Incidence of Canine Tick Vectors and Molecular Detection of Haemoparasites in Vectors and Hosts

Tick vectors are highly specialized obligatory haematophagous ectoparasites of mammals, birds and reptiles; these are of great importance as they transmit many diseases such as Lyme disease, West Nile disease, etc., some of which are zoonotic in nature (Otranto et al., 2009). Several characteristics of ticks make them outstanding vectors of pathogenic agents, the wide host range and slow feeding habit along with tendency to feed on several hosts during life cycle ensures ample opportunity to acquire and transmit pathogens; tough contoured robust body and incredible longevity have made them enable to survive long periods in unfavorable environmental conditions and their high reproductive potential along with high frequency of host-vector contact ensure maintenance of a large populations (Parola and Raoult, 2001; Minjauw and McLeod, 2003; Peter et al., 2005). Kolkata with its tropical geoclimatic condition is quite favorable for growth and multiplication of ticks. Among different species of ticks infesting dogs in India, the most commonly occurring one is the brown dog tick, Rhipicephalus sanguineus; (Jadhav et al., 2011; Sahu et al., 2013). The suburban localities, virtually poses a major source of infestation for the dogs due to its rich geographical flora. Hence, very rationally Kolkata is afflicted by some tropical zoonotic infections including parasitic infections and many of them are arthropod transmitted.
       
There had been a long journey involving a great deal of traditional studies related to prevalence and incidence of canine tick infestation and canine tick borne diseases (Kaur et al., 2012 and Singla et al., 2016). But, this study focuses on status of canine tick vectors and molecular detection of haemoparasites in these ticks and their host, in and around Kolkata.

The work was conducted in the Department of Veterinary Parasitology, Faculty of Veterinary and Animal Sciences, WBUAFS, Kolkata. The blood and tick samples of dogs were collected from Dog Ward, Department of Teaching Veterinary and Clinical Complex, Faculty of Veterinary and Animal Sciences, WBUAFS at Belgachia, Veterinary Clinic of Kolkata Police dog Squad at Alipore, Veterinary Clinic of Barrackpore Police Dog Squad and samples from stray dogs were also collected from inside the University campus through a period of one year from 1^st August, 2016 to 31^st July, 2017. The study was conducted randomly on 300 dogs and the ticks that were procured from them, but for PCR based detection 100 dogs were selected to represent the whole sample size. Out of these 300, 152 were female and 148 were male, which was further categorized into ≤ 6 months (36), 6 months to 1yr (46) and ≥ 1yr (218). Broadly the dogs were categorized according to their utility namely companion dogs (190), working dogs (89) and stray dogs (21). In each season 100 animals were examined with comprehensive details.
       
All three hundred dogs were thoroughly examined for presence of ticks. For collection of ticks, dogs were properly restrained and meticulously examined and with the help of forceps  freely moving ticks were collected; palpation was used to locate smaller specimens and immature ticks. When required, small hairbrush dipped in ethanol or chloroform or xylene as per availability was used for the collection of ticks in order to loosen and remove the firmly attached ticks. Ticks collected were transferred into properly labeled plastic containers having perforated stopper and brought to the Parasitology laboratory of WBUAFS, Kolkata for analysis (Soulsby, 1986; Sahu et al., 2013; Kaur et al., 2015).
 
Identification of ticks
 
Presumptive identification of samples was made under dissecting microscope. Final identification were made under 10X and 40X compound microscope according to keys and description given by Soulsby (1986); Walker et al., (2000); Walker et al., (2003); Zajac and Conboy (2007); Walker et al., (2007); http://www.bristoluniversitytickid.uk/.
 
DNA extraction
 
DNA was extracted from whole ticks and corresponding blood samples of dogs using a commercially available kit (QIAGEN DNeasy Blood and Tissue Kit; cat. no. 69504) specially designed for isolation of genomic DNA, using manufacturer’s protocol. Primer sets were selected for PCR amplification of specific genomic segments of different canine haemoparasitic infections under study (Hilpertshauser et al., 2006; Hatta et al., 2013; Das et al., 2015) :
 
a. Babesia genus specific
 
B18sF: 5' - TGG TTG ATC CTG CCA GTA - 3'
B18sR: 5' - CTT CTC CTT CCT TTA AGT GA - 3'
 
b. Babesia gibsoni
 
Bg.PD4: 5' - TCC TCC TCA TCA TCC TCA TTC G - 3'
Bg.PD3: 5' - TCC GTT CCC ACA ACA CCA GC - 3'
 
c. Universal rickettsial
 
ECC: 5' - AGA ACG AAC GCT GGC GGC AAG C - 3'
ECB: 5' - CGT ATT ACC GCG GCT GCT GGC A - 3'

d. Ehrlichia chaffensis and E. canis
 
ECAN5: 5' - CAA TTA TTT ATA GCC TCT GGC TAT AGG A - 3'
HE1: 5' - CAA TTG CTT ATA ACC TTT TGG TTA TAA AT - 3'
HE3: 5' - TAT AGG TAC CGT CAT TAT CTT CCC TAT - 3' 

e. Hepatozoon canis
 
HepF: 5'- ATA CAT GAG CAA AAT CTC AAC - 3'
HepR: 5'- CTT ATT ATT CCA TGC TGC AG - 3'
 
f. Mycoplasma haemocanis syn. Haemobartonella canis
 
HBT-F: 5' - ATA CGG CCC ATA TTC CTA CG - 3'
HBT-R: 5' - TGC TCC ACC ACT TGT TCA - 3'
 
g. Anaplasma platys
 
Platys: 5' - GAT TTT TGT CGT AGC TTG CTA TG - 3'
EHR16SR: 5' - TAG CAC TCA TCG TTT ACA GC - 3'
 
h. Theileria annae
 
5' - GAT ATG TAC CAA GAG CCA TTC TTA TG - 3'
5' - TGT TAC TCC ACT CAT AGC AGC AC - 3'
 
PCR assays
 
Universal canine Babesia-specific primers B18S-F and B18S-R were used to amplify 18S ribosomal DNA (18S rDNA) as described by Ikadi et al., (2004). Amplified DNA from this reaction is used as template for detection of Babesia gibsoni P18 gene. The reaction mixtures were subjected to 10 mins of initiation at 94^oC, 30 cycles each of denaturation at 94^oC for 30s, 1min of annealing at 54^oC and extension at 72^oC for 1 min and subsequent extension after final cycle at 72^oC for 10 mins (Ikadai et al., 2004).
       
In case of Ehrlichia canis and E. chaffensis, the extracted DNA samples were amplified with the Universal rickettsial primer ECC and ECB targeting 16S rRNA gene (Nakaghi et al., 2008). Then, the amplicons were subjected to subsequent separate amplifications by primers ECAN5/HE3 for E. canis and HE1/HE3 for E. chaffensis targeting 16S rRNA gene for both cases. A touchdown PCR technique was used. Amplification was performed under the following conditions: 94^oC for 3 min, two cycles of 94^oC for 30 s, 62^oC for 30 s, 72^oC for 30 s, two cycles of 94^oC for 30 s, 60^oC for 30 s, 72^oC for 30 s, two cycles of 94^oC for 30 s, 58^oC for 30 s, 72^oC for 30 s, two cycles of 94^oC for 30 s, 56^oC for 30 s 72^oC for 30 s, two cycles of 94^oC for 30 s, 54^oC for 30 s 72^oC for 30 s, thirty-nine cycles of 94^oC for 30 s, 52^oC for 30 s, 72^oC for 30 s and final extension at 72^oC for 3 min (Gal et al., 2008).
       
For the detection of Hepatozoon, PCR was performed using primers HEP-F and HEP-R to amplify 18S rRNA gene under the following conditions: 95^oC for 5 min (initial denaturation); 40 cycles of 95^oC for 30s (denaturation); 57^oC for 30s (annealing) and 72^oC for 90s (extension); then final extension at 72^oC for 5 min (Otranto et al., 2009).
       
Haemotropic Mycoplasma synonymously known as Haemobartonella canis was detected through PCR amplification of targeting 16S rRNA gene using the primers HBT-F and HBT-R. The reaction mixture was processed downstream for initial denaturation at 94^oC for 5 min; 40 cycles of denaturation at 95^oC for 30 s, annealing at 60^oC for 30 s and extension at 72^oC for 90 s; then final extension at 72^oC for 10 min (Brinson and Messick, 2001; Abd Rani et al., 2011).
       
A 678 bp fragment of 16S rRNA gene is amplified using the forward and reverse primers Platys and EHR16SR respectively under the conditions: 95oC for 5 mins (initial denaturation); 40 cycles of 94^oC for 30 s (denaturation), 55^oC for 30 s (annealing) and 72^oC for 90 s (extension); then final extension at 72^oC for 5 mins. (Abd Rani et al., 2011; Otranto et al., 2009).
       
Genomic DNA of different haemoprotozoons were isolated from infected blood samples (identified through blood smear examination) and utilized as positive control. For negative control, the reaction mixture was prepared without the template DNA.
       
The data recorded during this study was subjected to statistical analysis using Statistical Package for Social Science (SPSS) version 22.0 software (SPSS Inc., Chicago, IL, USA) as described by Snedecor and Cochran (1994). Using SPSS version 22.0 Chi Square test was calculated. The P values less than 0.05 were considered to be significant and the P values less than 0.01 were considered to be highly significant to be correlated. The P values more than 0.05 were having no significant relationship.

Overall incidence of ticks in dogs
 
For identification, ticks were mounted and observed under microscope (10X). The identification of ticks was done based on morphological characters of male and female ticks. Overall 123 (41%) out of 300 dogs examined were found positive (Table 1) for ticks. This finding is in accordance with earlier studies (Abd Rani et al., 2011; Sahu et al., 2013; Gondard et al., 2017; Basu and Charles, 2017). Rhipicephalus sanguineus was the only tick found in this study, which could be due the limitation of this tick to urban areas as was seen in case of Delhi and Mumbai (Abd Rani et al., 2011) and in urban areas of Japan (Shimada et al., 2003). These findings were again correlated with risk factors that may affect the incidence of tick infestation. The earlier studies have found slightly to much higher incidence rate of 53.00% all over India (Abd Rani et al., 2011); Totton et al., (2011) found a very high prevalence of 68.00% tick infestation at Jodhpur, India among 323 stray dogs and Sahu et al., (2013) recorded 46.39% incidence at Bhubaneswar from a combined study of both pet and stray dogs. The reason for slightly lower incidence in this study may be due to the smaller population size among the stray population, since it was recorded that stray dogs had the highest incidence of (98.48%) (Table 3) tick infestation with 19 out of 21 being found positive.
 

       
Among the twelve dog breeds taken in this study, Mongrels (25; 92.59%) were found to be highly susceptible for tick infestation. In this study (X² =52.841; P=0.00), the breeds of the dogs found to have significant influence on tick infestation. The order of susceptibility observed was Mongrels (25; 92.59%), German shepherd (42; 56%), Golden retriever (6; 46.15%), Dachshund (2; 33.33%), Pug (2; 33.33%), Bullmastiff (10; 33.33%) Spitz (8; 32%), Labrador (29; 28.71%), Doberman (5; 23.81%), Chihuahua (1; 16.67%) and St. Bernard with no incidence (Table 2). The second highest German shepherd has been seen to have higher incidences in earlier findings (Hadi et al., 2016; Jennett et al., 2013), but they found no significant relationship between breed and tick infestation. The reason for Mongrels or non-descript to be high could be due to the reason that most of these Mongrels were free roaming, scavenging or stray (owner less).
 

       
The grouping of dogs was made as companion, working and stray dogs keeping in mind the utility as a risk factor, which produced highly strong statistical evidence (X²=55.337; P=0.00) of relationship between utility and tick infestation. Highest incidence was recorded in stray dogs (19; 90.48%) followed by working (55; 61.80%) and companions dogs (49; 25.79%) (Table 3). This higher incidence in stray dogs is in accordance with Jafri and Rabbani, (1999), Ekanem et al., (2010), Abd Rani et al., (2011), Sahu et al., (2013) and Ayodhya (2014). The much higher incidence of tick infestation in this study, in stray dogs (90.48%) may be attributed to poor nutrition and unhygienic living condition coupled with stress (Totton et al., 2011).
 

       
The age wise incidence of tick infestation has been recorded in relation to utility dogs (Table 3). The stray dogs were indicated with very high level of incidence in all three groups i.e. <6 months (100%), 6 months to 1 yr (100%), >1 year (83.33%) but due to insufficient data the correlation could not be established among them. The overall incidence of tick infestation was found to be higher in 6 months to 1 year (27; 58.70%) age group, followed by <6 months (17; 47.22%) and > 1 year (79; 36.24%). This overall incidence between the different age groups was found highly significant (X² = 55.34; 2; P= 0.00). In working dogs, highest incidence was recorded in the age group of 6months to 1yr (10; 90.91%). Companion dogs in relation to age group also found highly significant correlation (X² = 8.08; 2; P=0.018) and the highest and lowest were from 6 months to 1yr (13; 41.49%) and >1yr (26; 19.85%) age groups, respectively. The pattern of statistically significant higher incidence of tick infestation in dogs of younger age group might be due to development of resistance and effective scratching activity in adult dogs than that of young ones (Sahu et al., 2013). The infestation among younger dogs, reported from Sahu et al., (2013); Hadi et al., (2016) and Krishnamurthy et al., (2017) corroborates with the present observations.
       
The overall season wise incidence was significantly (X² =20.17; P=0.00) higher in rainy (51%) and summer (49%) seasons, followed by winter (23%) (Table 3). This result corroborates the study of earlier workers (Raut et al., 2006); Ekanem et al., (2010); Abd Rani et al., (2011); Ayodhya (2014). Stray dogs were found to have higher incidence in all three seasons, i.e. rainy and summer at 100% and winter at 66.67%. Highly significant (X² = 24.17; P=0.00) correlation was found between working dogs and seasonal variations, with dogs being most susceptible in rainy (21; 91.30%) followed by summer (23; 74.19%) and winter (11; 31.43%). Similarly, significantly (X² = 6.88; P=0.032) higher incidence was recorded in rainy and summer seasons for that of companion dogs. The highest incidence was seen in rainy (51%) season while lowest was observed in winter (23%), can be explained as warm and humid climate being favorable for the breeding of ixodid ticks (Soulsby, 1986) but can be variable with geographical situation of any region (Moghaddar et al., 2001; Sahu et al., 2013; Debbarma et al., 2017).
       
The females had higher incidence rate (Table 3) (67; 44.08%) than males (56; 37.84%) and this is in agreement with James-Rugu and Jidayi (2004), Arong et al., (2011), Konto et al., (2014) and Ayodhya (2014), this study has found no significant correlation between age and incidence of ticks. But higher incidence in male was recorded by Moghaddar et al., (2001), Sahu et al., (2013), whereas some other authors stated no association (Agbo et al., 2007 and Ul-Hasan et al., 2012).
 
Comparative incidence of haemoparasites in tick vectors and dogs
 
For this study, the same 100 dogs were used (unbiased random basis) for diagnosis of haemoparasites from the earlier collected ticks, which have been already screened for presence of haemoparasites in blood samples (Table 4). The PCR assay was performed on tick found on these specific dogs, in which out of the total (n=100) dogs taken the dogs positive for tick infestation was 57 (57%) (Fig 1, 2, 3 and 4). In this study, PCR assay was considered to be the standard approach (O’Dwyeret_al2009). No relationships were possible to be established in between infections in ticks and in dogs due to a number of uncontrolled variables.
 

 

 

 

 

 
Incidence of haemoparasites in tick vectors using PCR assay
 
A total 57 dogs out of 100 were positive for tick infestation, of which 26 (45.61%) were positive for haemoparasites (Table 4; Fig 1, 2, 3 and 4). Out of these, Babesia spp. (18; 31.58%) had highest infection in ticks followed by E. canis (14; 24.56%) and H. canis (3; 5.26%) with 9 (15.79%) positive for mixed or co-infection. Apart from these, PCR assays were also conducted for the detection of Mycoplasma spp., E. chaffensis, B. gibsoni, A. platys and T. annae but none of these samples were screened positive. The higher rate of incidence seen in mixed or co-infection may be the reason why these ticks are able to spread a number of different diseases.
 
Correlation of haemoparasitic infections with incidence of tick infestation
 
The dogs which were diagnosed PCR positive for TBDs or haemoparasitic infections were correlated with tick infestation, if any (Table 4; Fig 1, 2, 3 and 4). For this statistical analysis, Mycoplasma spp. wasn’t considered, as it had insufficient statistical data. These data were then statistical analysed to obtain, X² = 40.68; P value=0.00. There is strong statistical evidence suggesting a relationship between dogs suffering from haemoparasitic diseases and their tick infestation. Findings of this study are in accordance with Abd Rani et al., (2011), in India found a correlation revealing that the dogs infested with ticks were 3.3 times more likely to be PCR positive for one or more of the canine tick borne diseases than dogs without tick infestation, neutered dogs were 1.9 times less likely to be PCR positive compared to intact dogs. They also found that dogs from refuges were 2.3 times less likely to be PCR positive for canine TBDs than stray dogs. But in similar studies conducted by other workers found no significant co-relation (O’Dwyer et al., 2009; Hadi et al., 2016).

The tick infestation was recorded at 41% with Brown dog tick or Rhipicephalus sanguineus, being the only tick to have been found in this study, which could be due to the limitation of this tick to urban areas or due to our smaller area of work. Females (44.08%) were found to be more susceptible than males (37.84%). The study of incidence of haemoparasites in tick vectors using PCR assay indicates 57 dogs out of 100 were positive for tick infestation, of which 26 (45.61%) were positive for haemoparasites. This study conclusively provides evidence of high rates of incidence of haemoparasitic infection or canine TBD infection and tick infestation, with at least four haemoparasites infecting the dog population and at least one tick species (Rhipicephalus sanguineus) infesting the dogs in and around Kolkata.