Optimising Guide RNA Production for Multiplexed Cas9-Targeted Nanopore Sequencing to Detect Pathogens.

In agriculture, biosecurity, and human health, the rapid and accurate detection of pathogens and pests is crucial. Our study investigates the sensitivity and practicality of six guide RNA (gRNA) production methods for use in Nanopore Cas9-targeted sequencing (nCATS), focusing on their implications for multiplexed pathogen and pest detection. Each production method generated a library of eight gRNAs capable of excising ~ 1.

Application of a Quantitative Real-Time PCR Assay for Early Detection of Serovar Enteritidis on Poultry Farms During an Outbreak in New South Wales, Australia (2018-2020).

spp. are a significant cause of human foodborne illness globally, with ingestion of contaminated eggs a major vehicle for infection. serovar Enteritidis (.

Genomic diversity and tracing of in Australia: implications for American foulbrood outbreak surveillance in low-diversity populations.

is the causative agent of American foulbrood (AFB) in honeybees () and a devastating pathogen for honey and pollination industries worldwide. Despite this threat, a genomic survey of has not been attempted within Australia. To examine the diversity of Australian populations, we sequenced 368 .

sp. nov., sp. nov. and sp. nov.: three members of group 1 .

Between 1976 and 2010, four bacterial isolates were collected in New South Wales and Queensland, Australia, and stored as part of routine biosecurity surveillance. Recently, these historic isolates were analysed as part of a larger project to enhance the taxonomic accuracy of our culture collection and improve Australia's biosecurity preparedness. Three isolates were collected from , initially identified as sp.

Characterisation of the genetic diversity of citrus viroid VII using amplicon sequencing.

Viroids occur in plants as swarms of sequence variants clustered around a dominant variant, leading to adoption of the term 'quasispecies' to describe the viroid population in an individual host. The composition of the quasispecies can potentially change according to the age of the infection, the position of the leaf or branch in the canopy, and the host species. The primary aim of this study was to investigate the quasispecies concept for citrus viroid VII (CVd-VII), a recently discovered member of the family Pospiviroidae.

Comparative genomic analysis of ESBL-selected and non-selected Escherichia coli in Australian wastewater: Elucidating differences in diversity, antimicrobial resistance, and virulence profiles.

Extended-spectrum β-lactamases (ESBLs)-producing E. coli have been proposed as an indicator bacterium for antimicrobial resistance (AMR) surveillance within a OneHealth framework. However, it is important to understand the effects and potential biases ESBL-selection has on E.

A Multilocus Sequence Typing Scheme for Rapid Identification of Based on Whole-Genome Sequencing Data.

is a plant-pathogenic bacterium associated with a diverse range of host plant species. It has undergone substantial reclassification and currently consists of 14 different subspecies or pathovars that are responsible for a wide range of plant diseases. Whole-genome sequencing (WGS) provides a cutting-edge advantage over other diagnostic techniques in epidemiological and evolutionary studies of .

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

Five bacterial isolates were isolated from in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, the results of biochemical characterisation indicated that these isolates represented members of the genus . To determine their species, further analysis was conducted using both phenotypic and genotypic approaches.

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

The Pacific oyster (PO), , 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).

Characterisation of the Piroplasm Proteome across Three Common Genotypes.

is an emerging apicomplexan pathogen of cattle occurring in areas populated by the principal vector tick, . Unlike transforming spp. that induce cancer-like proliferation of lymphocytes via their schizont stage, destroys host erythrocytes during its piroplasm phase resulting in anaemia.

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

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 using long-read sequencing technology.

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.

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

The draft genome sequence of a novel " Liberibacter" species detected in an unidentified species of (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 " 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.

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.

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.

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.

Detection of Theileria orientalis genotypes in Haemaphysalis longicornis ticks from southern Australia.

Background: Theileria are blood-borne intracellular protozoal parasites belonging to the phylum Apicomplexa. Previously considered a benign parasite in Australia, outbreaks of clinical disease resulting from Theileria orientalis genotypes have been reported in Australia since 2006. Since this time, outbreaks have become widespread in south-eastern Australia, resulting in significant adverse impacts on local dairy and beef industries.

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.

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.

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.

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.

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.

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).