Mosquito Identification From Bulk Samples Using DNA Metabarcoding: a Protocol to Support Mosquito-Borne Disease Surveillance in Canada.

Approximately 80 species of mosquitoes (Diptera: Culicidae) have been documented in Canada. Exotic species such as Aedes albopictus (Skuse) (Diptera: Culicidae) are becoming established. Recently occurring endemic mosquito-borne diseases (MBD) in Canada including West-Nile virus (WNV) and Eastern Equine Encephalitis (EEE) are having significant public health impacts. Here we explore the use of DNA metabarcoding to identify mosquitoes from CDC light-trap collections from two locations in eastern Canada. Two primer pairs (BF2-BR2 and F230) were used to amplify regions of the cytochrome c oxidase subunit I (CO1) gene. High throughput sequencing was conducted using an Illumina MiSeq platform and GenBank-based species identification was applied using a QIIME 1.9 bioinformatics pipeline. From a site in southeastern Ontario, Canada, 26 CDC light trap collections of 72 to >300 individual mosquitoes were used to explore the capacity of DNA metabarcoding to identify and quantify captured mosquitoes. The DNA metabarcoding method identified 33 species overall while 24 species were identified by key. Using replicates from each trap, the dried biomass needed to identify the majority of species was determined to be 76 mg (equivalent to approximately 72 mosquitoes), and at least two replicates from the dried biomass would be needed to reliably detect the majority of species in collections of 144-215 mosquitoes and three replicates would be advised for collections with >215 mosquitoes. This study supports the use of DNA metabarcoding as a mosquito surveillance tool in Canada which can help identify the emergence of new mosquito-borne disease potential threats.

Evolutionary aspects of emerging Lyme disease in Canada.

In North America, Lyme disease (LD) is a tick-borne zoonosis caused by the spirochete bacterium Borrelia burgdorferi sensu stricto, which is maintained by wildlife. Tick vectors and bacteria are currently spreading into Canada and causing increasing numbers of cases of LD in humans and raising a pressing need for public health responses. There is no vaccine, and LD prevention depends on knowing who is at risk and informing them how to protect themselves from infection. Recently, it was found in the United States that some strains of B. burgdorferi sensu stricto cause severe disease, whereas others cause mild, self-limiting disease. While many strains occurring in the United States also occur in Canada, strains in some parts of Canada are different from those in the United States. We therefore recognize a need to identify which strains specific to Canada can cause severe disease and to characterize their geographic distribution to determine which Canadians are particularly at risk. In this review, we summarize the history of emergence of LD in North America, our current knowledge of B. burgdorferi sensu stricto diversity, its intriguing origins in the ecology and evolution of the bacterium, and its importance for the epidemiology and clinical and laboratory diagnosis of LD. We propose methods for investigating associations between B. burgdorferi sensu stricto diversity, ecology, and pathogenicity and for developing predictive tools to guide public health interventions. We also highlight the emergence of B. burgdorferi sensu stricto in Canada as a unique opportunity for exploring the evolutionary aspects of tick-borne pathogen emergence.

Complex population structure of Borrelia burgdorferi in southeastern and south central Canada as revealed by phylogeographic analysis.

Lyme disease, caused by the bacterium Borrelia burgdorferi sensu stricto, is an emerging zoonotic disease in Canada and is vectored by the blacklegged tick, Ixodes scapularis. Here we used Bayesian analyses of sequence types (STs), determined by multilocus sequence typing (MLST), to investigate the phylogeography of B. burgdorferi populations in southern Canada and the United States by analyzing MLST data from 564 B. burgdorferi-positive samples collected during surveillance. A total of 107 Canadian samples from field sites were characterized as part of this study, and these data were combined with existing MLST data for samples from the United States and Canada. Only 17% of STs were common between both countries, while 49% occurred only in the United States, and 34% occurred only in Canada. However, STs in southeastern Ontario and southwestern Quebec were typically identical to those in the northeastern United States, suggesting a recent introduction into this region from the United States. In contrast, STs in other locations in Canada (the Maritimes; Long Point, Ontario; and southeastern Manitoba) were frequently unique to those locations but were putative descendants of STs previously found in the United States. The picture in Canada is consistent with relatively recent introductions from multiple refugial populations in the United States. These data thus point to a geographic pattern of populations of B. burgdorferi in North America that may be more complex than simply comprising northeastern, midwestern, and Californian groups. We speculate that this reflects the complex ecology and spatial distribution of key reservoir hosts.

Investigation of the population structure of the tick vector of Lyme disease Ixodes scapularis (Acari: Ixodidae) in Canada using mitochondrial cytochrome C oxidase subunit I gene sequences.

Genotyping of Ixodes scapularis Say (Acari: Ixodidae) ticks could enhance understanding of the occurrence and genotypes of I. scapularis-borne pathogens. We investigated the utility of mitochondrial (mt) Cytochrome C Oxidase subunit I gene (cox1) sequences as a tool for understanding the population structure of I. scapularis collected in Canada, where we also investigated the geographic occurrence of different cox1 haplotypes. Sequences obtained from 414 ticks were one of 55 unique haplotypes, most of which grouped into one of six clades. Demographic analysis suggested that cox1 sequences have haplotype and nucleotide diversity comparable to other mt genes. All haplotypes were connected in a single minimum spanning network tree. Despite low fixation index values there were significant differences in the frequency of occurrence of haplotypes of different clades among four geographic regions: 1) Alberta to western Ontario, 2) eastern Ontario, 3) Quebec, and 4) Atlantic Provinces; suggesting that cox1 sequences could reveal population structure differences between I. scapularis in geographically separated populations of northeastern and midwestern North America. Spatial clusters of ticks of the same haplotype identified in regions of southern Quebec and southern Ontario where I. scapularis is invading were consistent with population bottlenecks associated with founder events. These findings suggest that cox1 sequences are useful for the study of I. scapularis population structure, are of sufficient diversity that spatial analyses of haplotypes can be used to identify where I. scapularis is emerging in southern Canada, and may be useful for exploring differences between northeastern and midwestern populations of I. scapularis.

Characterization of the third most common genotype of hepatitis C virus in France.

We have previously identified the two major hepatitis C virus (HCV) genotypes prevalent in France (type I and type II). We report here the identification and partial characterization of a new HCV genotype with a highly divergent 5' noncoding (NC) region and a structural protein region. This genotype showed only 93-94% sequence identity with either type I or type II HCV in the 5' NC region. Sequence analysis of the structural protein region revealed extremely low sequence homology with all the four major HCV genotypes: 86-89% for the core protein and 56-69% for the envelope protein. However, further analysis revealed that this new genotype was very similar to the genotype 3a described most recently. Screening of 150 clinical samples with genotype-specific oligoprobes revealed prevalence of this genotype in 12% of the French samples with a significant association with drug addiction and a good response to interferon therapy. These results may have implications for the diagnosis of HCV infection and the design of HCV vaccines.

Hepatitis C virus genotypes in France: comparison of clinical features of patients infected with HCV type I and type II.

Two major hepatitis C virus genotypes, F1 and F2, corresponding to hepatitis C virus type I and type II respectively, were found in France. To investigate the correlation between infection with these genotypes (F1 and F2) and clinical features of patients, serum samples proven to be hepatitis C virus positive by polymerase chain reaction amplification on 5' non-coding region were further amplified in the NS3 region with nested polymerase chain reaction. The NS3-polymerase chain reaction products were Southern blotted and hybridized with specific probes to identify the genotype of hepatitis C virus. Of 70 samples 64 were NS3-polymerase chain reaction positive. Twenty-eight (40%) samples were hepatitis C virus type I (F1) and 34 (49%) were hepatitis C virus type II (F2), while one sample (HB) hybridized with both probes and another (HN) hybridized with neither. Some samples were sequenced, with results consistent with those of hybridization. The HB sample was related more to hepatitis C virus type II than to type I and the HN sample was divergent from both type I and type II genotypes. Clinical profiles of patients infected with hepatitis C virus type I and type II were compared. Type I infected patients were younger (p < 0.01) and more often male (p < 0.05) than those of the type II group. Nine of 28 patients in the type I infected group had a history of drug abuse, whereas none did in the type II group. Five of 22 (23%) type I infected patients and 19 of 32 (59%) type II infected patients had cirrhosis (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)