Influence of genomic variations on glanders serodiagnostic antigens using integrative genomic and transcriptomic approaches.

Glanders is a highly contagious and life-threatening zoonotic disease caused by Burkholderia mallei (B. mallei). Without an effective vaccine or treatment, early diagnosis has been regarded as the most effective method to prevent glanders transmission. Currently, the diagnosis of glanders is heavily reliant on serological tests. However, given that markedly different host immune responses can be elicited by genetically different strains of the same bacterial species, infection by B. mallei, whose genome is unstable and plastic, may result in various immune responses. This variability can make the serodiagnosis of glanders challenging. Therefore, there is a need for a comprehensive understanding and assessment of how B. mallei genomic variations impact the appropriateness of specific target antigens for glanders serodiagnosis. In this study, we investigated how genomic variations in the B. mallei genome affect gene content (gene presence/absence) and expression, with a special focus on antigens used or potentially used in serodiagnosis. In all the genome sequences of B. mallei isolates available in NCBI's RefSeq database (accessed in July 2023) and in-house sequenced samples, extensive small and large variations were observed when compared to the type strain ATCC 23344. Further pan-genome analysis of those assemblies revealed variations of gene content among all available genomes of B. mallei. Specifically, differences in gene content ranging from 31 to 715 genes with an average of 334 gene presence-absence variations were found in strains with complete or chromosome-level genome assemblies, using the ATCC 23344 strain as a reference. The affected genes included some encoded proteins used as serodiagnostic antigens, which were lost due mainly to structural variations. Additionally, a transcriptomic analysis was performed using the type strain ATCC 23344 and strain Zagreb which has been widely utilized to produce glanders antigens. In total, 388 significant differentially expressed genes were identified between these two strains, including genes related to bacterial pathogenesis and virulence, some of which were associated with genomic variations, particularly structural variations. To our knowledge, this is the first comprehensive study to uncover the impacts of genetic variations of B. mallei on its gene content and expression. These differences would have significant impacts on host innate and adaptive immunity, including antibody production, during infection. This study provides novel insights into B. mallei genetic variants, knowledge which will help to improve glanders serodiagnosis.

Special Issue "Advances in Rabies Research".

Rabies kills approximately 60,000 humans each year, with deaths mostly occurring in developing countries, where rabies lyssavirus (RABV) variants are maintained in dog populations [...].

Evaluation of the use of CRISPR loci for discrimination of Salmonella enterica subsp. enterica serovar Enteritidis strains recovered in Canada and comparison with other subtyping methods.

Salmonella enterica subsp. enterica serovar Enteritidis remains one of the most important foodborne pathogens worldwide. To minimise its public health impact when outbreaks of the disease occur, timely investigation to identify and recall the contaminated food source is necessary. Central to this approach is the need for rapid and accurate identification of the bacterial subtype epidemiologically linked to the outbreak. While traditional methods of S. Enteritidis subtyping, such as pulsed field gel electrophoresis (PFGE) and phage typing (PT), have played an important role, the clonal nature of this organism has spurred efforts to improve subtyping resolution and timeliness through molecular based approaches. This study uses a cohort of 92 samples, recovered from a variety of sources, to compare these two traditional methods for S. Enteritidis subtyping with recently developed molecular techniques. These latter methods include the characterisation of two clustered regularly interspaced short palindromic repeats (CRISPR) loci, either in isolation or together with sequence analysis of virulence genes such as fimH. For comparison, another molecular technique developed in this laboratory involved the scoring of 60 informative single nucleotide polymorphisms (SNPs) distributed throughout the genome. Based on both the number of subtypes identified and Simpson's index of diversity, the CRISPR method was the least discriminatory and not significantly improved with the inclusion of fimH gene sequencing. While PT analysis identified the most subtypes, the SNP-PCR process generated the greatest index of diversity value. Combining methods consistently improved the number of subtypes identified, with the SNP/CRISPR typing scheme generating a level of diversity comparable with that of PT/PFGE. While these molecular methods, when combined, may have significant utility in real-world situations, this study suggests that CRISPR analysis alone lacks the discriminatory capability required to support investigations of foodborne disease outbreaks.

Ampliseq for Illumina Technology Enables Detailed Molecular Epidemiology of Rabies Lyssaviruses from Infected Formalin-Fixed Paraffin-Embedded Tissues.

Whole genome sequencing of rabies lyssaviruses (RABVs) has enabled the generation of highly detailed phylogenies that reveal viral transmission patterns of disease in reservoir species. Such information is highly important for informing best practices with respect to wildlife rabies control. However, specimens available only as formalin fixed paraffin embedded (FFPE) samples have been recalcitrant to such analyses. Due to the damage inflicted by tissue processing, only relatively short amplicons can be generated by standard RT-PCR methods, making the generation of full-length genome sequences very tedious. While highly parallel shotgun sequencing of total RNA can potentially overcome these challenges, the low percentage of reads representative of the virus may be limiting. Ampliseq technology enables massively multiplex amplification of nucleic acids to produce large numbers of short PCR products. Such a strategy has been applied to the sequencing of entire viral genomes but its use for rabies virus analysis has not been reported previously. This study describes the generation of an Ampliseq for Illumina primer panel, which was designed based on the global sequence diversity of rabies viruses, and which enables efficient viral genome amplification and sequencing of rabies-positive FFPE samples. The subsequent use of such data for detailed phylogenetic analysis of the virus is demonstrated.

A comparison of fourteen fully characterized mammalian-associated Campylobacter fetus isolates suggests that loss of defense mechanisms contribute to high genomic plasticity and subspecies evolution.

Campylobacter fetus is currently classified into three main subspecies, but only two of these, C. fetus subspecies fetus and C. fetus subsp. venerealis originate principally from ruminants where they inhabit different niches and cause distinct pathogenicity. Their importance as pathogens in international trade and reporting is also different yet the criteria defining these properties have never been fully substantiated nor understood. The situation is further compromised because the ability to differentiate between these two closely related C. fetus subspecies has traditionally been performed by phenotypic characterisation of isolates, methods which are limited in scope, time-consuming, tedious, and often yield inconsistent results, thereby leading to isolate misidentification. The development of robust genetic markers that could enable rapid discrimination between C. fetus subsp. fetus and subsp. venerealis has also been challenging due to limited differences in the gene complement of their genomes, high levels of sequence repetition, the small number of closed genome sequences available and the lack of standardisation of the discriminatory biochemical tests employed for comparative purposes. To yield a better understanding of the genomic differences that define these C. fetus strains, seven isolates were exhaustively characterised phenotypically and genetically and compared with seven previously well characterised isolates. Analysis of these 14 C. fetus samples clearly illustrated that adaption by C. fetus subsp. venerealis to the bovine reproductive tract correlated with increasing genome length and plasticity due to the acquisition and propagation of several mobile elements including prophages, transposons and plasmids harbouring virulence factors. Significant differences in the repertoire of the CRISPR (clustered regularly interspersed short palindromic repeats)-cas system of all C. fetus strains was also found. We therefore suggest that a deficiency in this adaptive immune system may have permitted the emergence of extensive genome plasticity and led to changes in host tropism through gene disruption and/or changes in gene expression. Notable differences in the sub-species complement of DNA adenine methylase genes may also have an impact. These data will facilitate future studies to better understand the precise genetic differences that underlie the phenotypic and virulence differences between these animal pathogens and may identify additional markers useful for diagnosis and sub-typing.

Relationships between fox populations and rabies virus spread in northern Canada.

Rabies spreads in both Arctic (Vulpes lagopus) and red foxes (Vulpes vulpes) throughout the Canadian Arctic but limited wildlife disease surveillance, due to the extensive landmass of the Canadian north and its small widely scattered human population, undermines our knowledge of disease transmission patterns. This study has explored genetic population structure in both the rabies virus and its fox hosts to better understand factors that impact rabies spread. Phylogenetic analysis of 278 samples of the Arctic lineage of rabies virus recovered over 40 years identified four sub-lineages, A1 to A4. The A1 lineage has been restricted to southern regions of the Canadian province of Ontario. The A2 lineage, which predominates in Siberia, has also spread to northern Alaska while the A4 lineage was recovered from southern Alaska only. The A3 sub-lineage, which was also found in northern Alaska, has been responsible for virtually all cases across northern Canada and Greenland, where it further differentiated into 18 groups which have systematically evolved from a common predecessor since 1975. In areas of Arctic and red fox sympatry, viral groups appear to circulate in both hosts, but both mitochondrial DNA control region sequences and 9-locus microsatellite genotypes revealed contrasting phylogeographic patterns for the two fox species. Among 157 Arctic foxes, 33 mitochondrial control region haplotypes were identified but little genetic structure differentiating localities was detected. Among 162 red foxes, 18 control region haplotypes delineated three groups which discriminated among the Churchill region of Manitoba, northern Quebec and Labrador populations, and the coastal Labrador locality of Cartwright. Microsatellite analyses demonstrated some genetic heterogeneity among sampling localities of Arctic foxes but no obvious pattern, while two or three clusters of red foxes suggested some admixture between the Churchill and Quebec-Labrador regions but uniqueness of the Cartwright group. The limited population structure of Arctic foxes is consistent with the rapid spread of rabies virus subtypes throughout the north, while red fox population substructure suggests that disease spread in this host moves most readily down certain independent corridors such as the northeastern coast of Canada and the central interior. Interestingly the evidence suggests that these red fox populations have limited capacity to maintain the virus over the long term, but they may contribute to viral persistence in areas of red and Arctic fox sympatry.

Mobility of ß-Lactam Resistance Under Bacterial Co-infection and Ampicillin Treatment in a Mouse Model.

Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to the dissemination of antibiotic-resistance genes in the gut microbiota and the development of antibiotic-resistant bacterial infection, a significant threat to animal and public health. Food or water may be contaminated with multiple resistant bacteria, but animal models on gene transfer were mainly based on single-strain infections. In this study, we investigated the mobility of ß-lactam resistance following infection with single- versus multi-strain of resistant bacteria under ampicillin treatment. We characterized three bacterial strains isolated from food-animal production systems, Escherichia coli O80:H26 and Salmonella enterica serovars Bredeney and Heidelberg. Each strain carries at least one conjugative plasmid that encodes a ß-lactamase. We orally infected mice with each or all three bacterial strain(s) in the presence or absence of ampicillin treatment. We assessed plasmid transfer from the three donor bacteria to an introduced E. coli CV601gfp recipient in the mouse gut, and evaluated the impacts of the bacterial infection on gut microbiota and gut health. In the absence of ampicillin treatment, none of the donor or recipient bacteria established in the normal gut microbiota and plasmid transfer was not detected. In contrast, the ampicillin treatment disrupted the gut microbiota and enabled S. Bredeney and Heidelberg to colonize and transfer their plasmids to the E. coli CV601gfp recipient. E. coli O80:H26 on its own failed to colonize the mouse gut. However, during co-infection with the two Salmonella strains, E. coli O80:H26 colonized and transferred its plasmid to the E. coli CV601gfp recipient and a residential E. coli O2:H6 strain. The co-infection significantly increased plasmid transfer frequency, enhanced Proteobacteria expansion and resulted in inflammation in the mouse gut. Our findings suggest that single-strain infection models for evaluating in vivo gene transfer may underrepresent the consequences of multi-strain infections following the consumption of heavily contaminated food or water.

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.

A long-distance translocation initiated an outbreak of raccoon rabies in Hamilton, Ontario, Canada.

Despite proactive measures to prevent raccoon rabies entering Canada from the United States, several incursions of this disease have occurred. The largest outbreak, first reported in December 2015 in the city of Hamilton, Ontario, has resulted in the reporting of 449 animal cases as of December 31, 2018. Initial phylogenetic studies on the index case suggested that this outbreak was not due to local cross-border spread from the Niagara region of the United States where raccoon rabies has persisted for several years. Phylogenetic analysis of whole genome sequences of a viral collection from the Hamilton area and several US states indicates that a long-distance translocation of a diseased animal from southeastern New York State was responsible for this incursion. The role of the skunk as a potential secondary host supporting persistence and / or spread of the virus is also examined.

Population- and Variant-Based Genome Analyses of Viruses from Vaccine-Derived Rabies Cases Demonstrate Product Specific Clusters and Unique Patterns.

Rabies in wildlife has been successfully controlled in parts of Europe and North America using oral rabies vaccination, i.e., the distribution of baits containing live-attenuated virus strains. Occasionally, these vaccines caused vaccine virus-induced rabies cases. To elucidate the mechanisms of genetic selection and the effect of viral populations on these rabies cases, a next generation sequencing approach as well as comprehensive data analyses of the genetic diversity of Street Alabama Dufferin (SAD) and ERA vaccine virus strains and vaccine-induced rabies cases from Canada and several European countries were conducted. As a result, twelve newly generated sets of sequencing data from Canada and Poland were added to a pool of previously investigated samples. While the population-based analysis showed a segregation of viruses of ERA vaccine-induced rabies cases from those of SAD Bern original (SAD Bernorig)-derived rabies cases, the in-depth variant analysis revealed three distinct combinations of selected variants for the ERA vaccine-induced cases, suggesting the presence of multiple replication-competent haplotypes in the investigated ERA-BHK21 vaccine. Our findings demonstrate the potential of a deep sequencing approach in combination with comprehensive analyses on the consensus, population, and variant level.

Rabies virus infection in a 21-year-old male presenting with ascending paralysis after a bat scratch.

A 21-year-old, previously healthy male presented to hospital following 1 week of bilateral asymmetric ascending paralysis, odynophagia, and dysphagia. Initial magnetic resonance imaging (MRI) of the spine revealed an abnormal increased T2 signal with predominant dorsal column involvement and sparing of white matter throughout the cervical cord and extending to T5. The initial presumptive diagnosis was an acute infectious, versus inflammatory, myelitis. On reviewing the history, family members recalled a bat scratch on the left hand, sustained months prior, for which the patient did not seek or receive post-exposure prophylaxis (PEP). Rabies virus (RABV) RNA was detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two saliva samples, while nuchal skin biopsy and cerebrospinal fluid (CSF) were negative. Serum was negative for RABV neutralizing antibody. Sequencing and phylogenetic analyses identified the infecting RABV as a variant associated with silver-haired bats. Following risk assessment of exposure, 67 health care workers and several family members were offered PEP.

Un homme de 21 ans auparavant en santé a consulté à l'hôpital parce qu'il souffrait de paralysie ascendante, asymétrique et bilatérale, d'odynophagie et de dysphagie depuis une semaine. Une première imagerie par résonance magnétique (IRM) du rachis a révélé une augmentation anormale du signal T2 avec atteinte prédominante de la colonne dorsale et épargne de la matière blanche dans toute la colonne cervicale jusqu'à la vertèbre T5. Le diagnostic provisoire en était un de myélite infectieuse, et non inflammatoire. À la prise de l'histoire, les membres de la famille se sont souvenus d'une égratignure de chauve-souris sur la main gauche du patient plusieurs mois auparavant, qui n'a pas été suivie d'une prophylaxie postexposition (PPE). Les chercheurs ont décelé l'ARN du virus de la rage (RABV) par amplification en chaîne par polymérase quantitative de transcription inverse (RT-qPCR) dans deux échantillons de salive, mais constaté un résultat négatif de la biopsie de la peau nucale et du liquide céphalorachidien. Le sérum était négatif à l'anticorps neutralisant du RABV. Les analyses de séquençage et de phylogénétique ont confirmé une contamination par une variante du RABV associée aux chauves-souris argentées. Après une évaluation du risque d'exposition, 67 travailleurs de la santé et plusieurs membres de la famille se sont fait offrir une PPE.

Origins of the arctic fox variant rabies viruses responsible for recent cases of the disease in southern Ontario.

A subpopulation of the arctic fox lineage of rabies virus has circulated extensively in red fox populations of Ontario, Canada, between the 1960s and 1990s. An intensive wildlife rabies control program, in which field operations were initiated in 1989, resulted in elimination of the disease in eastern Ontario. However in southwestern Ontario, as numbers of rabid foxes declined the proportion of skunks confirmed to be infected with this rabies virus variant increased and concerted control efforts targeting this species were employed to eliminate the disease. Since 2012 no cases due to this viral variant were reported in southwestern Ontario until 2015 when a single case of rabies due to the arctic fox variant was reported in a bovine. Several additional cases have been documented subsequently. Since routine antigenic typing cannot discriminate between the variants which previously circulated in Ontario and those from northern Canada it was unknown whether these recent cases were the result of a new introduction of this variant or a continuation of the previous enzootic. To explore the origins of this new outbreak whole genome sequences of a collection of 128 rabies viruses recovered from Ontario between the 1990s to the present were compared with those representative of variants circulating in the Canadian north. Phylogenetic analysis shows that the variant responsible for current cases in southwestern Ontario has evolved from those variants known to circulate in Ontario previously and is not due to a new introduction from northern regions. Thus despite ongoing passive surveillance the persistence of wildlife rabies went undetected in the study area for almost three years. The apparent adaptation of this rabies virus variant to the skunk host provided the opportunity to explore coding changes in the viral genome which might be associated with this host shift. Several such changes were identified including a subset for which the operation of positive selection was supported. The location of a small number of these amino acid substitutions in or close to protein motifs of functional importance suggests that some of them may have played a role in this host shift.

ONRAB® oral rabies vaccine is shed from, but does not persist in, captive mammals.

ONRAB® is a human adenovirus rabies glycoprotein recombinant vaccine developed to control rabies in wildlife. To support licensing and widespread use of the vaccine, safety studies are needed to assess its potential residual impact on wildlife populations. We examined the persistence of the ONRAB® vaccine virus in captive rabies vector and non-target mammals. This research complements work on important rabies vector species (raccoon, striped skunk, and red fox) but also adds to previous findings with the addition of some non-target species (Virginia opossum, Norway rats, and cotton rats) and a prolonged period of post vaccination monitoring (41 days). Animals were directly inoculated orally with the vaccine and vaccine shedding was monitored using quantitative real-time PCR applied to oral and rectal swabs. ONRAB® DNA was detected in both oral and rectal swabs from 6 h to 3 days post-inoculation in most animals, followed by a resurgence of shedding between days 17 and 34 in some species. Overall, the duration over which ONRAB® DNA was detectable was shorter for non-target mammals, and by day 41, no animal had detectable DNA in either oral or rectal swabs. All target species, as well as cotton rats and laboratory-bred Norway rats, developed robust humoral immune responses as measured by competitive ELISA, with all individuals being seropositive at day 31. Similarly, opossums showed good response (89% seropositive; 8/9), whereas only one of nine wild caught Norway rats was seropositive at day 31. These results support findings of other safety studies suggesting that ONRAB® does not persist in vector and non-target mammals exposed to the vaccine. As such, we interpret these data to reflect a low risk of adverse effects to wild populations following distribution of ONRAB® to control sylvatic rabies.

In-depth genome analyses of viruses from vaccine-derived rabies cases and corresponding live-attenuated oral rabies vaccines.

Live-attenuated rabies virus strains such as those derived from the field isolate Street Alabama Dufferin (SAD) have been used extensively and very effectively as oral rabies vaccines for the control of fox rabies in both Europe and Canada. Although these vaccines are safe, some cases of vaccine-derived rabies have been detected during rabies surveillance accompanying these campaigns. In recent analysis it was shown that some commercial SAD vaccines consist of diverse viral populations, rather than clonal genotypes. For cases of vaccine-derived rabies, only consensus sequence data have been available to date and information concerning their population diversity was thus lacking. In our study, we used high-throughput sequencing to analyze 11 cases of vaccine-derived rabies, and compared their viral population diversity to the related oral rabies vaccines using pairwise Manhattan distances. This extensive deep sequencing analysis of vaccine-derived rabies cases observed during oral vaccination programs provided deeper insights into the effect of accidental in vivo replication of genetically diverse vaccine strains in the central nervous system of target and non-target species under field conditions. The viral population in vaccine-derived cases appeared to be clonal in contrast to their parental vaccines. The change from a state of high population diversity present in the vaccine batches to a clonal genotype in the affected animal may indicate the presence of a strong bottleneck during infection. In conclusion, it is very likely that these few cases are the consequence of host factors and not the result of the selection of a more virulent genotype. Furthermore, this type of vaccine-derived rabies leads to the selection of clonal genotypes and the selected variants were genetically very similar to potent SAD vaccines that have undergone a history of in vitro selection.

Rabies.

Rabies is a life-threatening neglected tropical disease: tens of thousands of cases are reported annually in endemic countries (mainly in Africa and Asia), although the actual numbers are most likely underestimated. Rabies is a zoonotic disease that is caused by infection with viruses of the Lyssavirus genus, which are transmitted via the saliva of an infected animal. Dogs are the most important reservoir for rabies viruses, and dog bites account for >99% of human cases. The virus first infects peripheral motor neurons, and symptoms occur after the virus reaches the central nervous system. Once clinical disease develops, it is almost certainly fatal. Primary prevention involves dog vaccination campaigns to reduce the virus reservoir. If exposure occurs, timely post-exposure prophylaxis can prevent the progression to clinical disease and involves appropriate wound care, the administration of rabies immunoglobulin and vaccination. A multifaceted approach for human rabies eradication that involves government support, disease awareness, vaccination of at-risk human populations and, most importantly, dog rabies control is necessary to achieve the WHO goal of reducing the number of cases of dog-mediated human rabies to zero by 2030.

Processes Underlying Rabies Virus Incursions across US-Canada Border as Revealed by Whole-Genome Phylogeography.

Disease control programs aim to constrain and reduce the spread of infection. Human disease interventions such as wildlife vaccination play a major role in determining the limits of a pathogen's spatial distribution. Over the past few decades, a raccoon-specific variant of rabies virus (RRV) has invaded large areas of eastern North America. Although expansion into Canada has been largely prevented through vaccination along the US border, several outbreaks have occurred in Canada. Applying phylogeographic approaches to 289 RRV whole-genome sequences derived from isolates collected in Canada and adjacent US states, we examined the processes underlying these outbreaks. RRV incursions were attributable predominantly to systematic virus leakage of local strains across areas along the border where vaccination has been conducted but also to single stochastic events such as long-distance translocations. These results demonstrate the utility of phylogeographic analysis of pathogen genomes for understanding transboundary outbreaks.

The phylogeography of Myotis bat-associated rabies viruses across Canada.

As rabies in carnivores is increasingly controlled throughout much of the Americas, bats are emerging as a significant source of rabies virus infection of humans and domestic animals. Knowledge of the bat species that maintain rabies is a crucial first step in reducing this public health problem. In North America, several bat species are known to be rabies virus reservoirs but the role of bats of the Myotis genus has been unclear due to the scarcity of laboratory confirmed cases and the challenges encountered in species identification of poorly preserved diagnostic submissions by morphological traits alone. This study has employed a collection of rabid bat specimens collected across Canada over a 25 year period to clearly define the role of particular Myotis species as rabies virus reservoirs. The virus was characterised by partial genome sequencing and host genetic barcoding, used to confirm species assignment of specimens, proved crucial to the identification of certain bat species as disease reservoirs. Several variants were associated with Myotis species limited in their Canadian range to the westernmost province of British Columbia while others were harboured by Myotis species that circulate across much of eastern and central Canada. All of these Myotis-associated viral variants, except for one, clustered as a monophyletic MYCAN clade, which has emerged from a lineage more broadly distributed across North America; in contrast one distinct variant, associated with the long-legged bat in Canada, represents a relatively recent host jump from a big brown bat reservoir. Together with evidence from South America, these findings demonstrate that rabies virus has emerged in the Myotis genus independently on multiple occasions and highlights the potential for emergence of new viral-host associations within this genus.

Application of high-throughput sequencing to whole rabies viral genome characterisation and its use for phylogenetic re-evaluation of a raccoon strain incursion into the province of Ontario.

Raccoon rabies remains a serious public health problem throughout much of the eastern seaboard of North America due to the urban nature of the reservoir host and the many challenges inherent in multi-jurisdictional efforts to administer co-ordinated and comprehensive wildlife rabies control programmes. Better understanding of the mechanisms of spread of rabies virus can play a significant role in guiding such control efforts. To facilitate a detailed molecular epidemiological study of raccoon rabies virus movements across eastern North America, we developed a methodology to efficiently determine whole genome sequences of hundreds of viral samples. The workflow combines the generation of a limited number of overlapping amplicons covering the complete viral genome and use of high throughput sequencing technology. The value of this approach is demonstrated through a retrospective phylogenetic analysis of an outbreak of raccoon rabies which occurred in the province of Ontario between 1999 and 2005. As demonstrated by the number of single nucleotide polymorphisms detected, whole genome sequence data were far more effective than single gene sequences in discriminating between samples and this facilitated the generation of more robust and informative phylogenies that yielded insights into the spatio-temporal pattern of viral spread. With minor modification this approach could be applied to other rabies virus variants thereby facilitating greatly improved phylogenetic inference and thus better understanding of the spread of this serious zoonotic disease. Such information will inform the most appropriate strategies for rabies control in wildlife reservoirs.

Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples.

A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella -specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non- Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

Spatio-temporal Analysis of the Genetic Diversity of Arctic Rabies Viruses and Their Reservoir Hosts in Greenland.

There has been limited knowledge on spatio-temporal epidemiology of zoonotic arctic fox rabies among countries bordering the Arctic, in particular Greenland. Previous molecular epidemiological studies have suggested the occurrence of one particular arctic rabies virus (RABV) lineage (arctic-3), but have been limited by a low number of available samples preventing in-depth high resolution phylogenetic analysis of RABVs at that time. However, an improved knowledge of the evolution, at a molecular level, of the circulating RABVs and a better understanding of the historical perspective of the disease in Greenland is necessary for better direct control measures on the island. These issues have been addressed by investigating the spatio-temporal genetic diversity of arctic RABVs and their reservoir host, the arctic fox, in Greenland using both full and partial genome sequences. Using a unique set of 79 arctic RABV full genome sequences from Greenland, Canada, USA (Alaska) and Russia obtained between 1977 and 2014, a description of the historic context in relation to the genetic diversity of currently circulating RABV in Greenland and neighboring Canadian Northern territories has been provided. The phylogenetic analysis confirmed delineation into four major arctic RABV lineages (arctic 1-4) with viruses from Greenland exclusively grouping into the circumpolar arctic-3 lineage. High resolution analysis enabled distinction of seven geographically distinct subclades (3.I - 3.VII) with two subclades containing viruses from both Greenland and Canada. By combining analysis of full length RABV genome sequences and host derived sequences encoding mitochondrial proteins obtained simultaneously from brain tissues of 49 arctic foxes, the interaction of viruses and their hosts was explored in detail. Such an approach can serve as a blueprint for analysis of infectious disease dynamics and virus-host interdependencies. The results showed a fine-scale spatial population structure in Greenland arctic foxes based on mitochondrial sequences, but provided no evidence for independent isolated evolutionary development of RABV in different arctic fox lineages. These data are invaluable to support future initiatives for arctic fox rabies control and elimination in Greenland.