Xanthomonas rydalmerensis sp. nov., a non-pathogenic member of Group 1 Xanthomonas.

Five bacterial isolates were isolated from Fragaria × ananassa in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, the results of biochemical characterisation indicated that these isolates represented members of the genus Xanthomonas. To determine their species, further analysis was conducted using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates represented members of the genus Xanthomonas but did not allow them to be classified with respect to species. Genome relatedness indices and the results of extensive phylogenetic analysis confirmed that the isolates were members of the genus Xanthomonas and represented a novel species. On the basis the minimal presence of virulence-associated factors typically found in genomes of members of the genus Xanthomonas, we suggest that these isolates are non-pathogenic. This conclusion was supported by the results of a pathogenicity assay. On the basis of these findings, we propose the name Xanthomonas rydalmerensis, with DAR 34855T = ICMP 24941 as the type strain.

Characterisation of the Theileria orientalis Piroplasm Proteome across Three Common Genotypes.

Theileria orientalis is an emerging apicomplexan pathogen of cattle occurring in areas populated by the principal vector tick, Haemaphysalis longicornis. Unlike transforming Theileria spp. that induce cancer-like proliferation of lymphocytes via their schizont stage, T. orientalis destroys host erythrocytes during its piroplasm phase resulting in anaemia. The underlying pathogenic processes of T. orientalis infection are poorly understood; consequently, there are no vaccines for prevention of T. orientalis infection and chemotherapeutic options are limited. To identify antigens expressed during the piroplasm phase of T. orientalis, including those which may be useful targets for future therapeutic development, we examined the proteome across three common genotypes of the parasite (Ikeda, Chitose and Buffeli) using preparations of piroplasms purified from bovine blood. A combination of Triton X-114 extraction, one-dimensional electrophoresis and LC-MS/MS identified a total of 1113 proteins across all genotypes, with less than 3% of these representing host-derived proteins. Just over three quarters of T. orientalis proteins (78%) identified were from the aqueous phase of the TX-114 extraction representing cytosolic proteins, with the remaining 22% from the detergent phase, representing membrane-associated proteins. All enzymes involved in glycolysis were expressed, suggesting that this is the major metabolic pathway used during the T. orientalis piroplasm phase. Proteins involved in binding and breakdown of haemoglobin were also identified, suggesting that T. orientalis uses haemoglobin as a source of amino acids. A number of proteins involved in host cell interaction were also identified which may be suitable targets for the development of chemotherapeutics or vaccines.

Complete Genomes of Theileria orientalis Chitose and Buffeli Genotypes Reveal within Species Translocations and Differences in ABC Transporter Content.

Theileria orientalis causes losses to cattle producers in Eastern Asia, Oceania and, more recently, North America. One pathogenic genotype (Ikeda) has been sequenced to the chromosomal level, while only draft genomes exist for globally distributed Chitose and Buffeli genotypes. To provide an accurate comparative gene-level analysis and help further understand their pathogenicity, we sequenced isolates of the Chitose and Buffeli genotypes of T. orientalis using long-read sequencing technology. A combination of several long-read assembly methods and short reads produced chromosomal-level assemblies for both Fish Creek (Chitose) and Goon Nure (Buffeli) isolates, including the first complete and circular apicoplast genomes generated for T. orientalis. Comparison with the Shintoku (Ikeda) reference sequence showed both large and small translocations in T. orientalis Buffeli, between chromosomes 2 and 3 and chromosomes 1 and 4, respectively. Ortholog clustering showed expansion of ABC transporter genes in Chitose and Buffeli. However, differences in several genes of unknown function, including DUF529/FAINT-domain-containing proteins, were also identified and these genes were more prevalent in Ikeda and Chitose genotypes. Phylogenetics and similarity measures were consistent with previous short-read genomic analysis. The generation of chromosomal sequences for these highly prevalent T. orientalis genotypes will also support future studies of population genetics and mixed genotype infections.

Phylogenomic diversity of Vibrio species and other Gammaproteobacteria isolated from Pacific oysters (Crassostrea gigas) during a summer mortality outbreak.

The Pacific oyster (PO), Crassostrea gigas, is an important commercial marine species but periodically experiences large stock losses due to disease events known as summer mortality. Summer mortality has been linked to environmental perturbations and numerous viral and bacterial agents, indicating this disease is multifactorial in nature. In 2013 and 2014, several summer mortality events occurred within the Port Stephens estuary (NSW, Australia). Extensive culture and molecular-based investigations were undertaken and several potentially pathogenic Vibrio species were identified. To improve species identification and genomically characterise isolates obtained from this outbreak, whole-genome sequencing (WGS) and subsequent genomic analyses were performed on 48 bacterial isolates, as well as a further nine isolates from other summer mortality studies using the same batch of juveniles. Average nucleotide identity (ANI) identified most isolates to the species level and included members of the Photobacterium, Pseudoalteromonas, Shewanella and Vibrio genera, with Vibrio species making up more than two-thirds of all species identified. Construction of a phylogenomic tree, ANI analysis, and pan-genome analysis of the 57 isolates represents the most comprehensive culture-based phylogenomic survey of Vibrios during a PO summer mortality event in Australian waters and revealed large genomic diversity in many of the identified species. Our analysis revealed limited and inconsistent associations between isolate species and their geographical origins, or host health status. Together with ANI and pan-genome results, these inconsistencies suggest that to determine the role that microbes may have in Pacific oyster summer mortality events, isolate identification must be at the taxonomic level of strain. Our WGS data (specifically, the accessory genomes) differentiated bacterial strains, and coupled with associated metadata, highlight the possibility of predicting a strain's environmental niche and level of pathogenicity.

Draft genomes of Perkinsus olseni and Perkinsus chesapeaki reveal polyploidy and regional differences in heterozygosity.

Perkinsus spp. parasites have significant impact on aquaculture and wild mollusc populations. We sequenced the genomes of five monoclonal isolates of Perkinsus olseni and one Perkinsus chesapeaki from international sources. Sequence analysis revealed similar levels of repetitive sequence within species, a polyploid genome structure, and substantially higher heterozygosity in Oceanian-sourced isolates. We also identified tandem replication of the rRNA transcriptional unit, with high strain variation. Characterized gene content was broadly similar amongst all Perkinsus spp. but P. olseni Oceanian isolates contained an elevated number of genes compared to other P. olseni isolates and cox3 could not be identified in any Perkinsus spp. sequence. Phylogenetics and average nucleotide identity scans were consistent with all P. olseni isolates being within one species. These are the first genome sequences generated for both P. olseni and P. chesapeaki and will allow future advances in diagnostic design and population genomics of these important aquatic parasites.

Draft Genome Sequence of a Novel "Candidatus Liberibacter" Species Detected in a Zanthoxylum Species from Bhutan.

The draft genome sequence of a novel "Candidatus Liberibacter" species detected in an unidentified species of Zanthoxylum (Rutaceae) collected in Bhutan is reported. The total length is 1,408,989 bp with 1,169 coding sequences in 96 contigs, a GC content of 37.3%, and 76 to 77% average nucleotide identity with several other "Ca Liberibacter" species.

An epizootic of Chlamydia psittaci equine reproductive loss associated with suspected spillover from native Australian parrots.

Chlamydia psittaci is an avian pathogen capable of spill-over infections to humans. A parrot C. psittaci strain was recently detected in an equine reproductive loss case associated with a subsequent cluster of human C. psittaci infections. In this study, we screened for C. psittaci in cases of equine reproductive loss reported in regional New South Wales, Australia during the 2016 foaling season. C. psittaci specific-PCR screening of foetal and placental tissue samples from cases of equine abortion (n = 161) and foals with compromised health status (n = 38) revealed C. psittaci positivity of 21.1% and 23.7%, respectively. There was a statistically significant geographical clustering of cases ~170 km inland from the mid-coast of NSW (P < 0.001). Genomic analysis and molecular typing of C. psittaci positive samples from this study and the previous Australian equine index case revealed that the equine strains from different studs in regional NSW were clonal, while the phylogenetic analysis revealed that the C. psittaci strains from both Australian equine disease clusters belong to the parrot-associated 6BC clade, again indicative of spill-over of C. psittaci infections from native Australian parrots. The results of this work suggest that C. psittaci may be a more significant agent of equine reproductive loss than thought. A range of studies are now required to evaluate (a) the exact role that C. psittaci plays in equine reproductive loss; (b) the range of potential avian reservoirs and factors influencing infection spill-over; and

Analysis of Theileria orientalis draft genome sequences reveals potential species-level divergence of the Ikeda, Chitose and Buffeli genotypes.

BACKGROUND: Theileria orientalis (Apicomplexa: Piroplasmida) has caused clinical disease in cattle of Eastern Asia for many years and its recent rapid spread throughout Australian and New Zealand herds has caused substantial economic losses to production through cattle deaths, late term abortion and morbidity. Disease outbreaks have been linked to the detection of a pathogenic genotype of T. orientalis, genotype Ikeda, which is also responsible for disease outbreaks in Asia. Here, we sequenced and compared the draft genomes of one pathogenic (Ikeda) and two apathogenic (Chitose, Buffeli) isolates of T. orientalis sourced from Australian herds. RESULTS: Using de novo assembled sequences and a single nucleotide variant (SNV) analysis pipeline, we found extensive genetic divergence between the T. orientalis genotypes. A genome-wide phylogeny reconstructed to address continued confusion over nomenclature of this species displayed concordance with prior phylogenetic studies based on the major piroplasm surface protein (MPSP) gene. However, average nucleotide identity (ANI) values revealed that the divergence between isolates is comparable to that observed between other theilerias which represent distinct species. Analysis of SNVs revealed putative recombination between the Chitose and Buffeli genotypes and also between Australian and Japanese Ikeda isolates. Finally, to inform future vaccine studies, dN/dS ratios and surface location predictions were analysed. Six predicted surface protein targets were confirmed to be expressed during the piroplasm phase of the parasite by mass spectrometry. CONCLUSIONS: We used whole genome sequencing to demonstrate that the T. orientalis Ikeda, Chitose and Buffeli variants show substantial genetic divergence. Our data indicates that future researchers could potentially consider disease-associated Ikeda and closely related genotypes as a separate species from non-pathogenic Chitose and Buffeli.

Factors associated with seroconversion to the major piroplasm surface protein of the bovine haemoparasite Theileria orientalis.

BACKGROUND: Bovine theileriosis caused by Theileria orientalis is an emerging disease of cattle in the Asia-Pacific region where it causes a significant economic burden to meat and milk production. While host immunological responses to the lymphocyte-transforming species of Theileria, T. parva and T. annulata, have been well studied, little is known about the immune response to this non-transforming species. METHODS: We developed a recombinant antigen ELISA based on the major piroplasm surface protein (MPSP) of T. orientalis and investigated whether seroconversion to the MPSP was associated with clinical factors (anaemia), parasite burden and parasite genotype. We also examined the dynamics of seroconversion in animals acutely infected with T. orientalis. RESULTS: In cattle testing qPCR positive for T. orientalis, seroconversion was more frequent in anaemic compared to normal cattle (P < 0.0001). The ELISA ratio (ER) was highly correlated with total parasite burden as measured by qPCR (r = 0.69; P < 0.0001); however when loads of individual genotypes of the parasite were examined, only the pathogenic Ikeda genotype was highly correlated with ER. Conversely, seroconversion was less frequently detected in the presence of benign T. orientalis genotypes. Temporal measurement of the serological response, parasite burden and packed cell volume (PCV) in acutely infected animals revealed that seroconversion to the MPSP occurs within 2-3 weeks of the initial qPCR detection of the parasite and coincides with a peak in infection intensity and a declining PCV. CONCLUSION: Whether the serological response to the MPSP is immunoprotective against re-infection or recrudescence requires further investigation; however the MPSP represents a promising target for a subunit vaccine given that genetic variability within the MPSP results in differential pathogenicity of T. orientalis.

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity.

Quantification of the pirimicarb resistance allele frequency in pooled cotton aphid (Aphis gossypii Glover) samples by TaqMan SNP genotyping assay.

BACKGROUND: Pesticide resistance monitoring is a crucial part to achieving sustainable integrated pest management (IPM) in agricultural production systems. Monitoring of resistance in arthropod populations is initially performed by bioassay, a method that detects a phenotypic response to pesticides. Molecular diagnostic assays, offering speed and cost improvements, can be developed when the causative mutation for resistance has been identified. However, improvements to throughput are limited as genotyping methods cannot be accurately applied to pooled DNA. Quantifying an allele frequency from pooled DNA would allow faster and cheaper monitoring of pesticide resistance. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate a new method to quantify a resistance allele frequency (RAF) from pooled insects via TaqMan assay by using raw fluorescence data to calculate the transformed fluorescence ratio k' at the inflexion point based on a four parameter sigmoid curve. Our results show that k' is reproducible and highly correlated with RAF (r >0.99). We also demonstrate that k' has a non-linear relationship with RAF and that five standard points are sufficient to build a prediction model. Additionally, we identified a non-linear relationship between runs for k', allowing the combination of samples across multiple runs in a single analysis. CONCLUSIONS/SIGNIFICANCE: The transformed fluorescence ratio (k') method can be used to monitor pesticide resistance in IPM and to accurately quantify allele frequency from pooled samples. We have determined that five standards (0.0, 0.2, 0.5, 0.8, and 1.0) are sufficient for accurate prediction and are statistically-equivalent to the 13 standard points used experimentally.

Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography.

UNLABELLED: Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF↓QQ(677), consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. IMPORTANCE: Vaccines used to control Mycoplasma hyopneumoniae infection provide only partial protection. Proteins of the P97/P102 families are highly expressed, functionally redundant molecules that are substrates of endoproteases that generate multifunctional adhesin fragments on the cell surface. We show that P146 displays a chimeric structure consisting of an N terminus, which shares sequence identity with P97, and novel central and C-terminal regions. P146 is endoproteolytically processed at multiple sites, generating at least nine fragments on the surface of M. hyopneumoniae. Dominant cleavage events occurred at S/T-X-F↓X-D/E-like sites generating P50(P146), P40(P146), and P85(P146). Recombinant proteins designed to mimic the major cleavage fragments bind porcine cilia, heparin, and plasminogen. P146 undergoes endoproteolytic processing events at multiple sites and with differential processing efficiency, generating combinatorial diversity on the surface of M. hyopneumoniae.

Mycoplasma hyopneumoniae Surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs.

P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site (761)L-N-V↓A-V-S(766) in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.

Mhp182 (P102) binds fibronectin and contributes to the recruitment of plasmin(ogen) to the Mycoplasma hyopneumoniae cell surface.

Mycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102 kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (K(D) ~ 76 nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with K(D) s of 26 and 33 nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence.

Sequence TTKF ↓ QE defines the site of proteolytic cleavage in Mhp683 protein, a novel glycosaminoglycan and cilium adhesin of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45(683), P48(683), and P50(683). A peptide sequence (TTKF↓QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48(683) and P50(683), determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45(683), P48(683), and P50(683) reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1(683)-F5(683), spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1(683)-F5(683) also bound porcine epithelial cilia, and antisera to F2(683) and F5(683) significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45(683), P48(683), and P50(683) each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.

Mass spectrometric characterization of the surface-associated 42 kDa lipoprotein JlpA as a glycosylated antigen in strains of Campylobacter jejuni.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in the developed world. Immunoproteomics highlighted a 42-45 kDa antigen that comigrated on two-dimensional (2-DE) gels with the C. jejuni major outer membrane protein (MOMP). Predictive analysis revealed two candidates for the identity of the antigen, the most likely of which was the surface-associated lipoprotein, JlpA. Recombinant JlpA (rJlpA) reacted with patient sera, confirming that JlpA is antigenic. Polyclonal antibodies raised against rJlpA reacted against 3 JlpA mass variants from multiple C. jejuni. These variants differed by approximately 1.5 kDa, suggesting the presence of the N-linked C. jejuni glycan on two sites. Soybean agglutinin affinity and 2-DE purified 2 JlpA glycoforms (43.5 and 45 kDa). Their identities were confirmed using mass spectrometry following trypsin digest. Glycopeptides within JlpA variants were identified by proteinase-K digestion, graphite micropurification and MS-MS. Sites of glycosylation were confirmed as asparagines 107 and 146, both of which are flanked by the N-linked sequon. Sequence analysis confirmed that the N146 sequon is conserved in all C. jejuni genomes examined to date, while the N107 sequon is absent in the reference strain NCTC 11168. Western blotting confirmed the presence of only a single JlpA glycoform in both virulent (O) and avirulent (GS) isolates of NCTC 11168. MS analysis showed that JlpA exists as 3 discrete forms, unmodified, glycosylated at N146, and glycosylated at both N(146/107), suggesting glycan addition at N146 is necessary for N107 glycosylation. Glycine extracts and Western blotting revealed that doubly glycosylated JlpA was the predominant form on the C. jejuni JHH1 surface; however, glycosylation is not required for antigenicity. This is the first study to identify N-linked glycosylation of a surface-exposed C. jejuni virulence factor and to show strain variation in glycosylation sites.