Molecular detection of Aphanomyces astaci - An improved species specific qPCR assay.

The parasitic oomycete Aphanomyces astaci is the causative agent of crayfish plague, a devastating disease for European freshwater crayfish. Species specific quantitative real-time PCR (qPCR) can offer rapid detection of the pathogen. However, the well established A. astaci qPCR assay recommended by the World Organization for Animal Health (WOAH) amplifies the recently described Aphanomyces fennicus. Consequently, false-positive results may occur. This calls for the improvement of the established species specific A. astaci qPCR assay in order to avoid amplifying A. fennicus while screening for A. astaci. We developed an improved species specific A. astaci qPCR assay and validated the assay across three laboratories, using established procedures including different qPCR master mixes for each respective laboratory. Genomic DNA from A. astaci, A. fennicus and closely related Aphanomyces spp. was analysed and compared with both the improved and established assay. Additionally, DNA from crayfish tissue and environmental samples were analysed with both assays. The improved assay showed similar sensitivity with the established assay for all sample types, while proving highly specific for A. astaci avoiding amplification of A. fennicus and the other tested Aphanomyces spp. Environmental DNA (eDNA) samples collected at River Lierelva in Norway amplified with the established assay, but not with the improved assay indicating false positive. We were able to sequence a 530 bp fragment of the ITS region from these eDNA samples and the consensus sequence showed 99.9-100 % pairwise identity with A. fennicus and 97.2-98 % pairwise identity with A. astaci, suggesting that the occurrence of A. fennicus is not limited to Finland, where it was first discovered.

Non-lethal sampling for the detection of Renibacterium salmoninarum by qPCR for diagnosis of bacterial kidney disease.

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), can be transmitted both horizontally and vertically and there is no available cure or prophylaxis. The control of BKD requires continuous surveillance, which is challenging in aquaculture as well as in programs for conservation and restoration of salmonid fish strains. BKD is a notifiable disease in Sweden and is monitored through the mandatory health control program using a polyclonal ELISA for detection of the Rs p57 protein in kidney. Fish must be killed for sampling, an obvious disadvantage especially regarding valuable broodfish. The present study shows that gill-/cloacal swabs collected in vivo for real-time PCR (qPCRgc ), allow a sensitive and specific detection of Rs. The sensitivity of qPCRgc was estimated to 97.8% (credible interval (ci) 93.8%-100%) compared to 98.3% (ci 92.7%-100%) and 48.8% (ci 38.8%-58.8%) of kidney samples for qPCR (qPCRk ) and ELISA (ELISAk ) respectively, by use of the Bayesian Latent Class Analysis (BLCA). Since the goal of the program is eradication of BKD the most sensitive test is preferrable. Using qPCRgc instead of ELISAk will result in a lower false negative rate and can be useful for surveillance in aquaculture and in breeding programs with valuable fish. However, a higher false positive rate warrants confirmatory lethal testing before a previously Rs negative farm is subject to restrictions.

Assessing the presence and spread of Renibacterium salmoninarum between farmed and wild fish in Sweden.

Bacterial kidney disease (BKD) can be a devastating bacterial infection in salmonids, and it is present in aquaculture throughout the world. BKD is caused by the Gram-positive facultative intracellular bacterium Renibacterium salmoninarum (R. salmoninarum) that is spread both horizontally and vertically. Disease signs include external ulcerations and blisters and internal signs such as organ swelling, granulomas, petechiae and ascites. In Sweden, BKD accounts for a significant income loss in aquacultures due to expensive decontamination of the facility and increased disease susceptibility for the immunocompromised fish leading to higher mortality rates. In addition, uncontrolled spread in aquaculture may threaten the survival of wild fish populations. The aim of our study was to investigate the prevalence of R. salmoninarum in wild salmonids caught in Swedish waters where net pen farms with a recent history of BKD are present. Four rivers with at least one BKD-positive or recently BKD-positive farm were selected. In addition, we evaluated the use of environmental DNA (eDNA) for surveillance and monitoring of ongoing infections at these locations. In total, 1058 fish were sampled from four different river systems, and of them 52 (4.9%) were positive for R. salmoninarum by antigen ELISA. Surprisingly, these fish were not evenly distributed between the four river systems, but 50 were caught in the same river (Ljungan). This accounts for an alarmingly high rate of 17% R. salmoninarum-positive samples in wild salmonids in this area. This number is far above what was expected and clearly shows the risk with an open farming system as well as the importance of effective health monitoring programmes to avoid an uncontrolled spread of the disease. The use of eDNA for monitoring BKD is somewhat difficult to evaluate. Few of the water samples analysed were PCR positive for R. salmoninarum (2 of 38) and those were collected where no ELISA positive fish were identified. In addition to water, sediment samples were collected under a net pen farm that had recently slaughtered all fish due to ongoing R. salmoninarum infections. Sediment samples are more promising than water as 4 of 5 samples at one farming facility where positive for R. salmoninarum. Thus, sediment samples may be valuable for monitoring potential ongoing BKD in farms, without the need to sacrifice valuable fish.

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.

A European inter-laboratory trial to evaluate the performance of three serological methods for diagnosis of Mycoplasma bovis infection in cattle using latent class analysis.

BACKGROUND: Mycoplasma bovis (M. bovis) is an emerging bovine pathogen, leading to significant economic losses in the livestock industry worldwide. Infection can result in a variety of clinical signs, such as arthritis, pneumonia, mastitis and keratoconjunctivitis, none of which are M. bovis-specific. Laboratory diagnosis is therefore important. Serological tests to detect M. bovis antibodies is considered an effective indicator of infection in a herd and often used as a herd test. Combined with clinical judgement, it can also be used to implement control strategies and/or to estimate the disease prevalence within a country. However, due to lack of harmonisation of approaches to testing, and serological tests used by different laboratories, comparisons of prevalence data between countries is often difficult. A network of researchers from six European countries designed and participated in an inter-laboratory trial, with the aim of evaluating the sensitivity (Se) and specificity (Sp) of two commercially available ELISA tests (ID Screen® ELISA (IDvet) and BIO K302 ELISA (BIO-X Diagnostics)) for diagnosis of M. bovis infection. Each laboratory received a blinded panel of bovine sera and tested independently, according to manufacturer's instructions. Western blot analyses (WB) performed by one of the participating laboratories was used as a third diagnostic test in the statistical evaluation of Se and Sp values using latent class analysis. RESULTS: The Se of WB, the ID Screen® ELISA and the BIO K302 ELISA were determined to be 91.8, 93.5 and 49.1% respectively, and corresponding Sp of the three tests were 99.6, 98.6 and 89.6%, respectively. CONCLUSIONS: The present study is, to our knowledge, the first to present an inter-laboratory comparison of the BIO K302 ELISA and the ID Screen® ELISA. Based on our results, the ID Screen® ELISA showed high consistency with WB and performed with higher precision and accuracy than the BIO K302 ELISA.

A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis.

BACKGROUND: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. RESULTS: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. CONCLUSION: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.

Prevalence of adhesin and toxin genes in E. coli strains isolated from diarrheic and non-diarrheic pigs from smallholder herds in northern and eastern Uganda.

BACKGROUND: Enterotoxigenic E. coli (ETEC) significantly contribute to diarrhea in piglets and weaners. The smallholder pig producers in Uganda identified diarrhea as one of the major problems especially in piglets. The aim of this study was to; i) characterize the virulence factors of E. coli strains isolated from diarrheic and non-diarrheic suckling piglets and weaners from smallholder herds in northern and eastern Uganda and ii) identify and describe the post-mortem picture of ETEC infection in severely diarrheic piglets. Rectal swab samples were collected from 83 piglets and weaners in 20 herds and isolated E. coli were characterized by PCR, serotyping and hemolysis. RESULTS: The E. coli strains carried genes for the heat stable toxins STa, STb and EAST1 and adhesins F4 and AIDA-I. The genes for the heat labile toxin LT and adhesins F5, F6, F18 and F41 were not detected in any of the E. coli isolates. Where the serogroup could be identified, E. coli isolates from the same diarrheic pig belonged to the same serogroup. The prevalence of EAST1, STb, Stx2e, STa, AIDA-I, and F4 in the E. coli isolates from suckling piglets and weaners (diarrheic and non-diarrheic combined) was 29, 26.5, 2.4, 1.2, 16, and 8.4 %, respectively. However the prevalence of F4 and AIDA-I in E. coli from diarrheic suckling piglets alone was 22.2 and 20 %, respectively. There was no significant difference in the prevalence of the individual virulence factors in E. coli from the diarrheic and non-diarrheic pigs (p > 0.05). The main ETEC strains isolated from diarrheic and non-diarrheic pigs included F4/STb/EAST1 (7.2 %), F4/STb (1.2 %), AIDA/STb/EAST1 (8 %) and AIDA/STb (8 %). At post-mortem, two diarrheic suckling piglets carrying ETEC showed intact intestinal villi, enterocytes and brush border but with a layer of cells attached to the brush border, suggestive of ETEC infections. CONCLUSION: This study has shown that the F4 fimbriae is the most predominant in E. coli from diarrheic piglets in the study area and therefore an F4-based vaccine should be considered one of the preventive measures for controlling ETEC infections in the piglets in northern and eastern Uganda.

Summer mortalities and detection of ostreid herpesvirus microvariant in Pacific oyster Crassostrea gigas in Sweden and Norway.

The Pacific oyster Crassostrea gigas has recently expanded its range in Scandinavia. The expansion is presumably a result of northwards larval drift. Massive settlements were recorded in many areas along the Swedish west coast and southern Norway in 2013 and 2014. After the spawning season in 2014, the temperature of the surface water peaked at 24-26°C. After this period, high and sudden mortalities occurred in a Swedish hatchery and in wild populations along the Swedish west coast and south coast of Norway. Surveys and collected data showed that mortalities mainly occurred during 3 wk in September. All size classes were affected, and affected populations displayed a patchy distribution with heavily affected and unaffected populations in close proximity. Flat oysters Ostrea edulis and blue mussels Mytilus edulis were unaffected. Ostreid herpesvirus (OsHV) was detected in moribund Pacific oyster spat as well as in surviving adults. The virus was identified as OsHV-1 µvar. This is the first detection of this variant in Scandinavia, showing that OsHV-1 µvar is present in areas with recent establishments of Pacific oysters, and where there is no aquaculture of this species.

Multilocus sequence typing identifies epidemic clones of Flavobacterium psychrophilum in Nordic countries.

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD), which affects a variety of freshwater-reared salmonid species. A large-scale study was performed to investigate the genetic diversity of F. psychrophilum in the four Nordic countries: Denmark, Finland, Norway, and Sweden. Multilocus sequence typing of 560 geographically and temporally disparate F. psychrophilum isolates collected from various sources between 1983 and 2012 revealed 81 different sequence types (STs) belonging to 12 clonal complexes (CCs) and 30 singleton STs. The largest CC, CC-ST10, which represented almost exclusively isolates from rainbow trout and included the most predominant genotype, ST2, comprised 65% of all isolates examined. In Norway, with a shorter history (<10 years) of BCWD in rainbow trout, ST2 was the only isolated CC-ST10 genotype, suggesting a recent introduction of an epidemic clone. The study identified five additional CCs shared between countries and five country-specific CCs, some with apparent host specificity. Almost 80% of the singleton STs were isolated from non-rainbow trout species or the environment. The present study reveals a simultaneous presence of genetically distinct CCs in the Nordic countries and points out specific F. psychrophilum STs posing a threat to the salmonid production. The study provides a significant contribution toward mapping the genetic diversity of F. psychrophilum globally and support for the existence of an epidemic population structure where recombination is a significant driver in F. psychrophilum evolution. Evidence indicating dissemination of a putatively virulent clonal complex (CC-ST10) with commercial movement of fish or fish products is strengthened.

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.

Comparison of Multiple-Locus Variable-Number Tandem Repeat Analysis Profiles of Enteropathogenic Yersinia spp. Obtained from Humans, Domestic Pigs, Wild Boars, Rodents, Pork and Dog Food.

The enteropathogenic Yersinia genus is commonly detected in wildlife including wild boars. Difficulties in its cultivation may hamper subsequent epidemiological studies and outbreak investigations. Multiple-locus variable-number tandem repeat analysis (MLVA) of Yersinia (Y.) enterocolitica and Y. pseudotuberculosis has proven useful in source attribution and epidemiological studies but has hitherto relied on the analysis of isolates. In the present study, MLVA profiles generated from 254 isolates of Y. enterocolitica indicated similarities between human, pig and rodent isolates. Further, MLVA analyses of 13 Y. pseudotuberculosis pure-cultured isolates were compared to MLVA analyses performed directly on the 14 PCR-positive enrichment broths from which the isolates originated, which showed matching MLVA profiles. This indicates that MLVA analysis performed directly on enrichment broths could be a useful method for molecular epidemiological investigations. In addition, 10 out of 32 samples of wild boar minced meat obtained from private hunters and from approved wild-game-handling establishments were PCR-positive for the presence of Y. enterocolitica and may indicate a risk for public health.

Presence of Anaplasma spp. and Their Associated Antibodies in the Swedish Goat Population.

Anaplasmosis is a tick-borne disease that has a severe impact on livestock production and welfare. The aim of this pilot study was to investigate the presence of Anaplasma spp. and associated antibodies in a subset of the Swedish goat population. In 2020, six goat herds located in different parts of Sweden were visited and whole blood and serum samples were collected. The whole blood samples (n = 40) were analysed for the presence of Anaplasma phagocytophilum, A. ovis and A. capra using quantitative and conventional polymerase chain reaction (PCR). The serum samples (n = 59) were analysed for the presence of antibodies to Anaplasma spp. using a commercial competitive enzyme-linked immunosorbent assay, and the same analysis was carried out on additional serum samples previously collected in 2018, 2019 and 2020 (n = 166). One goat (2.5%) tested positive for the presence of A. phagocytophilum genetic material, while the seropositivity rate ranged from 20 to 71%, depending on the surveyed year and area. These results indicate widespread exposure to Anaplasma spp. in the Swedish goat population. To inform future risk assessments and control efforts, further research is warranted to determine the prevalence of anaplasmosis and its impact on goat farming in Sweden.

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.

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.

Genetically similar strains of Escherichia coli O157:H7 isolated from sheep, cattle and human patients.

BACKGROUND: Comparatively little is known about the prevalence or the molecular characteristics of the zoonotic pathogen E. coli O157:H7 in the sheep reservoir. To investigate this and determine the host specificity of subclones of the bacterium, we have conducted a slaughterhouse prevalence study in sheep and compared the collected isolates to O157:H7 previously isolated from cattle and human patients. RESULTS: Verotoxin-producing O157:H7 was found in 11/597 (1.8%) of samples from sheep in Swedish slaughterhouses, 9/492 faecal (1.8%) and 2/105 ear samples (1.9%). All positive sheep were < 6 months old. Pulsed field gel electrophoresis typing revealed exact matches between isolates from the sheep prevalence study and human patients as well as between isolates from sheep and cattle. In one case, matching isolates were found in sheep, cattle, and a human patient in the same municipality. Identical PFGE profiles generally corresponded to similar but non-identical multi-locus VNTR profiles. In one sheep sample, SNP-typing found the highly virulent clade 8 variant of O157:H7. The virulence gene profiles of sheep isolates from the prevalence study and three sheep farms linked to cases of human illness were investigated by PCR detection (eaeA, hlyA, cdtV-B, vtx1), and partial sequencing of vtx2. The observed profiles were similar to those of cattle strains investigated previously. CONCLUSIONS: The same pathogenic subtypes of VTEC O157:H7, including the highly virulent clade 8, appear to be present in both sheep and cattle in Sweden, suggesting strains can circulate freely between ruminant reservoirs.

Repeated nasopharyngeal lavage predicts freedom from silent carriage of Streptococcus equi after a strangles outbreak.

BACKGROUND: The value of repeated nasopharyngeal lavage (NPL) to detect silent carriers of Streptococcus equi has not been investigated. HYPOTHESIS/OBJECTIVES: Determine if results of serial testing for S. equi by NPL predicts subsequent true carrier status as determined by both NPL and guttural pouch lavage. ANIMALS: An outbreak of strangles with 100% morbidity in 41 mature Icelandic horses was followed prospectively to investigate development of silent carriers. All were initially positive to S. equi on NPL. The farm was closed to horse movement during the entire study. METHODS: Prospective observational study. Testing for S. equi was performed by NPL at weeks 18, 28, 29, and 30 postindex case and subsequently at week 45 by both NPL and guttural pouch lavage. Carrier status at week 45 was compared to results obtained at weeks 18, 28, 29, and 30. Descriptive statistics were calculated. Comparisons were made using Fisher's exact test or the Freeman-Halton extension with a P < .05 level of significance. RESULTS: Of 24 noncarriers at week 45, only 4 horses were negative on all 3 consecutive weekly NPL samples at weeks 28 to 30. However, 10 of the 11 horses with at least 3 negative NPL obtained from weeks 18, 28, 29, and 30 were S. equi-free at week 45 (P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Repeated NPL on at least 3 separate occasions can assist in predicting S. equi carrier-free status in horses after recovery from a strangles outbreak.

Differences in the genome, methylome, and transcriptome do not differentiate isolates of Streptococcus equi subsp. equi from horses with acute clinical signs from isolates of inapparent carriers.

Streptococcus equi subsp. equi (SEE) is a host-restricted bacterium that causes the common infectious upper respiratory disease known as strangles in horses. Perpetuation of SEE infection appears attributable to inapparent carrier horses because it neither persists long-term in the environment nor infects other host mammals or vectors, and infection results in short-lived immunity. Whether pathogen factors enable SEE to remain in horses without causing clinical signs remains poorly understood. Thus, our objective was to use next-generation sequencing technologies to characterize the genome, methylome, and transcriptome of isolates of SEE from horses with acute clinical strangles and inapparent carrier horses-including isolates recovered from individual horses sampled repeatedly-to assess pathogen-associated changes that might reflect specific adaptions of SEE to the host that contribute to inapparent carriage. The accessory genome elements and methylome of SEE isolates from Sweden and Pennsylvania revealed no significant or consistent differences between acute clinical and inapparent carrier isolates of SEE. RNA sequencing of SEE isolates from Pennsylvania demonstrated no genes that were differentially expressed between acute clinical and inapparent carrier isolates of SEE. The absence of specific, consistent changes in the accessory genomes, methylomes, and transcriptomes of acute clinical and inapparent carrier isolates of SEE indicates that adaptations of SEE to the host are unlikely to explain the carrier state of SEE. Efforts to understand the carrier state of SEE should instead focus on host factors.

Molecular characterization and comparison of Clostridium botulinum type C avian strains.

Type C botulinum neurotoxin (BoNT/C)-producing Clostridium botulinum causes animal botulism worldwide and has become a serious problem in poultry flocks and waterfowl in Sweden. The objectives of the present study were to isolate, characterize and subtype C. botulinum type C avian isolates in order to increase the knowledge of the genetic diversity. Isolates from 13 birds were identified by 16S rRNA sequencing and BoNT/C gene detection by real-time polymerase chain reaction (PCR). Conventional PCR was used to distinguish a chimeric BoNTC/D gene, often associated with avian botulism, from the BoNT/C gene. The isolates analysed all contained the gene coding for a chimeric toxin type C/D. Two fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA analysis (RAPD), were optimized and used to investigate the epidemiological relatedness among the strains. The isolates were divided into three different pulsotypes based upon their restriction profiles for SmaI and SalI. The RAPD system proved to be as discriminative as PFGE. This study reveals a small genetic diversity among Swedish type C strains, with a high similarity between strains from broilers and herring gulls.

Verotoxigenic Escherichia coli O157:H7 from Swedish cattle; isolates from prevalence studies versus strains linked to human infections--a retrospective study.

BACKGROUND: Several cases of human infection caused by verotoxin-producing Escherichia coli (VTEC) O157:H7 in Sweden have been connected with cattle farm visits. Between 1996 and 2002, 18 farms were classified as the source of human cases with isolation of EHEC (Enterohaemorrhagic Escherichia coli) after VTEC O157:H7 had been isolated from cattle on those farms. RESULTS: Characterization by phage typing and molecular methods of the strains isolated from these 18 farms, including PCR for virulence genes (vtx1, vtx2 and variants thereof, eaeA and EHEC-hlyA) and Pulsed-Field Gel Electrophoresis (PFGE), demonstrated a cluster of very similar strains from 16 farms. All were of phage type 4, carried the genes encoding the verotoxins VT2 and VT2c, intimin, EHEC-haemolysin and flagellin H7 as shown by PCR, and had identical or very similar PFGE patterns. When analysing strains in a prevalence study of VTEC O157:H7 from cattle at slaughter as well as from an on-farm prevalence study of dairy cattle, using the same typing methods, a rather wide variation was observed among the isolated VTEC O157:H7 strains. CONCLUSIONS: In Sweden, a limited group of genetically similar and highly pathogenic VTEC O157:H7 strains seem to predominate in direct or indirect transmission from cattle to man.

Streptococcus suis in Swedish grower pigs: occurrence, serotypes, and antimicrobial susceptibility.

BACKGROUND: Streptococcus suis is a major cause of meningitis, arthritis, and pneumonia in pigs worldwide, and an emerging pathogen in humans. In Sweden, S. suis has previously received little attention but has in recent years become increasingly recognized as affecting the pig production. The aim of the present study was to investigate the occurrence, serotypes and antimicrobial susceptibility of S. suis in Swedish grower pigs from herds with and without reported S. suis associated disease, as well as possible associations between S. suis associated disease and selected environmental and production factors. Swab samples were taken from the tonsils of clinically healthy 8-13-week-old grower pigs from ten case herds and ten control herds. Isolates were cultured, identified using MALDI-TOF MS, and serotyped using latex agglutination. The antimicrobial susceptibility of 188 isolates was tested using broth microdilution. Production data was gathered and environmental parameters were measured on the farms. RESULTS: Streptococcus suis was isolated from 95% of the sampled pigs in both the case and the control herds. Serotypes 3, 4, 5, 7, 9, 10, 11, 15, 16, and 17-34 were detected, although a majority of the isolates (81.5%) were non-typeable. There was less diversity among the serotypes isolated from the case herds than among those from the control herds; four and nine different serotypes, respectively. Isolates resistant to penicillin (3.8%) were reported for the first time in Sweden. Tetracycline resistance was common (88.4%). No association was noted between the production and the environmental factors investigated, and the carriership of S. suis. CONCLUSIONS: The carriership of S. suis was found to be higher in clinically healthy Swedish pigs than previously estimated, and for the first time, the presence of Swedish isolates resistant to penicillin was reported. Many of the most commonly disease-associated serotypes, e.g. serotypes 2, 9, 3, and 7, were detected in healthy grower pigs although further studies are needed to investigate the virulence of these isolates.