Brachyspira suanatina sp. nov., an enteropathogenic intestinal spirochaete isolated from pigs and mallards: genomic and phenotypic characteristics.

BACKGROUND: The genus Brachyspira currently encompasses seven valid species that colonize the intestines of mammals and birds. In a previous study a group of strongly haemolytic isolates from pigs and mallards was provisionally described as a new species within genus Brachyspira, "B. suanatina", and enteropathogenic properties were demonstrated in a porcine challenge model. METHODS: In the current study characterization of B. suanatina was performed on the basis of cell morphology, growth characteristics, enzyme profiles, DNA-DNA hybridization (DDH) and whole genome comparisons. The draft genome sequence of B. suanatina strain AN4859/03 was determined and compared with the available genomes of all valid species of Brachyspira. RESULTS: According to morphological traits, growth characteristics and enzymatic profiles, B. suanatina was similar to the type strain of B. hyodysenteriae, but using the recommended threshold value of 70% similarity by DDH it did not belong to any of the recognized Brachyspira species (range 16-64% similarity). This was further supported by average nucleotide identity values. Phylogenetic analysis performed using housekeeping genes and core genomes of all valid Brachyspira sp. and "B. hampsonii" revealed that B. suanatina and B. intermedia formed a clade distinct from B. hyodysenteriae. By comparing the genomes of the three closely related species B. intermedia, B. hyodysenteriae and B. suanatina similar profiles of general genomic features and distribution of genes in different functional categories were obtained. However, the genome size of B. hyodysenteriae was smallest among the species, suggesting the possibility of reductive evolution in the divergence of this species. A bacteriophage region and a putative plasmid sequence were also found in the genome of B. suanatina strain AN4859/03. CONCLUSIONS: The results of our study suggest that despite being similar to B. hyodysenteriae phenotypically, B. suanatina should be regarded as a separate species based on its genetic characteristics. Based on characteristics presented in this report we propose that strains AN4859/03, AN1681:1/04, AN2384/04 and Dk12570-2 from pigs in Sweden and Denmark, and strains AN3949:2/02 and AN1418:2/01 isolated from mallards in Sweden, represent a unique species within genus Brachyspira. For this new species we propose the name B. suanatina for which the type strain is AN4859/03T (=ATCC® BAA-2592™=DSM 100974T).

The prevalence of rodent-borne zoonotic pathogens in the South Gobi desert region of Mongolia.

The alpine ecosystems and communities of central Asia are currently undergoing large-scale ecological and socio-ecological changes likely to affect wildlife-livestock-human disease interactions and zoonosis transmission risk. However, relatively little is known about the prevalence of pathogens in this region. Between 2012 and 2015 we screened 142 rodents in Mongolia's Gobi desert for exposure to important zoonotic and livestock pathogens. Rodent seroprevalence to Leptospira spp. was >1/3 of tested animals, Toxoplasma gondii and Coxiella burnetii approximately 1/8 animals, and the hantaviruses being between 1/20 (Puumala-like hantavirus) and <1/100 (Seoul-like hantavirus). Gerbils trapped inside local dwellings were one of the species seropositive to Puumala-like hantavirus, suggesting a potential zoonotic transmission pathway. Seventeen genera of zoonotic bacteria were also detected in the faeces and ticks collected from these rodents, with one tick testing positive to Yersinia. Our study helps provide baseline patterns of disease prevalence needed to infer potential transmission between source and target populations in this region, and to help shift the focus of epidemiological research towards understanding disease transmission among species and proactive disease mitigation strategies within a broader One Health framework.

Detection of Leptospira in Urban Swedish Rats: Pest Control Interventions as a Promising Source of Rats Used for Surveillance.

Rat carcasses obtained from pest control interventions can potentially be used for an efficient surveillance of zoonotic diseases such as leptospirosis. To evaluate the performance of different laboratory methods for detection of pathogenic Leptospira spp., heart and kidney samples from wild Norway rats were analyzed by microscopic agglutination test (MAT, the gold standard), a commercial IgG enzyme-linked immunosorbent assay, and by an optimized quantitative PCR (secY qPCR, followed by sequencing). We found secY qPCR to be as sensitive as MAT for screening of Leptospira infection in pest control rats and selected secY qPCR for a larger screening of rats from urban and rural areas in central and southern Sweden. We identified secY qPCR positive rats from the cities Stockholm, Gothenburg, and Malmö, which were further confirmed by sequencing.

Identification and genetic fingerprinting of Brachyspira species.

Six Brachyspira type and reference strains, and 14 well characterized porcine field isolates representing all recognised porcine Brachyspira spp. were compared by different molecular methods. Sequence analysis of the 16S rRNA and the nox genes, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were used in the study. In addition the isolates were analysed by five species-specific PCR systems. The topologies of the dendrograms obtained from each of the four typing systems were different. The B. pilosicoli isolates formed monophyletic clusters in all dendrograms, but with different sister lines. All five porcine Brachyspira species formed monophyletic clusters in the nox gene-based dendrogram only. All five PCR systems accurately identified their targets, except for the nox gene-based B. intermedia-specific system, by which it was not possible to identify one of the presumed B. intermedia isolates, and the other B. intermedia-specific system, based on the 23S rRNA gene, gave a positive reaction for one B. innocens isolate. In an extended study, 46 additional isolates and the original eight isolates with the phenotypes of B. hyodysenteriae or B. intermedia were compared by PFGE and PCR. The PFGE results indicated a high genetic diversity of isolates with the phenotype of B. intermedia. Thirty-three of 34 tested isolates could be identified by one or both of the two B. intermedia-specific PCR systems used, however, only 19 of the 34 isolates were positive in both systems.

Prevalence of human pathogenic Yersinia enterocolitica in Swedish pig farms.

BACKGROUND: Pigs are the most important reservoir for human pathogenic Yersinia enterocolitica. We investigated the herd prevalence of human pathogenic Y. enterocolitica in Swedish pig farms by analysing pen faecal samples using a cold enrichment of 1 week and thereafter subsequent plating onto chromogenic selective media (CAY agar). RESULTS: Pathogenic Y. enterocolitica was found in 32 (30.5%) of the 105 sampled farms with finisher pigs. Bioserotype 4/O:3 was identified at all but one farm, where 2/O:9 was identified. Pen-prevalence within the positive herds varied from 1/4 to 4/4 pens. The calculated intra-class correlation coefficient ICC (0.89) from a model with a random effect for grouping within herd indicated a very high degree of clustering by herd. None of the explored risk factors, including herd size, herd type, pig flow, feed type, access to outdoors, evidence of birds and rodents in the herd, usage of straw, number of pigs in sampled pen and age of pigs in pen were significantly associated with Y. enterocolitica status of the pen. The use of high pressure washing with cold water was significantly associated with Y. enterocolitica in the pen (OR = 84.77, 4.05-1772). Two culture methods were assessed for detection of Y. enterocolitica, one of which included the use of a chromogenic agar (CAY agar) intended for detection of human pathogenic Y. enterocolitica. The chromogenic media was found equal or superior to traditional methods and was used in this study. The isolates obtained were characterised by biotyping, serotyping, mass spectrometry (MALDI-TOF) and PCR. Characterisation by MALDI-TOF gave identical results to that of conventional bioserotyping. All porcine isolates were positive for the ail and inv genes by PCR, indicating that the isolates were most likely pathogenic to humans. CONCLUSIONS: Human pathogenic Y. enterocolitica was found in nearly one-third of the Swedish pig farms with finisher pigs. The use of high pressure washing with cold water was associated with the presence of Y. enterocolitica in the pen. A modified culturing method using a chromogenic agar was efficient for detection of pathogenic Y. enterocolitica in pig faeces. The use of masspectrometry for identification and subtyping was in agreement with conventional biotyping and serotyping methods.

Immunological alterations during the clinical and recovery phases of experimental swine dysentery.

The aim of this study was to examine changes in the systemic immune response during the incubation period and following the onset of clinical swine dysentery, including the recovery period. Ten healthy conventional pigs were inoculated with Brachyspira hyodysenteriae. Blood was sampled at pre-inoculation, at days 4 and 14 post-inoculation, during the first 4 days with clinical signs of dysentery and at days 1, 3, 7, 11 and 15 of the recovery period. Eight pigs developed haemorrhagic diarrhoea. Flow-cytometric analyses of lymphocyte subpopulations showed that all animals, including the two that remained healthy, had an increase in CD8alpha+ CD4- cells and gammadelta T cells at days 4 and 14 post-inoculation. In addition, an increase in CD4+ CD8alpha+ cells and CD8alpha+ CD8beta+ cells was observed at days 4 and 14 post-inoculation in animals that developed dysentery. During clinical signs of dysentery, the acute-phase protein serum amyloid A was increased. There was a two- to threefold increase in both neutrophils and monocytes during signs of dysentery and at the beginning of the recovery period. The numbers of CD8alpha+ CD8beta- CD4-, CD45RA- lymphocytes also increased during the dysentery period. Circulating CD21+ cells and CD21+ CD45RA- cells decreased at the end of the incubation period, during signs of dysentery and at the beginning of the recovery period. The dysentery-affected animals developed antibodies to B. hyodysenteriae-specific antigens (approximately 16 kDa and approximately 30 kDa) from the first day of recovery, and gammadelta T cells showed an increase during the recovery period. In comparison with pre-inoculation, increased numbers of monocytes, neutrophils, CD8alpha+ CD8beta- CD4- lymphocytes and CD45RA- lymphocytes were observed during clinical dysentery. Increased numbers of neutrophils, gammadelta T cells and specific antibodies were seen during the recovery period.

Highly Pathogenic Leptospira Found in Urban Brown Rats (Rattus norvegicus) in the Largest Cities of Sweden.

Leptospirosis is an emerging zoonosis of global concern; however, its contemporary occurrence in Sweden, a European country partly located north of the Arctic Circle, is poorly known. Four out of 30 brown rats, captured within urban districts in Sweden, were found to be positive for antibodies to Leptospira interrogans serovar Icterohaemorrhagiae. This serovar causes Weil's disease in humans, a severe infection with jaundice, renal failure, and hemorrhage. Our study is the first finding of this highly pathogenic serovar in Swedish rats since the 1930s.

Brachyspira hyodysenteriae and other strongly beta-haemolytic and indole-positive spirochaetes isolated from mallards (Anas platyrhynchos).

The aims of the current study were to collect intestinal spirochaetes (genus Brachyspira) from farmed and wild mallards (Anas platyrhynchos) and to identify and classify those isolates that phenotypically resembled Brachyspira hyodysenteriae, an enteric pathogen of pigs. The isolation rate of Brachyspira spp. was high from both farmed (93 %) and wild mallards (78 %). In wild mallards, it appeared that Brachyspira spp. were more likely to be found in migratory birds (multivariate analysis: RR = 1.8, 95 % CI 1.1-3.1) than in mallards sampled in a public park. Pure cultures of putative B. hyodysenteriae were obtained from 22 birds. All five isolates from farmed mallards and ten randomly selected isolates with this phenotype were used for further studies. All isolates from farmed mallards and two of the isolates from wild mallards were PCR-positive for the tlyA gene of B. hyodysenteriae. Two isolates from farmed mallards were selected for pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis. These isolates clustered with the type and reference strains of B. hyodysenteriae. 16S rDNA sequence analysis performed on 11 of the strains showed that they were all closely related to each other and to the B. hyodysenteriae-Brachyspira intermedia cluster. Three of the mallard isolates had 16S rDNA sequences that were identical to those of B. hyodysenteriae strains R1 and NIV-1 previously isolated from common rheas (Rhea americana). To conclude, the isolates from farmed mallards and two isolates from wild mallards were classified as B. hyodysenteriae based on the fact that they could not be differentiated by any of the applied methods from type, reference and field strains of B. hyodysenteriae. The remaining isolates could not be assigned irrefutably to any of the presently recognized Brachyspira species. These results point to a broader host spectrum of B. hyodysenteriae than is generally recognized, and to the presence in mallards of strongly beta-haemolytic and indole-producing spirochaetes that possess many, but not all, of the currently recognized characteristics of B. hyodysenteriae.

A novel enteropathogenic, strongly haemolytic spirochaete isolated from pig and mallard, provisionally designated 'Brachyspira suanatina' sp. nov.

Atypical, strongly haemolytic porcine isolates of intestinal spirochaetes differing genetically from Brachyspira hyodysenteriae were identified and characterized. The isolates were subjected to culture and biochemical tests, antimicrobial susceptibility testing and molecular analyses. None of four species-specific polymerase chain reaction systems targeting genes of B. hyodysenteriae gave a positive reaction. All the atypical porcine isolates were identical in their partial 16S rRNA and nox gene sequences with a previously described isolate from a mallard (Anas platyrhynchos), and differed only slightly from another mallard isolate. All these isolates were distinctly different from all currently recognized Brachyspira species. A challenge study was carried out using recently weaned pigs. Clinical signs and macroscopic changes consistent with swine dysentery were seen both in pigs given the atypical porcine isolate and in control pigs given the reference strain of B. hyodysenteriae (B204(R)). Pigs given the genetically similar isolate from a mallard became colonized and diarrhoea was observed. This is the first study indicating that Brachyspira isolates from mallard can infect pigs and induce diarrhoea. We propose that this atypical spirochaete genotype should be regarded as a new species within the genus Brachyspira, and be provisionally designated 'Brachyspira suanatina' sp. nov.