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Comprehensive study of ticks and tick-borne diseases in dogs in Nepal: molecular identification, risk analysis and hematological alterations

BMC Veterinary Research volume 21, Article number: 309 (2025) Cite this article

Abstract

Background

Ticks are responsible for the transmission of various viral, bacterial, and protozoal pathogens through their mouthparts while feeding on the blood of the host. Owing to the increasing trend of tick-borne diseases, they are considered major emerging public health issues throughout the globe. In South Asia, the major important canine tick-borne pathogens are Babesia spp., Hepatozoon canis, Ehrlichia canis, Anaplasma platys, and Borrelia burgdorferi. Among various diagnostic tests, molecular techniques are considered the gold standard for the detection of tick-borne diseases. A total of 341 canine blood samples were collected from Kathmandu, Pokhara, and Chitwan, Nepal. The collected blood samples were subjected to hematological analysis, DNA extraction, and conventional polymerase chain reaction to detect the presence of tick-borne pathogens. Additionally, a total of 219 ticks were collected from the sampled dogs and identified via morphometry.

Results

PCR assays revealed four tick-borne pathogens, Babesia spp., Ehrlichia canis, Hepatozoon canis, and Anaplasma platys, with prevalence rates of 26.09%, 5.87%, 3.52%, and 2.93%, respectively, and an overall prevalence of 31.09% (95% CI: 26.27–36.34%). However, this study could not identify the prevalence of Borrelia burgdorferi. During the hematological analysis, anemia and thrombocytopenia in the sampled dogs were significantly associated with the presence of Babesia spp. (p < 0.01 and p < 0.05, respectively), lymphocytosis was significantly associated with Hepatozoon canis (p < 0.001), and thrombocytopenia was significantly associated with Ehrlichia canis (p < 0.05). Among the ticks infesting dogs at the study sites, the Rhipicephalus genus was the most prevalent, followed by Haemaphysalis and Dermacentor, with an overall tick infestation rate of 27.86%. Geographic location, type of dog (stray or pet), and body condition score were determined as potential risk factors by multiple logistic regression analysis (OR = 0.40, 2.16, 0.73; p < 0.01, p < 0.05, p < 0.05, respectively) for the presence of canine tick-borne pathogens.

Conclusions

This study identified at least four species of canine tick-borne pathogens and three genera in dogs from study area. Findings of this study highlights the importance of robust treatment, control and preventive measures to mitigate the transmission of these pathogens.

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Background

Ticks are considered vectors of worldwide veterinary and public health importance. Owing to their hematophagous nature, ticks are the second most efficient vectors for the transmission of various viral, bacterial, and protozoal pathogens after mosquitoes [1]. In addition to the transmission of diseases while feeding on the host, the secure attachment of ticks to the host contributes to the spread of microorganisms into different geographical areas with increasing animal transport and migration [2]. Additionally, the transstadial and transovarian transmission of pathogens within tick populations leads to the maintenance and increase of these pathogens in terms of geographical distribution and host coverage. Similarly, owing to the high fecundity and distribution of ticks, there is widespread dissemination of tick-borne diseases in animal and human populations in different geographical areas [3]. Tick-borne diseases in canine species are among the major emerging issues in veterinary medicine that may also affect public health. There is an increasing trend in the incidence of tick-borne diseases in canines in tropical, subtropical, temperate, and urban areas. This increased incidence of canine tick-borne diseases threatens human infections, as dogs act as reservoir hosts for these diseases. The major tick-borne diseases of dogs in the South Asian region are babesiosis (B. gibsoni, B. vogeli); hepatozoonosis (Hepatozoon canis); ehrlichiosis (Ehrlichia canis); borrelosis (Borrelia garinii, B. afzelii, Borrelia burgdorferi); and anaplasmosis (Anaplasma platys) [2, 4].

Tick-borne pathogens produce various clinical presentations, including fever and life-threatening illness, which depend upon the characteristics of the pathogens, their coinfection, and host-related factors [5]. One of the most important clinical aspects of canine tick-borne infection is direct and/or indirect effects on hematological indices [6,7,8]. However, these profound clinical findings and hematological changes are not specific to different etiological agents, which limits their use in exact diagnosis [9]. Thin blood smear examination under a microscope is the major way to diagnose canine tick-borne diseases in veterinary hospitals and clinics in the Indian subcontinent, including Nepal [10]. The use of serological tests for the diagnosis of tick-borne pathogens has major limitations due to the high number of false positive [11] and false negative results [12]. Owing to the lack of proper tools for the species-specific diagnosis of canine tick-borne pathogens and their variation in pathogenicity, the use of molecular diagnostic techniques is the most efficient tool for investigating tick-borne diseases. Molecular techniques such as polymerase chain reaction (PCR) have high levels of sensitivity, specificity, and precision in the detection of tick-borne pathogens [13].

In the context of Nepal, rabies is the major public health issue related to the dog population [14]. Various efforts and policies are in place for the control of rabies in humans transmitted via dog bites in Nepal. However, information and research on the prevalence and epidemiology of canine tick-borne diseases are generally neglected or are less explored in the present conditions of Nepal [15]. Díaz-Regañón and his colleagues explored the prevalence of canine vector-borne pathogens via real-time PCR in Kathmandu Metropolitan City [16]. In that research, a total of 81.43% of the dogs were positive for infection, with at least one or more vector-borne pathogens investigated. Similarly, another study by Abd Rani et al. reported a 49.7% prevalence of canine tick-borne diseases in India [4]. There is a lack of information about canine tick-borne disease prevalence and risk factors in pet and stray dogs in Nepal. Hence, this study aims to perform a comprehensive investigation of major ticks and the prevalence of canine tick-borne pathogens in stray and pet dogs of Kathmandu, Pokhara and Chitwan along with associated risk factors and hematological alterations.

Materials and methods

Ethical statement

The Institutional Review Board (IRB) of the Directorate of Research & Extension (DoREX), Agriculture and Forestry University approved the ethical statement for this study as follows: Ref: Protocol #2023–001.

Study area

The research project was carried out in three major cities of Nepal, namely, Kathmandu, Pokhara, and Chitwan, which are located geographically in the lesser Himalayan region, Himalayan region, and Central Plain region of Nepal, respectively as shown in Fig. 1. Chitwan is located at 27°40’59.99"N, 84°25’59.99"E and coordinates with a mean altitude of 208 masl (meters above sea level). Similarly, Kathmandu is located at 27°42’6.08"N, 85°19’14.16"E, with a mean altitude of 1400 masl and Pokhara is located at 28°16’0.8"N, 83°58’6.64"E, with a mean altitude of 822 masl. All 3 cities have both pet and stray dog populations, which constitute the major portion of canine species in Nepal. The exact populations of pets and stray dogs have not yet been documented in these cities.

Fig. 1
figure 1

Study area and sampling sites for blood and ticks from canines

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Sample collection

Dogs were restrained while considering the animal welfare standards, and no harm was produced to the dogs. Informed verbal consent was obtained from dog owners before the sample collection in accordance with the ethical statement. After proper restraint, a 2 ml venous blood sample was collected from the saphenous or cephalic vein of the stray and pet dogs in Kathmandu, Pokhara and Chitwan into sterile K3EDTA tubes. A total of 341 samples of venous blood from each individual dog were collected from 1st May 2023 to 15th January 2024. The blood samples were stored at 4°C in an icebox until arrival at the laboratory and were transported to the laboratory within 24 h. The blood samples were transferred to a − 20°C refrigerator until the DNA was extracted. The age of the dogs was determined either by interviewing the owner or by observing the dentition pattern based on previously established procedure [17]. The dogs were divided into puppies, juveniles, adults, and geriatrics according to age: younger than 6 months, 6 months to 1 year, 1 year to 7 years, and older than 7 years, respectively. The body condition score of each dog was assessed via a 9-integer scale according to the established procedure [18]. If there were any ticks present while searching for more than 1 min per dog, 2 to 5 tick samples were collected from tick-infested dogs and preserved in 70% ethanol. The common areas from where ticks were collected were ear, neck, body, under the legs, head, and abdominal areas. The collected ticks were identified on the basis of the morphological features with the help of a stereo-zoom microscope at 4X and 10X magnification [19, 20].

Blood parameter estimation

A complete blood count of the collected sample was performed via a Spincell 3 Automatic Hematology Analyzer Ref. 5,006,101 at the Center for Biotechnology, Agriculture and Forestry University. A thin blood smear was prepared for each sample, and differential leucocyte counting was performed manually via the Giemsa staining protocol, followed by observation of the blood smear under a microscope at 100X oil immersion magnification at the Faculty of Animal Science, Veterinary Science and Fisheries, Department of Microbiology and Parasitology, Agriculture and Forestry University.

DNA extraction and quantification

DNA extraction from blood was performed using Gene Direx Genomic DNA Isolation Kit (Blood/Cultured Cell/Fungus) (Cat. No. NA023–0100) according to the manufacturer’s instructions. All the obtained DNA samples were stored at -80°C until use. The purity and concentration of extracted DNA was measured using Multiskan Sky (ESW version 1.00.58 Serial number 1530–801345 C) spectrophotometer.

PCR assay

Conventional PCR amplification was conducted to target and identify selected species, including babesiosis (Babesia spp.), hepatozoonosis (Hepatozoon canis), ehrlichiosis (Ehrlichia canis), borreliosis (Borrelia burgdorferi), and anaplasmosis (Anaplasma platys) using the specific primers in mentioned in Table 1.

Table 1 Primer sets for PCR amplification of canine tick-borne diseases
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For Babesia spp. and E. canis, a 25 μl reaction mixture was prepared by mixing 100 ng of extracted gDNA, 12.5 pmol of each primer, 0.625 U of Taq DNA polymerase, 400 μM dATP, dCTP, dGTP, dTTP, and 4 mM MgCl2 (12.5 μl of Thermo Scientific™ PCR Master Mix (2X) Cat no. K0172) with nuclease-free water. Similarly, for H. canis, the reaction mixture contained 5 μl of extracted gDNA and 25 pmol of each primer. For A. platys, 5 μl of extracted gDNA and 20 pmol of each primer were used. Similarly, for Borrelia burgdorferi, 5 μl of extracted gDNA and 10 pmol of each primer were used for the reaction mixture. PCR amplification was performed in a Bio-Rad T100™ Thermal Cycler (Bio-Rad, USA). The reaction mixture was subjected to PCR according to the conditions mentioned in Table 2 for each species.

Table 2 PCR conditions for each pathogens investigated
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Gel electrophoresis

The obtained PCR products were run on 2% agarose gels in Tris acetic EDTA (TAE) buffer at 100 V and 100 A for 60 min. Thus, the obtained gel was visualized via a UVITEC CAMBRIDGE gel documentation system. The PCR product length of the positive sample was visualized with the help of the GeneRuler 100 bp DNA Ladder (Cat no. SM0242). Each PCR run was regarded as positive after the demonstration of a PCR product band corresponding to the amplicon size shown in Table 1.

Statistical analysis

Chi-square tests or Fisher’s exact tests were used to analyze the relationships between canine tick-borne pathogens and the descriptive data of the dogs. The continuous data were analyzed with two sample t test with welch’s correction where appropriate. The prevalence rate was calculated for the PCR result of each canine tick-borne disease. A confidence interval of 95% was used to determine the significance of the statistical test. The odds ratio with a 95% confidence interval was calculated via univariable regression analysis of each risk factor. The risk factors were analyzed via multiple logistic regression analysis, and risk factors that were significant at p < 0.25 were considered for multiple regression. Stepwise forward multiple regression analysis was performed based on the lowest value of the Akaike information criterion (AIC) to identify the significant risk factors. Multiple collinearities were assessed via variance inflation factors (VIFs) and collinearity analysis.

Results

Dog population description

A total of 341 dogs were sampled during this study, which consisted of 131 blood samples from Chitwan, 127 blood samples from Kathmandu and 83 blood samples from Pokhara. The Table 3 summarizes the descriptives associated with the samples which consists of 162 (47.51%) male dogs and 179 (52.49%) female dogs, among which 54.32% (88) were castrated and 59.22% (106) were spayed. Among the 341 dogs, 76.83% (262) were stray, and the remaining 23.17% (79) were pets. Based on their external appearance, 81.23% (277) of the dogs were non-descriptive local breeds, whereas 11.44% (39) were Japanese Spitz, 3.81% (13) were German Shepherd, 1.17% (4) were Labrador Retriever, 0.88% (3) were Cocker Spaniel, 0.59% (2) were Golden Retriever, and 0.29% (1) were Boxer, Husky & Pug each.

The average body condition score of the sampled dogs was found to be 4.07 ± 1.22. The age of the dogs, as determined by their dentition pattern, was as follows: adults (64.52%), followed by geriatrics (17.01%), juveniles (15.25%), and puppies (3.23%). At the time of sample collection, only 17.30% (59 out of 341) of the dogs had various clinical signs on clinical examination. The clinical signs include skin infection, paralysis, epistaxis, wounds, ocular discharge, coughing, cloudy eyes, and other nonspecific signs.

Table 3 Descriptive statistics of the sample population
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Distribution and identification of tick genera

On external examination, 27.86% of the dogs were infested with ticks. In Chitwan, 58.02% of the dogs were infested with ticks, whereas dogs in Kathmandu and Pokhara presented significantly lower infestation rates of 11.81% and 4.82%, respectively (p < 0.000). The tick infestation rates among pets and stray dogs were 22.78% and 29.39%, respectively, with no significant difference. Among the 219 ticks obtained from the sampled dog population, Rhipicephalus was the most common dog tick (80.37%) present in this study, followed by the genera Haemaphysalis (18.26%) and Dermacentor (1.37%). The sex ratios of males to females were 0.93 and 0.14 for the Rhipicephalus and Haemaphysalis genera, respectively. The most common tick species found in dogs was Rhipicephalus sanguineus (71.23%), followed by Haemaphysalis leachi (7.31%), as shown in the Table 4. A pictorial representation of the ticks is given in the Fig. 2.

Table 4 Distribution and prevalence of different tick species infesting dogs in the study area
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Fig. 2
figure 2

Morphological identification of collected ticks. Dermacentor spp. Male (A-B), Rhipicephalus spp. Male (C-D), and Haemaphysalis spp. Female (E-F)

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PCR assay results

The PCR results revealed that 106 (31.09%, 95% CI: 26.27–36.34%) dogs were positive for at least one canine tick-borne pathogen investigated. PCR results, as visualized in Fig. 3 and Fig. 4, revealed that the prevalence of Babesia spp. was 26.09%, followed by Ehrlichia canis, Hepatozoon canis and Anaplasma platys at 5.87%, 3.52%,,and 2.93%, respectively as shown in Table 5. The conventional PCR assay did not detect the presence of Borrelia burgdorferi at any location. Coinfection with the different canine tick-borne pathogens investigated was also found in lower proportions, with the most common coinfection being Babesia and E. canis infection (1.76%). No dog tested positive for all the pathogens investigated.

Fig. 3
figure 3

Babesia spp. (A) (800 bp) and Hepatozoon canis (B) (666 bp) amplicons visualized by agarose gel electrophoresis

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Fig. 4
figure 4

Ehrlichia canis (C) (400 bp) and Anaplasma platys (D) (678 bp) amplicons visualized by agarose gel electrophoresis

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Table 5 Canine tick-borne pathogens and their co-infection rate in Nepal with 95% confidence interval (lower, upper intervals) abbreviation: CI, confidence interval
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The Table 6 describes the prevalence of each canine tick-borne pathogens investigated based on the sampling location. The prevalence of Babesia spp. was significantly greater in Chitwan than in other locations. A similar trend was shown by H. canis and A. platys infection. Interestingly, infection with H. canis and A. platys in Kathmandu was not detected in the sampled dogs. The prevalence of E. canis was not significantly different among the three locations.

Table 6 Prevalence of canine tick-borne pathogens in the sampling location. NS = non-significant, *= significant at p < 0.05; **= significant at p < 0.01, ***=significant at p < 0.001
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Hematological analysis

Hematological analysis of the blood revealed a significantly lower RBC count (5.18 ± 1.67^106/ml) in the Babesia-infected dogs (t = 2.11, p < 0.05) than in the Babesia-negative dogs (5.62 ± 1.84^106/ml), as shown in the Table 7. Similarly, H. canis infection significantly decreased the platelet count (129.42 ± 59.99^103/ml) and significantly increased the mean platelet volume (9.36 ± 1.04 fL) in H. canis-infected dogs compared with H. canis-negative dogs (t = 2.46, -2.18; p < 0.05). The haemoglobin content was significantly lower in Babesia-infected dogs (14.52 ± 4.89 g/dl, t = 2.90, p < 0.01) and dogs infected with at least one canine tick-borne pathogen (14.87 ± 4.73 g/dl, t = 2.45, p < 0.05). Similarly, E. canis-infected dogs had significantly lower eosinophil counts (0.71 ± 0.59^103/ml) than did E. canis-negative dogs (t = 2.59, p < 0.05).

Table 7 Mean values of hematological parameter of dogs infected with canine tick-borne pathogens *= significant at p < 0.05; **= significant at p < 0.01, ***=significant at p < 0.001, abbreviations: WBC, white blood cells; RBC, red blood cells; PLT, platelet; HGB, hemoglobin; HCT, hematocrit; RDW, red cell distribution; MPV, mean platelet volume; lymph, lymphocyte; mono, monocyte; Eosino, eosinophil; Neutro, neutrophil; Baso, basophils
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The mean WBC counts of H. canis and E. canis infected dogs were found to be higher than the normal range. Similarly, the infection by canine tick-borne pathogen decreased the mean RBC counts, platelet counts, and hematocrits below the normal range, whereas the mean neutrophil counts were increased above the normal range.

The Table 8 describes the abnormal hematological parameters of sampled dogs infected with different canine tick-borne pathogen investigated in this study. A total of 57.30% of the dogs with canine babesiosis presented low RBC counts, and 78.65% of the dogs presented low hematocrit values, with significant differences (p < 0.05 and p < 0.01, respectively). Similar findings revealed that 78.65% of the Babesia-infected dogs presented thrombocytopenia (p < 0.05), but only 19.10% of the dogs presented an increase in the MPV. Other interesting findings associated with canine babesiosis were leukocytosis (47.19%), eosinophilia (31.36%), and neutrophilia (50.56%), which were not significantly different. Dogs infected with the Hepatozoon canis showed a significant increase in lymphocyte count (p < 0.001). 58.33% of the Hepatozoon canis infected dogs showed increased lymphocyte counts. Similarly, Hepatozoon canis infection was associated with anemia in 66.67% of infected dogs, thrombocytopenia in 83.33% of infected dogs, and neutrophilia in 58.33% of dogs, but this difference was not statistically significant. In terms of E. canis infection, 65.00% of the infected dogs suffered from thrombocytopenia, with a statistically significant difference (p < 0.05). 70% of dogs with canine ehrlichiosis presented anemia, but the difference was not statistically significant. Anaplasma platys infection in 80.00% of the dogs was associated with thrombocytopenia, but the difference was not statistically significant.

Table 8 Interpretation of abnormal hematological parameter in dogs infected with canine tick-borne pathogens; *= significant at p < 0.05; **= significant at p < 0.01, ***=significant at p < 0.001, abbreviations: WBC, white blood cells; RBC, red blood cells; PLT, platelet; HGB, hemoglobin; HCT, hematocrit; RDW, red cell distribution; MPV, mean platelet volume; lymph, lymphocyte; mono, monocyte; Eosino, eosinophil; Neutro, neutrophil; Baso, basophils
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Risk factor analysis

Univariable logistic regression analysis, as shown in Table 9, revealed that the odds of males being infected with A. platys were significantly lower than those of females (OR = 0.12, p < 0.05). Similarly, geriatric dogs had a greater chance of Babesia infection (OR- 1.88, p < 0.05). Tick infestations were associated with greater odds of having Babesia, E. canis or H. canis. (OR = 3.13, p < 0.00; OR = 2.78, p < 0.05; OR = 3.83, p < 0.05, respectively). Compared with pet dogs, stray dogs were 2.07 times more likely to have babesiosis (p < 0.05). Tick infestation appeared to be a potential risk factor, as it increased the odds of having canine tick-borne diseases by 2.91 times (p < 0.001).

Table 9 Risk factors associated with canine tick-borne diseases with univariable logistic regression analysis, *= significant at p < 0.05; **= significant at p < 0.01, ***=significant at p < 0.001, abbreviations: CI, confidence interval; OR, odds ratio
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Multiple logistic regression analysis of the risk factors associated with at least one or more canine tick-borne disease infections investigated (R-squared = 0.119) revealed that location, type of dog, and BCS had significant association with canine tick-borne pathogen infection. Dogs from Kathmandu showed a 2.52 times lower chance of infection by canine tick-borne pathogens as compared to other study area (OR = 0.40, 95% CI: 0.20–0.77, p = 0.006). Similarly, the stray dogs had 2.16 times more chance of infection as compared to the pet dogs (95% CI: 1.13–4.25, p = 0.022). Similarly, a unit increase in the BCS of a dog would decrease its chance of being infected with canine tick-borne pathogens by 0.73 time (95% CI: 0.57–0.92, p = 0.010). Similarly, multiple logistic regression analysis of association of risk factors with Babesia infection revealed age of dogs, tick infestation and castration had signification association (R-squared = 0.171). The geriatric dogs were 3.22 times more likely to acquire Babesia infection (95% CI: 1.27–8.39, p = 0.015), tick infestation increases the odds of having Babesia infection by 3.53 times (95% CI: 1.48–8.62, p = 0.005), and castration of male dogs increases the odds of having Babesia infection by 2.77 times (OR = 2.77, 95% CI: 1.16–7.06, p = 0.026). Multiple logistic regression analysis of association of different risk factors with Hepatozoon canis, Ehrlichia canis, and Anaplasma platys did not reveal any significant relationships.

Discussion

The prevalence of canine tick-borne pathogens in this study was high among pet and stray dog populations in Nepal. This finding suggests that canine tick-borne diseases circulate among stray and pet dog populations more frequently in Nepal. A previous study of prevalence of vector-borne pathogen in stray dogs of Kathmandu showed overall prevalence of 81.43% which was higher than the overall prevalence of canine tick-borne pathogens in this study [16]. The higher prevalence in this previous study is due to difference in sampling technique where only stray dogs were sampled, and among the sampled dogs, 65.71% presented clinical signs related to canine tick-borne diseases [16]. In contrast, our study included both pet and stray dogs, and only 17.30% of the dogs presented clinical signs that were not specific to canine tick-borne diseases. Similar studies in India reported overall prevalence rates of 49.7% [4], 52.00% [21], and 67.8% [22], which are comparable with the findings of this study when we consider differences in the climatic conditions of India compared with Nepal.

This study revealed Babesia spp. as the most prevalent canine tick-borne pathogen in the studied dog population, which is similar to the findings of previous studies in Northeast India [21] and Punjab, India [23], but in contrast with the other studies in India [4] and Kathmandu, Nepal [16], where H. canis was reported as the most prevalent pathogen. The prevalence of Babesia in this study is lower than the prevalence of Babesia/Theileria spp. (28.57%) in Kathmandu [16], which can be explained by the difference in the sampling population, as the samples from that study had only stray dogs, with a greater percentage of dogs showing clinical signs [16]. More previous studies of Babesia spp. in dogs reported a prevalence of 57.5% in Kerala, India [24], 56.75% in Assam [25], and 43% of B. gibsoni & 3% of B. vogeli in Northeast India [21]. As India has a tropical environment, the higher infection rate of canine babesiosis can be attributed to the more suitable environmental conditions for pathogen and tick infestations. Previous study in Shenzhen, China showed 11.0% of pet dogs tested positive for canine babesiosis [26]. This discrepancy in the prevalence may be attributed to the sampling of only pet dogs as pet dogs typically receive better veterinary care and have lower exposure to infective agents than stray dogs do.

Similarly, the prevalence of H. canis was lower than the previous report from Kathmandu (31.43%) [16], and was higher than the previous from Punjab, India (0.26%) [23]. Similarly, prevalence of H. canis in this study was lower as compared to other reports from Tamil Nadu [22], Northeast India [21], and Punjab [27], where it was reported as of 37.80%, 38.00%, and 13.78% respectively. The difference of H. canis prevalence as compared to the previous reports may be due to differences in the climatic conditions of the study areas, sample dog populations and tick infestation rates. The prevalence of E. canis in this study was lower than the previously reported prevalence from Kathmandu [16] but higher than that reported from Punjab (0.39%) [23] and Myanmar (0.75%) [28]. Similar studies have reported that the prevalence of E. canis is 16.1% in Tamil Nadu [22], 30% in Haryana [29], and 5.8% in India [30]. The findings from India [30] were similar to the findings of this study, whereas the findings from Haryana and Tamil Nadu revealed a higher prevalence of E. canis, which may be attributed to differences in geographical location, climatic conditions and sample dog populations. The prevalence of A. platys in this survey was much lower than that reported in Kathmandu [16]. The findings of this study were similar to the prevalence of A. platys reported in Northeast India (3%) [21], and was higher than the prevalence reported in Myanmar (0.25%) [28], and lower than the prevalence reported in Tamil Nadu (22.6%) [22]. This may be due to differences in geoclimatic conditions. In this study, we could not detect Borrelia burgdorferi, which is supported by a study from Italy in which only one dog (0.3%) was found to be positive via real-time PCR [31]. A study in Greece reported 0.1% seroprevalence for (B) burgdorferi [32], and only one dog (1.67%) was positive for B. burgdorferi infection in a study in Egypt [33], supporting our findings.

The canine-tick borne pathogens included in this study have a significant impact on different hematological parameters according to several studies. We compared the different hematological parameters of the sampled dogs with respect to each pathogen we studied. In this study, Babesia-positive dogs presented significant reductions in erythrocyte count, hematocrit, and hemoglobin levels. Different previous studies revealed that anemia was associated with Babesia spp. infection in dogs, which supports our findings in which 70.79% of infected dogs experienced anemia [8, 34,35,36]. The pathogenesis of anemia in Babesia-positive dogs is attributed to three main mechanisms that cause hemolysis, namely, mechanical disruption of the RBC structure, which leads to increased osmotic fragility; immune-mediated hemolysis of infected RBCs; and the production of hemolytic factors by the parasite [35,36,37]. Similarly, thrombocytopenia was also associated with Babesia-infected dogs, which was supported by reports in which 99.5% & 86.03% of infected dogs showed thrombocytopenia [34, 38]. The thrombocytopenia in dogs is caused by immune-mediated destruction of platelets which explains a high incidence of thrombocytopenia in canine babesiosis [35]. Similarly, eosinophilia among Babesia-infected dogs is supported by similar findings in previous studies [8, 39], which may be caused due to host immune response to tick the infestation. Similarly, the finding of leukocytosis among Babesia-infected dogs in this study aligns with the report from Warsaw, Poland in which 14.9% of infected dogs exhibited leukocytosis [38], but it contrasts with few other previous reports where there was significant association of leukopenia with the Babesia spp. infection [8, 34, 35]. Leukocytosis may suggest chronicity associated with Babesia infection because one of the most prevalent Babesia species known as Babesia gibsoni in dogs is regarded as a chronic infection.

Platelet counts and mean platelet volume showed a significant decrease when the dogs were positive for H. canis infection similar to the few previous findings in Thailand and Brazil [8, 34]. This finding suggests that following the decrease of platelet count due to H. canis infection, there is a bone marrow response in infected dogs to replenish the decreased platelets evident by increased mean platelet volume. H. canis-infected dogs showed leukocytosis, anemia and neutrophilia, as reported by several studies in Thailand, Brazil and India [8, 34, 40]. The leukocytosis in H. canis-infected dogs is due to the inflammatory response of the dogs towards the entry and invasion by H. canis. The anemia in H. canis-infected dogs is due to the result of bone marrow depression or immune-mediated hemolysis induced by H. canis whereas the neutrophilia can be described as a primary response from the immune system of the host towards the H. canis.

In this study, thrombocytopenia was associated with the E. canis-infection in dogs, which is a typical finding in clinical and subclinical ehrlichiosis, in accordance with the previous findings in Thailand and Indonesia where 90.6% & 82.3% of E. canis-infected dogs presented with thrombocytopenia [8, 41]. The platelet destruction or decrease in case of E. canis infection is attributed to the acute phase of infection, during which the inflammation process causes increased platelet utilization [42]. Similarly, anemia was associated with 70% of E. canis-infected dogs, which is similar to the previous findings in Thailand and Indonesia [8, 41]. The cause of anemia in E. canis infected dogs is due to either antibody production against surface RBC molecules or immune-mediated hemolytic anemia or bone marrow dysfunction [8, 43]. The increase in the mean WBC count in E. canis-infected dogs was similar to the study from Thailand [8], but in contrast with another study from Indonesia where leukopenia was reported to be associated with E. canis-infection [41]. Similarly, this study revealed an association between neutrophilia and E. canis infection, which is similar to the findings from India [44] but in contrast to the study from Indonesia [41]. Inflammation during the acute or subacute stage of infection by E. canis may be responsible for neutrophilia. A. platys infection showed a decreasing trend in the mean RBC count, mean hematocrit, and mean platelet count and an increasing trend in the mean neutrophil count without significance, which is similar to the findings of previous study in which there was thrombocytopenia in 81.0% of dogs, anemia in 81.0% of dogs, and leukocytosis in 33.33% of dogs [45].

The rate of tick infestation in dogs was higher in Chitwan as compared to Kathmandu & Pokhara. The modified Koppen-Geiger climate classification described the enviroment of Chitwan as tropical environment [46] which favors the growth and maintenance of ticks, increasing the infestation rate. Similarly, the prevalence of canine tick-borne diseases was significantly greater in Chitwan than in Pokhara and Kathmandu. This could also be explained by the favorable climatic conditions for the causative agent, with a relatively high tick infestation rate in Chitwan. This study showed Rhipicephalus spp. as most prevalent tick in dogs which is similar to the studies from Sri Lanka and India where R. sanguineus is the most prevalent tick species in dogs [4, 47]. The second most prevalent ticks in dogs from study area was identified as Haemaphysalis spp. which is supported by the studies where Haemaphysalis longicornis and H. leachi was reported to infest the dogs [48, 49]. The identification of Dermacentor spp. from Nepal is supported by a study in the Hindkush Mountain range in which Dermacentor spp. were identified by molecular methods [50] and by a study in Pakistan, India and Bangladesh, who reported the prevalence of Dermacentor genera across different species [51].

In this study, tick infestation in dogs was associated with increased odds of Babesia spp, E. canis, and H. canis infection which is similar to previous findings [4, 29, 44]. This may be due to increased exposure to the causative factors through tick bites because Rhipicephalus sanguineus acts as the vector of Babesia, H. canis & E. canis, which was found to be the most prevalent tick in dogs in the study area. Similarly, the Geriatric dogs and castrated male dogs were found to be more prone to Babesia spp. Higher prevalence of Babesia spp. in geriatric dogs is may be due to deterioration in the immune status [52]. The high rate of Babesia spp. infection in castrated males is may be due to changes in the hormonal status of the dogs [53]. This study showed a higher prevalence of A. platys in female dogs which may be associated with a decrease in immunity due to stress during reproductive activities or due to increased exposure to ticks during their movement for mating [54]. Again, The lower incidence of canine tick-borne pathogens in the temperate area like Kathmandu as compared to tropical area like Chitwan found in this study was in accordance with a study in India, where the incidence of infection was greater in the tropical part of the study area [4]. A lower body condition score is associated with a deterioration in the normal physiological response of dogs, which may increase the chance of being infected with canine tick-borne pathogens. This study showed stray dogs were more susceptible to canine tick-borne pathogens due to the greater exposure of stray dogs to causative agents and vectors, a lack of veterinary care and proper nutrition, which is similar to previous study in India [4].

Conclusion

This study is one of the first attempts to investigate the prevalence of canine tick-borne pathogens in both pet and stray populations in Nepal using molecular tests. This study identified at least four species of canine tick-borne pathogens, namely, Babesia spp., Hepatozoon canis, Ehrlichia canis and Anaplasma platys, in pets and stray dogs of Nepal. Additionally, at least three tick genera was identified from dogs in Nepal. Babesia spp. was the most prevalent canine tick-borne pathogen in study area and Rhipicephalus spp. the most prevalent tick in dogs in the study area. Similarly, this study showed association between Babesia spp. infection with anemia & thrombocytopenia, H. canis infection with lymphocytosis, and E. canis with thrombocytopenia. Similarly, the risk factors associated with tick-borne pathogen infection were identified as geographic location, type of dog, and body condition score. This study represents a step toward more robust and systematic future investigations of canine tick-borne and vector-borne diseases in Nepal. A proper vector control and disease eradication strategy should be formulated and implemented to avoid the transmission of canine tick-borne diseases. Proper population awareness, vaccination campaigns, parasite control, and birth control camps would help minimize the risk of canine babesiosis in stray and pet dogs. Further research with increased geographical reach and larger numbers of samples would help to obtain a more comprehensive understanding of important canine tick-borne diseases in Nepal.

Limitations

This study highlighted epidemiological importance and molecular identification canine-tick borne pathogens. This study couldn’t perform sequencing and phylogenetic analysis of identified canine tick-borne pathogens due to limited resources. The molecular identification of tick species would have helped to draw more valuable conclusions. Also, the study area could have been expanded to the western and eastern regions of Nepal to give a comprehensive representation of whole country’s dog population.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

%:

Percent

<:

Less Than

>:

Greater Than

AIC:

Akaike Information Criterion

bp:

Base Pair

CI:

Confidence Interval

dATP:

Deoxyadenosine Triphosphate

dCTP:

Deoxycytidine Triphosphate

dGTP:

Deoxyguanosine Triphosphate

dTTP:

Deoxythymidine Triphosphate

DNA:

Deoxyribonucleic Acid

DoREX:

Directorate of Research and Extension

HCT:

Hematocrit

HGB:

Hemoglobin

IRB:

Institutional Review Board

K3EDTA:

Tripotassium Ethylenediaminetetraacetic Acid

Masl:

Meters Above Sea Level

MPV:

Mean Platelet Volume

PCR:

Polymerase Chain Reaction

PLT:

Platelet Count

RBC:

Red Blood Cell

RDW:

Red Cell Distribution Width

TAE:

Tris–Acetate–EDTA Buffer

VIFs:

Variance Inflation Factors

WBC:

White Blood Cell

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Acknowledgements

The authors gratefully acknowledge the receipt of a Michael J. Day Scholarship for this project. This scholarship is administered by the World Small Animal Veterinary Association and generously funded by MSD Animal Health. Special thanks to the Himalayan Animal Rescue Trust (HART), Animal Nepal, Sneha’s Care, Veterinary Teaching Hospital (VTH), and Veterinary Hospital. and Livestock Service Expert Center (VHLSEC) Chitwan & Pokhara for assisting in sample collection.

Funding

Michael J. Day Scholarship by the World Small Animal Veterinary Association and generously funded by MSD Animal Health. The funding body only supported financially for this study.

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  1. Faculty of Animal Science, Veterinary Science and Fisheries, Agriculture and Forestry University, Rampur, 44209, Chitwan, Nepal

    Somnath Aryal, Rebanta Kumar Bhattarai & Kamana Thapa

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Contributions

SA performed conceptualization, experimental design, methodology, formal analysis, investigation, visualization, data curation, data analysis, writing- original draft, writing – review & editing, funding acquisition. RB performed supervision, conceptualization, resources, project administration, data analysis, writing – review & editing. KT performed methodology, data curation, investigation, visualization, writing – original draft. All authors read and approved the final manuscript.

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Correspondence to Somnath Aryal.

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The Institutional Review Board (IRB) of the Directorate of Research & Extension (DoREX), Agriculture and Forestry University approved the ethical statement for this study as follows: Ref: Protocol #2023–001.

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Aryal, S., Bhattarai, R.K. & Thapa, K. Comprehensive study of ticks and tick-borne diseases in dogs in Nepal: molecular identification, risk analysis and hematological alterations. BMC Vet Res 21, 309 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04777-x

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