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.

Climate change and infectious diseases: What can we expect?

Global climate change, driven by anthropogenic greenhouse gas emissions, is being particularly felt in Canada, with warming generally greater than in the rest of the world. Continued warming will be accompanied by changes in precipitation, which will vary across the country and seasons, and by increasing climate variability and extreme weather events. Climate change will likely drive the emergence of infectious diseases in Canada by northward spread from the United States and introduction from elsewhere in the world via air and sea transport. Diseases endemic to Canada are also likely to re-emerge. This special issue describes key infectious disease risks associated with climate change. These include emergence of tick-borne diseases in addition to Lyme disease, the possible introduction of exotic mosquito-borne diseases such as malaria and dengue, more epidemics of Canada-endemic vector-borne diseases such as West Nile virus, and increased incidence of foodborne illnesses. Risk is likely to be compounded by an aging population affected by chronic diseases, which results in greater sensitivity to infectious diseases. Identifying emerging disease risks is essential to assess our vulnerability, and a starting point to identify where public health effort is required to reduce the vulnerability and exposure of the Canadian population.

Weather-based forecasting of mosquito-borne disease outbreaks in Canada.

Early warning systems to predict infectious disease outbreaks have been identified as a key adaptive response to climate change. Warming, climate variability and extreme weather events associated with climate change are expected to drive an increase in frequency and intensity of mosquito-borne disease (MBD) outbreaks globally. In Canada, this will mean an increased risk of endemic and emerging MBD outbreaks such as West Nile virus and other MBDs. The availability of timely information on the risk of impending MBD outbreaks has important public health implications, by allowing implementation of mosquito control measures and targeted communications regarding the need for increased personal protective measures-before an outbreak occurs. In Canada, both mechanistic and statistical weather-based models have been developed to predict West Nile virus outbreaks. These include models for different species of mosquitoes that transmit West Nile virus in different geographical areas of Canada. Although initial results have been promising, further validation and assessment of forecasting skill are needed before wide scale implementation. Weather-based forecasting for other emerging MBDs in Canada, such as Eastern equine encephalitis, may also be feasible.

Quebec's Multi-Party Observatory on Zoonoses and Adaptation to Climate Change.

Climate change has been linked with the establishment and geographical expansion of zoonotic diseases, an example of which is the well-documented increase in human cases of Lyme disease in Quebec, Canada. As temperatures continue to increase in Quebec, it is anticipated that several zoonotic diseases will be affected. In response to the growing zoonotic issues facing public health authorities, Quebec's Multi-Party Observatory on Zoonoses and Adaptation to Climate Change (Observatoire multipartite québécois sur les zoonoses et l'adaptation aux changements climatiques) (the Observatory) was founded in 2015 as part of the Quebec government's Climate Change Action Plan (Plan d'action 2013-2020 sur les changements climatiques). The Observatory was designed to bring together agencies involved in formulating public policy and experts from the disciplines of human health, animal health and environmental sciences, in a manner similar to the innovative "One World, One Health" approach. The Observatory provides a platform for knowledge sharing and consensus building among representatives of public policy decision makers and scientists. Its main objectives are to anticipate and prioritize potential issues associated with zoonotic diseases in Quebec, in order to support applicable risk management and climate change adaptation. This article describes what the Observatory is, what it does and outlines its plans for the future.

What is the real number of Lyme disease cases in Canada?

BACKGROUND: Lyme disease is emerging in Canada due to expansion of the range of the tick vector Ixodes scapularis from the United States. National surveillance for human Lyme disease cases began in Canada in 2009. Reported numbers of cases increased from 144 cases in 2009 to 2025 in 2017. It has been claimed that few (< 10%) Lyme disease cases are reported associated with i) supposed under-diagnosis resulting from perceived inadequacies of serological testing for Lyme disease, ii) expectation that incidence in Canadian provinces and neighbouring US states should be similar, and iii) analysis of serological responses of dogs to the agent of Lyme disease, Borrelia burgdorferi. We argue that performance of serological testing for Lyme disease is well studied, and variations in test performance at different disease stages are accounted for in clinical diagnosis of Lyme disease, and in surveillance case definitions. Extensive surveillance for tick vectors has taken place in Canada providing a clear picture of the emergence of risk in the Canadian environment. This surveillance shows that the geographic scope of I. scapularis populations and Lyme disease risk is limited but increasing in Canada. The reported incidence of Lyme disease in Canada is consistent with this pattern of environmental risk, and the differences in Lyme disease incidence between US states and neighbouring Canadian provinces are consistent with geographic differences in environmental risk. Data on serological responses in dogs from Canada and the US are consistent with known differences in environmental risk, and in numbers of reported Lyme disease cases, between the US and Canada. CONCLUSION: The high level of consistency in data from human case and tick surveillance, and data on serological responses in dogs, suggests that a high degree of under-reporting in Canada is unlikely. We speculate that approximately one third of cases are reported in regions of emergence of Lyme disease, although prospective studies are needed to fully quantify under-reporting. In the meantime, surveillance continues to identify and track the ongoing emergence of Lyme disease, and the risk to the public, in Canada.

Risk factors associated with seropositivity to California serogroup viruses in humans and pet dogs, Quebec, Canada.

Jamestown Canyon and snowshoe hare viruses are two emerging human pathogens associated with cases of neuroinvasive disease in North America. This study aimed to identify environmental and individual risk factors for seropositivity to these arboviruses in humans and pet dogs from Québec, Canada, 2012-2014. In humans, areas with moderate densities of white-tailed deer (Odocoileus virginianus) were associated with higher odds of seropositivity compared with areas with low densities of white-tailed deer (OR 2.50, P = 0.009) and odds of seropositivity were higher in males than in females (OR 2.03, P = 0.016). Among humans reporting more than 10 mosquito bites weekly, the odds of being seropositive were 4.44 times higher (P = 0.004) for people living in hardwood forested areas. Exposure to areas with coniferous forests was identified as the main environmental risk factor for seroconversion in dogs (OR 2.39, P = 0.04). These findings may help target further public health research, diagnostic and surveillance efforts in Canada.

Emerging arboviruses in Quebec, Canada: assessing public health risk by serology in humans, horses and pet dogs.

Periodic outbreaks of West Nile virus (WNV), Eastern equine encephalitis virus (EEEV) and to a lesser extent, California serogroup viruses (CSGV), have been reported in parts of Canada in the last decade. This study was designed to provide a broad assessment of arboviral activity in Quebec, Canada, by conducting serological surveys for these arboviruses in 196 horses, 1442 dogs and 485 humans. Sera were screened by a competitive enzyme linked immunosorbent assay and positive samples confirmed by plaque reduction neutralisation tests. The percentage of seropositive samples was 83·7%, 16·5%, 7·1% in horses, 18·8%, 0·6%, 0% in humans, 11·7%, 3·1%, 0% in adult dogs and 2·9%, 0·3%, 0% in juvenile dogs for CSGV, WNV and EEEV, respectively. Serological results in horses and dogs appeared to provide a meaningful assessment of risk to public health posed by multiple arboviruses.

Characterizing environmental risk factors for West Nile virus in Quebec, Canada, using clinical data in humans and serology in pet dogs.

The identification of specific environments sustaining emerging arbovirus amplification and transmission to humans is a key component of public health intervention planning. This study aimed at identifying environmental factors associated with West Nile virus (WNV) infections in southern Quebec, Canada, by modelling and jointly interpreting aggregated clinical data in humans and serological data in pet dogs. Environmental risk factors were estimated in humans by negative binomial regression based on a dataset of 191 human WNV clinical cases reported in the study area between 2011 and 2014. Risk factors for infection in dogs were evaluated by logistic and negative binomial models based on a dataset including WNV serological results from 1442 dogs sampled from the same geographical area in 2013. Forested lands were identified as low-risk environments in humans. Agricultural lands represented higher risk environments for dogs. Environments identified as impacting risk in the current study were somewhat different from those identified in other studies conducted in north-eastern USA, which reported higher risk in suburban environments. In the context of the current study, combining human and animal data allowed a more comprehensive and possibly a more accurate view of environmental WNV risk factors to be obtained than by studying aggregated human data alone.

Evaluating the submission of digital images as a method of surveillance for Ixodes scapularis ticks.

Widespread access to the internet is offering new possibilities for data collection in surveillance. We explore, in this study, the possibility of using an electronic tool to monitor occurrence of the tick vector of Lyme disease, Ixodes scapularis. The study aimed to compare the capacity for ticks to be identified in web-based submissions of digital images/photographs, to the traditional specimen-based identification method used by the provincial public health laboratory in Quebec, Canada. Forty-one veterinary clinics participated in the study by submitting digital images of ticks collected from pets via a website for image-based identification by an entomologist. The tick specimens were then sent to the provincial public health laboratory to be identified by the 'gold standard' method using a microscope. Of the images submitted online, 74·3% (284/382) were considered of high-enough quality to allow identification. The laboratory identified 382 tick specimens from seven different species, with I. scapularis representing 76% of the total submissions. Of the 284 ticks suitable for image-based species identification, 276 (97·2%) were correctly identified (Kappa statistic of 0·92, Z = 15·46, P < 0·001). This study demonstrates that image-based tick identification may be an accurate and useful method of detecting ticks for surveillance when images are of suitable quality.

Surveillance for Lyme disease in Canada: 2009-2015.

OBJECTIVE: To summarize seven years of surveillance data for Lyme disease cases reported in Canada from 2009 to 2015. METHODS: We describe the incidence over time, seasonal and geographic distribution, demographic and clinical characteristics of reported Lyme disease cases. Logistic regression was used to explore differences between age groups, sex and year to better understand potential demographic risk factors for the occurrence of Lyme disease. RESULTS: The number of reported Lyme disease cases increased more than six-fold, from 144 in 2009 to 917 in 2015, mainly due to an increase in infections acquired in Canada. Most locally acquired cases were reported between May and November. An increase in incidence of Lyme disease was observed in provinces from Manitoba eastwards. This is consistent with our knowledge of range expansion of the tick vectors in this region. In the western provinces the incidence has remained low and stable. All cases reported by Alberta, Saskatchewan and Newfoundland and Labrador were acquired outside of the province, either elsewhere in Canada or abroad. There was a bimodal distribution for Lyme disease by age with peaks at 5-9 and 45-74 years of age. The most common presenting symptom was a single erythema migrans rash (74.2%) and arthritis (35.7%). Variations in the frequency of reported clinical manifestations were observed among age groups and years of study. CONCLUSION: Lyme disease incidence continues to increase in Canada as does the geographic range of ticks that carry the Lyme disease bacteria. Ongoing surveillance, preventive strategies as well as early disease recognition and treatment will continue to minimize the impact of Lyme disease in Canada.

Emerging infectious diseases: prediction and detection.

Emerging infectious diseases (EIDs), including West Nile virus, severe acute respiratory syndrome (SARS) and Lyme disease, have had a direct effect within Canada, while many more EIDs such as Zika, chikungunya and Ebola are a threat to Canadians while travelling. Over 75% of EIDs affecting humans are, or were originally, zoonoses (infectious diseases transmitted from animals to humans). There are two main ways by which infectious diseases can emerge: by changes in their geographical ranges and by adaptive emergence, a genetic change in a microorganism that results in it becoming capable of invading a new niche, often by jumping to a new host species such as humans. Diseases can appear to emerge simply because we become capable of detecting and diagnosing them. Management of EID events is a key role of public health globally and a considerable challenge for clinical care. Increasingly, emphasis is being placed on predicting EID occurrence to "get ahead of the curve" - that is, allowing health systems to be poised to respond to them, and public health to be ready to prevent them. Predictive models estimate where and when EIDs may occur and the levels of risk they pose. Evaluation of the internal and external drivers that trigger emergence events is increasingly considered in predicting EID events. Currently, global changes are driving increasing occurrence of EIDs, but our capacity to prevent and deal with them is also increasing. Web-based scanning and analysis methods are increasingly allowing us to detect EID outbreaks, modern genomics and bioinformatics are increasing our ability to identify their genetic and geographical origins, while developments in geomatics and earth observation will enable more real-time tracking of outbreaks. EIDs will, however, remain a key, global public health challenge in a globalized world where demographic, climatic, and other environmental changes are altering the interactions between hosts and pathogen in ways that increase spillover from animals to humans and global spread.

Characterizing areas of potential human exposure to eastern equine encephalitis virus using serological and clinical data from horses.

Eastern equine encephalitis (EEE) is a rare but severe emerging vector-borne disease affecting human and animal populations in the northeastern United States where it is endemic. Key knowledge gaps remain about the epidemiology of EEE virus (EEEV) in areas where its emergence has more recently been reported. In Eastern Canada, viral activity has been recorded in mosquitoes and horses throughout the 2000s but cases of EEEV in humans have not been reported so far. This study was designed to provide an assessment of possible EEEV human exposure by modelling environmental risk factors for EEEV in horses, identifying high-risk environments and mapping risk in the province of Quebec, Canada. According to logistic models, being located near wooded swamps was a risk factor for seropositivity or disease in horses [odds ratio (OR) 4·15, 95% confidence interval (CI) 1·16-14·8) whereas being located on agricultural lands was identified as protective (OR 0·75, 95% CI 0·62-0·92). A better understanding of the environmental risk of exposure to EEEV in Canada provides veterinary and public health officials with enhanced means to more effectively monitor the emergence of this public health risk and design targeted surveillance and preventive measures.

Response to letter regarding article "How far north are migrant birds transporting the tick Ixodes scapularis in Canada? Insights from stable hydrogen isotope analyses of feathers".

In this letter we respond to correspondence and clarify the intent and importance of our article.

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.

How far north are migrant birds transporting the tick Ixodes scapularis in Canada? Insights from stable hydrogen isotope analyses of feathers.

Lyme disease is emerging in Canada because of northward range expansion of the tick vector Ixodes scapularis. It is hypothesised that I. scapularis feeding on passerine birds migrating north in spring are important in founding new I. scapularis populations leading to northward range expansion. However, there are no studies on how far north I. scapularis may be carried, only inferences from passive tick surveillance. We used stable hydrogen isotope (δ(2)H) analysis of rectrices collected from northward migrating, I. scapularis-carrying, passerine birds captured in Canada to estimate how far north I. scapularis may be carried. Rectrices are usually grown close to breeding sites and their δ(2)H values reflect those in the environment, which vary strongly with latitude in North America. Passerines usually return to their breeding or natal sites so δ(2)H values of rectrices of northward migrating birds can identify the likely latitudinal bands of their intended destinations. In 2006 we analysed δ(2)H from rectrices of 73 I. scapularis-carrying birds captured at five migration monitoring stations, mainly from southern Ontario. Values of δ(2)H ranged from -33 to -124‰, suggesting 19/71 (26.7%) birds were destined for latitude band B (the most southerly part of Ontario), 40/71 (56.3%) birds were destined for band C (which extends from southern Ontario, Quebec and the Maritimes to southern James Bay) and 12/71 (16.9%) birds were destined for bands D and E (which extend from northern Ontario and Quebec into the southern Canadian Arctic). This indicates that many I. scapularis-carrying migratory birds in spring have destinations far north in Canada, including some farther north than the current region of climatic suitability for I. scapularis. These findings support the hypothesis that I. scapularis may continue to be spread north by spring migrating passerines. Some thrush species may be particularly implicated in far northward dispersion of I. scapularis.

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.

Travel-related chikungunya cases in Canada, 2014.

Since the spring of 2014, there has been a large increase in travel-related chikungunya cases diagnosed in Canada. As of December 9, 2014, 320 confirmed and 159 probable cases have been diagnosed in Canada, with the majority of provinces identifying at least one imported case. This surge in Canadian infections has been associated with the incursion of chikungunya virus into the Caribbean and the expansion of the virus in the Americas. Ongoing outbreaks in the Asia-Pacific region have also contributed to imported cases among Canadian travellers. Heightened awareness of chikungunya among clinicians is key to diagnosis. This highlights the need to ask for a travel history from anyone who presents with fever or recent onset of polyarthralgia, and to consider testing by provincial laboratories and the National Microbiology Laboratory for chikungunya virus and other diseases as indicated. Also essential is continued communication with travellers regarding the use of preventative measures to decrease the risk of exposure to mosquitoes when travelling to endemic areas.