Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs.

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.

A national study confirms that Escherichia coli from Australian commercial layer hens remain susceptible to critically important antimicrobials.

Controlling the use of the most critically important antimicrobials (CIAs) in food animals has been identified as one of the key measures required to curb the transmission of antimicrobial resistant bacteria from animals to humans. Expanding the evidence demonstrating the effectiveness of restricting CIA usage for preventing the emergence of resistance to key drugs amongst commensal organisms in animal production would do much to strengthen international efforts to control antimicrobial resistance (AMR). As Australia has strict controls on antimicrobial use in layer hens, and internationally comparatively low levels of poultry disease due to strict national biosecurity measures, we investigated whether these circumstances have resulted in curtailing development of critical forms of AMR. The work comprised a cross-sectional national survey of 62 commercial layer farms with each assessed for AMR in Escherichia coli isolates recovered from faeces. Minimum inhibitory concentration analysis using a panel of 13 antimicrobials was performed on 296 isolates, with those exhibiting phenotypic resistance to fluoroquinolones (a CIA) or multi-class drug resistance (MCR) subjected to whole genome sequencing. Overall, 53.0% of isolates were susceptible to all antimicrobials tested, and all isolates were susceptible to cefoxitin, ceftiofur, ceftriaxone, chloramphenicol and colistin. Resistance was observed for amoxicillin-clavulanate (9.1%), ampicillin (16.2%), ciprofloxacin (2.7%), florfenicol (2.4%), gentamicin (1.0%), streptomycin (4.7%), tetracycline (37.8%) and trimethoprim/sulfamethoxazole (9.5%). MCR was observed in 21 isolates (7.0%), with two isolates exhibiting resistance to four antimicrobial classes. Whole genome sequencing revealed that ciprofloxacin-resistant (fluoroquinolone) isolates were devoid of both known chromosomal mutations in the quinolone resistance determinant regions and plasmid-mediated quinolone resistance genes (qnr)-other than in one isolate (ST155) which carried the qnrS gene. Two MCR E. coli isolates with ciprofloxacin-resistance were found to be carrying known resistance genes including aadA1, dfrA1, strA, strB, sul1, sul2, tet(A), blaTEM-1B, qnrS1 and tet(A). Overall, this study found that E. coli from layer hens in Australia have low rates of AMR, likely due to strict control on antimicrobial usage achieved by the sum of regulation and voluntary measures.

Comparative genomic analysis of Colistin resistant Escherichia coli isolated from pigs, a human and wastewater on colistin withdrawn pig farm.

In this study, genomic and plasmid characteristics of Escherichia coli were determined with the aim of deducing how mcr genes may have spread on a colistin withdrawn pig farm. Whole genome hybrid sequencing was applied to six mcr-positive E. coli (MCRPE) strains isolated from pigs, a farmworker and wastewater collected between 2017 and 2019. Among these, mcr-1.1 genes were identified on IncI2 plasmids from a pig and wastewater, and on IncX4 from the human isolate, whereas mcr-3 genes were found on plasmids IncFII and IncHI2 in two porcine strains. The MCRPE isolates exhibited genotypic and phenotypic multidrug resistance (MDR) traits as well as heavy metal and antiseptic resistance genes. The mcr-1.1-IncI2 and IncX4 plasmids carried only colistin resistance genes. Whereas, the mcr-3.5-IncHI2 plasmid presented MDR region, with several mobile genetic elements. Despite the MCRPE strains belonged to different E. coli lineages, mcr-carrying plasmids with high similarities were found in isolates from pigs and wastewater recovered in different years. This study highlighted that several factors, including the resistomic profile of the host bacteria, co-selection via adjunct antibiotic resistance genes, antiseptics, and/or disinfectants, and plasmid-host fitness adaptation may encourage the maintenance of plasmids carrying mcr genes in E. coli.

Differential Impacts of Cereal and Protein Sources Fed to Pigs after Weaning on Diarrhoea and Faecal Shedding of Escherichia coli, Production, and Total Tract Apparent Digestibility.

Different cereal types, in combination with different protein sources, are fed to pigs after weaning, but their interactions and possible implications are not well researched. In this study, 84 male weaned piglets were used in a 21-day feeding trial to investigate the effects of feeding either medium-grain or long-grain extruded rice or wheat, in a factorial combination with protein sources of either vegetable or animal origin, on postweaning performance, shedding of ß-haemolytic Escherichia coli, and the coefficient of total tract apparent digestibility (CTTAD). Pigs fed either rice type performed the same (p > 0.05) as wheat-fed pigs after weaning. The use of vegetable protein sources reduced growth rate (p < 0.001) and feed intake (p = 0.007) and deteriorated the feed conversion ratio (p = 0.028) in weeks two and three compared to pigs fed animal protein sources. The number of antibiotic treatments given for clinical diarrhoea was similar (p > 0.05). However, the faecal E. coli score showed a trend for the main effect of protein source, with pigs fed animal proteins showing a higher E. coli score than pigs fed vegetable proteins (0.63 vs. 0.43, p = 0.057). There was also a tendency for an interaction (p = 0.069) between cereal type and protein source (p = 0.069), with this difference being associated with a greater faecal score in pigs fed diets with long-grain rice plus animal proteins and wheat plus animal proteins. Significant interactions occurred for the CTTAD when assessed in week three. In general, pigs fed diets with medium-grain rice or long-grain rice with animal proteins had a higher (p < 0.001) CTTAD for dietary components than pigs fed all other diets, and vegetable proteins depressed (p < 0.001) CTTAD compared to animal proteins (main effect of protein: p < 0.001). In summary, pigs tolerated the extruded rice-based diets well and performed equivalently to pigs fed wheat as the sole cereal, and the use of vegetable proteins decreased the E. coli score.

Proximity to human settlement is directly related to carriage of critically important antimicrobial-resistant Escherichia coli and Klebsiella pneumoniae in Silver Gulls.

Human population and activities play an important role in dissemination of antimicrobial resistant bacteria. This study investigated the relationship between carriage rates of critically important antimicrobial-resistant (CIA-R) Escherichia coli and Klebsiella pneumoniae by Silver Gulls and their proximity to human populations. Faecal swabs (n = 229) were collected from Silver Gulls across 10 southern coastline locations in Western Australia (WA) traversing 650 kms. The sampling locations included main town centres and remote areas. Fluoroquinolone and extended-spectrum cephalosporin-resistant E. coli and K. pneumoniae were isolated and tested for antimicrobial sensitivity. Genome sequencing was performed on n = 40 subset out of 98 E. coli and n = 14 subset out of 27 K. pneumoniae isolates to validate phenotypic resistance profiles and determine the molecular characteristics of strains. CIA-R E. coli and K. pneumoniae were detected in 69 (30.1 %) and 20 (8.73 %) of the faecal swabs respectively. Two large urban locations tested positive for CIA-R E. coli (frequency ranging from 34.3 % to 84.3 %), and/or for CIA-R K. pneumoniae (frequency ranging from 12.5 % to 50.0 %). A small number of CIA-R E. coli (3/31, 9.7 %) were identified at a small tourist town, but no CIA-R bacteria were recovered from gulls at remote sites. Commonly detected E. coli sequence types (STs) included ST131 (12.5 %) and ST1193 (10.0 %). Five K. pneumoniae STs were detected which included ST4568, ST6, ST485, ST967 and ST307. Resistance genes including blaCTX-M-3, blaCTX-M-15 and blaCTX-M-27 were identified in both bacterial species. High-level colonisation of CIA-R E. coli and K. pneumoniae in Silver Gulls in and around urban areas compared to remote locations substantiates that anthropogenic activities are strongly associated with acquisition of resistant bacteria by gulls.

Antimicrobial Resistance of and Genomic Insights into Pasteurella multocida Strains Isolated from Australian Pigs.

Infection with Pasteurella multocida represents a significant economic threat to Australian pig producers, yet our knowledge of its antimicrobial susceptibilities is lagging, and genomic characterization of P. multocida strains associated with porcine lower respiratory disease is internationally scarce. This study utilized high-throughput robotics to phenotypically and genetically characterize an industry-wide collection of 252 clinical P. multocida isolates that were recovered between 2014 and 2019. Overall, antimicrobial resistance was found to be low, with clinical resistance below 1% for all tested antimicrobials except those from the tetracycline class. Five dominant sequence types, representing 64.8% of all isolates, were identified; they were disseminated across farms and had previously been detected in various animal hosts and countries. P. multocida in Australian farms remain controllable via current antimicrobial therapeutic protocols. The identification of highly dominant, interspecies-infecting strains provides insight into the epidemiology of the opportunistic pathogen, and it highlights a biosecurity threat to the Australian livestock industry. IMPORTANCE Pasteurellosis is rated by the World Animal Health Organisation (OIE) as a high-impact disease in livestock. Although it is well understood in many host-disease contexts, our understanding of the organism in porcine respiratory disease is limited. Given its high frequency of involvement in porcine respiratory disease complex (PRDC), it is important that we are aware of its antimicrobial susceptibilities so that we can respond quickly and appropriately with antimicrobial therapy. Genetic insights about the organism can help us to better understand its epidemiology and inform our biosecurity practices and prophylactic management.

Metagenomic analysis of the gut microbiota in piglets either challenged or not with enterotoxigenic Escherichia coli reveals beneficial effects of probiotics on microbiome composition, resistome, digestive function and oxidative stress responses.

This study used metagenomic analysis to investigate the gut microbiota and resistome in piglets that were or were not challenged with enterotoxigenic Escherichia coli (ETEC) and had or had not received dietary supplementation with microencapsulated probiotics. The 72 piglets belonged to six groups that were either non-ETEC challenged (groups 1-3) or ETEC challenged (receiving 5ml of 109 CFU/ml pathogenic ETEC strain L3.2 one week following weaning at three weeks of age: groups 4-6). On five occasions at 2, 5, 8, 11, and 14 days of piglet age, groups 2 and 5 were supplemented with 109 CFU/ml of multi-strain probiotics (Lactiplantibacillus plantarum strains 22F and 25F, and Pediococcus acidilactici 72N) while group 4 received 109 CFU/ml of P. acidilactici 72N. Group 3 received 300mg/kg chlortetracycline in the weaner diet to mimic commercial conditions. Rectal faecal samples were obtained for metagenomic and resistome analysis at 2 days of age, and at 12 hours and 14 days after the timing of post-weaning challenge with ETEC. The piglets were all euthanized at 42 days of age. The piglets in groups 2 and 5 were enriched with several desirable microbial families, including Lactobacillaceae, Lachnospiraceae and Ruminococcaceae, while piglets in group 3 had increases in members of the Bacteroidaceae family and exhibited an increase in tetW and tetQ genes. Group 5 had less copper and multi-biocide resistance. Mobile genetic elements IncQ1 and IncX4 were the most prevalent replicons in antibiotic-fed piglets. Only groups 6 and 3 had the integrase gene (intl) class 2 and 3 detected, respectively. The insertion sequence (IS) 1380 was prevalent in group 3. IS3 and IS30, which are connected to dietary intake, were overrepresented in group 5. Furthermore, only group 5 showed genes associated with detoxification, with enrichment of genes associated with oxidative stress, glucose metabolism, and amino acid metabolism compared to the other groups. Overall, metagenomic analysis showed that employing a multi-strain probiotic could transform the gut microbiota, reduce the resistome, and boost genes associated with food metabolism.

Microencapsulated probiotic Lactiplantibacillus plantarum and/or Pediococcus acidilactici strains ameliorate diarrhoea in piglets challenged with enterotoxigenic Escherichia coli.

Lactiplantibacillus plantarum (strains 22F and 25F) and Pediococcus acidilactici (strain 72N) have displayed antibacterial activity in vitro, suggesting that they could be used to support intestinal health in pigs. The aim of this study was to determine if microencapsulated probiotics could reduce the severity of infection with enterotoxigenic Escherichia coli (ETEC) in weaned pigs. Sixty healthy neonatal piglets were cross-fostered and separated into five groups. Piglets to be given the microencapsulated probiotics received these orally on days 0, 3, 6, 9, and 12. Only piglets in groups 1 and 5 did not receive probiotics: those in groups 2 and 4 received the three microencapsulated probiotic strains (multi-strain probiotic), and piglets in group 3 received microencapsulated P. acidilactici strain 72N. After weaning, the pigs in groups 3-5 were challenged with 5 mL (at 109 CFU/mL) of pathogenic ETEC strain L3.2 carrying the k88, staP, and stb virulence genes. The multi-strain probiotic enhanced the average daily gain (ADG) and feed conversion ratio (FCR) of weaned piglets after the ETEC challenge (group 4), whilst supplementing with the single-strain probiotic increased FCR (group 3). Piglets in groups 3 and 4 developed mild to moderate diarrhoea and fever. In the probiotic-fed piglets there was an increase in lactic acid bacteria count and a decrease in E. coli count in the faeces. By using real-time PCR, virulence genes were detected at lower levels in the faeces of pigs that had received the probiotic strains. Using the MILLIPLEX MAP assay, probiotic supplementation was shown to reduce pro-inflammatory cytokines (IL-1α, IL-6, IL-8, and TNFα), while group 4 had high levels of anti-inflammatory cytokine (IL-10). Challenged piglets receiving probiotics had milder intestinal lesions with better morphology, including greater villous heights and villous height per crypt depth ratios, than pigs just receiving ETEC. In conclusion, prophylactic administration of microencapsulated probiotic strains may improve outcomes in weaned pigs with colibacillosis.

Antimicrobial resistance and genomic relationships of Salmonella enterica from Australian cattle.

The aim of this study was to evaluate phenotypic and genotypic AMR characteristics of Salmonella enterica isolates from Australian cattle collected through a structured national survey utilizing 1001 faecal samples collected from healthy cattle at slaughter. A total of 184 Salmonella isolates were subsequently derived and subjected to microbroth dilution to 16 drugs from 11 classes with interpretation of minimum inhibitory concentrations (MICs) using epidemiological cut off (ECOFF) values to distinguish between wild-type and non-wild-type populations. Most isolates were susceptible (wild type) to all antimicrobials tested, with no resistance (non-wild type) detected for colistin, nalidixic acid, meropenem, gentamicin, florfenicol or chloramphenicol. Low rates of resistance were detected for ampicillin (2.2%), cefoxitin (2.2%), ceftiofur (2.2%), ceftriaxone (2.2%), ciprofloxacin (0.5%), streptomycin (3.3%) and tetracycline (0.5%). Isolates resistant to ceftriaxone (a critically important antimicrobial, CIA) carried the extended spectrum cephalosporin gene blaCMY-2 while no known mutation in the QRDR region or qnrS genes were detected for the CIA ciprofloxacin-resistant isolate. Thirty-six serovars were detected among the 184 Salmonella isolates using whole genome sequencing, dominated by Typhimurium (n = 36), Saintpaul (n = 22) and Anatum (n = 16). Genomic analysis clustered the cattle isolates based on serovar, with the majority of serovars containing a single sequence type (ST). Further analysis of the bovine Typhimurium isolates (ST19) and comparison with publicly available data from human Typhimurium isolates from Australia, revealed the majority of cattle isolates were unrelated to human isolates. In conclusion, this study demonstrates the continued low prevalence of AMR among Salmonella within the beef, dairy and veal industries in Australia. Salmonella Typhimurium from cattle were genetically distinct from isolates sourced from human infections. Further investigations are warranted to expand on the potential clinical and public health relevance of these findings to inform risk-management of this key pathogen.

Longitudinal Monitoring Reveals Persistence of Colistin-Resistant Escherichia coli on a Pig Farm Following Cessation of Colistin Use.

Colistin-resistant bacteria harboring plasmid-mediated mcr genes are of concern as they may be a cause of serious nosocomial infections. It is hypothesized that cessation of colistin use as a feed additive for pigs will reduce the occurrence and distribution of mcr genes in farms. The aim of this study was to investigate this hypothesis by longitudinal monitoring and characterizing of mcr positive Escherichia coli (MCRPE) isolates after colistin was withdrawn on a central Thailand pig farm that previously had a high frequency of MCRPE. Colistin use ceased at the beginning of 2017, and subsequently 170 samples were collected from farrowing sows and suckling piglets (n = 70), wastewater (n = 50) and farm workers (n = 50) over a 3.5-year period. Bacteria were identified by MALDI-TOF mass spectrometry and minimal inhibitory concentrations were determined by broth microdilution. The antibiogram of mcr positive E. coli isolates was determined using the Vitek2 automated susceptibility machine, and multiplex and simplex PCRs were performed for mcr-1-8 genes. MCRPE containing either mcr-1 or mcr-3 were isolated from pigs throughout the investigation period, but with a declining trend, whereas MCRPE isolates were recovered from humans only in 2017. MCRPE were still being recovered from wastewater in 2020. Most MCRPE isolates possessed the virulence genes Stap, Stb, or Stx2e, reflecting pathogenic potential in pigs, and showed high rates of resistance to ampicillin, gentamicin and tetracycline. Pulsed-field gel electrophoresis and multi-locus sequence typing showed that diverse MCRPE clones were distributed on the farm. The study identified a decline of pathogenic MCRPE following withdrawal of colistin, with pigs being the primary source, followed by wastewater. However, short-term therapeutic usage of other antibiotics could enhance the re-occurrence of mcr-carrying bacteria. Factors including the environment, management, and gene adaptations that allow maintenance of colistin resistance require further investigation, and longer-term studies are needed.

Reducing the Risk of Transmission of Critical Antimicrobial Resistance Determinants From Contaminated Pork Products to Humans in South-East Asia.

Antimicrobial resistance (AMR) is a critical challenge worldwide as it impacts public health, especially via contamination in the food chain and in healthcare-associated infections. In relation to farming, the systems used, waste management on farms, and the production line process are all determinants reflecting the risk of AMR emergence and rate of contamination of foodstuffs. This review focuses on South East Asia (SEA), which contains diverse regions covering 11 countries, each having different levels of development, customs, laws, and regulations. Routinely, here as elsewhere antimicrobials are still used for three indications: therapy, prevention, and growth promotion, and these are the fundamental drivers of AMR development and persistence. The accuracy of detection of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG) depends on the laboratory standards applicable in the various institutes and countries, and this affects the consistency of regional data. Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the standard proxy species used for indicating AMR-associated nosocomial infections and healthcare-associated infections. Pig feces and wastewater have been suspected as one of the hotspots for spread and circulation of ARB and ARG. As part of AMR surveillance in a One Health approach, clonal typing is used to identify bacterial clonal transmission from the production process to consumers and patients - although to date there have been few published definitive studies about this in SEA. Various alternatives to antibiotics are available to reduce antibiotic use on farms. Certain of these alternatives together with improved disease prevention methods are essential tools to reduce antimicrobial usage in swine farms and to support global policy. This review highlights evidence for potential transfer of resistant bacteria from food animals to humans, and awareness and understanding of AMR through a description of the occurrence of AMR in pig farm food chains under SEA management systems. The latter includes a description of standard pig farming practices, detection of AMR and clonal analysis of bacteria, and AMR in the food chain and associated environments. Finally, the possibility of using alternatives to antibiotics and improving policies for future strategies in combating AMR in a SEA context are outlined.

Porcine enterotoxigenic Escherichia coli: Antimicrobial resistance and development of microbial-based alternative control strategies.

Strains of enterotoxigenic Escherichia coli (ETEC) causing post-weaning diarrhoea (PWD) in piglets have a widespread and detrimental impact on animal health and the economics of pork production. Traditional approaches to control and prevention have placed a strong emphasis on antimicrobial use (AMU) to the extent that current prevalent porcine ETEC strains have developed moderate to severe resistance. This complicates treatment of ETEC infection by limiting therapeutic options, increasing diagnostic costs and increasing mortality rates. Management factors, the use of supra-physiological levels of zinc oxide and selected feed additives have all been documented to lower the incidence of ETEC infection in pigs; however, each intervention has its own limitations and cannot solely be relied upon as an alternative to AMU. Consequently, treatment options for porcine ETEC are moving towards the use of newer antimicrobials of higher public health significance. This review focuses on microorganisms and microbial-derived products that could provide a naturally evolved solution to ETEC infection and disease. This category holds a plethora of yet to be explored possibilities, however studies based around bacteriophage therapy, probiotics and the use of probiotic fermentation products as postbiotics have demonstrated promise. Ultimately, pig producers and veterinarians need these solutions to reduce the reliance on critically important antimicrobials (CIAs), to improve economic and animal welfare outcomes, and to lessen the One Health threat potentiated by the dissemination of AMR through the food chain.

Evidence of homologous recombination as a driver of diversity in Brachyspira pilosicoli.

The enteric, pathogenic spirochaete Brachyspira pilosicoli colonizes and infects a variety of birds and mammals, including humans. However, there is a paucity of genomic data available for this organism. This study introduces 12 newly sequenced draft genome assemblies, boosting the cohort of examined isolates by fourfold and cataloguing the intraspecific genomic diversity of the organism more comprehensively. We used several in silico techniques to define a core genome of 1751 genes and qualitatively and quantitatively examined the intraspecific species boundary using phylogenetic analysis and average nucleotide identity, before contextualizing this diversity against other members of the genus Brachyspira. Our study revealed that an additional isolate that was unable to be species typed against any other Brachyspira lacked putative virulence factors present in all other isolates. Finally, we quantified that homologous recombination has as great an effect on the evolution of the core genome of the B. pilosicoli as random mutation (r/m=1.02). Comparative genomics has informed Brachyspira diversity, population structure, host specificity and virulence. The data presented here can be used to contribute to developing advanced screening methods, diagnostic assays and prophylactic vaccines against this zoonotic pathogen.

First identification and characterisation of Brachyspira hyodysenteriae in pigs in Hong Kong.

Swine dysentery (SD) is an important endemic disease of pigs throughout the world. The most common aetiological agent is the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. The related spirochaete Brachyspira pilosicoli causes a milder form of colitis. We report the first isolation of B. hyodysenteriae and B. pilosicoli from a pig farm in Hong Kong. Faecal samples containing mucus or fresh blood were collected from the ground where finisher pigs had just been loaded into a truck for transport to the abattoir. The samples were subjected to selective anaerobic culture and PCR for B. hyodysenteriae and B. pilosicoli, and two isolates of both species were obtained. The B. hyodysenteriae isolates showed clinical resistance to tylosin and lincomycin, whilst the B. pilosicoli isolates were resistant to tylosin and showed intermediate susceptibility to lincomycin. The B. hyodysenteriae isolates were subjected to multilocus sequence typing and a single previously undescribed sequence type (ST250) was identified. Disease was not recorded in other pigs on the farm, but it may have been masked by the use of antimicrobials. Further work is required to examine the distribution of these two pathogens in this and other farms in Hong Kong and in adjoining mainland China.

Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing.

The pig colon is the habitat of diverse Brachyspira species, of which only a few are of clinical importance. Methods for identification have shifted from phenotypic to molecular testing over the last two decades. Following the emergence of B. hampsonii it became evident that relying on species-specific PCRs carries the risk of overlooking important new species. Consequently, sequencing was proposed as an unbiased alternative for identification of isolates. So far, the main target for identification across species has been the NADH oxidase gene (nox). However, multiple copies of this gene in the genome and potential lateral gene transfer reduce confidence when using this gene. This study compared identification and phylogentic relationship inferred from nox sequencing to that inferred from sequencing of the cpn60 universal target using a collection of 168 isolates from different Brachyspira species. The majority of isolates had an identical identification with both methods. There were a few outliers in the trees with uncertain assignment to a species by BLAST analysis. A few major discrepancies pertained to the pathogenic species B. hampsonii (2), B. pilosicoli (1) and B. suanatina (1). Weakly haemolytic variants of B. hyodysenteriae were assigned to the correct species by both methods. Some of the isolates identified as B. hampsonii also had a weakly haemolytic phenotype.

Correction to: The pathogenic intestinal spirochaete Brachyspira pilosicoli forms a diverse recombinant species demonstrating some local clustering of related strains and potential for zoonotic spread.

[This corrects the article DOI: 10.1186/1757-4749-5-24.].

Brachyspira catarrhinii sp. nov., an anaerobic intestinal spirochaete isolated from vervet monkeys may have been misidentified as Brachyspira aalborgi in previous studies.

To date nine species of anaerobic intestinal spirochaetes have been validly assigned to the genus Brachyspira. These include both pathogenic and non-pathogenic species. In the current study a genomic analysis of a novel spirochaete isolate was undertaken to determine whether it is a distinct species that previously has been misidentified as Brachyspira aalborgi. The genome of spirochaete strain Z12 isolated from the faeces of a vervet monkey was sequenced and compared to the genomes of the type strains of the nine assigned Brachyspira species. Genome to Genome Distance (GGD) values and Average Nucleotide Identity (ANI) values were determined. Single nucleotide polymorphisms (SNP) were used to create a phylogenetic tree to assess relatedness. The 16S rRNA gene sequences of the strains were aligned and the similarity amongst the Brachyspira species was recorded. Multilocus sequence typing (MLST) using five loci was conducted on Z12 and results compared with those for other Brachyspira isolates. Assembly of the Z12 sequences revealed a 2,629,108 bp genome with an average G + C content of 31.3%. The GGD, ANI, 16S rRNA gene sequence comparisons and the MLST results all indicated that Z12 represents a distinct species within the genus Brachyspira, with its nearest neighbour being B. aalborgi. Spirochaete strain Z12T was assigned as the type strain of a new species, Brachyspira catarrhinii sp. nov. The diagnostic PCR currently in use to detect B. aalborgi cross-reacts with Z12, but RFLP analysis of PCR product can be used to distinguish the two species. Previous reports of non-human primates being colonised by B. aalborgi based on PCR results may have been incorrect. The development of an improved diagnostic method will allow future studies on the distribution and possible clinical significance of these two anaerobic spirochaete species.

Chronology of emergence of the genus Leptospira and over-representation of gene families enriched by vitamin B2, B12 biosynthesis, cell adhesion and external encapsulating structure in L. interrogans isolates from asymptomatic dogs.

The spirochete species Leptospira interrogans is the most common cause of leptospirosis, producing acute to chronic infections in most mammalian species. This pathogenic bacterium has an ability to evolve in many ways to occupy various environments and hosts. In this study, we performed chronology analysis to look for insight into the emergence of Leptospira species, focusing on L. interrogans, and investigated gene gain and loss related to their adaptation in strains isolated from asymptomatic dogs. Chronology analysis revealed that the emergence of L. interrogans was around 53.7 million years ago (MYA), corresponding to the Paleogene period that coincided with an optimal climatic temperature and the evolution of suitable mammalian hosts. Gene families encoding for vitamin B2, B12 biosynthesis, cell adhesion and external encapsulating structure were found to be enriched in L. interrogans isolated from the urine of asymptomatic dogs. The activity of these gene families may have favored adaptations resulting in chronic infections.

Correction to: Genomic analysis of Leptospira interrogans serovar Paidjan and Dadas isolates from carrier dogs and comparative genomic analysis to detect genes under positive selection.

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Genomic analysis of Leptospira interrogans serovar Paidjan and Dadas isolates from carrier dogs and comparative genomic analysis to detect genes under positive selection.

BACKGROUND: Leptospirosis is an emerging infectious disease worldwide that can cause high morbidity and mortality rates in humans and animals. The causative spirochetes have reservoirs in mammalian hosts, but there has been limited analysis of the genomes of isolates recovered from animals. The aims of this study were to characterize genomic features of two Leptospira interrogans strains recently isolated from asymptomatic dogs in Thailand (strains CUDO5 and CDUO8), and to perform comparative genome analyses with other strains. Molecular adaptive evolution in L. interrogans as signaled by positive selection also was analyzed. RESULTS: Whole genome sequence analysis revealed that strains CUDO5 and CUDO8 had genome sizes of approximately 4.9 Mbp with 35.1% GC contents. Using monoclonal antibodies, strains CUDO5 and CUDO8 were identified as serovars Paidjan and Dadas, respectively. These strains harbored genes known to be associated with acute and chronic infections. Using Single Nucleotide Polymorphisms phylogeny (SNPs) with 97 L. interrogans strains, CUDO5 and CUDO8 had closest genetic relatedness with each other. Nevertheless, the serovar determinant region (rfb locus) showed variations in the genes encoding sugar biosynthesis. Amongst 13 representative L. interrogans strains examined for molecular adaptive evolution through positive selection under the site-model of Phylogenetic Analysis of Maximum Likelihood, genes responsible for iron acquisition (tlyA and hbpA), motility (fliN2, flgK, and flhB) and thermal adaptation (lpxD1) were under increased selective pressure. CONCLUSIONS: L. interrogans serovar Paidjan strain CUDO5 and serovar Dadas strain CUDO8 had close genetic relatedness as analyzed by SNPs phylogeny. They contained genes with established roles in acute and chronic leptospirosis. The rfb locus in both serovars showed gene variation associated with sugar biosynthesis. Positive selection analysis indicated that genes encoding factors involved in motility, temperature adaptation, and iron acquisition were under strong positive selection in L. interrogans. These may be associated with adaptation in the early stages of infection.