Complete Genome Sequence of an Endemic Mycobacterium bovis Strain from Wood Bison in Wood Buffalo National Park, Canada.

Mycobacterium bovis is the primary causative agent of bovine tuberculosis, a zoonotic infectious disease of concern for human health, livestock, and wildlife conservation. We report a complete genome sequence of an endemic Mycobacterium bovis strain affiliated with a wildlife reservoir of bovine tuberculosis found in wood bison in Wood Buffalo National Park, Canada.

Complete Genome Sequences of Mycobacterium bovis Strains Affiliated with Bovine Tuberculosis Outbreaks in Canada in 2016 and 2018.

Mycobacterium bovis is the primary causative agent of bovine tuberculosis, a zoonotic infectious disease that presents a risk to public health, livestock, and wildlife. Here, we report complete genome sequences of two Mycobacterium bovis strains affiliated with bovine tuberculosis outbreaks in Canadian cattle farms in 2016 and 2018.

High female mortality caused by an atypical Mycobacterium species closely related to the Mycobacterium ulcerans-marinum complex in a colony of bearded dragons (Pogona vitticeps).

A commercial breeding colony of bearded dragons (Pogona vitticeps) experienced an increase in mortality that affected females only. Before death, the animals had lost appetite and weight, were dehydrated, and some had labored breathing. Necropsy revealed granulomas in many organs (ovaries, lungs, liver, kidneys, heart, bone marrow) in which numerous acid-fast bacteria were identified. Bacterial isolation confirmed Mycobacterium spp., which was identified by whole genome sequencing as closely related to the Mycobacterium ulcerans-marinum complex. Due to the zoonotic potential of this bacterium and the poor prognosis for the remaining sick animals, the entire colony was culled and 7 animals were evaluated. The possible routes for introduction of this bacterium, the female predisposition to the disease, as well as the zoonotic potential of this microorganism are discussed. Key clinical message: An atypical Mycobacterium species closely related to Mycobacterium ulcerans-marinum complex can cause high female morality in captive bearded dragons.

Mortalité élevée de femelles dans une colonie de dragons barbus (Pogona vitticeps) causée par une mycobactérie atypique étroitement reliée au complexe Mycobacterium ulcerans-marinum . Une augmentation de la mortalité affectant uniquement les femelles est survenue dans une colonie de dragons barbus reproducteurs. Avant leur mort, ces animaux étaient anorexiques, amaigris, déshydratés et certains respiraient la gueule ouverte. Leur nécropsie révéla la présence de granulomes dans plusieurs viscères (poumons, coeur, reins, foie, ovaires, moelle osseuse), dans lesquels des bacilles acido-alcoolo-résistants étaient visibles à l'examen microscopique. L'isolement bactérien a permis de confirmer qu'il s'agissait bien de Mycobacterium spp. et les analyses moléculaires ont démontré que cette mycobactérie était étroitement reliée au complexe Mycobacterium ulcerans-marinum. À cause du potentiel zoonotique de cette infection et du pronostic sombre, la colonie entière fut euthanasiée et sept (7) animaux soumis pour nécropsie. Les causes potentielles d'introduction de cette bactérie dans la colonie, la prédisposition particulière des femelles à cette infection et le risque zoonotique qui y est associé seront discutés.Message clinique clé :Une mycobactérie étroitement associée au complexe Mycobacterium ulcerans-marinum peut causer une mortalité élevée chez les dragons barbus en captivité et cibler tout particulièrement les femelles.(Traduit par les auteurs).

Diagnostic and public health investigation of Mycobacterium tuberculosis infection in a dog in Ontario, Canada.

A 4-y-old, female mixed-breed dog was presented to the Ontario Veterinary College for further evaluation of multiple pulmonary and hepatic masses, intrathoracic lymphadenitis, and recent development of a pyogranulomatous pleural effusion. Along with other comprehensive tests, a thoracic lymph node biopsy was performed, and Mycobacterium tuberculosis complex infection was confirmed by real-time PCR. The dog's condition declined post-operatively, and euthanasia was elected. Postmortem examination confirmed severe granulomatous pneumonia, hepatitis, intrathoracic and intraabdominal lymphadenitis, omentitis, and nephritis. Line-probe assays performed on samples collected postmortem confirmed the species as M. tuberculosis. 24-loci MIRU-VNTR genotyping, spoligotyping, and whole-genome sequencing revealed relations to known human isolates, but no epidemiologic link to these cases was investigated. Given the concern for potential human exposure during this animal's disease course, a public health investigation was initiated; 45 individuals were tested for M. tuberculosis exposure, and no subsequent human infections related to this animal were identified. Our case highlights the need for more readily available, minimally invasive testing for the diagnosis of canine mycobacteriosis, and highlights the ability of canid species to act as potential contributors to the epidemiology of M. tuberculosis infections.

An Unusual Salmonella Enteritidis Strain Carrying a Modified Virulence Plasmid Lacking the prot6e Gene Represents a Geographically Widely Distributed Lineage.

This study identifies a strain of Salmonella enterica subspecies enterica serovar Enteritidis that harbors a highly unusual virulence plasmid. During the characterisation of a group of S. Enteritidis isolates, 10 isolates recovered from Canadian duck production facilities, of which seven were phage type 9b and three were closely related atypical phage types, failed detection by a PCR targeting the prot6e gene, a marker located on the virulence plasmid often employed for identification of this serovar. Comparison to prot6e+ isolates by several standard genetic typing tools, further revealed their distinctive genomic makeup. Both short read and long read whole genome sequencing were completed on six of these isolates. In addition to loss of the prot6e gene, the virulence plasmid of each isolate was found to be exceptionally large (86.5 Kb) due to a 28 Kb insertion of S. Typhimurium plasmid sequence that encodes multiple genes of the incF operon. Interrogation of the chromosome sequence data of these isolates using a SNP-based typing tool and MLST both indicated their close genetic relatedness. One additional isolate carrying this plasmid was identified in an in-house collection of S. Enteritidis isolates. Finally, the identification of this unusual plasmid sequence in additional isolates submitted to public repositories of Salmonella sequence data was explored. All these analyses indicated that a very distinctive but rarely reported strain of S. Enteritidis was widely distributed across North America and the United Kingdom with one additional report involving a case from Brazil. With increased use of genetic methods for Salmonella identification, the loss of the prot6e sequence may confound correct identification of this serovar while also potentially altering the mode of transmission to humans given the gene's role in facilitating propagation of this bacterium in eggs. Accordingly, this strain may present certain challenges with respect to public health investigations. Our studies also suggest this strain is often associated with duck hosts thereby providing a possible mechanism by which this strain has spread over an extensive geographical area.

Genome Sequences of Five Mycobacterium bovis Strains Isolated from Farmed Animals and Wildlife in Canada.

Mycobacterium bovis is the causative agent of bovine tuberculosis, an infectious disease that affects both animals and humans and thus presents a risk to public health and the livestock industry. Here, we report the genome sequences of five Mycobacterium bovis strains that represent major genotype clusters observed in farmed animals and wildlife in Canada.

Genotypes of Mycobacterium bovis strains isolated from domestic animals and wildlife in Canada in 1985-2015.

Two internationally recognised and standardised genotyping methods, mycobacterial interspersed repetitive unit and variable number tandem repeat analysis (MIRU-VNTR) and spoligotyping, were applied to characterise genetic variations among 137 Mycobacterium bovis isolates recovered from Canadian domestic and wild animals during 1985-2015. Spoligotyping generated seven types that were discriminated further into12 MIRU-VNTR types. The discriminatory power indexes were estimated as 0.71 and 0.77 for spoligotyping and MIRU-VNTR typing approaches, respectively. In total, 6 prominent clusters of isolates were observed by the genotyping schemes. Four genotype clusters were exclusively observed in farmed animals. Three of these four clusters were affiliated with localised tuberculosis outbreaks, and each cluster corresponded to a single specific spoligotype (SB0140, SB0673, and SB1069) and a MIRU-VNTR profile. The fourth genotype cluster, with spoligotype SB0265 which segregated into two MIRU-VNTR types, was associated with bovine tuberculosis outbreaks in several farms across Canada during 1990-2002. Two genotype clusters of M. bovis stains were associated with wildlife reservoirs: a spoligotype SB0130 with 3 unique MIRU-VNTR profiles were observed in wood bison in Wood Buffalo National Park, and unique spoligotypes SB1070 and 1071 represented by four MIRU-VNTR profiles were recovered from cervidae species in and around the Riding Mountain National Park of Manitoba. Genotyping data confirmed M. bovis transmission between wildlife and livestock in Manitoba in 1990-2008. Overall, notwithstanding the low level of genetic diversity of Canadian M. bovis strains, the spoligotyping and MIRU-VNTR typing were useful tools in monitoring transmission of endemic strains and defining new introductions to Canada. The majority of genotypes were most likely introduced into domestic animals through live animal trade, and subsequently eliminated as a result of bovine tuberculosis outbreak investigation and eradication activities.

A novel approach for scrapie-associated prion (PrPSc) detection in blood using the competitive affinity of an aggregate-specific antibody and streptavidin to PrPSc.

Scrapie is a fatal neurodegenerative disorder affecting sheep and goats, originating from exposure to disease-associated prions (PrPSc). An ante-mortem screening test that can detect native PrPSc in body fluids remains unavailable due to insufficient sensitivity of current detection methods that involve proteinase or denaturation treatments. We adopted an approach to detect PrPSc in whole blood using a simple proteinase- and denaturation-independent immunoassay, based on the competitive affinity of an aggregate-specific monoclonal antibody and streptavidin to PrPSc. First, we demonstrated the ability of native PrPSc to bind to streptavidin and the inhibition of this interaction by 15B3 antibody (P<0.05). This led to a new two-step assay that involved capturing native prions from infected blood on a solid-state matrix and detection of PrPSc aggregates by evaluating the conformation-dependent conjugate catalytic activity ratio in samples against a pre-determined threshold. This test showed capacity for detecting scrapie prions in 500µl of sheep whole blood spiked with scrapie brain homogenate containing approximately 5ng of total brain protein, and estimated to have 500fg of PrPSc. The test also discriminated between blood samples from scrapie-negative (6 sheep, 4 goats) and scrapie-infected animals (3 experimentally infected sheep, 7 naturally infected goats). Collectively, with the proposed high-throughput sample-processing platform, these initial studies provide insights into the development of a large-scale screening test for the routine diagnosis of scrapie.

Detection of bovine central nervous system tissues in rendered animal by-products by one-step real-time reverse transcription PCR assay.

Contamination of rendered animal byproducts with central nervous system tissues (CNST) from animals with bovine spongiform encephalopathy is considered one of the vehicles of disease transmission. Removal from the animal feed chain of CNST originated from cattle of a specified age category, species-labeling of rendered meat products, and testing of rendered products for bovine CNST are tasks associated with the epidemiological control of bovine spongiform encephalopathy. A single-step TaqMan real-time reverse transcriptase (RRT) PCR assay was developed and evaluated for specific detection of bovine glial fibrillary acidic protein (GFAP) mRNA, a biomarker of bovine CNST, in rendered animal by-products. An internal amplification control, mammalian b -actin mRNA, was coamplified in the duplex RRT-PCR assay to monitor amplification efficiency, normalize amplification signals, and avoid false-negative results. The functionality of the GFAP mRNA RRT-PCR was assessed through analysis of laboratory-generated binary mixtures of bovine central nervous system (CNS) and muscle tissues treated under various thermal settings imitating industrial conditions. The assay was able to detect as low as 0.05 % (wt/wt) bovine brain tissue in binary mixtures heat treated at 110 to 130°C for 20 to 60 min. Further evaluation of the GFAP mRNA RRT-PCR assay involved samples of industrial rendered products of various species origin and composition obtained from commercial sources and rendering plants. Low amounts of bovine GFAP mRNA were detected in several bovine-rendered products, which was in agreement with declared species composition. An accurate estimation of CNS tissue content in industrial-rendered products was complicated due to a wide range of temperature and time settings in rendering protocols. Nevertheless, the GFAP mRNA RRT-PCR assay may be considered for bovine CNS tissue detection in rendered products in combination with other available tools (for example, animal age verification) in inspection programs.

Binding of bovine T194A PrP(C) by PrP(Sc)-specific antibodies: potential implications for immunotherapy of familial prion diseases.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that are based on the misfolding of a cellular prion protein (PrP(C)) into an infectious, pathological conformation (PrP(Sc)). There is proof-of-principle evidence that a prion vaccine is possible but this is tempered with concerns of the potential dangers associated with induction of immune responses to a widely-expressed self-protein. By targeting epitopes that are specifically exposed upon protein misfolding, our group developed a vaccine that induces PrP(Sc)-specific antibody responses. Here we consider the ability of this polyclonal antibody (SN6b) to bind to a mutant of PrP(C) associated with spontaneous prion disease. Polyclonal antibodies were selected to mimic the vaccination outcome and also explore all possible protein conformations of the recombinant bovine prion protein with mutation T194A [bPrP(T194A)]. This mutant is a homolog of the human T183A mutation of PrP(C) that is associated with early onset of familial dementia. With nanopore analysis, under non-denaturing conditions, we observed binding of the SN6b antibody to bPrP(T194A). This interaction was confirmed through ELISAs as well as immunoprecipitation of the recombinant and cellularly expressed forms of bPrP(T194A). This interaction did not promote formation of a protease resistant conformation of PrP in vitro. Collectively, these findings support the disease-specific approach for immunotherapy of prion diseases but also suggest that the concept of conformation-specific immunotherapy may be complicated in individuals who are genetically predisposed to PrP(C) misfolding.

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

Nanopore analysis of the interaction of metal ions with prion proteins and peptides.

Nanopore analysis can be used to study conformational changes in individual peptide or protein molecules. Under an applied voltage there is a change in the event parameters of blockade current or time when a molecule bumps into or translocates through the pore. If a molecule undergoes a conformational change upon binding a ligand or metal ion the event parameters will be altered. The objective of this research was to demonstrate that the conformation of the prion protein (PrP) and prion peptides can be modulated by binding divalent metal ions. Peptides from the octarepeat region (Octa2, (PHGGGWGQ)2 and Octa 4, (PHGGGWGQ)4), residues 106-126 (PrP106-126), and the full-length Bovine recombinant prion (BrecPrP) were studied with an alpha-hemolysin pore. Octa2 readily translocated the pore but significant bumping events occurred on addition of Cu(II) and to a lesser extent Zn(II), demonstrating that complex formation was occurring with concomitant conformational changes. The binding of Cu(II) to Octa4 was more pronounced and at high concentrations only a small proportion of the complex could translocate. Addition of Zn(II) also caused significant changes to the event parameters but Mg(II) and Mn(II) were inert. Addition of Cu(II) to PrP106-126 caused the formation of a very tight complex, which could not translocate the pore. Small changes were observed with Zn(II), but not with Mg(II) or Mn(II). Analysis of BrecPrP showed that about 37% were translocation events, but on addition of Cu(II) or Zn(II) these disappeared and only bumping events were recorded. Suprisingly, addition of Mn(II) caused an increase in translocation events to about 64%. Thus, conformational changes to prions upon binding metal ions are readily observed by nanopore analysis.

Nanopore detection of antibody prion interactions.

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an alpha-hemolysin pore embedded in a lipid membrane. A prion peptide, PrP(143-169), can readily translocate through the pore, but on the addition of monoclonal antibody M2188, which binds the peptide, the number of translocations is reduced because the complex is too large to translocate. At a peptide-to-immunoglobulin G (IgG) ratio of 2:1, only bumping events were observed. The event profile of a control peptide that does not bind the antibody was unchanged. Similarly, the presence of the antibody prevents translocation of the full-length prion protein. Because a nanopore can detect a single molecule, these experiments represent an important first step towards the development of a sensitive prion detector.

Prion protein in sheep urine.

The misfolded form of cellular prion protein (PrP(C)) is the main component of the infectious agent of transmissible spongiform encephalopathies and the validated biomarker for these diseases. The expression of PrP(C) is highest in the central nervous system and has been found in peripheral tissues. Soluble PrP(C) has been detected in cerebrospinal fluid, urine, serum, milk, and seminal plasma. In this study, attempts were made to characterize prion protein in urine samples from normal and scrapie-infected sheep. Urine samples from scrapie-infected sheep and age-matched healthy sheep were collected and analyzed by Western blot following concentration. A protease K-sensitive protein band with a molecular weight of approximately 27-30 kDa was visualized after immunoblotting with anti-PrP monoclonal antibodies to a C-terminal part of PrP(C), but not after immunoblotting with monoclonal antibodies to an N-terminal epitope of PrP(C) or with secondary antibodies only. The amount of PrP(C) in the urine of 49 animals (control group: n = 16; naturally scrapie-infected group: n = 33) was estimated by comparison with known amounts of ovine recombinant PrP in the immunoblot. Background concentration of PrP(C) in urine was found to be 0-0.16 ng/ml (adjusted to the initial nonconcentrated volume of the urine samples). Seven out of 33 naturally scrapie-infected animals had an elevated level (0.3-4.7 ng/ml) of PrP(C) in urine. The origin of PrP(C) in urine and the reason for the increased level of PrP(C) in scrapie-infected sheep urine has yet to be explored.

Binding of bovine prion protein to heparin: a fluorescence polarization study.

Glycosaminoglycans (GAGs) are believed to be associated with prion disease pathology and also with metabolism of the prion protein. Fluorescence polarization assay (FPA) of binding between bovine recombinant prion protein (brecPrP) and heparin labelled with AlexaFluor488 was used in model experiments to study glycosaminoglycan-prion protein interaction. Heparin binding to brecPrP was a rapid reversible event which occurred under defined conditions. The interaction of brecPrP with fluorophore-labelled heparin was inhibited by the presence of Cu(2+) ions and was sensitive to competition with heparin, heparan sulphate, and dextran. The dissociation constant of the heparin-brecPrP complex was 73.4+/-3.7 nM. Circular dichroism (CD) experiments indicated that the structure of brecPrP was less helical in the presence of heparin.

Generation of antibodies against bovine recombinant prion protein in various strains of mice.

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, belong to a group of neurodegenerative disorders affecting humans and animals. To date, definite diagnosis of prion disease can only be made by analysis of tissue samples for the presence of protease-resistant misfolded prion protein (PrP(Sc)). Monoclonal antibodies (MAbs) to the prion protein provide valuable tools for TSE diagnosis, as well as for basic research on these diseases. In this communication, the development of antibodies against recombinant bovine prion protein (brecPrP) in four strains of mice (BALB/c, ND4, SJL, and NZB/NZW F(1)) is described. Immunization of autoimmunity-prone NZB/NZW F(1) and SJL mice with brecPrP was applied to overcome self-tolerance against the prion protein. ND4 and SJL mice did not develop an immune response to brecPrP. BALB/c mice produced antibody titers of 1:1,000 to 1:1,500 in an enzyme-linked immunosorbent assay (ELISA), while NZB/NZW F(1) mice responded with titers of 1:7,000 to 1:11,000. A panel of 71 anti-brecPrP MAbs recognizing continuous and discontinuous epitopes was established from BALB/c and NZB/NZW F(1) mice. Seven anti-brecPrP MAbs reacted with both the cellular form of PrP and protease K-resistant PrP(Sc) from sheep brain in Western blot assays. The epitope specificity of these MAbs was determined, and applicability to immunohistochemical detection of prions was studied. The MAbs generated will be useful tools in the development of TSE immunochemical diagnosis and for research. This is the first report of the development of anti-PrP MAbs by use of autoimmune NZB/NZW F(1) mice as an alternative approach for the generation of PrP-specific MAbs.