Rapid typing of infectious laryngotracheitis virus directly from tracheal tissues based on next-generation sequencing.

Infectious laryngotracheitis virus (ILTV) is the causative agent of an economically important disease of chickens causing upper respiratory tract infection. Strains of ILTV are commonly identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and/or PCR high resolution melt (PCR-HRM) curve analysis targeting several genes. However, these techniques examine only a limited number of mutations present inside the target regions and may generate unreliable results when the sample contains more than one strain. Here, we attempted to sequence the whole genome of ILTV with known identity (class 9) directly from tracheal scrapings to circumvent in vitro culturing, which can potentially introduce variations into the genome. Despite the large number of quality reads, mapping was compromised by poor overlapping and gaps, and assembly of the complete genome sequence was not possible. In a map-to-reference alignment, the regions with low coverage were deleted, those with high coverage were concatenated and a genome sequence of 139,465 bp was obtained, which covered 91% of the ILTV genome. Sixteen single-nucleotide polymorphisms (SNPs) were found between the ILTV isolate examined and ILTV class 9 (JN804827). Despite only 91% genome coverage, using sequence analysis and comparison with previously sequenced ILTVs, we were able to classify the isolate as class 9. Therefore, this technique has the potential to replace the current PCR-HRM technique, as it provides detailed information about the ILTV isolates.

Nebulization as a more efficient method than atomizer for experimental reproduction of avian colibacillosis in young chickens.

Infection and immunity studies involving genetically modified organisms (GMOs), such as gene knockout bacterial mutants, require stringent physical containment to prevent the accidental spread of these organisms into the environment. Experimental respiratory tract infection models often require the animals, for example birds, to be transported several times between a negative pressure housing isolator and a bespoke aerosol exposure chamber under positive pressure. While the exposure chamber is sealed and fitted with HEPA filters, the repeated movements of infected animals and opening of the chamber can still pose a serious risk of breaching containment of the organism in the experimental facility. In the current study, the ability of two aerosol infection protocols that expose birds to avian pathogenic E. coli (APEC) aerosols directly within the housing isolator was evaluated. Young chicks were exposed to APEC E956 within the negative pressure housing isolators using either a nebulizer or an atomizer. Birds exposed twice (days 1 and 4) to aerosols of APEC E956 produced by the nebulizer developed a rapidly progressing disease mimicking field cases of avian colibacillosis. However, birds exposed to aerosols of APEC E956 produced by an atomizer did not develop colibacillosis even after three exposures to APEC E956 on days 1, 4 and 7. Consequently, the current study reports the nebulizer was more efficacious in producing avian colibacillosis under stricter bacterial containment settings.RESEARCH HIGHLIGHTS Two aerosol exposure methods were evaluated to develop avian colibacillosis.Nebulizer method found to be more efficient in reproducing avian colibacillosis.Refined infection method can be used to study genetically modified organisms (GMOs).

Mycoplasmas under experimental antimicrobial selection: The unpredicted contribution of horizontal chromosomal transfer.

Horizontal Gene Transfer was long thought to be marginal in Mycoplasma a large group of wall-less bacteria often portrayed as minimal cells because of their reduced genomes (ca. 0.5 to 2.0 Mb) and their limited metabolic pathways. This view was recently challenged by the discovery of conjugative exchanges of large chromosomal fragments that equally affected all parts of the chromosome via an unconventional mechanism, so that the whole mycoplasma genome is potentially mobile. By combining next generation sequencing to classical mating and evolutionary experiments, the current study further explored the contribution and impact of this phenomenon on mycoplasma evolution and adaptation using the fluoroquinolone enrofloxacin (Enro), for selective pressure and the ruminant pathogen Mycoplasma agalactiae, as a model organism. For this purpose, we generated isogenic lineages that displayed different combination of spontaneous mutations in Enro target genes (gyrA, gyrB, parC and parE) in association to gradual level of resistance to Enro. We then tested whether these mutations can be acquired by a susceptible population via conjugative chromosomal transfer knowing that, in our model organism, the 4 target genes are scattered in three distinct and distant loci. Our data show that under antibiotic selective pressure, the time scale of the mutational pathway leading to high-level of Enro resistance can be readily compressed into a single conjugative step, in which several EnroR alleles were transferred from resistant to susceptible mycoplasma cells. In addition to acting as an accelerator for antimicrobial dissemination, mycoplasma chromosomal transfer reshuffled genomes beyond expectations and created a mosaic of resistant sub-populations with unpredicted and unrelated features. Our findings provide insights into the process that may drive evolution and adaptability of several pathogenic Mycoplasma spp. via an unconventional conjugative mechanism.

Mycoplasma bovis mbfN Encodes a Novel LRR Lipoprotein That Undergoes Proteolytic Processing and Binds Host Extracellular Matrix Components.

Mycoplasma bovis causes serious infections in ruminants, leading to huge economic losses. Lipoproteins are key components of the mycoplasma membrane and are believed to function in nutrient acquisition, adherence, enzymatic interactions with the host, and induction of the host's immune response to infection. Many genes of M. bovis have not been assigned functions, in part because of their low sequence similarity with other bacteria, making it difficult to extrapolate gene functions. This study examined functions of a surface-localized leucine-rich repeat (LRR) lipoprotein encoded by mbfN of M. bovis PG45. Homologs of MbfN were detected as 48-kDa peptides by Western blotting in all the strains of M. bovis included in this study, with the predicted 70-kDa full-length polypeptide detected in some strains. Sequence analysis of the gene revealed the absence in some strains of a region encoding the carboxyl-terminal 147 amino acids found in strain PG45, which could account for the variation detected by immunoblotting. In silico analysis of MbfN suggested that it may have an adhesion-related function. In vitro binding assays confirmed MbfN to be a fibronectin and heparin-binding protein. Disruption of mbfN in M. bovis PG45 significantly reduced (P = 0.033) the adherence of M. bovis PG45 to MDBK cells in vitro, demonstrating the role of MbfN as an adhesin.IMPORTANCE Experimental validation of the putative functions of genes in M. bovis will advance our understanding of the basic biology of this economically important pathogen and is crucial in developing prevention strategies. This study demonstrated the extracellular matrix binding ability of a novel immunogenic lipoprotein of M. bovis, and the role of this protein in adhesion by M. bovis suggests that it could play a role in virulence.

Mycoplasma bovis Membrane Protein MilA Is a Multifunctional Lipase with Novel Lipid and Glycosaminoglycan Binding Activity.

The survival, replication, and virulence of mycoplasmas depend on their ability to capture and import host-derived nutrients using poorly characterized membrane proteins. Previous studies on the important bovine pathogen Mycoplasma bovis demonstrated that the amino-terminal end of an immunogenic 226-kDa (P226) protein, encoded by milA (the full-length product of which has a predicted molecular weight of 303 kDa), had lipase activity. The predicted sequence of MilA contains glycosaminoglycan binding motifs, as well as multiple copies of a domain of unknown function (DUF445) that is also found in apolipoproteins. We mutagenized the gene to facilitate expression of a series of regions spanning the gene in Escherichia coli Using monospecific antibodies against these recombinant proteins, we showed that MilA was proteolytically processed into 226-kDa and 50-kDa fragments that were both partitioned into the detergent phase by Triton X-114 phase fractionation. Trypsin treatment of intact cells showed that P226 was surface exposed. In vitro, the recombinant regions of MilA bound to 1-anilinonaphthalene-8-sulfonic acid and to a variety of lipids. The MilA fragments were also shown to bind heparin. Antibody against the carboxyl-terminal fragment inhibited the growth of M. bovisin vitro This carboxyl end also bound and hydrolyzed ATP, suggestive of a potential role as an autotransporter. Our studies have demonstrated that DUF445 has lipid binding activity and that MilA is a multifunctional protein that may play multiple roles in the pathogenesis of infection with M. bovis.

Preliminary comparative analysis of the genomes of selected field reisolates of the Mycoplasma synoviae vaccine strain MS-H reveals both stable and unstable mutations after passage in vivo.

BACKGROUND: Genomic comparison of Mycoplasma synoviae vaccine strain MS-H and the MS-H parental strain 86,079/7NS established a preliminary profile of genes related to attenuation of MS-H. In this study we aimed to identify the stability of mutations found in MS-H after passage in experimental or field chickens, and to evaluate if any reverse mutation may be associated with changes in characteristics of MS-H in vitro or in vivo. RESULTS: Whole genome sequence analysis of 5 selected MS-H field reisolates revealed that out of 32 mutations reported previously in MS-H, 28 remained stable, while four found to be reversible to the wild-type. Each isolate possessed mutations in one to three of the genes obg, oppF1 and gap and/or a non-coding region. Examination of the 4 reversible mutations by protein modeling predicted that only two of them (in obg and oppF1 genes) could potentially restore the function of the respective protein to that of the wild-type. CONCLUSIONS: These results suggest that the majority of the MS-H mutations are stable after passage in vaccinated chickens. Characterisation of stable mutations found in MS-H could be utilised to develop rapid diagnostic techniques for differentiation of vaccine from field strains or ts- MS-H reisolates.

Correction to: Preliminary comparative analysis of the genomes of selected field reisolates of the Mycoplasma synoviae vaccine strain MS-H reveals both stable and unstable mutations after passage in vivo.

An amendment to this paper has been published and can be accessed via the original article.

Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type oppF1 influences its growth characteristics.

The Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF1 (oligopeptide permease transporter) which results in expression of a truncated OppF1. The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF1 gene in the MS-H genome. Wild-type oppF1 was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF1-tetM or with a similar vector lacking oppF1 coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF1-tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF1 detected bands of expected size for full-length OppF1 in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF1-tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF1-tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF1-tetM (passage 27) showed that the chromosomal copy of the mutated oppF1 had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF1 impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species.RESEARCH HIGHLIGHTS The full-length OppF1 was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF1 impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF1 in MS-H was replaced with wild-type oppF1.

Comparative genomic analyses of Mycoplasma synoviae vaccine strain MS-H and its wild-type parent strain 86079/7NS: implications for the identification of virulence factors and applications in diagnosis of M. synoviae.

Mycoplasma synoviae is an economically important avian pathogen worldwide, causing respiratory disease, infectious synovitis, airsacculitis and eggshell apex abnormalities in commercial chickens. Despite the widespread use of MS-H as a live attenuated vaccine over the past two decades, the precise molecular basis for loss of virulence in this vaccine is not yet fully understood. To address this, the whole genome sequence of the vaccine parent strain, 86079/7NS, was obtained and compared to that of the MS-H vaccine. Except for the vlhA expressed region, both genomes were nearly identical. Thirty-two single nucleotide polymorphisms (SNPs) were identified in MS-H, including 11 non-synonymous mutations that were predicted, by bioinformatics analysis, to have changed the secondary structure of the deduced proteins. One of these mutations caused truncation of the oppF-1 gene, which encodes the ATP-binding protein of an oligopeptide permease transporter. Overall, the attenuation of MS-H strain may be caused by the cumulative and complex effects of several mutations. The SNPs identified in MS-H were further analyzed by comparing the MS-H and 86079/7NS sequences with the strains WVU-1853 and MS53. In the genomic regions conserved between all strains, 30 SNPs were found to be unique to MS-H lineage. These results have provided a foundation for developing novel biomarkers for the detection of virulence in M. synoviae and also for designing new genotyping assays for discrimination of MS-H from field strains.

Autoimmune-Disease-Prone NOD Mice Help To Reveal a New Genetic Locus for Reducing Pulmonary Disease Caused by Mycoplasma pulmonis.

Mycoplasmas are bacterial pathogens of a range of animals, including humans, and are a common cause of respiratory disease. However, the host genetic factors that affect resistance to infection or regulate the resulting pulmonary inflammation are not well defined. We and others have previously demonstrated that nonobese diabetic (NOD) mice can be used to investigate disease loci that affect bacterial infection and autoimmune diabetes. Here we show that NOD mice are more susceptible than C57BL/6 (B6) mice to infection with Mycoplasma pulmonis, a natural model of pulmonary mycoplasmosis. The lungs of infected NOD mice had higher loads of M. pulmonis and more severe inflammatory lesions. Moreover, congenic NOD mice that harbored different B6-derived chromosomal intervals enabled identification and localization of a new mycoplasmosis locus, termed Mpr2, on chromosome 13. These congenic NOD mice demonstrated that the B6 allele for Mpr2 reduced the severity of pulmonary inflammation caused by infection with M. pulmonis and that this was associated with altered cytokine and chemokine concentrations in the infected lungs. Mpr2 also colocalizes to the same genomic interval as Listr2 and Idd14, genetic loci linked to listeriosis resistance and autoimmune diabetes susceptibility, respectively, suggesting that allelic variation within these loci may affect the development of both infectious and autoimmune disease.

Genome analysis of Mycoplasma synoviae strain MS-H, the most common M. synoviae strain with a worldwide distribution.

BACKGROUND: The bacterial pathogen Mycoplasma synoviae can cause subclinical respiratory disease, synovitis, airsacculitis and reproductive tract disease in poultry and is a major cause of economic loss worldwide. The M. synoviae strain MS-H was developed by chemical mutagenesis of an Australian isolate and has been used as a live attenuated vaccine in many countries over the past two decades. As a result it may now be the most prevalent strain of M. synoviae globally. Differentiation of the MS-H vaccine from local field strains is important for epidemiological investigations and is often required for registration of the vaccine. RESULTS: The complete genomic sequence of the MS-H strain was determined using a combination of Illumina and Nanopore methods and compared to WVU-1853, the M. synoviae type strain isolated in the USA 30 years before the parent strain of MS-H, and MS53, a more recent isolate from Brazil. The vaccine strain genome had a slightly larger number of pseudogenes than the two other strains and contained a unique 55 kb chromosomal inversion partially affecting a putative genomic island. Variations in gene content were also noted, including a deoxyribose-phosphate aldolase (deoC) fragment and an ATP-dependent DNA helicase gene found only in MS-H. Some of these sequences may have been acquired horizontally from other avian mycoplasma species. CONCLUSIONS: MS-H was somewhat more similar to WVU-1853 than to MS53. The genome sequence of MS-H will enable identification of vaccine-specific genetic markers for use as diagnostic and epidemiological tools to better control M. synoviae.

pSTM6-275, a Conjugative IncHI2 Plasmid of Salmonella enterica That Confers Antibiotic and Heavy-Metal Resistance under Changing Physiological Conditions.

Detailed annotation of an IncHI2 plasmid, pSTM6-275, from Salmonella enterica serotype 1,4,5,12:i:- strain TW-Stm6 revealed a composite structure, including antimicrobial resistance genes on mobile genetic elements. The plasmid was thermosensitive for transfer to Escherichia coli and conferred reduced susceptibility to antibiotics, copper sulfate, and silver nitrate. Metal ion susceptibility was dependent on physiological conditions, giving an insight into the environments where this trait might confer a fitness advantage.

A Novel Glaesserella sp. Isolated from Pigs with Severe Respiratory Infections Has a Mosaic Genome with Virulence Factors Putatively Acquired by Horizontal Transfer.

An unknown member of the family Pasteurellaceae was repeatedly isolated from 20- to 24-week-old pigs with severe pulmonary lesions reared on the same farm in Victoria, Australia. The etiological diagnosis of the disease was inconclusive. The complete genome sequence analysis of one strain, 15-184, revealed some phylogenic proximity to Glaesserella (Haemophilus) parasuis, the cause of Glasser's disease. However, the sequences of the 16S rRNA and housekeeping genes, as well as the average nucleotide identity scores, differed from those of all other known species in the family Pasteurellaceae The protein content of 15-184 was composite, with 60% of coding sequences matching known G. parasuis products, while more than 20% had a closer relative in the genera Actinobacillus, Mannheimia, Pasteurella, and Bibersteinia Several putative virulence genes absent from G. parasuis but present in other Pasteurellaceae were also found, including the apxIII RTX toxin gene from Actinobacillus pleuropneumoniae, ABC transporters from Actinobacillus minor, and iron transporters from various species. Three prophages and one integrative conjugative element were present in the isolate. Horizontal gene transfers might explain the mosaic genomic structure and atypical metabolic and virulence characteristics of 15-184. This organism has not been assigned a taxonomic position in the family, but this study underlines the need for a large-scale epidemiological and clinical characterization of this novel pathogen in swine populations, as a genomic analysis suggests it could have a severe impact on pig health.IMPORTANCE Several species of Pasteurellaceae cause a range of significant diseases in pigs. A novel member of this family was recently isolated from Australian pigs suffering from severe respiratory infections. Comparative whole-genome analyses suggest that this bacterium represents a new species, which possesses a number of virulence genes horizontally acquired from a diverse range of other Pasteurellaceae While the possible contribution of other coinfecting noncultivable agents to the disease has not been ruled out in this study, the repertoire of virulence genes found in this organism may nevertheless explain some aspects of the associated pathology observed on the farm. The prevalence of this novel pathogen within pig populations is currently unknown. This finding is of particular importance for the pig industry, as this organism can have a serious impact on the health of these animals.

Pathological and microbiological investigations into cases of bacterial chondronecrosis and osteomyelitis in broiler poultry.

Bacterial chondronecrosis and osteomyelitis (BCO) is increasingly recognized as a major cause of lameness in commercial broilers chickens worldwide, but the pathogenesis of the condition is incompletely understood. This was a longitudinal study of 20 commercial broiler farms in Victoria, Australia, to investigate the aetiology and pathology of BCO. Thorough postmortem examination was performed on culled and dead birds (n = 325) from 20 different flocks at either 1 week, 4 weeks or 5 weeks of age and samples were analysed by conventional bacteriology, molecular identification of infectious organisms detected, serology and histopathology. BCO occurs throughout the life of broiler flocks at a very high rate, with lesions detected in 28% (95% CI 23-34%) of the mortalities and culls. The condition occurs with similar prevalence in both the femur and tibiotarsus. BCO is an infectious process that appears to result from bacteraemia and haematological spread of bacterial pathogens, especially Escherichia coli, to the bones, with 65.3% bacterial isolates from histologically confirmed BCO identified as E. coli, 11.5% as Staphylococcus and the remainder composed of mixed infections or a range of other minor isolates. We observed that almost all E. coli isolated from cases of BCO are avian pathogenic E. coli, suggesting that preventative measures should be directed at this organism.

Integrative conjugative elements are widespread in field isolates of Mycoplasma species pathogenic for ruminants.

Comparative genomics have revealed massive horizontal gene transfer (HGT) between Mycoplasma species sharing common ruminant hosts. Further results pointed toward an integrative conjugative element (ICE) as an important contributor of HGT in the small-ruminant-pathogen Mycoplasma agalactiae. To estimate the prevalence of ICEs in ruminant mycoplasmas, we surveyed their occurrence in a collection of 166 field strains representing 4 (sub)species that are recognized as major pathogens. Based on available sequenced genomes, we first defined the conserved, minimal ICE backbone as composed of 4 coding sequences (CDSs) that are evenly distributed and predicted to be essential for ICE chromosomal integration-excision and horizontal transfer. Screening of the strain collection revealed that these 4 CDSs are well represented in ruminant Mycoplasma species, suggesting widespread occurrence of ICEs. Yet their prevalence varies within and among species, with no correlation found with the individual strain history. Extrachromosomal ICE forms were also often detected, suggesting that ICEs are able to circularize in all species, a first and essential step in ICE horizontal transfer. Examination of the junction of the circular forms and comparative sequence analysis of conserved CDSs clearly pointed toward two types of ICE, the hominis and spiroplasma types, most likely differing in their mechanism of excision-integration. Overall, our data indicate the occurrence and maintenance of functional ICEs in a large number of field isolates of ruminant mycoplasmas. These may contribute to genome plasticity and gene exchanges and, presumably, to the emergence of diverse genotypes within pathogenic mycoplasmas of veterinary importance.

ICEA of Mycoplasma agalactiae: a new family of self-transmissible integrative elements that confers conjugative properties to the recipient strain.

Horizontal gene transfer (HGT) is a major force of microbial evolution but was long thought to be marginal in mycoplasmas. In silico detection of exchanged regions and of loci encoding putative Integrative Conjugative Elements (ICE) in several mycoplasma genomes challenged this view, raising the prospect of these simple bacteria being able to conjugate. Using the model pathogen Mycoplasma agalactiae, we demonstrated for the first time that one of these elements, ICEA, is indeed self-transmissible. As a hallmark of conjugative processes, ICEA transfers were DNase resistant and required viable cells. ICEA acquisition conferred ICE-negative strains with the new ability to conjugate, allowing the spread of ICEA. Analysis of transfer-deficient mutants indicated that this process requires an ICEA-encoded lipoprotein of unknown function, CDS14. Formation of a circular extrachromosomal intermediate and the subsequent chromosomal integration of ICEA involved CDS22, an ICEA-encoded product distantly related to the ISLre2 transposase family. Remarkably, ICEA has no specific or no preferential integration site, often resulting in gene disruptions. Occurrence of functional mycoplasma ICE offers these bacteria with a means for HGT, a phenomenon with far-reaching implications given their minute-size genome and the number of species that are pathogenic for a broad host-range.

Detection and Differentiation of Entomopathogenic Serratia spp. to Inform Reintroduction of the Critically Endangered Lord Howe Island Stick Insect Dryococelus australis.

Once rodents have been successfully eradicated from Lord Howe Island, Australia, the critically endangered Lord Howe Island stick insect (Dryococelus australis (Montrouzier)) may be reintroduced, a century after it was thought to have become extinct. In captive populations of D. australis, elevated mortalities have been associated with bacterial pathogens. To better define the infectious risk posed by entomopathogens to the reintroduction program, we investigated the bacteria isolated from captive D. australis kept at Melbourne Zoo and on Lord Howe Island and from environmental samples and free-living invertebrates collected on various parts of the island. At Melbourne Zoo, Serratia and Pseudomonas spp. were the bacteria most frequently isolated between 2013 and 2019. Serratia spp. were also the organisms most frequently isolated from insects sampled in April 2019 from the captive population on Lord Howe Island. In addition, Serratia spp. were isolated from a range of environmental samples collected on Lord Howe Island during March-April 2019. These environmental isolates had a broader range of biochemical and molecular characteristics than those obtained from the captive insect populations. A large proportion of these isolates were urease positive and had biochemical profiles previously not described for Serratia spp. This study highlights the need for better surveillance for potential pathogens in understudied regions and sites. We conclude that infections caused by Serratia spp. might pose a problem to the captive breeding program for D. australis but that the risk of introducing novel pathogens to Lord Howe Island through infected insects is low. Our study explores some of the potential risks involved in captive breeding and provides a valuable example of using pathogen surveillance to better inform an invertebrate conservation program.

Pasteurella multocida ST20 is widespread in Australian poultry farms and may infect wild waterbirds.

The bacterial agent that causes fowl cholera, Pasteurella multocida, was isolated from two deceased wild waterbirds in Victoria, Australia, in 2013. Whole genome sequence analysis placed the isolates into ST20, a subtype described in farmed chickens from Queensland, Australia and more recently in feedlot cattle and in pigs across a broader area of the continent. This study also found ST20 between 2009 and 2022 on three chicken farms and two turkey farms located in four Australian states. The sequences of 25 of these ST20 isolates were compared to 280 P. multocida genomes from 23 countries and to 94 ST20 Illumina datasets from Queensland that have been deposited in public databases. The ST20 isolates formed a single phylogenetic clade and were clustered into four sub-groups with highly similar genomes, possessing either LPS type 1 or type 3 loci. Various repertoires of mobile genetic elements were present in isolates from farmed, but not wild birds, suggesting complex histories of spill-over between avian populations and gene acquisition within farm environments. No major antimicrobial resistance was predicted in any of the ST20 isolates by the genomic analysis. The closest relative of these isolates was a ST394 bovine respiratory tract isolate from Queensland, which differed from ST20 by only one allele and carried beta-lactam and tetracycline resistance genes. These findings underline the importance of understanding the role of wild and commercial birds in the maintenance of fowl cholera, and of implementing regular epidemiological surveillance and biosecurity management programmes in wildlife, as well as free-range poultry farms.

The faecal microbiome of the Australian silver gull contains phylogenetically diverse ExPEC, aEPEC and Escherichia coli carrying the transmissible locus of stress tolerance.

Wildlife are implicated in the dissemination of antimicrobial resistance, but their roles as hosts for Escherichia coli that pose a threat to human and animal health is limited. Gulls (family Laridae) in particular, are known to carry diverse lineages of multiple-antibiotic resistant E. coli, including extra-intestinal pathogenic E. coli (ExPEC). Whole genome sequencing of 431 E. coli isolates from 69 healthy Australian silver gulls (Chroicocephalus novaehollandiae) sampled during the 2019 breeding season, and without antibiotic selection, was undertaken to assess carriage in an urban wildlife population. Phylogenetic analysis and genotyping resolved 123 sequence types (STs) representing most phylogroups, and identified diverse ExPEC, including an expansive phylogroup B2 cluster comprising 103 isolates (24 %; 31 STs). Analysis of the mobilome identified: i) widespread carriage of the Yersinia High Pathogenicity Island (HPI), a key ExPEC virulence determinant; ii) broad distribution of two novel phage elements, each carrying sitABCD and iii) carriage of the transmissible locus of stress tolerance (tLST), an element linked to sanitation resistance. Of the 169 HPI carrying isolates, 49 (48 %) represented diverse B2 isolates hosting FII-64 ColV-like plasmids that lacked iutABC and sitABC operons typical of ColV plasmids, but carried the serine protease autotransporter gene, sha. Diverse E. coli also carried archetypal ColV plasmids (52 isolates; 12 %). Clusters of closely related E. coli (<50 SNVs) from ST58, ST457 and ST746, sourced from healthy gulls, humans, and companion animals, were frequently identified. In summary, anthropogenically impacted gulls host an expansive E. coli population, including: i) putative ExPEC that carry ColV virulence gene cargo (101 isolates; 23.4 %) and HPI (169 isolates; 39 %); ii) atypical enteropathogenic E. coli (EPEC) (17 isolates; 3.9 %), and iii) E. coli that carry the tLST (20 isolates; 4.6 %). Gulls play an important role in the evolution and transmission of E. coli that impact human health.

Combination of differential growth at two different temperatures with a quantitative real-time polymerase chain reaction to determine temperature-sensitive phenotype of Mycoplasma synoviae.

Mycoplasma synoviae infections result in significant economic losses in the chicken and turkey industries. A commercially available live temperature-sensitive (ts (+)) vaccine strain MS-H has been found to be effective in controlling M. synoviae infections in commercial layer and broiler breeder farms in various countries, including Australia. Detection and differentiation of MS-H from field strains (ts (-)) and from ts (-) MS-H reisolates in vaccinated flocks is vital in routine flock status monitoring. At present microtitration is the only available technique to determine the ts phenotype of M. synoviae. This technique is time consuming and not amenable to automation. In the present study, a quantitative real-time polymerase chain reaction (Q-PCR) was combined with simultaneous culturing of M. synoviae at two different temperatures (33°C and 39.5°C) to determine the ts phenotype of 22 Australian M. synoviae strains/isolates. The M. synoviae type strain WVU-1853 was also included for comparison. A ratio of the copy numbers of the variable lipoprotein haemagglutinin (vlhA) gene at the two temperatures was calculated and a cut-off value was determined and used to delineate the ts phenotype. In all M. synoviae strains/isolates tested in this study, the ts phenotype determined using Q-PCR was in agreement with that determined using conventional microtitration. Combination of Q-PCR with differential growth at two different temperatures is a rapid, reliable and accurate technique that could be used as an effective tool in laboratories actively involved in ts phenotyping of M. synoviae strains/isolates.