Beak and feather disease virus detected in the endangered Red Goshawk (Erythrotriorchis radiatus).

We report the first detection and prevalence of Beak and feather disease virus (BFDV) in Australia's Red Goshawk (Erythrotriorchis radiatus). This is a new host for this pervasive pathogen amongst a growing list of non-psittacine species including birds of prey from the orders Accipitriformes (hawks, eagles, kites), Falconiformes (falcons and caracas), and Strigiformes (owls). The Red Goshawk is the first non-psittacine species listed as Endangered to be diagnosed with BFDV. We report an initial case of infection discovered post-mortem in a dead nestling and subsequent surveillance of birds from across northern Australia. We reveal BFDV prevalence rates in a wild raptor population for the first time, with detections in 25% (n = 7/28) of Red Goshawks sampled. Prevalence appears higher in juveniles compared to adults, although not statistically significant, but is consistent with studies of wild psittacines. BFDV genotypes were associated with the Loriinae (lorikeets, budgerigar, and fig parrots), Cacatuini (Cockatoos), and Polytelini (long-tailed parrots) tribes; species which are preyed upon by Red Goshawks. A positive BFDV status may be associated with lower body mass but small sample sizes precluded robust statistical analysis. We postulate the possible impacts of the virus on Red Goshawks and discuss future research priorities given these preliminary observations.

Recombinantly expressed virus-like particles (VLPs) of canine circovirus for development of an indirect ELISA.

Canine circovirus (CanineCV) is an emerging pathogen in domestic dogs, detected in multiple countries in association with varying clinical and pathological presentations including diarrhoea, vasculitis, granulomatous inflammation, and respiratory signs. Understanding the pathology of CanineCV is confounded by the fact that it has been detected in asymptomatic dogs as well as in diseased dogs concurrently infected with known pathogens. Recombinantly expressed self-assembling Virus-like particles (VLPs) lack viral genomic material but imitate the capsid surface conformations of wild type virion, allowing arrays of biological applications including subunit vaccine development and immunodiagnostics. In this study, full length CanineCV capsid gene was expressed in Escherichia coli followed by two-step purification process to yield soluble capsid protein in high concentration. Transmission electron microscopy (TEM) confirmed the capsid antigen self-assembled into 17-20 nm VLPs in glutathione S-transferase (GST) buffer, later utilised to develop an indirect enzyme-linked immunosorbent assay (iELISA). The respective sensitivity and specificity of the proposed iELISA were 94.10% and 88.40% compared with those obtained from Western blot. The mean OD450 value for western blot positive samples was 1.22 (range 0.12-3.39) and negative samples was 0.21 (range 0.07-0.41). An optimal OD450 cut-off of 0.35 was determined by ROC curve analysis. Median inter-assay and intra-assay validation revealed that the iELISA test results were reproducible with coefficients of variation 7.70 (range 5.6-11.9) and 4.21 (range 1.2-7.4). Our results demonstrated that VLP-based iELISA is a highly sensitive method for serological diagnosis of CanineCV infections in dogs, suitable for large-scale epidemiological studies.

High prevalence of ciprofloxacin resistance in Escherichia coli isolated from chickens, humans and the environment: An emerging one health issue.

The emergence of antimicrobial resistance in commensal bacteria poses a serious public health burden worldwide. Commensals can disseminate the resistance genes to pathogenic bacteria causing life-threatening infections. This cross-sectional study was designed to investigate the antimicrobial resistance pattern and molecular mechanism(s) of ciprofloxacin resistance in commensal E. coli from three major one health components (humans, animals and the environment) in Bangladesh. Samples were randomly collected from broiler chickens, broiler farm environments and hospitalized human patients from the same geographical area. Isolation and identification of E. coli were performed following standard bacteriological techniques. Antimicrobial susceptibility testing (AST) was performed by disk diffusion and broth microdilution methods. Mutation at the quinolone-resistance determining region (QRDR) was analyzed by sequencing. Of 450 samples, a total of 287 (63.8%; 95% CI 59.2-68.1%) E. coli strains was isolated, where 240 (83.6%; 95% CI 78.9-87.5%) strains were phenotypically resistant to ciprofloxacin. The prevalence of ciprofloxacin-resistant E. coli in broiler chicken, broiler farm environments and hospitalized human patients are 77.6%, 88.8% and 89% respectively. In AST against nine antimicrobials, all the isolates were found to be multidrug-resistant (MDR). The minimum inhibitory concentration (MIC) of ciprofloxacin was ranged from 4 to >128mg/L. Point mutations were detected in several sites of QRDR, specifically at 83 and 87 amino acid positions in gyrA gene, and 56, 57, 78, 80 and 84 amino acid positions in parC gene. Mutations resulted in amino acid substitutions. Phylogenetic analysis of gyrA and parC gene sequences showed a close relationship between the strains isolated from different sources. This study demonstrates a high prevalence of ciprofloxacin resistance in commensal E. coli in humans, animals and environment interface and their genealogically similarity poses an alarming public health consequence.

Australasian Pigeon Circoviruses Demonstrate Natural Spillover Infection.

Pigeon circovirus (PiCV) is considered to be genetically diverse, with a relatively small circular single-stranded DNA genome of 2 kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Australasia is known to be the origin of diverse species of the Order Columbiformes, but limited data on the PiCV genome sequence has hindered phylogeographic studies in this species. To fill this gap, this study was conducted to investigate PiCV in 118 characteristic samples from different birds across Australia using PCR and sequencing. Eighteen partial PiCV Rep sequences and one complete PiCV genome sequence were recovered from reservoir and aberrant hosts. Phylogenetic analyses revealed that PiCV circulating in Australia was scattered across three different subclades. Importantly, one subclade dominated within the PiCV sequenced from Australia and Poland, whereas other PiCV sequenced in this study were more closely related to the PiCV sequenced from China, USA and Japan. In addition, PiCV Rep sequences obtained from clinically affected plumed whistling duck, blue billed duck and Australian magpie demonstrated natural spillover of PiCV unveiled host generalist characteristics of the pigeon circovirus. These findings indicate that PiCV genomes circulating in Australia lack host adapted population structure but demonstrate natural spillover infection.

Visualizing neutrophil extracellular traps in septic equine synovial and peritoneal fluid samples using immunofluorescence microscopy.

Septic synovitis and peritonitis are routinely diagnosed in horses based on clinical examination findings and laboratory assessment of synoviocentesis and abdominocentesis samples, respectively. Diagnosis is difficult in some cases because of an overlap in laboratory results for septic and non-septic inflammation. Neutrophil extracellular trap (NET) formation is part of the innate immune response against pathogens. Identifying and quantifying NETs, which have not been explored in clinical samples from horses with septic synovitis and peritonitis, to our knowledge, may be helpful in detecting infectious processes. Our main objective was to determine whether NETs could be visualized in septic equine synovial and peritoneal fluid cytology samples using immunofluorescence with antibodies against citrullinated histone H3 (Cit-H3) and myeloperoxidase (MPO). We analyzed 9 synovial and 4 peritoneal fluid samples. NET percentages were quantified using a simple counting technique, which is suitable for high-quality, well-preserved, and stained cytospin smears. NETs were evident in all septic samples and were absent in a non-septic sample; NETs were better visualized with Cit-H3 than with MPO immunolabeling. Overall, we believe that there is the potential for NETs and associated markers to be used to investigate and understand septic inflammation in horses.

A Multicentre Epidemiologic Study of Sudden and Unexpected Death in Adult Cats and Dogs in Australia.

Sudden and unexpected death (SUD) is a common reason for animals to undergo post-mortem examination. There is limited literature examining the causes of SUD in cats and dogs, and no research specific to Australia. The purpose of this study was to investigate the epidemiology and pathology of SUD in cats and dogs in a multicentric study across Australia. Retrospective post-mortem reports of SUD in cats and dogs were obtained from four veterinary schools in Australia distributed across four states. The frequency of SUD between institutes ranged from 2.1% to 6.5%. Dogs composed the majority of the study population (76%), and males outnumbered females, particularly in the feline subpopulation. After necropsy, 37% of SUD remained cause unknown, the largest category in both cats and dogs. When cause was identified, cardiovascular disease was most common in both species, followed by gastrointestinal disease in dogs, and trauma in cats. In dogs, multinomial logistic regression identified age as a risk factor significantly associated with the four largest categories of SUD. This study identified causes of SUD in Australian cats and dogs, including novel causes not previously reported. Further, this study revealed a higher rate of unsolved SUD in Australia than can be found in the literature from other countries.

Multiplex Immunoassay Approaches Using Luminex® xMAP® Technology for the Study of COVID-19 Disease.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has been one of the most severe outbreaks of respiratory illness in history. The clinical symptoms of COVID-19 may be similar to flu, although they can be life-threatening, particularly in the elderly and immunocompromised population. Together with nucleic acid detection, serological testing has been essential for the diagnosis of SARS-CoV-2 infection but has been critically important for studying the epidemiology, serosurveillance, and for vaccine research and development. Multiplexed immunoassay technologies have a particular advantage as they can simultaneously measure multiple analytes from a single sample. xMAP technology is a multiplex analysis platform that can measure up to 500 analytes at the same time from the same sample. It has been shown to be an important tool for studying immune response to the various SARS-CoV-2 antigens, as well as for measuring host protein biomarker levels as prognostic indicators of COVID-19. In this chapter, we describe several key studies where xMAP technology was used for multiplexed analysis of SARS-COV-2 antibody responses and host protein expression in COVID-19 patients.

Lesions and viral loads in racing pigeons naturally coinfected with pigeon circovirus and columbid alphaherpesvirus 1 in Australia.

Columbid alphaherpesvirus 1 (CoHV1) is associated with oral or upper respiratory tract lesions, encephalitis, and occasional fatal systemic disease in naive or immunosuppressed pigeons. Clinical disease is often reported with CoHV1 and coinfecting viruses, including pigeon circovirus (PiCV), which may cause host immunosuppression and augment lesion development. A natural outbreak of CoHV1 and PiCV coinfection occurred in a flock of 60 racing rock pigeons (Columba livia), in which 4 pigeons succumbed within 7 d of clinical onset. Lesions included suppurative stomatitis, pharyngitis, cloacitis, meningitis, and tympanitis, with eosinophilic intranuclear inclusion bodies consistent with herpesviral infection. In addition, large numbers of botryoid intracytoplasmic inclusion bodies were present in the skin, oral mucosa, and bursa of Fabricius, suggestive of circoviral infection, which was confirmed by immunohistochemistry. The concurrent viral load of CoHV1 and PiCV was high in liver, oropharynx, and bursa of Fabricius. We found PiCV in oro-cloacal swabs from 44 of 46 additional birds of variable clinical status, PiCV alone in 23 birds, and coinfection with CoHV1 in 21 birds. Viral copy numbers were significantly higher (p < 0.0001) for both viruses in clinically affected pigeons than in subclinical qPCR-positive birds. The CoHV1-induced lesions might have been exacerbated by concomitant PiCV infection.

Development and applications of a TaqMan based quantitative real-time PCR for the rapid detection of Pigeon circovirus (PiCV).

TaqMan probe based quantitative polymerase reaction (TaqMan qPCR) is a robust and reliable technique for detecting and quantifying target DNA copies. Quantitative molecular diagnosis of genetically diverse single stranded DNA (ssDNA) virus such as Pigeon circovirus (PiCV) can be challenging owing to difficulties in primer binding or low abundance of template DNA copies in clinical specimens. Several methods have been described for the detection of PiCV, being qPCR the most simple and reliable. As far as is known, two qPCR systems described until now are based on SYBR green. This study reports development and validation of a highly sensitive TaqMan qPCR targeted to Rep for the detection of highly diverse PiCV in pigeon samples with excellent reproducibility, specificity, and sensitivity. The limit of detection was determined as low as 2 (two) plasmid copies. Estimations of 100 % specificity and 100 % sensitivity were obtained based on the qPCR results with panel of 60 samples (known PiCV positive, n = 30; known PiCV negative, n = 20; samples positive to Beak and feather disease virus (BFDV), n = 5 and samples positive to canine circovirus, n = 5). Co-efficient of variation (CV) for Ct values ranged between 0.27 % and 0.78 % in the same assay and 1.84-2.87 % in different assays.

Detection of IgG Antibodies to SARS-CoV-2 and Neutralizing Capabilities Using the Luminex® xMAP® SARS-CoV-2 Multi-Antigen IgG Assay.

Serological assays have been a useful tool for detection of antibodies to SARS-CoV-2 during the COVID-19 pandemic. These assays are used for epidemiology and serosurveillance to monitor the progression of the pandemic, to identify and differentiate individuals who have developed antibodies from natural infection versus vaccine-induced immunity, and to identify potential donors of convalescent plasma for therapeutic purposes. In this chapter, we describe a commercially available bead-based serological assay, the Luminex® xMAP® SARS-CoV-2 Multi-Antigen IgG Assay, that detects and identifies antibodies against three SARS-CoV-2 antigens. In addition to the assay principle and workflow, we describe modifications that may be used to evaluate alternate sample types, antibody isotypes, and potential neutralizing antibody responses.

Diagnosis and Management of Bloodstream Infections With Rapid, Multiplexed Molecular Assays.

Bloodstream infection is a major health concern, responsible for considerable morbidity and mortality across the globe. Prompt identification of the responsible pathogen in the early stages of the disease allows clinicians to implement appropriate antibiotic therapy in a timelier manner. Rapid treatment with the correct antibiotic not only improves the chances of patient survival, but also significantly reduces the length of hospital stay and associated healthcare costs. Although culture has been the gold standard and most common method for diagnosis of bloodstream pathogens, it is being enhanced or supplanted with more advanced methods, including molecular tests that can reduce the turnaround time from several days to a few hours. In this article, we describe two rapid, molecular bloodstream infection panels that identify the most common pathogens and associated genetic determinants of antibiotic resistance - the Luminex® VERIGENE® Gram-Positive Blood Culture Test and the VERIGENE® Gram-Negative Blood Culture Test. We conducted a search on PubMed to retrieve articles describing the performance and impact of these tests in the clinical setting. From a total of 48 articles retrieved, we selected 15 for inclusion in this review based on the type and size of the study and so there would be minimum of three articles describing performance and three articles describing the impact post-implementation for each assay. Here we provide a comprehensive review of these publications illustrating the performance and clinical utility of these assays, demonstrating how genotypic tests can benefit diagnostic and antimicrobial stewardship efforts.

The COVID-19 Pandemic - A Diagnostic Industry Perspective.

The COVID-19 pandemic has brought about unprecedented changes to all facets of the healthcare system, including the diagnostic industry. The pandemic has highlighted both the challenges and strengths of the industry and has also provided valuable insights on how to be better prepared for future pandemics. In this perspective article, we describe the challenges faced by the diagnostic industry in general, particularly the difficulties encountered by Luminex Corporation, a diagnostic assay development and manufacturing company located in Austin, Texas, USA, as well as the mitigation strategies employed. In addition to discussion of the key challenges, the article provides insights on the lessons learned and steps that can be undertaken to better prepare for future outbreaks.

Retrospective Genotyping and Whole Genome Sequencing of a Canine Parvovirus Outbreak in Bangladesh.

Canine parvovirus 2 (CPV-2) outbreaks in close quarters such as kennels or shelters can cause substantial case fatality. Thirteen dead Labradors from a secluded kennel of security dogs presented with typical clinical signs and gross pathology of parvovirus infection. Whole genome shotgun sequencing from tissue-extracted genomic DNA detected new CPV-2a as the contributing antigenic variant. Further genotyping using polymerase chain reaction coupled with high-resolution melt assays (PCR-HRM) confirmed new CPV-2a infection in all deceased dogs. PCR-HRM of additional thirty-four clinically suspected dogs suggested that this variant is in wider community circulation, at least in the southeastern part of Bangladesh. We present complete genome sequence of the new CPV-2a variant circulating in the domestic canine population of Bangladesh.

Lumpy skin disease outbreak in cattle population of Chattogram, Bangladesh.

BACKGROUND: Lumpy skin disease (LSD) is an important viral disease causing significant economic losses in commercial livestock production. In mid-2019, an outbreak of LSD has been reported in cattle population from different parts of Bangladesh including Chattogram division. A cross-sectional surveillance study was undertaken from August 2019 to December 2019 to investigate the prevalence and associated risk factors of LSD in cattle in Chattogram district. METHODS: A total of 3,327 cattle from 19 commercial farms were examined for the LSD specific skin lesions and associated risk factors. A total of 120 skin biopsies were collected from the suspected animal for the confirmation of the disease using molecular detection and histopathological examination. Partial genome sequencing and phylogenetic analyses were performed on selected viral isolates. RESULTS: The overall clinical prevalence of LSD in the study population was 10% (95% confidence interval [CI]: 9.4%-11%) where the highest farm level outbreak frequency was 63.33% (95% CI: 45.51%-78.13%) and the lowest 4.22% (95% CI: 3.39%-5.25%). Crossbred and female cattle showed a significantly higher prevalence of the disease compared to their counterparts. Introduction of new animals in farms was found to be one of the most significant risk factors in the transmission of the disease. All suspected skin biopsies were positive for LSD virus (LSDV) infection with granulomatous and pyogranulomatous dermatitis was revealed on histopathology. Phylogenetic analysis based on the inverted terminal repeat region of the LSDV gene suggested that the locally circulating strain was closely related to the strains isolated from the Middle East and North African countries. CONCLUSIONS: The data generated in this study would be beneficial to the field veterinarians and animal health decision makers in the country as well as it will aid in taking appropriate measures to prevent further relapse or outbreak of this disease in future.

Structural Perspectives of Beak and Feather Disease Virus and Porcine Circovirus Proteins.

Circoviruses represent a rapidly expanding group of viruses that infect both vertebrate and invertebrate hosts. Members are responsible for diseases of veterinary and economic importance, including postweaning multisystemic wasting syndrome in pigs, and beak and feather disease (BFD) in birds. These viruses are associated with lymphoid depletion and immunosuppressive conditions in infected animals leading to systemic illness. Circoviruses are small nonenveloped DNA viruses containing a single-stranded circular genome, encoding two major proteins: the capsid-associated protein (Cap), comprising the entirety of the viral capsid, and the replication-associated protein (Rep). Cap is the only protein component of the virion and plays crucial roles throughout the virus replication cycle, including viral attachment, cell entry, genome uncoating, and packaging of newly formed viral particles. Rep mediates recognition of replication origin motifs in the viral genome sequence and is responsible for endonuclease activity enabling nicking of the circular DNA and initiation of rolling-circle replication (RCR). Porcine circovirus 2 (PCV2) was the first circovirus capsid structure to be solved at atomic resolution using X-ray crystallography. The structure revealed an assembly comprising 60 monomeric subunits to form virus-like particles. Each Cap monomer harbors a canonical viral jelly roll domain composed of two, four-stranded antiparallel ß-sheets. Crystal structures of two distinct macromolecular assemblies from BFD virus Cap were also resolved at high resolution. In these structures, the exposure of the N-terminal arginine-rich motif, responsible for DNA binding and nuclear localization is reversed. Additional structural investigations have also elucidated a PCV2 type-specific neutralizing epitope, and interaction between the PCV2 capsid and polymers such as heparin. In this review, we provide a snapshot of the structural and functional aspects of circovirus proteins.

Molecular and non-molecular approaches to etiologic diagnosis of gastroenteritis.

Gastroenteritis is a major cause of mortality and morbidity globally and rapid identification of the causative pathogen is important for appropriate treatment and patient management, implementation of effective infection control measures, reducing hospital length of stay, and reducing overall medical costs. Although stool culture and microscopic examination of diarrheal stool has been the primary method for laboratory diagnosis, culture-independent proteomic and genomic tests are receiving increased attention. Antigen tests for stool pathogens are routinely implemented as rapid and simple analytics whereas molecular tests are now available in various formats from high complexity to waived point-of-care tests. In addition, metagenomic next-generation sequencing stands poised for use as a method for both diagnosis and routine characterization of the gut microbiome in the very near future. Analysis of host biomarkers as indicators of infection status and pathogenesis may also become important for prediction, diagnosis, and monitoring of gastrointestinal infection. Here we review current methods and emerging technologies for the etiologic diagnosis of gastroenteritis in the clinical laboratory. Benefits and limitations of these evolving methods are highlighted.

REPEAT SPILLOVER OF BEAK AND FEATHER DISEASE VIRUS INTO AN ENDANGERED PARROT HIGHLIGHTS THE RISK ASSOCIATED WITH ENDEMIC PATHOGEN LOSS IN ENDANGERED SPECIES.

Conservation efforts for the orange-bellied parrot (Neophema chrysogaster), one of the world's most critically endangered bird species, have been hampered by beak and feather disease virus (BFDV) spillover infection. To understand the vulnerability of orange-bellied parrots to potential reservoirs of infection we investigated geographic versus taxonomic structure in 160 full-genome and 319 partial Rep gene BFDV sequences from captive and wild orange-bellied parrots and other wild parrot species in Australia. We found that Australian BFDV populations are structured by host taxonomy. By identifying genetic stratification of BFDV in reservoir hosts we characterized three separate recent incursions of BFDV into orange-bellied parrots from other wild parrots, which demonstrates the susceptibility of critically endangered species to multiple threats of pathogen re-emergence. Our study highlighted how loss of endemic circulating BFDV in orange-bellied parrots precipitated repeated spillover into an immunologically naïve population, causing significant disease.

Molecular characterisation of a novel pathogenic avipoxvirus from the Australian magpie (Gymnorhina tibicen).

Avipoxviruses are significant pathogens infecting a wide range of wild and domestic bird species globally. Here, we describe a novel genome sequence of magpiepox virus (MPPV) isolated from an Australian magpie. In the present study, histopathologically confirmed cutaneous pox lesions were used for transmission electron microscopic analysis, which demonstrated brick-shaped virions with regular spaced thread-like ridges, indicative of likely infectious particles. Subsequent analysis of the recovered MPPV genome positioned phylogenetically to a distinct sub-clade with the recently isolated avipoxvirus genome sequences from shearwater and canary bird species, and demonstrates a high degree of sequence similarity with CNPV (96.14%) and SWPV-2 (95.87%). The novel MPPV complete genome is missing 19 genes with a further 41 genes being truncated/fragmented compared to SWPV-2 and contains nine predicted unique genes. This is the first avipoxvirus complete genome sequence that infects Australian magpie.

Diverse Trichomonas lineages in Australasian pigeons and doves support a columbid origin for the genus Trichomonas.

Trichomonas is a significant protist genus, and includes T. vaginalis, the most prevalent sexually transmitted non-viral infection of humans, and T. gallinae of rock doves (Columba livia), one of the earliest known avian pathogens. New Trichomonas genotypes, including T. vaginalis-like isolates, have been discovered in American columbid hosts, suggesting geographically widespread cryptic diversity of Trichomonas in pigeons and doves. We sampled 319 birds from 22 columbid species in Australia, Papua New Guinea, New Zealand and southern Africa and uncovered 15 novel lineages of Trichomonas, more than doubling the known diversity of this parasite genus and providing evidence for frequent host-switching that eventually gave rise to T. vaginalis in humans. We show that Trichomonas has a columbid origin and likely underwent Miocene expansion out of Australasia. Our chronological topology for Trichomonas is calibrated on the evolution of a host phenotypic trait associated with ecological entrapment of the most basal extant lineage of Trichomonas in Ptilinopus fruit-doves.

Assessing circovirus gene flow in multiple spill-over events.

The establishment of viral pathogens in new host environments following spillover events probably requires adaptive changes within both the new host and pathogen. After many generations, signals for ancient cross-species transmission may become lost and a strictly host-adapted phylogeny may mimic true co-divergence while the virus may retain an inherent ability to jump host species. The mechanistic basis for such processes remains poorly understood. To study the dynamics of virus-host co-divergence and the arbitrary chances of spillover in various reservoir hosts with equal ecological opportunity, we examined structural constraints of capsid protein in extant populations of Beak and feather disease virus (BFDV) during known spillover events. By assessing reservoir-based genotype stratification, we identified co-divergence defying signatures in the evolution BFDV which highlighted primordial processes of cryptic host adaptation and competing forces of host co-divergence and cross-species transmission. We demonstrate that, despite extensive surface plasticity gathered over a longer span of evolution, structural constraints of the capsid protein allow opportunistic host switching in host-adapted populations. This study provides new insights into how small populations of endangered psittacine species may face multidirectional forces of infection from reservoirs with apparently co-diverging genotypes.