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Keywords:

  • Ixodes scapularis;
  • Ixodes kingi;
  • Dermacentor;
  • Thomomys talpoides;
  • single strand conformation polymorphism;
  • Saskatchewan

ABSTRACT:

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Morphological examination of ticks feeding on northern pocket gophers, Thomomys talpoides, near Clavet (Saskatchewan, Canada) revealed the presence of two genera, Ixodes and Dermacentor. All adult ticks collected were identified as I. kingi. Single strand conformation polymorphism (SSCP) analyses and DNA sequencing of the mitochondrial 16S rRNA gene confirmed the species identity of most Ixodes immatures as I. kingi (two nymphs and 82 larvae), and the Dermacentor immatures as D. variabilis (one nymph and one larva) and D. andersoni (three larvae). Six Ixodes larvae feeding on three T. talpoides individuals were identified as four different 16S haplotypes of I. scapularis, which was unexpected because there are no known established populations of this species in Saskatchewan. However, flagging for questing ticks and further examination of the ticks feeding on T. talpoides in two subsequent years failed to detect the presence of I. scapularis near Clavet, suggesting that there is no established population of I. scapularis in this area. Nonetheless, since I. scapularis is a vector of pathogenic agents, passive and active surveillance needs to be conducted in Saskatchewan on an ongoing basis to determine if this tick species and its associated pathogens become established within the province.


INTRODUCTION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Ticks are important vectors of human and animal pathogens in North America. For example, the blacklegged tick, Ixodes scapularis, is the vector of Borrelia burgdorferi, the causative agent of Lyme borreliosis in the Midwest and northeastern U.S.A. (Thompson et al. 2001, Bacon et al. 2008). Lyme borreliosis is also an emerging vector-borne disease in Canada (Ogden et al. 2008, 2009) given that several populations of I. scapularis have recently established in southern Ontario, Nova Scotia, southeastern Manitoba, and New Brunswick (Ogden et al. 2009). Ixodes scapularis is also a vector of Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (Thompson et al. 2001). Another two common tick species, the Rocky Mountain wood tick, Dermacentor andersoni, and the American dog tick, D. variabilis, are vectors of Anaplasma marginale, the bacterium that causes bovine anaplasmosis in North America (Kocan et al. 2010). They are also vectors for Rickettsia rickettsii and Francisella tularensis, bacteria that are responsible for Rocky Mountain spotted fever and tularemia, respectively (Burgdorfer 1975, Foley and Nieto 2010). All three of these tick species, as well as a number of other tick species in North America, use a variety of rodents (e.g., mice, voles, shrews, ground squirrels, and pocket gophers) as hosts (Wilkinson 1967, Keirans et al. 1996, Allan 2001, Salkeld et al. 2006), some of which are important reservoirs for tick-borne pathogens (Allan 2001, Oliver et al. 2006, Foley and Nieto 2010).

The northern pocket gopher, Thomomys talpoides, which comprises a number of subspecies, has a broad distributional range in North America that includes the northern parts of central and western U.S.A., some mountainous valleys of British Columbia in Canada, and the Canadian prairie provinces of Alberta, Saskatchewan, and Manitoba (Hall and Kelson 1959). Although there is information as to which tick species (i.e., Ixodes and Dermacentor spp.) parasitize pocket gophers (Cooley and Kohls 1945, Miller and Ward 1960, Gregson 1971, Allan 2001, Salkeld et al. 2006), these records are limited to certain parts of the geographical range of T. talpoides. In some cases, the species identity of larval ticks feeding on pocket gophers could not be determined by morphological examination (Miller and Ward 1960).

Molecular approaches, using a variety of genetic markers, have been shown to be useful in the identification of individual ticks and for examining the population genetics and phylogenetic relationships of different tick species (Norris et al. 1996, Qiu et al. 2002, Guglielmone et al. 2006, Dergousoff and Chilton 2007, Patterson et al. 2009, Krakowetz et al. 2010, 2011). In the present study, molecular tools were used to identify, to the species level, ticks feeding on T. talpoides from a locality in central Saskatchewan. We report the unexpected detection of I. scapularis larvae on T. talpoides and discuss the implications of this finding.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Northern pocket gophers (T. talpoides) were kill-trapped periodically between spring and early autumn (May-October) in 2007 on an acreage situated 8 km southwest of Clavet in Saskatchewan (51.9519N, 106.4473W) using Victor® BlackBox Gopher traps (model #0635). This site was composed of mostly mixed grass prairie, with occasional shrub cover. A large slough was located adjacent to the property. Each T. talpoides was placed separately into a sealed metal container and transported to the laboratory where they were transferred into individual plastic bags and stored at -20° C.

Ectoparasites were removed from the body and fur of thawed hosts using fine forceps, and the ticks were identified morphologically to genus (Clifford et al. 1961, Keirans and Litwak 1989) as part of an undergraduate parasitology laboratory exercise. Ticks were then stored in 70% ethanol for future molecular examination. The four adult ticks collected were all identified as Ixodes kingi Bishopp, 1911 using the morphological key of Keirans and Litwak (1989). Many of the engorged immature ticks were identified as belonging to the genus Ixodes based on the presence of an anal groove (see Kleinjan and Lane 2008); however, the species identity of most these individuals could not be determined unequivocally. Therefore, a molecular approach was used to determine the species identity of all ticks feeding on T. talpoides.

The mitochondrial (mt) 16S rRNA gene was used as the target to determine the species identity of each tick, and to examine the magnitude of genetic variation among the four I. kingi adults and 98 putative I. kingi immatures feeding on T. talpoides. This gene was selected because it has been used as a genetic marker to examine the population structure of I. scapularis (e.g., Norris et al. 1996, Qiu et al. 2002, Krakowetz et al. 2011), and of the phylogenetic relationships of species within the genus Ixodes (e.g., Guglielmone et al. 2006). Genomic DNA (gDNA) was extracted and purified from the legs of adults and the complete bodies of each larva and nymph using the DNeasy Blood and Tissue Kit (Qiagen) as described by Dergousoff and Chilton (2007). Part (∼410 bp) of the mt 16S rDNA was amplified by PCR using the primers 16S-1 (5′-CCACAGCAATTTAAAAAATCATTGAGCAG-3′) and 16S+1 (5′-CCGGTCTGAACTCAGATCAAGT-3′) (Norris et al. 1996) and the conditions described previously by Krakowetz et al. (2010). All amplicons were subjected to single strand conformation polymorphism (SSCP) analyses using the methodology of Krakowetz et al. (2010). DNA sequencing was performed on column-purified (MinElute PCR Purification kit, Qiagen) amplicons of representative individuals of the different SSCP banding patterns. BLAST searches (GenBank) were performed on sequences to determine the identity of each sample. Given that there was no sequence data for I. kingi on GenBank, the species identity of the putative I. kingi immatures was confirmed by comparing their mt 16S rDNA sequences to those of the morphologically identified I. kingi adults. This approach was feasible since mt DNA sequences are maternally inherited, and different species of Ixodes have different mt 16S rDNA sequences (Guglielmone et al. 2006). Nucleotide sequence data have been deposited in GenBank under the accession numbers FR854227-FR854232.

RESULTS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Morphological examination of the ticks collected from eight of 27 T. talpoides revealed the presence of two genera, Ixodes and Dermacentor. Of the 102 ticks identified morphologically as Ixodes, amplicons of the mt 16S rDNA were produced for a total of 94 individuals. There were no differences in the size (∼450 bp) of amplicons on agarose gels (data not shown). A comparison of the SSCP profiles of the 94 individuals revealed the presence of more than one banding pattern (Figure 1). Five different sequence types of the mt 16S rDNA were obtained for representative samples of the different banding patterns. There was no genetic variation in the 413 bp fragment of the 16S sequences of one male and three female I. kingi. There were 82 larval and two nymphal Ixodes with the same SSCP banding patterns as the I. kingi adults. The sequences of 13 of these larvae were also identical to the sequences of the four adult I. kingi. However, there were another six larvae from three hosts with different SSCP banding patterns to those of the I. kingi adults. These six larvae also had different 16S sequences when compared to those of I. kingi (Table 1). A BLAST search of the six sequences (405–406 bp) revealed that they were identical or genetically most similar to the 16S sequences of I. scapularis. The species identity of one of these larvae (Tick #CA32) was examined further by sequencing the second internal transcribed spacer (ITS-2) rDNA. The 677 bp ITS-2 sequence of this individual was 99.9% similar (i.e., 1 bp difference) to that of an ITS-2 sequence of I. scapularis (accession number X63868). Three of the six I. scapularis larvae from T. talpoides had the same 16S sequence as haplotype F of Qiu et al. (2002), while the other three individuals each had a unique haplotype that differed from haplotype F individuals at one or two alignment positions (Table 1). The four variable sequence differences among the I. scapularis individuals represented two purine transitions, one pyrimidine transition, and one indel. A comparison of the aligned 16S sequences (416 bp) of I. scapularis and I. kingi revealed 64 (15%) nucleotide differences; 17 purine transitions, three pyrimidine transitions, 30 transversions and 14 indels (Table 1).

image

Figure 1. SSCP profiles of mitochondrial 16S rDNA amplicons for representative specimens of larval I. kingi (lanes 1–9 and 11–10) and I. scapularis (lane 10).

Download figure to PowerPoint

Table 1.  Variable nucleotide positions in the aligned mitochondrial 16S rDNA sequences of the Ixodes specimens examined in the present study. A dot indicates the same nucleotide as in the sequence of I. scapularis. Thumbnail image of

A comparison of the mt 16S rDNA sequences of the Dermacentor specimens with sequence data on GenBank revealed that they represented one D. variabilis nymph, one D. variabilis larva and three D. andersoni larvae. The D. variabilis nymph and larva had the same 16S sequences as those of haplotypes 1 and 7, respectively (accession numbers FN665376 and FN665382), as defined by Krakowetz et al. (2010). Each D. andersoni larva had a different mt 16S rDNA sequence (Table 2). The 16S sequences of two individuals were identical to those of haplotypes P and T (accession numbers FM955611 and FM955615, respectively), while the third had a unique haplotype compared to those of D. andersoni from two populations in Saskatchewan and Alberta (Patterson et al. 2009).

Table 2.  Variable nucleotide positions in the aligned mitochondrial 16S rDNA sequences of the three D. andersoni individuals feeding on northern pocket gophers. A dot indicates the same nucleotide as in the sequence of haplotype. Thumbnail image of

DISCUSSION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

In the present study, 107 ticks representing four tick species were collected from T. talpoides near Clavet, Saskatchewan. A small proportion (5%) of these were D. variabilis and D. andersoni. The presence of two D. variabilis immatures (one nymph and one larva) on T. talpoides near Clavet was not unusual given that we have collected questing adults at this locality. However, immatures of D. variabilis are more commonly found feeding on other smaller rodents (e.g., Clethrionomys gapperi, Microtus pennsylvanicus, and Peromyscus maniculatus) in this region2. In contrast, the discovery of three D. andersoni larvae near Clavet, each representing a different 16S haplotype and feeding on a different host individual, was surprising because this species has not been recorded previously this far east in Saskatchewan (see Wilkinson 1967). Although the distributional range of D. andersoni has expanded eastwards in Saskatchewan since the 1970s, which may have implications for transmission of tick-borne pathogens to livestock and humans, the closest known population of D. andersoni to the study site near Clavet is situated ∼70 km to the southeast2. A population of D. andersoni has not yet established at our study site because no D. andersoni immatures have been subsequently collected from T. talpoides, and flagging vegetation for questing adults has only revealed the presence of D. variabilis and not D. andersoni.

The majority of the ticks collected from T. talpoides were identified as I. kingi based on morphological and molecular analyses. This tick species has been recorded from 40 species of rodent (including Thomomys spp.), four species of lagomorph, 17 species of carnivore, domestic dogs and cats, and humans in North America (Cooley and Kohls 1945, Gregson 1971, Salkeld et al. 2006). However, in the study by Miller and Ward (1960) on the ectoparasites of pocket gophers from Colorado, I. sculptus and not I. kingi were collected from T. talpoides, whereas both these tick species were collected from Botta's pocket gopher, T. bottae. In the present study, all life cycle stages of I. kingi were found on T. talpoides near Clavet, including adults of both sexes, whereas no I. sculptus were detected on northern pocket gophers. Larvae accounted for 93% of the I. kingi collected. Gregson (1971) reported that there were differences in the types of hosts used by I. kingi in different geographical regions. On the western slopes of the Rocky Mountains, pocket gophers (Thomomys spp.), kangaroo rats (Dipodomys spp.), and mice (Peromyscus spp.) were the hosts most commonly used, whereas east of the Rocky Mountains the principal hosts were sciurid rodents (e.g., Sphermophilus, Urocitellus, and Cynomys spp.) and carnivores (Gregson 1971). Although the presence of I. kingi on T. talpoides from central Saskatchewan (i.e., east of the Rocky Mountains) is not consistent with the findings of Gregson (1971), there were no records of I. kingi occurring within Saskatchewan in the paper by Gregson (1971). Additional studies are needed to determine if sciurid rodents, such as 13-lined ground squirrels (S. tridecemlineatus) and Richardson's ground squirrels (U. richardsonii), which occur in the Clavet area, are common hosts for I. kingi.

Gregson (1971) also noted morphological differences between I. kingi from western populations and those in the eastern populations, which may be a reflection of evolutionary divergence (Oliver et al. 1974). Genetic studies of I. scapularis (e.g., Norris et al. 1996, Qiu et al. 2002, Krakowetz et al. 2011) and Dermacentor spp. (e.g., Patterson et al. 2007, Krakowetz et al. 2010) in North America using the mt 16S rRNA gene reported the presence of multiple haplotypes (i.e., genetic variants) within tick populations. In the present study, no genetic variation was detected in the 16S gene of I. kingi individuals based on SSCP analyses and DNA sequencing, which may be a consequence of sampling ticks from hosts over a relatively small area. Therefore, the usefulness of the mt 16S rRNA gene as a population genetic marker for I. kingi needs to be assessed further using individuals from different geographical localities, including both sides of the Rocky Mountains.

The SSCP banding patterns and DNA sequences of the 16S rDNA of I. kingi were distinct from those of the D. variabilis and D. andersoni found on T. talpoides. The SSCP analyses also revealed that the banding patterns of six Ixodes larvae, collected from three hosts, were distinct from those of I. kingi. A comparison of the mt 16S rDNA sequences of these six individuals revealed that they were I. scapularis. Half of these individuals were of haplotype F, which is consistent with studies on haplotype frequencies in populations of this tick in the U.S.A. (e.g., Qiu et al. 2002) and in Canada (Krakowetz et al. 2011). The species identity of one individual was further verified by its ITS-2 rDNA sequence. The presence of I. scapularis larvae feeding on three T. talpoides individuals near Clavet was totally unexpected because there appear to be no previous published reports of T. talpoides as a host for I. scapularis larvae, and there are no known established populations of I. scapularis in Saskatchewan. Although there have been genetic studies conducted on I. scapularis adults in our laboratory (Krakowetz et al. 2011), the results of the molecular work of the present study are not the consequence of a potential contamination of gDNA because the 16S rDNA sequences of two of the six larvae were different to those of all I. scapularis adults examined previously.

Numerous adventitious ticks have been recorded from Saskatchewan in the west to Newfoundland in the east (Ogden et al. 2006a), however, only a small number of I. scapularis populations have become established in Canada thus far (Ogden et al. 2009). A population of I. scapularis is considered established at a given locality when larvae, nymphs, and adults have all been collected while feeding on resident animals or questing in the environment for at least two consecutive years (Ogden et al. 2008). Therefore, on this basis, there is no evidence for an established population of I. scapularis near Clavet because only larvae (i.e., no nymphs or adults) of I. scapularis were found feeding on resident mammals in 2007. In addition, subsequent trapping of T. talpoides and flagging for questing ticks in 2009 and 2010 failed to detect the presence of any life cycle stage of I. scapularis. Although deer mice (P. maniculatus), a common host of I. scapularis immatures (e.g., Oliver et al. 2006), were not trapped at this specific locality, they have been trapped from a nearby area (i.e., Blackstrap Lake, situated 18 km to the south), but were not found to be parasitized by any species of Ixodes2. The absence of I. scapularis from the study site since 2007 suggests that individuals of this species were unable to successfully complete their life cycle. Populations of I. scapularis may be unable to establish in new areas because of a combination of factors including the incremental risk of mortality at each life cycle stage from egg to adult, unfavorable climatic conditions and habitat types, a relative low abundance of suitable hosts and a small number of colonizing adult individuals (Lindsay et al. 1995, 1998).

Given the maternal inheritance of mt DNA, the detection of four different 16S haplotypes among the six I. scapularis larvae indicates that they are the progeny of at least four adult females, each of which had been mated by a conspecific male, fed on a suitable host, and laid viable eggs; some of which hatched successfully. Although only a small number of I. scapularis adults have been collected from Saskatchewan by passive surveillance (Ogden et al. 2006a, Chilton et al., unpublished data), three I. scapularis females were collected in 2008 from two dogs on a single property located 29 km north of the study site near Clavet. Another I. scapularis female had also been collected from the same property a year earlier (Chilton et al., unpublished data). Therefore, it is possible for multiple adult ticks to have been present at our study site. These adult ticks were probably introduced into the area as immature stages because migratory passerines are known to carry I. scapularis larvae and nymphs from the U.S.A. into Canada each spring (Ogden et al. 2008). Fed larvae and nymphs dispersed by birds would had to have molted to the next life stage (nymphs and adults, respectively) prior to finding suitable hosts on which to feed. Questing nymphs may have used T. talpoides and other species of resident small rodent, such as shrews, mice, and voles as hosts, while adults may have used white-tailed deer (Odocoileus virginianus). These large mammals are the preferred hosts of adult I. scapularis (Keirans et al. 1996) and are common in the area around Clavet. At Long Point in southern Ontario, the life cycle of I. scapularis may take three or four years to complete and involves overwintering by all active life cycle stages, including fed females (Lindsay et al. 1995, 1998). Females then lay eggs in late May to mid-April and larvae emerge from eggs in late July or early August (Lindsay et al. 1995, 1998). If the timing of larval emergence at the site near Clavet was similar to that for ticks at Long Point, then questing larvae could have encountered T. talpoides during the summer months. This is possible because T. talpoides are known to forage above ground in summer, even though they have primarily a subterranean lifestyle (Hansen and Reid 1973). Nonetheless, the presence of I. scapularis larvae feeding on T. talpoides near Clavet, followed by the absence of individuals of any life cycle stage, either in the environment or on hosts, suggests a failed colonization attempt. Other failed colonization attempts by I. scapularis have also been seen at sites in Manitoba and Nova Scotia (L.R. Lindsay, personal communication).

Although there are no known established populations of I. scapularis in Saskatchewan, it has been predicted that by the 2020s, environmental conditions may become suitable for this species to become established in the province as a consequence of climate change (Ogden et al. 2006b). Given the discovery of I. scapularis larvae feeding on T. talpoides at one locality in Saskatchewan, the occasional occurrence of adventitious ticks, and that animal and/or human pathogens such as B. burgdorferi can establish following the formation of resident I. scapularis populations in Canada (Ogden et al. 2010), it is essential that passive and active surveillance be conducted within Saskatchewan on an ongoing basis to assess the potential risk of human exposure to pathogens. The findings of the present study also highlight the value of PCR-based techniques such as SSCP in combination with DNA sequencing to distinguish among tick species, particularly for engorged larvae, where it is often more difficult to determine species identity based on morphological examination alone, and/or to verify the species identity of immature ticks collected by passive and active surveillance.

Acknowledgments

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Funding for this work was provided to N.B.C. from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Foundation for Innovation. A Margaret McKay scholarship provided financial support to C.A.A. We are grateful to Dr. C. Todd, S. Dergousoff, A. McMurtry, and K. Sim for their assistance in the field, and to Dr. L.R. Lindsay and two anonymous reviewers for their valuable comments on the manuscript. This work was approved by the University of Saskatchewan's Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines for humane use.

Footnotes
  • 2

    Dergousoff, S.J. 2011. Comparison of the bacteria within ticks from allopatric and sympatric populations of Dermacentor andersoni and Dermacentor variabilis near their northern distributional limits in Canada. Ph.D. Thesis, University of Saskatchewan, Saskatoon, pp. 238.

REFERENCES CITED

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  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
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