transcriptional activity of E. tenella during schizogony in ceacal tissue and of the local host responses to parasite invasion during this phase of infection. Results indicate a role for IFN-γ and IFN-stimulated genes in the innate defence against Eimeria replication.

Whole-Genome Sequencing Evaluation of MALDI-TOF MS as a Species Identification Tool for Streptococcus suis.

Streptococcus suis is an important bacterial pathogen in pigs that may also cause zoonotic disease in humans. The aim of the study was to evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification of S. suis case isolates from diseased pigs and tonsil isolates from healthy pigs and wild boar using sequence analysis methods. Isolates (n = 348) that had been classified as S. suis by MALDI-TOF MS were whole-genome sequenced and investigated using analyses of (i) the 16S rRNA gene, (ii) the recN gene, and (iii) whole-genome average nucleotide identity (ANI). Analysis of the 16S rRNA gene indicated that 82.8% (288 out of 348) of the isolates were S. suis, while recN gene analysis indicated that 75.6% (263 out of 348) were S. suis. ANI analysis classified 44.3% (154 out of 348) as S. suis. In total, 44% (153 out of 348) of the investigated isolates were classified as S. suis by all of the species identification methods employed. The mean MALDI-TOF MS score was significantly higher for the S. suis case isolates than for the tonsil isolates; however, the difference is of limited practical use. The results show that species confirmation beyond MALDI-TOF MS is needed for S. suis isolates. Since the resolution of 16S rRNA gene analysis is too low for Streptococcus spp., ANI analysis with a slightly lowered cutoff of 94% may be used instead of, or in addition to, recN gene analysis. Supplementation of the MALDI-TOF MS reference library with mass spectra from S. orisratti, S. parasuis, S. ruminantium, and additional S. suis serotypes should be considered in order to produce more accurate classifications.

Benzylpenicillin-producing Trichophyton erinacei and methicillin resistant Staphylococcus aureus carrying the mecC gene on European hedgehogs - A pilot-study.

BACKGROUND: A high carriage rate of methicillin-resistant Staphylococcus aureus with the mecC gene (mecC-MRSA) has been described among Wild European hedgehogs (Europeaus erineaus). Due to this frequent occurrence, it has been suggested that hedgehogs could be a natural reservoir for mecC-MRSA. However, the reason why hedgehogs carry mecC-MRSA remains unknown, but it has been hypothesized that mecC-MRSA could have evolved on the skin of hedgehogs due to the co-occurrence with antibiotic producing dermatophytes. The aim of this pilot-study was therefore to investigate if hedgehogs in Sweden carry Trichophyton spp. and to provide evidence that these dermatophytes are able to produce penicillin or similar substances. In addition, the study aimed to identify if dermatophytes co-occurred with mecC-MRSA. METHODS: Samples were collected from hedgehogs (Europeaus erineaus) that were euthanized or died of natural causes. All samples were screened for dermatophytes and mecC-MRSA using selective cultivation methods. Suspected isolates were characterized using PCR-based methods, genome sequencing and bioinformatic analyses. Identification of penicillin was performed by ultra-high-performance liquid chromatography-tandem mass spectrometry. RESULTS: In total 23 hedgehogs were investigated, and it was shown that two carried Trichophyton erinacei producing benzyl-penicillin, and that these hedgehogs also carried mecC-MRSA. The study also showed that 60% of the hedgehogs carried mecC-MRSA. CONCLUSION: The pilot-study demonstrated that Trichophyton erinacei, isolated from Swedish hedgehogs, can produce benzylpenicillin and that these benzylpenicillin-producing T. erinacei co-occurred with mecC-MRSA. The study also reconfirmed the high occurrence of mecC-MRSA among hedgehogs.

Cryptosporidium chipmunk genotype I - An emerging cause of human cryptosporidiosis in Sweden.

Most cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or Cryptosporidium hominis. However, more uncommon species are increasingly being recognised to cause infection in humans. Here we report that Cryptosporidium chipmunk genotype I, which has various rodents as its natural host, is the third most common source of human cryptosporidiosis in Sweden. We also describe the first small outbreak of cryptosporidiosis caused by Cryptosporidium chipmunk genotype I and report the first case of zoonotic transmission of Cryptosporidium chipmunk genotype I from a red squirrel to a human. Cryptosporidium chipmunk genotype I was identified in 20 human cases, including 16 sporadic cases, three outbreak-related cases, and one zoonotic case, as well as in two squirrel samples. Gp60 subtyping which was successful for 19 human cases and two squirrel samples showed that all samples harboured the same subtype, XIVaA20G2T1. The work presented here suggests that red squirrel is a natural host of Cryptosporidium chipmunk genotype I and that infection with Cryptosporidium chipmunk genotype I is an emerging cause of domestic cryptosporidiosis in Sweden and a potential source of outbreaks.

Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella.

The study aimed to monitor parasite and host gene expression during the early stages of Eimeria tenella infection of chicken cells using dual RNA-Seq analysis. For this, we used chicken macrophage-like cell line HD11 cultures infected in vitro with purified E. tenella sporozoites. Cultures were harvested between 2 and 72 h post-infection and mRNA was extracted and sequenced. Dual RNA-Seq analysis showed clear patterns of altered expression for both parasite and host genes during infection. For example, genes in the chicken immune system showed upregulation early (2­4 h), a strong downregulation of genes across the immune system at 24 h and a repetition of early patterns at 72 h, indicating that invasion by a second generation of parasites was occurring. The observed downregulation may be due to immune self-regulation or to immune evasive mechanisms exerted by E. tenella. Results also suggested pathogen recognition receptors involved in E. tenella innate recognition, MRC2, TLR15 and NLRC5 and showed distinct chemokine and cytokine induction patterns. Moreover, the expression of several functional categories of Eimeria genes, such as rhoptry kinase genes and microneme genes, were also examined, showing distinctive differences which were expressed in sporozoites and merozoites.

Immune responses upon experimental Erysipelothrix rhusiopathiae infection of naïve and vaccinated chickens.

Erysipelas, a disease caused by Erysipelothrix rhusiopathiae (ER), is an increasing problem in laying hens housed in cage-free systems. This study aimed to monitor immune responses during ER infection of naïve chickens and chickens vaccinated intra muscularly with a commercial inactivated ER vaccine. Chickens were infected intra muscularly with ER at 30 days of age and blood leukocyte counts, serum levels of mannose binding lectin (MBL) and ER-specific IgY were monitored until the experiment was terminated at day 15 after infection. ER was detected in blood from more chickens and at higher bacterial counts in the naïve group (day 1: 1 of 7 chickens; day 3: 6 of 6 chickens) than in the vaccinated group (day 1: 0 of 7 chickens; day 3: 1 of 6 chickens). During the acute phase of infection transient increases in circulating heterophil numbers and serum MBL levels were detected in all ER infected chickens but these responses were prolonged in chickens from the naïve group compared to vaccinated chickens. Before infection IgY titers to ER in vaccinated chickens did not differ significantly from those of naïve chickens but vaccinated chickens showed significantly increased IgY titers to ER earlier after infection compared to chickens in the naïve group. In conclusion, the ER infection elicited prompt acute innate responses in all chickens. Vaccinated chickens did not have high IgY titers to ER prior to infection but did however show lower levels of bacteraemia and their acute immune responses were of shorter duration.

Comparative genome analysis of Erysipelothrix rhusiopathiae isolated from domestic pigs and wild boars suggests host adaptation and selective pressure from the use of antibiotics.

The disease erysipelas caused by Erysipelothrix rhusiopathiae (ER) is a major concern in pig production. In the present study the genomes of ER from pigs (n=87), wild boars (n=71) and other sources (n=85) were compared in terms of whole-genome SNP variation, accessory genome content and the presence of genetic antibiotic resistance determinants. The aim was to investigate if genetic features among ER were associated with isolate origin in order to better estimate the risk of transmission of porcine-adapted strains from wild boars to free-range pigs and to increase our understanding of the evolution of ER. Pigs and wild boars carried isolates representing all ER clades, but clade one only occurred in healthy wild boars and healthy pigs. Several accessory genes or gene variants were found to be significantly associated with the pig and wild boar hosts, with genes predicted to encode cell wall-associated or extracellular proteins overrepresented. Gene variants associated with serovar determination and capsule production in serovars known to be pathogenic for pigs were found to be significantly associated with pigs as hosts. In total, 30 % of investigated pig isolates but only 6 % of wild boar isolates carried resistance genes, most commonly tetM (tetracycline) and lsa(E) together with lnu(B) (lincosamides, pleuromutilin and streptogramin A). The incidence of variably present genes including resistance determinants was weakly linked to phylogeny, indicating that host adaptation in ER has evolved multiple times in diverse lineages mediated by recombination and the acquisition of mobile genetic elements. The presented results support the occurrence of host-adapted ER strains, but they do not indicate frequent transmission between wild boars and domestic pigs. This article contains data hosted by Microreact.

The prevalence and genomic context of Shiga toxin 2a genes in E. coli found in cattle.

Shiga toxin-producing Escherichia coli (STEC) that cause severe disease predominantly carry the toxin gene variant stx2a. However, the role of Shiga toxin in the ruminant reservoirs of this zoonotic pathogen is poorly understood and strains that cause severe disease in humans (HUSEC) likely constitute a small and atypical subset of the overall STEC flora. The aim of this study was to investigate the presence of stx2a in samples from cattle and to isolate and characterize stx2a-positive E. coli. In nationwide surveys in Sweden and Norway samples were collected from individual cattle or from cattle herds, respectively. Samples were tested for Shiga toxin genes by real-time PCR and amplicon sequencing and stx2a-positive isolates were whole genome sequenced. Among faecal samples from Sweden, stx1 was detected in 37%, stx2 in 53% and stx2a in 5% and in skin (ear) samples in 64%, 79% and 2% respectively. In Norway, 79% of the herds were positive for stx1, 93% for stx2 and 17% for stx2a. Based on amplicon sequencing the most common stx2 types in samples from Swedish cattle were stx2a and stx2d. Multilocus sequence typing (MLST) of 39 stx2a-positive isolates collected from both countries revealed substantial diversity with 19 different sequence types. Only a few classical LEE-positive strains similar to HUSEC were found among the stx2a-positive isolates, notably a single O121:H19 and an O26:H11. Lineages known to include LEE-negative HUSEC were also recovered including, such as O113:H21 (sequence type ST-223), O130:H11 (ST-297), and O101:H33 (ST-330). We conclude that E. coli encoding stx2a in cattle are ranging from strains similar to HUSEC to unknown STEC variants. Comparison of isolates from human HUS cases to related STEC from the ruminant reservoirs can help identify combinations of virulence attributes necessary to cause HUS, as well as provide a better understanding of the routes of infection for rare and emerging pathogenic STEC.

Linked seasonal outbreaks of Salmonella Typhimurium among passerine birds, domestic cats and humans, Sweden, 2009 to 2016.

In 2016, an outbreak of Salmonella Typhimurium (STm) with multilocus variable-number tandem repeat analysis (MLVA) profiles historically associated with passerine birds (2-[11-15]-[3-4]-NA-212) occurred among passerines, cats and humans in Sweden. Our retrospective observational study investigated the outbreak and revisited historical data from 2009-16 to identify seasonality, phylogeography and other characteristics of this STm variant. Outbreak isolates were analysed by whole-genome single nucleotide polymorphism (SNP) typing. The number of notified cases of passerine-associated STm among passerines, cats and humans per month and county, and their MLVA profiles, were compared to birdwatchers' counts of passerines. Seasonal trend decomposition and correlation analysis was performed. Outbreak isolates did not cluster by host on SNP level. Passerine-associated STm was seasonal for birds, cats and humans, with a peak in March. Cases and counts of passerines at bird feeders varied between years. The incidence of passerine-associated STm infections in humans was higher in the boreal north compared with the southern and capital regions, consistent with passerine population densities. Seasonal mass migration of passerines appears to cause STm outbreaks among cats certain years in Sweden, most likely via predation on weakened birds. Outbreaks among humans can follow, presumably caused by contact with cats or environmental contamination.

Risk factors and dynamics of verotoxigenic Escherichia coli O157:H7 on cattle farms: An observational study combining information from questionnaires, spatial data and molecular analyses.

The increasing number of human cases infected with a highly virulent type of verotoxigenic Escherichia coli (VTEC) O157:H7 in Sweden is the result of domestic transmission originating in regional clusters of infected cattle farms. To control the spread of the bacteria a comprehensive picture of infection dynamics, routes of transmission between farms and risk factors for persistence is urgently needed. The aim of the study was to investigate different aspects of the epidemiology of VTEC O157:H7 on the Swedish island of Öland by combining information from environmental sampling of VTEC O157:H7 from 80 farms with information from farmer questionnaires, spatial and molecular analyses. The farms were sampled in the spring and fall of 2014 and on four of them additional samples were collected during summer and winter. The results show a high prevalence of VTEC O157:H7 and a high proportion of strains belonging to the virulent clade 8. Farms that became infected between samplings were all located in an area with high cattle density. The most important risk factors identified are generally associated with biosecurity and indicate that visitors travelling between farms may be important for transmission. In addition, whole genome sequencing of a subset of isolates from the four farms where additional sampling was performed revealed ongoing local transmission that cannot be observed with a lower resolution typing method. Our observations also show that VTEC O157:H7 may persist in the farm environment for extended periods of time, suggesting that specific on-farm measures to reduce environmental prevalence and spread between groups of animals may be required in these cases.

Detection and quantification of Erysipelothrix rhusiopathiae in blood from infected chickens - addressing challenges with detection of DNA from infectious agents in host species with nucleated red blood cells.

PURPOSE: The present study aimed to establish pretreatment protocols as well as real-time and droplet digital polymerase chain reaction (PCR) methodologies to detect and quantify Erysipelothrix rhusiopathiae (ER) DNA in blood samples from infected chickens, as tools for routine diagnostics and monitoring of experimental infections. Chicken blood is a problematic matrix for PCR analysis because nucleated erythrocytes contribute large amounts of host DNA that inhibit amplification. METHODOLOGY: Using artificially spiked samples of fresh chicken blood, as well as blood samples from three experimental infection studies, the performance of pretreatment protocols, including choice of blood stabilization agent, centrifugation speeds and Ficoll gradient separation, was evaluated. The results were compared with those from traditional culture-based protocols combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Results/Key findings. Simple preparations producing cell-free samples performed well on artificial spike-in samples, providing high sensitivity. However, performance was poor in clinical samples or artificial samples where the bacteria were incubated for 4 h or more in fresh blood prior to DNA extraction. In these samples, a Ficoll separation protocol that creates samples rich in lymphocytes, monocytes and thrombocytes prior to DNA extraction was far more effective. CONCLUSIONS: Our results indicate that ER bacteria undergo rapid phagocytosis in chicken blood and that analysis of a blood fraction enriched for phagocytic cells is necessary for reliable detection and quantification. The presented results explain the poor performance of PCR detection reported in previously published experimental ER infection studies, and the proposed solutions are likely to have broader implications for PCR-based veterinary diagnostics in non-mammalian host species such as poultry and fish.