Comparison of the supercooling points of questing Dermacentor variabilis adults in two populations on the Canadian prairies and implications for overwinter survival.

A comparison was made of the supercooling points (SCPs) of questing Dermacentor variabilis adults from two populations located ca. 800 km apart on the Canadian prairies. This is also the first study to examine whether there are seasonal fluctuations in the SCP of questing D. variabilis adults. The SCPs of adult ticks from Lizard Lake Community Pasture, a recently established population in west-central Saskatchewan, varied over spring and summer, with the median SCP warming over time. In addition, the SCPs of ticks from Lizard Lake Community Pasture were significantly higher than those of adult ticks collected from Sandy Hook in Manitoba, a population that has been established for decades. The off-host survival of adults from Sandy Hook between summer and spring has been shown previously to be significantly greater than that of adults from Lizard Lake Community Pasture. The findings of the present study suggest that there may be geographical variation in the SCPs of D. variabilis adults which may be associated with differences in overwinter survival. The relatively low SCPs of questing D. variabilis adults, and the ability of some adults to survive off-host during winter, may be factors contributing to the range expansion of this tick species in Canada.

Effect of snow cover on the off-host survival of Dermacentor variabilis (Acari: Ixodidae) larvae.

The geographical range of the American dog tick, Dermacentor variabilis (Say), in Canada continues to expand northwards into areas with colder winter temperatures. Understanding what influences the off-host survival of D. variabilis over winter is important for predicting the northern distributional limit of this species. A field experiment was conducted to determine the effect of snow cover on the off-host survival of unfed D. variabilis larvae from a population situated near the northern distributional limit in Saskatchewan, Canada. Ticks were placed in tubes within 1 of 9 field enclosures (tickaria) in early fall. The 9 tickaria were divided into 3 equal treatment groups: no snow cover, natural snow cover (maximum depth of 25 cm) and extra snow cover (maximum depth of 32 cm). Tick survival was measured in mid-winter and in early spring (i.e., ~17 and ~26 wk after start of experiment). The results showed that snow cover had a significant impact on the relative humidity, but not temperature, in the tickaria. Larvae in tickaria with no snow cover had a higher mortality rate (27%) than larvae in tickaria with natural snow cover (6%) or extra snow cover (4%). Although snow cover enhanced off-host survival, many individuals were able to survive in sub-zero temperatures under leaf litter in the absence of continuous snow cover for a period of 108 days. These findings have implications for the construction of predictive models to determine the northern distributional limits of D. variabilis, a species that is an important vector of several pathogens.

Differences in the reproductive output and larval survival of Rocky Mountain wood ticks (Dermacentor andersoni) and American dog ticks (Dermacentor variabilis) from prairie populations near their northern distributional limits in western Canada.

The effects of temperature and relative humidity (RH) on female reproductive output, egg development and larval survival were determined for Rocky Mountain wood ticks (Dermacentor andersoni) from a prairie population (Chin Lakes, Alberta, Canada) near the northern distribution limit of this species. The responses of D. andersoni eggs and unfed larvae to different temperature (25 or 32 °C) and RH (35, 55, 75, 85 or 95%) regimes were compared to our previously published data (Diyes et al. 2021) for a northern prairie population of American dog ticks (Dermacentor variabilis). Oviposition by D. andersoni females took 21-30 days at 25 °C and 95% RH compared to 10-21 days for D. variabilis. The number of eggs laid by female ticks was strongly dependent on their engorgement weight, and D. andersoni females produced more eggs than D. variabilis females of an equivalent body weight. Eggs of D. andersoni took less time to develop at 32 °C than 25 °C with ≥ 85% RH, and hatched faster than those of D. variabilis. Larval survival times declined as temperature increased and RH decreased, but D. andersoni survived longer at 32 °C and ≤ 75% RH than D. variabilis. The interspecific differences in responses to the same temperature and humidity regimes indicate that D. andersoni is xerophilic, whereas D. variabilis is hydrophilic. Hence, 'prairie' populations of the Rocky Mountain wood tick occur in the drier grassland ecoregions but are absent in Aspen Parklands Ecoregion which is located to the north and east of the distributional range of D. andersoni.

Detection of Ehrlichia minasensis in a female winter tick (Dermacentor albipictus) feeding on a moose (Alces alces) in western Canada.

Ehrlichia minasensis, a pathogen of cattle in Brazil, was detected in genomic DNA of a female Dermacentor albipictus feeding on a moose (Alces alces) in west-central Saskatchewan, Canada. This bacterium has not previously been confirmed in ticks in Canada. It is unknown whether moose are reservoirs for E. minasensis.

Off-host survival of Dermacentor variabilis (Acari: Ixodidae) adults near their northern distributional limit in Saskatchewan, Canada.

Dermacentor variabilis (Say) is expanding its range northwards in Saskatchewan and Manitoba (Canada). Knowledge of the relative ability of different life cycle stages to survive off-host over winter is important for assessing the potential for further range expansion and risk of pathogen transmission. In the present study, the off-host survival of D. variabilis adults was determined in outdoor terraria within a field plot close to Lizard Lake Community Pasture (LLCP) situated near the northern distributional limit in Saskatchewan. Only a small proportion (7%) of adult ticks in this relatively recently established population survived from mid-summer to late winter. Off-host survival had declined further to <4% by mid-spring. A significantly greater proportion of female ticks (6%) survived from mid-summer to mid-spring than did male ticks (1%). The supercooling points (SCPs), the temperature below 0 °C when body fluids spontaneously freeze, of the 17 ticks that survived through to mid-spring did not differ significantly from the SCPs of questing ticks in the previous summer, suggesting adult D. variabilis do not undergo cold hardening during winter diapause. The off-host survival of D. variabilis adults near LLCP was also much lower than that previously reported for adults from Sandy Hook in Manitoba, a population that has been established for decades. This finding suggests that there is geographical variation in the ability of D. variabilis in southern Canada to survive from mid-summer to the subsequent spring. However, the ecological and genetic factors influencing the survival of northern populations of D. variabilis requires further investigation.

Anaplasma bovis-Like Infections in Humans, United States, 2015-2017.

We detected the DNA of an Anaplasma bovis-like bacterium in blood specimens from 4 patients from the United States with suspected tickborne illnesses. Initial molecular characterization of this novel agent reveals identity to A. bovis-like bacteria detected in Dermacentor variabilis ticks collected from multiple US states.

Traceback of the Psoroptes outbreak in British Columbian bighorn sheep (Ovis Canadensis).

Psoroptes are a non-burrowing, ectoparasitic, mange-causing mite that has been documented in American bighorn sheep populations throughout the 19th and 20th centuries; however, it was not seen on Canadian bighorn sheep until 2006. The aim of this study was to determine the potential source of the Psoroptes outbreak in Canadian bighorn sheep. Morphological and molecular analyses were used to compare mites recovered from outbreak-associated bighorn sheep, pet rabbits in Canada, and on historically infested bighorn sheep in the USA. The results revealed that Psoroptes acquired from the Canadian and outbreak-associated American bighorn sheep were morphologically more similar to those collected from rabbits than mites on historically infested bighorn sheep. Outer opisthosomal setae lengths measured an average of 81.7 µm (±7.7 µm) in outbreak associated bighorn mites, 88.9 µm (±12.0 µm) in rabbit mites and 151.2 µm (±16.6 µm) in historically infested bighorn mites. The opisthosomal lobe morphology of bighorn mites in the outbreak herds was also more similar to that of rabbit mites, previously described as P. cuniculi, than historically infested bighorn mites, which match previous descriptions of P. ovis. This finding was supported by DNA sequence data of the mitochondrial cytochrome B gene. This is the first report of Psoroptes of the rabbit ecotype on bighorn sheep. The morphological and molecular data therefore support the hypothesis that the source of Psoroptes outbreak in Canadian bighorn sheep represented a disease spillover event from rabbits rather than transmission from infested American bighorn sheep populations.

Distribution of Dermacentor andersoni (Acari: Ixodidae) in Grassland Regions of Alberta, Canada.

The geographic distribution of the Rocky Mountain wood tick, Dermacentor andersoni Stiles, was determined in Alberta, Canada, by drag sampling at 86 and 89 sites during 2011 and 2012, respectively. Tick density and prevalence varied between years, averaging (range) 1.0 (0-26.2) and 5.9 (0-110) ticks/1,000 m2 in 2011 and 2012, respectively. Ticks were detected at 24.4% and 42.7% of the sites sampled in each respective year. Tick density and presence declined in a northerly direction to 51.6°N and in a westerly direction to ca. 113°W, except for a small area of high density at the edge of the Rocky Mountains in the southeastern portion of the province. Ticks were most abundant in the Dry Mixedgrass and Montane natural subregions and in areas with Brown Chernozemic, Regosol, and Solodized Solonetzic great soil groups. A logistic regression model indicated that tick presence was increased in the Dry Mixedgrass natural subregion and in regions with greater temperatures during the previous summer and normal winter precipitation but was reduced in areas with Dark Brown Chernozemic soils. The model will be useful for predicting tick presence and the associated risk of tick-borne diseases in the province.

Molecular Differentiation of Four Species of Oropsylla (Siphonaptera: Ceratophyllidae) Using PCR-Based Single Strand Conformation Polymorphism Analyses and DNA Sequencing.

It is often difficult to distinguish morphologically between closely related species of fleas (Siphonaptera). Morphological identification of fleas often requires microscopic examination of internal structures in specimens cleared using caustic solutions. This process degrades DNA and/or inhibits DNA extraction from specimens, which limits molecular-based studies on individual fleas and their microbiomes. Our objective was to distinguish between Oropsylla rupestris (Jordan), Oropsylla tuberculata (Baker), Oropsylla bruneri (Baker), and Oropsylla labis (Jordan & Rothschild) (Ceratophyllidae) using PCR-based single strand conformation polymorphism (SSCP) analyses and DNA sequencing. A 446 bp region of the nuclear 28S ribosomal RNA (rRNA) gene was used as the genetic marker. The results obtained for 36 reference specimens (i.e., fleas that were morphologically identified to species) revealed no intraspecific variation in DNA sequence, whereas the DNA sequences of the four species of Oropsylla differed from one another at two to six nucleotide positions. Each flea species also had a unique SSCP banding pattern. SSCP analyses were then used to identify another 84 fleas that had not been identified morphologically. DNA sequencing data confirmed the species identity of fleas subjected to SSCP. This demonstrates that PCR-SSCP combined with DNA sequencing of the 28S rRNA gene is a very effective approach for the delineation of four closely related species of flea.

Reproductive output and larval survival of American dog ticks (Dermacentor variabilis) from a population at the northern distributional limit.

Female reproductive output and larval survival were determined for American dog ticks, Dermacentor variabilis (Say), from a recently established population near the northern distributional limit in Saskatchewan (Canada). Oviposition took 10-21 days at 25 °C and 95% relative humidity (RH). Temperature and relative humidity had a marked effect on egg development time and larval survival. Unfed larvae survived more than 100 days at 32 °C (with 95% RH) and 25 and 5 °C (with ≥ 85% RH). However, survival times declined markedly at lower relative humidities. In addition, 95% of the larvae placed in field enclosures survived for 140 days over winter during which they were exposed to sub-zero temperatures and 95-100% RH, while covered with snow. The median survival times (LT50) of unfed larvae submerged underwater was 68 days. These results show that D. variabilis larvae in populations near the periphery of the northern distributional limit are adapted to cope with sub-zero temperatures in winter, and can survive in the temporary pools of water created by the spring snow melt.

Supercooling Points of Adult Dermacentor variabilis (Acari: Ixodidae) From a Population Near the Northern Distribution Limit.

The northern distributional limit of Dermacentor variabilis Say, the American dog tick, is expanding in Saskatchewan and Manitoba (western Canada). The ability of D. variabilis to continue to expand its range northwards will depend upon the ability of individuals within populations at the species distributional edge to withstand very low temperatures during winter. One component of cold hardiness is the supercooling point (SCP), the temperature below 0°C at which an individual freezes. In this study, the SCP was determined for 94 questing D. variabilis adults (44 females and 50 males) from an established population near Blackstrap Provincial Park in Saskatchewan. SCP values ranged from -18.2 to -6.7°C, with a median of -13.3°C. This suggests that host-seeking D. variabilis adults differ in their ability to survive exposure to subzero temperatures, for at least a short period of time, without freezing. The distribution of SCPs was bimodal, but there was no significant difference in SCP values between female and male ticks, and no relationship between SCP and tick body weight. It remains to be determined what factors contribute to the variation in SCP values among questing D. variabilis adults.

Molecular detection and characterization of Hepatozoon canis in stray dogs from Cuba.

Canine hepatozoonosis caused by Hepatozoon canis is a worldwide distributed tick-borne disease of domestic and wild canids that is transmitted by ingestion of Rhipicephalus sanguineus sensu lato (s.l.) ticks. The present study was aimed to determine the prevalence of Hepatozoon infections in 80 stray dogs from Havana Province in Cuba, and to confirm the species identity and phylogenetic relationships of the causative agent. Samples were screened by microscopical examination of thin blood smears for the presence of Hepatozoon spp. gamonts and by genus-specific SYBR green-based real-time PCR assay targeting the 18S rRNA gene. Direct microscopy examination revealed Hepatozoon gamonts in the peripheral blood of 8 dogs (10.0%; 95% CI: 4.80-18.0%), while 38 animals (47.5%; 95% CI: 36.8-58.4%) were PCR-positive, including all microscopically positive dogs. Hence, the agreement between the two detection methods was 'poor' (κ = 0.20). Hematological parameters did not differ significantly between PCR-positive and PCR-negative dogs (p > 0.05). The DNA sequences of the 18S rRNA gene of the Hepatozoon spp. from Cuban dogs showed a nucleotide identity >99% with those of 18S rRNA sequences of Hepatozoon canis isolates from Czech Republic, Brazil and Spain. Phylogenetic analysis revealed that obtained sequences clustered within the Hepatozoon canis clade, different from the Hepatozoon felis or Hepatozoon americanum clades. The present study represents the first molecular characterization of Hepatozoon canis in stray dogs within Cuba.

Molecular identification of Stylops advarians (Strepsiptera: Stylopidae) in western Canada.

Strepsiptera are an enigmatic order of insects with extreme sexual dimorphism which makes it difficult to "match-up" free-living adult males with parasitic conspecific females of the Stylopidia, and free-living females of the Mengenillidae using morphological characters. Species identification is further complicated for the Stylopidia because adult females are endoparasitic and neotenic. Therefore, we used DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) to confirm the species identity of adult strepsipterans that were morphologically identified as Stylops advarians. These specimens, collected from Saskatoon (Saskatchewan, Canada), included one adult male, and eight females, the latter of which had been collected from solitary bees (Andrena milwaukeensis). Also included in the analyses were three pools of first-instar larvae that had emerged from three of the females. The results of the molecular analyses revealed that all specimens had an identical cox1 sequence, and belonged to a clade, with total statistical support (bootstrap value of 100%), that contained specimens of S. advarians from New York and Maine (USA). Hence, the results were consistent with the morphological identification of S. advarians. This study demonstrates the usefulness of a molecular approach for the identification of endoparasitic adult female and larval strepsipterans, life cycle stages that lack significant morphological characters for species identification.

Hopping species and borders: detection of Bartonella spp. in avian nest fleas and arctic foxes from Nunavut, Canada.

BACKGROUND: In a warmer and more globally connected Arctic, vector-borne pathogens of zoonotic importance may be increasing in prevalence in native wildlife. Recently, Bartonella henselae, the causative agent of cat scratch fever, was detected in blood collected from arctic foxes (Vulpes lagopus) that were captured and released in the large goose colony at Karrak Lake, Nunavut, Canada. This bacterium is generally associated with cats and cat fleas, which are absent from Arctic ecosystems. Arctic foxes in this region feed extensively on migratory geese, their eggs, and their goslings. Thus, we hypothesized that a nest flea, Ceratophyllus vagabundus vagabundus (Boheman, 1865), may serve as a vector for transmission of Bartonella spp. METHODS: We determined the prevalence of Bartonella spp. in (i) nest fleas collected from 5 arctic fox dens and (ii) 37 surrounding goose nests, (iii) fleas collected from 20 geese harvested during arrival at the nesting grounds and (iv) blood clots from 57 adult live-captured arctic foxes. A subsample of fleas were identified morphologically as C. v. vagabundus. Remaining fleas were pooled for each nest, den, or host. DNA was extracted from flea pools and blood clots and analyzed with conventional and real-time polymerase chain reactions targeting the 16S-23S rRNA intergenic transcribed spacer region. RESULTS: Bartonella henselae was identified in 43% of pooled flea samples from nests and 40% of pooled flea samples from fox dens. Bartonella vinsonii berkhoffii was identified in 30% of pooled flea samples collected from 20 geese. Both B. vinsonii berkhoffii (n = 2) and B. rochalimae (n = 1) were identified in the blood of foxes. CONCLUSIONS: We confirm that B. henselae, B. vinsonii berkhoffii and B. rochalimae circulate in the Karrak Lake ecosystem and that nest fleas contain B. vinsonii and B. henselae DNA, suggesting that this flea may serve as a potential vector for transmission among Arctic wildlife.

American Dog Ticks (Dermacentor variabilis) as Biological Indicators of an Association between the Enteric Bacterium Moellerella wisconsensis and Striped Skunks (Mephitis mephitis) in Southwestern Manitoba, Canada.

Total genomic (g)DNA from 100 American dog ticks (Dermacentor variabilis) collected from humans, dogs, raccoons, and skunks near Minnedosa (Manitoba, Canada) in 2005 was tested for the presence of Moellerella wisconsensis (Gammaproteobacteria: Enterobacteriales) using PCR. Although two gDNA samples derived from ticks attached to two striped skunks (Mephitis mephitis) contained M. wisconsensis DNA, it is unlikely that D. variabilis is a vector of this bacterium. Genomic DNA prepared from the washes of the external surfaces of these two ticks (i.e., before DNA extraction from the whole tick) and another two ticks attached to same skunks were also PCR positive for M. wisconsensis. This suggests that ticks acquired the bacterium by physical contact with contaminated or infected skunks. However, it does not exclude the possibility that the ticks may have also imbibed the bacterium from their host blood and lymph. Nonetheless, the results of this molecular study suggest that the four adult D. variabilis represent biological indicators of the presence of M. wisconsensis in association with their vertebrate hosts (i.e., striped skunks). Additional work is needed to determine if M. wisconsensis is present in the blood and lymph of striped skunks in southwestern Manitoba and if there are potential health risks for persons coming into contact with infected animals.

Identification of bacteria in the Rocky Mountain wood tick, Dermacentor andersoni, using single-strand conformation polymorphism (SSCP) and DNA sequencing.

PCR-based single-strand conformation polymorphism (SSCP) analyses combined with DNA sequencing of the prokaryotic 16S ribosomal (r) RNA gene encompassing the hypervariable V4 region was used to determine the bacterial composition of Rocky Mountain wood ticks (Dermacentor andersoni) attached to Richardson's ground squirrels (Urocitellus richardsonii) and questing on vegetation in southern Saskatchewan, Canada. The bacteria present in questing adult ticks from Saskatchewan Landing Provincial Park included Rickettsia peacockii, a Francisella-like endosymbiont (FLE) and an Arsenophonus-like endosymbiont. Bacteria in the adult and nymphal ticks attached to U. richardsonii collected from Beechy included R. peacockii, a FLE, and several other genera (e.g., Ralstonia, Sphingobium, Comamonas and Pseudomonas). The bacteria detected in D. andersoni in the present study are consistent with the findings of other studies that have characterized the microbiome of this tick species in the USA using next generation sequencing. This result demonstrates that the SSCP-based approach used in this study is cost- and time-effective for examining bacterial composition in ticks.

Passive and Active Surveillance for Ixodes scapularis (Acari: Ixodidae) in Saskatchewan, Canada.

Passive and active surveillance for the blacklegged tick, Ixodes scapularis Say, in the Canadian province of Saskatchewan was conducted over a 9-yr period (2009-2017). More than 26,000 ixodid ticks, representing 10 species, were submitted through passive surveillance. Most (97%) of these were the American dog tick, Dermacentor variabilis (Say). Of the 65 I. scapularis adults submitted, 75% were collected from dogs. Infection rates of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti in I. scapularis were 12%, 8%, and 0%, respectively. Although the I. scapularis submitted by passive surveillance were collected from five of seven ecoregions in central and southern Saskatchewan, they were most frequent in the Moist Mixed Grassland and Aspen Parklands. In contrast, no I. scapularis were collected from the extensive field sampling conducted at multiple sites in different ecoregions across the province. Hence, there is no evidence of I. scapularis having established a breeding population in Saskatchewan. Nonetheless, continued surveillance for blacklegged ticks is warranted given their important role as a vector of medically and veterinary important pathogens, and because they have recently become established across much of the southern portions of the neighboring province of Manitoba.

Genetic variation within the genus Macropostrongyloides (Nematoda: Strongyloidea) from Australian macropodid and vombatid marsupials.

The genetic variation and taxonomic status of the four morphologically-defined species of Macropostrongyloides in Australian macropodid and vombatid marsupials were examined using sequence data of the ITS+ region (=first and second internal transcribed spacers, and the 5.8S rRNA gene) of the nuclear ribosomal DNA. The results of the phylogenetic analyses revealed that Ma. baylisi was a species complex consisting of four genetically distinct groups, some of which are host-specific. In addition, Ma. lasiorhini in the common wombat (Vombatus ursinus) did not form a monophyletic clade with Ma. lasiorhini from the southern hairy-nosed wombat (Lasiorhinus latifrons), suggesting the possibility of cryptic (genetically distinct but morphologically similar) species. There was also some genetic divergence between Ma. dissimilis in swamp wallabies (Wallabia bicolor) from different geographical regions. In contrast, there was no genetic divergence among specimens of Ma. yamagutii across its broad geographical range or between host species (i.e. Macropus fuliginosus and M. giganteus). Macropostrongyloides dissimilis represented the sister taxon to Ma. baylisi, Ma. yamagutii and Ma. lasiorhini. Further morphological and molecular studies are required to assess the species complex of Ma. baylisi.

Prevalence of Anaplasma bovis in Canadian populations of the Rocky Mountain wood tick, Dermacentor andersoni.

PCR and DNA sequencing were used to determine the prevalence of Anaplasma bovis in Rocky Mountain wood ticks (Dermacentor andersoni) collected in Alberta and Saskatchewan, Canada. These analyses revealed that A. bovis DNA was present in 35 (2.1%) of 1679 ticks collected at 12 of the 24 localities. The discovery of A. bovis in host-seeking female and male D. andersoni from multiple locations in southern Canada may have important implications for diagnosis of anaplasmosis in Canada.

Three genetically distinct clades of Anaplasma phagocytophilum in Ixodes scapularis.

Human granulocytic anaplasmosis (HGA) is an emerging disease in Canada because of range expansion by the arthropod vector, Ixodes scapularis. These ticks carry the Ap-ha variant of Anaplasma phagocytophilum (Ap-ha), which has been implicated in causing HGA, and the Ap-variant 1, which is not associated with human infection. We report the detection of 13 genotypes of the ankyrin (ankA) gene among 76 infected blacklegged ticks. Haplotype network and phylogenetic analyses revealed that the ankA genotypes corresponding to the Ap-ha variant did not form a monophyletic assemblage. They formed two distinct clades (Clades I and III), one of which was genetically more similar in nucleotide and amino acid sequences to genotypes of Ap-variant 1 that comprised Clade II. Additional work is needed to explore the evolutionary history of A. phagocytophilum in North America, and to determine if there are differences in pathogenicity or clinical symptoms associated with the two divergent groups of the Ap-ha variant given the significant differences in ankA amino acid sequence.