Veterinary Biosciences - Research Publications

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    Global Phylogeny and F Virulence Plasmid Carriage in Pandemic Escherichia coli ST1193
    Wyrsch, ER ; Bushell, RN ; Marenda, MS ; Browning, GF ; Djordjevic, SP ; Andam, CP (AMER SOC MICROBIOLOGY, 2022-12-21)
    Lower urinary tract, renal, and bloodstream infections caused by phylogroup B2 extraintestinal pathogenic Escherichia coli (ExPEC) are a leading cause of morbidity and mortality. ST1193 is a phylogroup B2, multidrug-resistant sequence type that has risen to prominence globally, but a comprehensive analysis of the F virulence plasmids it carries is lacking. We performed a phylogenomic analysis of ST1193 (n = 707) whole-genome sequences from EnteroBase using entries with comprehensive isolation metadata. The data set comprised isolates from humans (n = 634 [90%]), including 339 (48%) from extraintestinal infection sites, and isolates from companion animals, wastewater, and wildlife. Phylogenetic analyses combined with gene detection and genotyping resolved an ST1193 clade structure segregated by serotype and F plasmid carriage. Most F plasmids fell into one of three related plasmid subtypes: F-:A1:B10 (n = 444 [65.97%]), F-:A1:B1 (n = 84 [12.48%]), and F-:A1:B20 (n = 80 [11.89%]), all of which carry the virulence genes cjrABC colocalized with senB (cjrABC-senB), a trademark signature of F29:A-:B10 subtype plasmids (pUTI89). To examine the phylogenetic relationship of these plasmids with pUTI89, complete sequences of F-:A1:B1 and F-:1:B20 plasmids were resolved. Unlike pUTI89, the most dominant and widely disseminated F plasmid that carries cjrABC-senB, F plasmids in ST1193 often carry a complex resistance region with an integron truncation (intI1Δ745) signature embedded within a structure assembled by IS26. Plasmid analysis shows that ST1193 has F plasmids that carry cjrABC-senB and ARG-encoding genes but lack tra regions and are likely derivatives of pUTI89. Further epidemiological investigation of ST1193 should seek to confirm its presence in human-associated environments and identify any potential agricultural links, which are currently lacking. IMPORTANCE We have generated an updated ST1193 phylogeny using publicly available sequences, reinforcing previous assertions that Escherichia coli ST1193 is a human-associated lineage, with many examples sourced from human extraintestinal infections. ST1193 from urban-adapted birds, wastewater, and companion animals are frequent, but isolates from animal agriculture are notably absent. Phylogenomic analysis identified several clades segregated by serogroup, all noted to carry highly similar F plasmids and antimicrobial resistance (AMR) signatures. Investigation of these plasmids revealed virulence regions with similarity to pUTI89, a key F virulence plasmid among dominant pandemic extraintestinal pathogenic E. coli lineages, and encoding a complex antibiotic resistance structure mobilized by IS26. This work has uncovered a series of F virulence plasmids in ST1193 and shows that the lineage mimics the host range and virulence attributes of other E. coli strains that carry pUTI89. These observations have significant ramifications for epidemiological source tracking of emerging and established pandemic ExPEC lineages.
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    Measures of tracheal lesions are more discriminatory and reproducible indications of chronic respiratory disease caused by Mycoplasma gallisepticum in poultry
    Kulappu Arachchige, SN ; Underwood, GJ ; Andrews, DM ; Abeykoon, AMH ; Wawegama, NK ; Browning, GF (TAYLOR & FRANCIS LTD, 2022-11-02)
    Mycoplasma gallisepticum is the primary causative agent of chronic respiratory disease in poultry, and vaccination is the measure most commonly used for its control. Pathological changes caused by M. gallisepticum are mainly observed in the trachea and air sacs, but assessment of air sac lesions is subjective. Standardized parameters for evaluation of pathological changes, and their reproducibility and discrimination in uninfected and infected groups, are critical when assessing the efficacy of M. gallisepticum vaccination. This study reviewed and critically appraised the published literature on evaluation of vaccine efficacy against pathological changes caused by M. gallisepticum in poultry in the trachea and air sacs. A search of four electronic databases, with subsequent manual filtering, identified 23 eligible papers published since 1962 describing the assessment of histopathological changes in the trachea using tracheal lesion scores and/or measurement of tracheal mucosal thicknesses and assessment of gross air sac lesions using lesion scores. Measurement of tracheal lesions proved a more reliable and robust method of assessing disease induced by M. gallisepticum when compared to assessment of air sac lesions, highlighting the importance of including assessment of tracheal lesions as the primary outcome variable in vaccine efficacy studies. In addition, this study also identified the necessity for use of a standardized model for evaluation and reporting on M. gallisepticum vaccines to minimize variations between vaccine efficacy studies and to allow direct comparisons between them.RESEARCH HIGHLIGHTS Tracheal and air sac lesions have been used to assess M. gallisepticum vaccine efficacy.The specific parameters and statistical tests used to compare tracheal and air sac lesions vary greatly.Measures of tracheal lesions are more discriminatory than measures of air sac lesions.A standardized model is needed to evaluate vaccines against infection with M. gallisepticum.
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    Water consumption and wastage behaviour in pigs: implications for antimicrobial administration and stewardship
    Little, SB ; Browning, GF ; Woodward, AP ; Billman-Jacobe, H (ELSEVIER, 2022-08)
    Daily water use and wastage patterns of pigs have major effects on the efficacy of in-water antimicrobial dosing events when conducted for metaphylaxis or to treat clinical disease. However, daily water use and wastage patterns of pigs are not routinely quantified on farms and are not well understood. We conducted a prospective, observational 27-day study of the daily water use and wastage patterns of a pen group of 15 finisher pigs reared in a farm building. We found that the group of pigs wasted a median of 36.5% of the water used per day. We developed models of the patterns of water used and wasted by pigs over each 24-h period using a Bayesian statistical method with the brm() function in the brms package. Both patterns were uni-modal, peaking at 1400-1700, and closely aligned. Wastage was slightly greater during hours of higher water use. We have shown that it is feasible to quantify the water use and wastage patterns of pigs in farm buildings using a system that records and aggregates data, and analyses them using hierarchical generalised additive models. This system could support more efficacious in-water antimicrobial dosing on farms, and better antimicrobial stewardship, by helping to reduce the quantities of antimicrobials used and disseminated into the environment.
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    Attitudes towards Use of High-Importance Antimicrobials-A Cross-Sectional Study of Australian Veterinarians
    Sri, A ; Bailey, KE ; Gilkerson, JR ; Browning, GF ; Hardefeldt, LY (MDPI, 2022-11)
    The timely implementation of antimicrobial stewardship interventions could delay or prevent the development of higher levels of antimicrobial resistance in the future. In food-producing animals in Australia, high-importance antimicrobials, as rated by the Australian Strategic and Technical Advisory Group (ASTAG), include virginiamycin and third-generation cephalosporins (in individual pigs or cattle). The use of high-importance antimicrobials in companion animals is more widespread and less regulated. There is no national antimicrobial use surveillance system for animals in Australia. Consequently, there is a gap in the knowledge about reasonable use across all sectors of veterinary practice. This study explored attitudes towards the use in veterinary medicine of antimicrobials with high importance to human health, and determined levels of agreement about the introduction of restrictions or other conditions on this use. An online survey was distributed via social media and email from June to December 2020 to veterinarians working in Australia. Of the 278 respondents working in clinical practice, 49% had heard of the ASTAG rating system, and 22% used a traffic light system for antimicrobial importance in their practice. Overall, 61% of participants disagreed that veterinarians should be able to prescribe high-importance antimicrobials without restrictions. If there were to be restrictions, there was most agreement amongst all respondents for only restricting high-importance antimicrobials (73%). There is a need for education, guidance, and practical support for veterinarians for prescribing high-importance antimicrobials alongside any restrictions.
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    Whole genome sequence analysis of equid gammaherpesvirus-2 field isolates reveals high levels of genomic diversity and recombination
    Onasanya, AE ; El-Hage, C ; Diaz-Mendez, A ; Vaz, PK ; Legione, AR ; Browning, GF ; Devlin, JM ; Hartley, CA (BMC, 2022-08-30)
    BACKGROUND: Equid gammaherpesvirus 2 (EHV2) is a gammaherpesvirus with a widespread distribution in horse populations globally. Although its pathogenic significance can be unclear in most cases of infection, EHV2 infection can cause upper respiratory tract disease in foals. Co-infection of different strains of EHV2 in an individual horse is common. Small regions of the EHV2 genome have shown considerable genetic heterogeneity. This could suggest genomic recombination between different strains of EHV2, similar to the extensive recombination networks that have been demonstrated for some alphaherpesviruses. This study examined natural recombination and genome diversity of EHV2 field isolates. RESULTS: Whole genome sequencing analysis of 18 EHV2 isolates, along with analysis of two publicly available EHV2 genomes, revealed variation in genomes sizes (from 173.7 to 184.8 kbp), guanine plus cytosine content (from 56.7 to 57.8%) and the size of the terminal repeat regions (from 17,196 to 17,551 bp). The nucleotide sequence identity between the genomes ranged from 86.2 to 99.7%. The estimated average inter-strain nucleotide diversity between the 20 EHV2 genomes was 2.9%. Individual gene sequences showed varying levels of nucleotide diversity and ranged between 0 and 38.1%. The ratio of nonsynonymous substitutions, Ka, to synonymous substitutions, Ks, (Ka/Ks) suggests that over 50% of EHV2 genes are undergoing diversifying selection. Recombination analyses of the 20 EHV2 genome sequences using the recombination detection program (RDP4) and SplitsTree revealed evidence of viral recombination. CONCLUSIONS: Analysis of the 18 new EHV2 genomes alongside the 2 previously sequenced genomes revealed a high degree of genetic diversity and extensive recombination networks. Herpesvirus genome diversification and virus evolution can be driven by recombination, and our findings are consistent with recombination being a key mechanism by which EHV2 genomes may vary and evolve.
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    Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques
    Armat, M ; Vaz, PK ; Browning, GF ; Noormohammadi, AH ; Hartley, CA ; Devlin, JM (SPRINGER, 2022-12)
    In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.
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    Genomic and Temporal Trends in Canine ExPEC Reflect Those of Human ExPEC
    Elankumaran, P ; Cummins, ML ; Browning, GF ; Marenda, MS ; Reid, CJ ; Djordjevic, SP ; Andam, CP (AMER SOC MICROBIOLOGY, 2022-06)
    Companion animals and humans are known to share extraintestinal pathogenic Escherichia coli (ExPEC), but the extent of E. coli sequence types (STs) that cause extraintestinal diseases in dogs is not well understood. Here, we generated whole-genome sequences of 377 ExPEC collected by the University of Melbourne Veterinary Hospital from dogs over an 11-year period from 2007 to 2017. Isolates were predominantly from urogenital tract infections (219, 58.1%), but isolates from gastrointestinal specimens (51, 13.5%), general infections (72, 19.1%), and soft tissue infections (34, 9%) were also represented. A diverse collection of 53 STs were identified, with 18 of these including at least five sequences. The five most prevalent STs were ST372 (69, 18.3%), ST73 (31, 8.2%), ST127 (22, 5.8%), ST80 (19, 5.0%), and ST58 (14, 3.7%). Apart from ST372, all of these are prominent human ExPEC STs. Other common ExPEC STs identified included ST12, ST131, ST95, ST141, ST963, ST1193, ST88, and ST38. Virulence gene profiles, antimicrobial resistance carriage, and trends in plasmid carriage for specific STs were generally reflective of those seen in humans. Many of the prominent STs were observed repetitively over an 11-year time span, indicating their persistence in the dogs in the community, which is most likely driven by household sharing of E. coli between humans and their pets. The case of ST372 as a dominant canine lineage observed sporadically in humans is flagged for further investigation. IMPORTANCE Pathogenic E. coli that causes extraintestinal infections (ExPEC) in humans and canines represents a significant burden in hospital and veterinary settings. Despite the obvious interrelationship between dogs and humans favoring both zoonotic and anthropozoonotic infections, whole-genome sequencing projects examining large numbers of canine-origin ExPEC are lacking. In support of anthropozoonosis, we found that most STs from canine infections are dominant human ExPEC STs (e.g., ST73, ST127, ST131) with similar genomic traits, such as plasmid carriage and virulence gene burden. In contrast, we identified ST372 as the dominant canine ST and a sporadic cause of infection in humans, supporting zoonotic transfer. Furthermore, we highlight that, as is the case in humans, STs in canine disease are consistent over time, implicating the gastrointestinal tract as the major community reservoir, which is likely augmented by exposure to human E. coli via shared diet and proximity.
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    Genomics and pathogenesis of the avian coronavirus infectious bronchitis virus
    Quinteros, JA ; Noormohammadi, AH ; Lee, SW ; Browning, GF ; Diaz-Mendez, A (WILEY, 2022-10)
    Infectious bronchitis virus (IBV) is a member of the family Coronaviridae, together with viruses such as SARS-CoV, MERS-CoV and SARS-CoV-2 (the causative agent of the COVID-19 global pandemic). In this family of viruses, interspecies transmission has been reported, so understanding their pathobiology could lead to a better understanding of the emergence of new serotypes. IBV possesses a single-stranded, non-segmented RNA genome about 27.6 kb in length that encodes several non-structural and structural proteins. Most functions of these proteins have been confirmed in IBV, but some other proposed functions have been based on research conducted on other members of the family Coronaviridae. IBV has variable tissue tropism depending on the strain, and can affect the respiratory, reproductive, or urinary tracts; however, IBV can also replicate in other organs. Additionally, the pathogenicity of IBV is also variable, with some strains causing only mild clinical signs, while infection with others results in high mortality rates in chickens. This paper extensively and comprehensibly reviews general aspects of coronaviruses and, more specifically, IBV, with emphasis on protein functions and pathogenesis. The pathogenicity of the Australian strains of IBV is also reviewed, describing the variability between the different groups of strains, from the classical to the novel and recombinant strains. Reverse genetic systems, cloning and cell culture growth techniques applicable to IBV are also reviewed.
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    Whole genome sequence analysis of equid herpesvirus type 2 field isolates reveals high levels of genomic diversity and recombination
    Onasanya, AE ; Diaz-Méndez, A ; El-Hage, C ; Vaz, PK ; Legione, AR ; Browning, GF ; Devlin, JM ; Hartley, CA ( 2022-04-28)
    BackgroundEquid gammaherpesvirus 2 (EHV2) is a gammaherpesvirus with a widespread distribution in horse populations globally. Although its pathogenic significance can be unclear in most cases of infection, EHV2 infection can cause upper respiratory tract disease in foals. Co-infection of different strains of EHV2 in an individual horse is common. Small regions of the EHV2 genome have shown considerable genetic heterogeneity. This could suggest genomic recombination between different strains of EHV2, similar to the extensive recombination networks that have been demonstrated for some alphaherpesviruses. This study examined natural recombination and genome diversity of EHV2 field isolates. ResultsWhole genome sequencing analysis of 18 EHV2 isolates, along with analysis of two publicly available EHV2 genomes, revealed variation in genomes sizes (from 173.7 to 184.8 kbp), guanine plus cytosine content (from 56.7 to 57.8 %) and the size of the terminal repeat regions (from 17,196 to 17,551 bp). The nucleotide sequence identity between the genomes ranged from 86.2 to 99.7%. The estimated average inter-strain nucleotide diversity between the 20 EHV2 genomes was 2.9%. Individual gene sequences showed varying levels of nucleotide diversity and ranged between 0 and 3.8%. The ratio of nonsynonymous substitutions, Ka, to synonymous substitutions, Ks, (Ka/Ks) suggests that over 50% of EHV2 genes are undergoing diversifying selection. Recombination analyses of the 20 EHV2 genome sequences using the recombination detection program (RDP4) and SplitsTree revealed evidence of viral recombination. ConclusionsAnalysis of the 18 new EHV2 genomes alongside the 2 previously sequenced genomes revealed a high degree of genetic diversity and extensive recombination networks. Herpesvirus genome diversification and virus evolution can be driven by recombination, and our findings are consistent with recombination being a key mechanism by which EHV2 genomes may vary and evolve.
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    Mycoplasma bovis mbfN Encodes a Novel LRR Lipoprotein That Undergoes Proteolytic Processing and Binds Host Extracellular Matrix Components
    Adamu, JY ; Mitiku, F ; Hartley, CA ; Sansom, FM ; Marenda, MS ; Markham, PF ; Browning, GF ; Tivendale, KA ; Comstock, LE (AMER SOC MICROBIOLOGY, 2021-01)
    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.