Kym S. Antonation

Ferrets Infected with Bundibugyo Virus or Ebola Virus Recapitulate Important Aspects of Human Filovirus Disease

ABSTRACT Bundibugyo virus (BDBV) is the etiological agent of a severe hemorrhagic fever in humans with a case-fatality rate ranging from 25 to 36%. Despite having been known to the scientific and medical communities for almost 1 decade, there is a dearth of studies on this pathogen due to the lack of a small animal model. Domestic ferrets are commonly used to study other RNA viruses, including members of the order Mononegavirales. To investigate whether ferrets were susceptible to filovirus infections, ferrets were challenged with a clinical isolate of BDBV. Animals became viremic within 4 days and succumbed to infection between 8 and 9 days, and a petechial rash was observed with moribund ferrets. Furthermore, several hallmarks of human filoviral disease were recapitulated in the ferret model, including substantial decreases in lymphocyte and platelet counts and dysregulation of key biochemical markers related to hepatic/renal function, as well as coagulation abnormalities. Virological, histopathological, and immunohistochemical analyses confirmed uncontrolled BDBV replication in the major organs. Ferrets were also infected with Ebola virus (EBOV) to confirm their susceptibility to another filovirus species and to potentially establish a virus transmission model. Similar to what was seen with BDBV, important hallmarks of human filoviral disease were observed in EBOV-infected ferrets. This study demonstrates the potential of this small animal model for studying BDBV and EBOV using wild-type isolates and will accelerate efforts to understand filovirus pathogenesis and transmission as well as the development of specific vaccines and antivirals. IMPORTANCE The 2013-2016 outbreak of Ebola virus in West Africa has highlighted the threat posed by filoviruses to global public health. Bundibugyo virus (BDBV) is a member of the genus Ebolavirus and has caused outbreaks in the past but is relatively understudied, likely due to the lack of a suitable small animal model. Such a model for BDBV is crucial to evaluating vaccines and therapies and potentially understanding transmission. To address this, we demonstrated that ferrets are susceptible models to BDBV infection as well as to Ebola virus infection and that no virus adaptation is required. Moreover, these animals develop a disease that is similar to that seen in humans and nonhuman primates. We believe that this will improve the ability to study BDBV and provide a platform to test vaccines and therapeutics.

Bacillus cereus Biovar Anthracis Causing Anthrax in Sub-Saharan Africa-Chromosomal Monophyly and Broad Geographic Distribution.

Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats) in West and Central Africa (Côte d’Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo). The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans.

Verification of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens

ABSTRACT We examined the utility of a single matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry method for the identification of security-sensitive biological agents (risk group 3 bacterial pathogens). The goal was 2-fold: to verify a method for inclusion into our scope of accreditation, and to assess the biological safety of extractions. We developed our sample flow to include a tube-based chemical extraction, followed by filtration, before processing on MALDI-TOF MS instruments in a containment level 2 laboratory.

Custom database development and biomarker discovery methods for MALDI-TOF mass spectrometry-based identification of high-consequence bacterial pathogens.

A high-quality custom database of MALDI-TOF mass spectral profiles was developed with the goal of improving clinical diagnostic identification of high-consequence bacterial pathogens. A biomarker discovery method is presented for identifying and evaluating MALDI-TOF MS spectra to potentially differentiate biothreat bacteria from less-pathogenic near-neighbour species.

Sylvatic Plague in a Canadian Black-Tailed Prairie Dog (Cynomys ludovicianus)

Abstract In 2010, a black-tailed prairie dog (Cynomys ludovicianus) was found dead in Grasslands National Park, Saskatchewan, Canada. Postmortem gross and histologic findings indicated bacterial septicemia, likely due to Yersinia pestis, which was confirmed by molecular analysis. This is the first report of Y. pestis in the prairie dog population within Canada.

Melioidosis in Trinidad and Tobago

To the Editor: Melioidosis refers to infection caused by the facultative intracellular gram-negative bacterium Burkholderia pseudomallei. The clinical manifestations of melioidosis span a wide spectrum, from asymptomatic exposure or localized cutaneous infection to septic shock with multiorgan failure. Melioidosis usually occurs in residents of or travelers to disease-endemic areas in northern Australia and Southeast Asia; however, an increasing number of confirmed melioidosis cases are being reported from the Caribbean. We report a case of melioidosis acquired in Trinidad and Tobago. In February 2014, a 17-year-old male student was admitted to a tertiary care hospital in Vancouver, British Columbia, Canada, with catecholaminergic polymorphic ventricular tachycardia and electrical storm. He had a 9-month history of dry cough that was unresponsive to multiple and prolonged courses of treatment for community-acquired pneumonia. During the 6 months before his admission, the patient had hemoptysis and radiologic evidence of pneumonia that were treated with courses of cephalosporins without resolution of symptoms. Bronchoscopy and culture of lavage samples had revealed infection with Staphylococcus aureus and an organism most closely related to Actinomyces graevenitzii . The patient had no history indicative of risk factors for recurrent sinusitis or pneumonia (e.g., cystic fibrosis, chronic granulatomous disease, Job syndrome), and no risk factors for tuberculosis or infection with dimorphic fungi. He was up to date on his vaccinations and had no pets. He was born in Jamaica, had moved to Canada at age 4, and had not traveled anywhere other than Trinidad and Tobago, Canada, and England. He had traveled to visit family in Trinidad for 2 months during the rainy season in 2012, at which time he also visited Tobago. On day 5 of hospital admission, the patient became febrile (39.6°C), and an infectious diseases specialist was consulted. Examination revealed that the patient was clinically stable but emaciated at 45 kg. His oxygen saturation while breathing room air was 98%. Physical examination, including cardiorespiratory examination, was unremarkable. Laboratory results showed a normal hemoglobin concentration of 133 g/L; elevated leukocyte count of 22.8 × 109 cells/L; neutrophils 19.4 × 109 cells/L; normal platelet count of 295 × 109/L; and normal creatinine of 54 μmol/L. Test results for HIV-1 and blood cultures were negative. Computed tomography scan showed dilated bronchi and dense consolidation of the right and left lower lobes. Piperacillin/tazobactam was started for presumed hospital-acquired pneumonia. The patient underwent diagnostic bronchoscopy with bronchoalveolar lavage. Gram staining of specimens showed occasional gram-negative bacilli, and aerobic cultures grew gram-negative bacilli. Further testing with the Vitek 2 (bioMerieux, Laval, Quebec, Canada) (96%) and RapID NF (Oxoid, Nepean, Ontario, Canada) (99.9%) systems identified B. pseudomallei, but matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Vitek MS, bioMerieux) did not. Phenotypic confirmation was performed at the provincial public health and reference laboratory. Antimicrobial drug susceptibility testing performed by broth microdilution according to Clinical and Laboratory Standards Institute recommendations (1) and by Etest (bioMerieux) showed susceptibility to amoxicillin/clavulanic acid, ceftazidime, imipenem, doxycycline, and trimethoprim/sulfamethoxazole. The patient’s condition improved after 2 weeks of intravenous meropenem, and antimicrobial therapy was changed to oral trimethoprim/sulfamethoxazole. The B. pseudomallei isolate was sent to the Public Health Agency of Canada’s National Microbiology Laboratory for molecular typing. Query of 7 standard multilocus sequence typing loci (http://bpseudomallei.mlst.net/) identified the isolate as a novel multilocus sequence type. The sequence type (1,1,2,1,5,6,1) closely resembled that of B. pseudomallei previously isolated from the Caribbean (2). Although melioidosis was first described in the Caribbean in 1947 (3), most case reports of the disease in the area are from the past 2 decades. This case report suggests progression of the range of melioidosis to include Trinidad and Tobago. A recent study documented the presence of B. pseudomallei in soil samples and high seroprevalence rates among contacts of persons with melioidosis in Puerto Rico (4). If examined, this pattern of regional melioidosis endemicity may also be found on other Caribbean islands. Increased clinical awareness of and improved surveillance for B. pseudomallei infection may partly explain emergence. Nonetheless, underascertainment probably occurs in rural areas with limited access to advanced diagnostic support and in urban areas when B. pseudomallei infection is not suspected because of lack of travel to classic disease-endemic areas. Because B. pseudomallei is a Biosafety Level 3 agent, when infectious disease specialists consider melioidosis in their differential diagnoses, they should alert the microbiology laboratory to confirm species identification and ensure that staff use proper biosafety measures. A total of 19 cases of melioidosis acquired in the Caribbean have been reported (Table). Nine of these were travel related, suggesting that melioidoisis may be emerging as a travel health issue. Travelers with known risk factors for melioidosis, such as diabetes mellitus and chronic lung disease, should be informed of their increased infection risk. Physicians should include B. pseudomallei in the differential diagnosis of travelers with pneumonia or sepsis who are returning from the Caribbean, particularly when they have a history of travel during the rainy season, soil-contaminated wounds, or known risk factors for melioidosis. Table Published case reports of melioidosis from the Caribbean*

Multiple-locus variable-number tandem-repeat analysis of Francisella tularensis from Quebec, Canada.

Francisella tularensis is ubiquitous in the Northern Hemisphere. Yet, little is known about the disease and its ecology within Canada as few serological studies have shown exposure to the disease and fewer case studies have been reported. This report is the first to describe the molecular subtyping of F. tularensis isolates within eastern Canada using multiple‐locus variable‐number tandem‐repeat analysis. From 1998 to 2011, a total of 73 specimens were isolated from unique human and animal sources. As expected, F. tularensis subsp. tularensis AI and F. tularensis subsp. holarctica subtypes were observed, corresponding to the known geographical division within this species. The majority of human isolates (78%) and all animal (hare) isolates were of the more virulent, AI type. Half of the B isolates were isolated from patients living in a region of Quebec where muskrat densities are known to be high. A relatively high level of marker diversity was found, suggestive of multiple introductions of the organism to the region, or more likely ongoing endemicity. There was no evidence of ongoing outbreaks or transmission, and the bulk of cases were likely due to interaction between human activity and the environment (e.g. hunting/trapping activities).

ECO-EPIZOOTIOLOGIC STUDY OF FRANCISELLA TULARENSIS, THE AGENT OF TULAREMIA, IN QUEBEC WILDLIFE

Abstract In Canada, Francisella tularensis, the zoonotic bacterial agent of tularemia, affects mostly snowshoe hares (Lepus americanus), muskrats (Ondatra zibethicus), and beavers (Castor canadensis). Despite numerous studies, the ecologic cycle and natural reservoirs of F. tularensis are not clearly defined. We conducted a cross-sectional study to estimate the prevalence of F. tularensis in snowshoe hares, muskrats, and coyotes (Canis latrans) in four regions of Québec, Canada, and to describe the risk of infection in relation to host and environmental characteristics at three spatial scales. Between October 2012 and April 2013, trappers captured 345 snowshoe hares, 411 muskrats, and 385 coyotes. Blood samples were tested by microagglutination tests, and DNA extracts of liver, kidney, lung, and spleen of snowshoe hares and muskrats were tested by real-time PCR to detect past and active infection to F. tularensis, respectively. Individual host characteristics, including body condition, age, and sex, were evaluated as risk factors of infection, along with ecologic characteristics of the location of capture extracted from geographic databases. Prevalences of antibody to F. tularensis and 95% confidence intervals were 2.9% (1.4–5.1%) in coyotes, 0.6% (0.1–2.1%) in hares, and 0% (0.0–0.9%) in muskrats. Francisella tularensis DNA was not detected by real-time PCR in the pools of four organs from muskrats and hares, but F. tularensis type AI was detected during testing of the individual organs of two antibody-positive hares. Exact logistic regression analyses showed that age was a significant predictor of antibody detection in coyotes, as were the proportion of forest and the proportion of area considered as suitable habitat for hares in the environment around the location of capture of the coyotes. Our results suggest a terrestrial cycle of F. tularensis in the regions studied.

Evaluation of Oxford Nanopore’s MinION Sequencing Device for Microbial Whole Genome Sequencing Applications

The MinION sequencer (Oxford Nanopore Technologies) is a paradigm shifting device allowing rapid, real time long read sequencing of nucleic acids. Yet external benchmarking of this technologies’ capabilities has not been extensively reported, nor has thorough evaluation of its utility for field-based analysis with sub-optimal sample types been described. The aim of this study was to evaluate the capability of the MinION sequencer for bacterial genomic and metagenomic applications, with specific emphasis placed on the quality, yield, and accuracy of generated sequence data. Two independent laboratories at the National Microbiology Laboratory (Public Health Agency of Canada), sequenced a set of microbes in replicate, using the currently available flowcells, sequencing chemistries, and software available at the time of the experiment. Overall sequencing yield and quality improved through the course of this set of experiments. Sequencing alignment accuracy was high reaching 97% for all 2D experiments, though was slightly lower for 1D sequencing (94%). 1D sequencing provided much longer sequences than 2D. Both sequencing chemistries performed equally well in constructing genomic assemblies. There was evidence of barcode cross-over using both the native and PCR barcoding methods. Despite the sub-optimal nature of samples sequenced in the field, sequences attributable to B. anthracis the target organism used in this scenario, could none-the-less be detected. Together, this report showcases the rapid advancement in this technology and its utility in the context of genomic sequencing of microbial isolates of importance to public health.

Application of whole genome sequence analysis to the study of Mycobacterium tuberculosis in Nunavut, Canada

Canada has one of the lowest rates of tuberculosis (TB) in the world, however, among certain sub-populations, disease incidence rates approach those observed in sub-Saharan Africa, and other high incidence regions. In this study, we applied mycobacterial interspersed repetitive unit (MIRU) variable number of tandem repeat (VNTR) and whole genome sequencing (WGS) to the analysis of Mycobacterium tuberculosis isolates obtained from Northern communities in the territory of Nunavut. WGS was carried out using the Illumina MiSeq, with identified variants used to infer phylogenetic relationships and annotated to infer functional implications. Additionally, the sequencing data from these isolates were augmented with publically available WGS to evaluate data from the Nunavut outbreak in the broader Canadian context. In this study, isolates could be classified into four major clusters by MIRU-VNTR analysis. These could be further resolved into sub-clusters using WGS. No evidence for antimicrobial resistance, either genetic or phenotypic, was observed in this cohort. Among most subjects with multiple samples, reactivation/incomplete treatment likely contributed to recurrence. However, isolates from two subjects appeared more likely to have occurred via reinfection, based on the large number of genomic single nucleotide variants detected. Finally, although quite distinct from previously reported Canadian MTB strains, isolates obtained from Nunavut clustered most closely with a cohort of samples originating in the Nunavik region of Northern Quebec. This study demonstrates the benefit of using WGS for discriminatory analysis of MTB in Canada, especially in high incidence regions. It further emphasizes the importance of focusing epidemiological intervention efforts on interrupting transmission chains of endemic TB throughout Northern communities, rather than relying on strategies applied in regions where the majority of TB cases result from importation of foreign strains.