Abstract
Background
Canine babesiosis is a significant and emerging tick-borne disease caused by Babesia spp. This study investigated the molecular prevalence, hematobiochemical profiles, and risk factors for B. vogeli and B. gibsoni infections in dogs in northern India, addressing the molecular research on babesiosis prevalence in this region.
Results
An overall prevalence of canine babesiosis by microscopy and PCR was 12.5% (27/216) and 27.78% (60/216), respectively. The prevalence rates for B. vogeli by microscopy and conventional PCR were 10.64% and 22.22%, respectively, whereas the corresponding rates for B. gibsoni were 1.85% and 5.55%, respectively. Results revealed that B. vogeli was the predominant species. Non-descript dogs were significantly more susceptible to B. vogeli infection (OR: 27.491; 95% CI: 1.092–692.018; P < 0.05). Additionally, dogs with enlarged lymph nodes were three times more likely to be B. vogeli-positive (OR = 3.338; 95% CI: 1.345–8.287; P < 0.05). Dogs under one year had higher odds of B. vogeli infection, but this was not statistically significant (OR = 1.765; 95% CI: 0.663–4.703). Similarly, B. gibsoni infection was associated with dogs over four years old (P < 0.001) and males (P < 0.05). Vital parameters (temperature, pulse rate, and respiration rate) showed no significant association with B. vogeli and B. gibsoni infections. Hematological parameters, viz., hemoglobin (Hb), total erythrocyte count (TEC), packed cell volume (PCV), and platelet count, were significantly decreased in both B. vogeli and B. gibsoni affected dogs (P < 0.05). Babesia gibsoni-infected dogs had significantly lower TEC, PCV, and platelet count than B. vogeli-infected dogs. Biochemical parameters, viz., alanine aminotransferase (ALT), total bilirubin (TB), direct bilirubin (DB), indirect bilirubin (IDB), and blood urea nitrogen (BUN), were significantly increased, with decreased albumin levels (P < 0.05). Babesia gibsoni-infected dogs had significantly higher BUN, TB, and IDB levels than B. vogeli-infected dogs. Treatment was based on the Babesia species, with imidocarb dipropionate for B. vogeli and a combination therapy (diminazene aceturate, imidocarb dipropionate, and clindamycin) for B. gibsoni, which showed promising efficacy.
Conclusions
The study highlighted the importance of accurate diagnosis and species-specific treatment for canine babesiosis, with B. vogeli being more prevalent (22.22%) than B. gibsoni (5.55%), and distinct risk factors and clinical implications for each Babesia species in northern India. These findings contribute to the understanding of Babesia epidemiology in the region and highlight the need for further studies to inform the development of targeted control strategies.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s12917-026-05657-8.
Keywords: Molecular prevalence, Canine babesiosis, Risk factors, Hematological, Biochemical, Treatment
Background
Canine babesiosis is a prevalent haemoprotozoan disease that can lead to severe clinical symptoms in dogs, caused by several Babesia species [1]. To date, eight Babesia species have been reported in dogs, namely Babesia canis, B. vogeli, B. rossi, B. coco, B. negevi, B. gibsoni, B. conradae, and B. vulpes. Based on morphology, B. canis, B. vogeli, B. rossi, and B. coco are classified as large Babesia, whereas B. gibsoni, B. conradae, and B. vulpes are considered small Babesia. Babesia negevi, described recently, exhibits intermediate morphology and does not fit clearly into either group [2]. In India, B. vogeli and B. gibsoni are the primary species responsible for the disease [3–5]. Factors such as international travel with dogs, climate change, and ecosystem alterations have facilitated the transcontinental spread of Babesia species [6, 7]. India's diverse climate and large stray dog population create an ideal environment for tick survival and disease transmission [8].
Within the vertebrate host, Babesia spp. undergo merogony within erythrocytes, causing hemolytic anemia through both direct parasitic damage and host-mediated mechanisms. Red blood cell destruction results from erythrophagocytosis, oxidative membrane damage, complement activation, and immune-mediated cytotoxicity affecting both infected and uninfected erythrocytes [9, 10]. Clinically, affected dogs commonly exhibit fever, pale mucous membranes, lymphadenopathy, lethargy, tachypnea, hemoglobinuria, and icterus. In severe cases, canine babesiosis may trigger systemic inflammatory response syndrome (SIRS), progressing to severe sepsis, septic shock, and multiple organ dysfunction [11]. Disease severity is influenced by the infecting Babesia species, parasite burden, host age, breed, immune status, and concurrent infections [1].
Canine babesiosis has been studied sporadically in various regions of India, primarily among stray dogs and pet dogs attending veterinary clinics [12–21]. Few reports are available on the association between risk factors and the occurrence of canine babesiosis [22–26]. The most common hematological abnormalities associated with canine babesiosis include anemia, thrombocytopenia, leukopenia, and neutropenia, although their severity varies with the infecting Babesia species and disease stage [7, 18, 27]. Similarly, the common biochemical abnormalities include hyperbilirubinemia, hypoalbuminemia, and azotemia, characterized by increased serum urea and creatinine concentrations, particularly in severe disease and cases with renal dysfunction [28].
Diagnosis of canine babesiosis typically relies on microscopic examination of stained blood smears, but this method may miss low-level infections [12, 16, 19]. Serological tests, such as indirect immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) offer high sensitivity but moderate specificity due to cross-reactions. Polymerase chain reaction (PCR)-based assays have improved detection, offering high sensitivity and specificity for accurate species identification. Nuclear markers are used for species-level diagnosis due to their conserved nature [29], while mitochondrial markers are used for studying genetic diversity and phylogenetics due to greater sequence variability [30].
This research aimed to bridge the knowledge gaps in canine babesiosis in the North Indian states, where comprehensive studies on clinical observations, molecular prevalence, and therapeutic management are scarce despite the disease's prevalence. The lack of sufficient data on risk factors and their association with the disease hinders the development of effective control strategies. Furthermore, existing studies on hematological changes are limited, and more extensive research on hematobiochemical alterations in dogs across different Indian regions is essential. This study investigated the molecular prevalence of B. vogeli and B. gibsoni infections, assessed clinical observations, hematobiochemical changes, and risk factors, and explored therapeutic management strategies to provide valuable insights into the diagnosis, treatment, and control of canine babesiosis in India.
Material and methods
Sample collection
This study investigated 216 dogs suspected of canine babesiosis from three North Indian states (Haryana, Rajasthan, and Uttar Pradesh) and one Union Territory (New Delhi) over a one-year period (May 2022–April 2023; Supplementary Table 1). Dogs were considered suspected cases if they had a history of current or past tick infestation and exhibited one or more clinical signs commonly associated with canine babesiosis, including fever, pale mucous membranes, anorexia, lethargy, tachypnea, tachycardia, jaundice, or petechial hemorrhages. Dogs meeting these criteria were prospectively enrolled as they were presented to the participating veterinary facilities during the study period and were not selected based on disease severity. A comprehensive clinical examination was performed, owner consent was obtained, and data on sampling location, sex, age, breed, purpose (pet/working), tick history, exposure to other animals, and previous history of tick fever were recorded. Institutional Animal Ethics Committee (IAEC) approval was secured (VCC/IAEC/2022/1679–1705).
Approximately 3 mL of blood was collected aseptically from the cephalic or saphenous vein. One milliliter was placed in dipotassium ethylenediaminetetraacetic acid (K2EDTA) tubes for hematology and deoxyribonucleic acid (DNA) extraction, while 2 mL was used for serum biochemical analysis. Thin blood smears were prepared from EDTA-anticoagulated blood, and samples stored at 4 °C for DNA extraction. Blood from B. vogeli-infected dogs was collected on day 1 (pre-treatment) and day 15 (post-treatment), and from B. gibsoni-infected dogs on day 1 and day 45 for hematobiochemical analyses and PCR. Control samples were collected from 10 apparently healthy dogs (five males and five females), aged 1–2 years, comprising Labradors, German Shepherds, and non-descript dogs from the same geographical region as the study population. All control dogs were clinically healthy and had no history or clinical evidence of tick-borne disease at the time of sampling. The dogs tested negative for canine haemoparasitic infections, including B. vogeli [3, 4], B. gibsoni [5, 31], Ehrlichia canis [32], Anaplasma platys [33], Hepatozoon canis [32], and Trypanosoma evansi [34, 35]. Infection-free status was confirmed by microscopic examination of Giemsa-stained blood smears and species-specific PCR assays, as described previously [5]. Blood samples were collected in tubes with and without anticoagulant for hematological and biochemical analyses, respectively.
Preparation of thin blood smears for microscopic examination
To study the disease prevalence, thin blood smears (at least three per sample) were prepared on clean, grease-free glass slides and stained with Giemsa stain according to the standard protocol, as described previously [34]. The smears were examined under a 100 × oil immersion objective to detect intraerythrocytic piroplasms. The results were compared with those obtained from a PCR assay.
Hematology
Whole blood with anticoagulant was used for estimation of hemoglobin (Hb), total erythrocyte count (TEC), total leukocyte count (TLC), differential leukocyte count (DLC), thrombocytes, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) using a hematology analyzer MS4S (HD Consortium India Limited, Kolkata, India). Additionally, differential leukocyte count (DLC) was reconfirmed by microscopic examination of stained thin blood smears.
Biochemistry
Serum samples were analyzed for various biochemical parameters, viz., alanine aminotransferase (ALT), blood glucose, total protein (TP), albumin, total bilirubin (TB), direct bilirubin (DB), indirect bilirubin (IB), blood urea nitrogen (BUN), and creatinine using the EM 200 automated clinical chemistry analyzer (Erba Mannheim, Transasia Biomedicals Limited, Germany) with internal controls. The analyses were conducted following the manufacturer's guidelines for each parameter, utilizing standard diagnostic kits.
Genomic DNA extraction and PCR amplification
Genomic DNA was extracted from 200 μL of whole blood using the QIAamp DNA Mini Kit (Qiagen, Germany) as per the manufacturer’s protocol and stored at − 20 °C. The purity, concentration, and quality (A260/A280) of DNA were assessed via spectrophotometry (NanoDrop ND-2000, Thermo Fisher Scientific, USA) as described previously [36]. Positive controls included DNA from confirmed B. vogeli [3] and B. gibsoni [31] cases, while negative controls included DNA from a known negative dog and nuclease-free water as a no-template control.
Conventional PCR (cPCR) assays were used to screen each sample for B. vogeli and B. gibsoni infections. Specifically, the assays targeted the nuclear internal transcribed spacer region (ITS1–5.8S–ITS2) and mitochondrial cytochrome b (cytb) gene for B. vogeli, and the nuclear thrombospondin-related adhesive protein (BgTRAP) and mitochondrial cytochrome oxidase subunit I (coxI) gene for B. gibsoni. These targets were selected because they are well-characterized genetic markers that provide sensitive and species-specific detection and confirmation of the respective parasites [3–5, 31]. All samples were tested using all four PCR assays. Species identification was based on concordant amplification of the corresponding nuclear and mitochondrial targets. In the event of discordant results, repeat PCR would have been undertaken, however, no discordant results were observed in the present study. Primer details are provided in Supplementary Table 2.
Each PCR reaction (n = 216) was carried out in a 25 μL volume for every genetic marker, using 10 ng of genomic DNA, 12.5 μL of 2X GoTaq Green Master Mix (Promega, USA), and 1.0 μL each of forward and reverse primers (10 pmol/μL). The final volume of the reaction mixture was made up with nuclease-free water. Amplifications were performed using a T100 thermal cycler (Bio-Rad, USA). The thermal cycling conditions for each genetic marker are listed in Supplementary Table 2. The amplified PCR products were analyzed by electrophoresis on a 1.2–1.5% agarose gel stained with ethidium bromide and visualized using a gel documentation system (Gel Doc Go, Bio-Rad, USA). The sequence and phylogenetic analyses of the selected B. vogeli and B. gibsoni isolates have already been published [3, 4, 31].
Therapeutic management of clinical cases of canine babesiosis
The treatment of infected cases was tailored according to the Babesia species detected. For large form (B. vogeli) infections, imidocarb dipropionate was administered subcutaneously @ 6.6 mg/kg, with a repeat dose given two weeks later if necessary [37]. For small form (B. gibsoni) infections, a combination therapy was administered consisting of diminazene aceturate @ 3.5 mg/kg intramuscularly on day one, followed by imidocarb dipropionate @ 6.6 mg/kg subcutaneously on day two, and then clindamycin @ 30 mg/kg orally every 12 h for 45 days [38]. Symptomatic and supportive treatments were also provided as needed, based on individual clinical case requirements.
Statistical analysis
SPSS version 23.0 was used for statistical analyses. The Pearson Chi-Square test was used to evaluate associations between infection status and various risk factors and clinical variables. Multivariate logistic regression modelling was performed to assess the likelihood of infection associated with different risk factors and clinical variables, and odds ratios (OR) with 95% confidence intervals (CI) were calculated. A P-value of less than 0.05 was considered statistically significant.
Before comparative analyses, the data were assessed for normality and homogeneity of variance using the Shapiro–Wilk and Levene's tests, respectively. Comparative analyses of hematological and biochemical parameters were performed using independent samples t-tests between healthy controls and dogs infected with B. vogeli or B. gibsoni, as well as between the two infected groups. Additionally, analysis of variance (ANOVA) was used to evaluate differences among control, diseased, and recovered dogs, providing insights into parameter changes across disease states and treatment outcomes [39].
Results
Clinical manifestations of babesiosis in the affected dogs
Common clinical signs included loss of appetite, fever, lethargy, tick infestation, anemia (indicated by pale mucous membranes), enlargement of popliteal and submandibular lymph nodes, and tachypnea. Less frequent signs were vomition, hind limb weakness, icterus, melena, and hemoglobinuria (Supplementary Fig. 1.). Chronically infected dogs exhibited nonspecific symptoms.
The study population was predominantly male (64%). The age range was broad, spanning from one month to 12 years, with 39% of dogs under one year age. The canine cases represented 16 breeds, with German Shepherd (n = 57) and non-descript dogs (n = 55) being the most common, followed by Labrador (n = 38), American Pitbull Terrier (n = 14), Pomeranian (n = 9), Pakistani Bully (n = 8), Golden Retriever (n = 8), Crossbred (n = 7), Rottweiler (n = 6), Pug (n = 4), American Bully (n = 4), Cane Corso (n = 2), Cocker Spaniel (n = 1), Dobermann (n = 1), Great Dane (n = 1) and Grey Hound (n = 1). Notably, German Shepherd and non-descript dogs comprised over 50% of the total cases.
Microscopic examination of blood smears
Out of 216 Giemsa-stained blood smears examined for the presence of small and large forms of Babesia, only four (1.85%) and 23 samples (10.64%) were found to be positive under light microscopy for small and large intraerythrocytic piroplasms similar to B. gibsoni and B. vogeli, respectively. None of the animals tested positive for a mixed infection with both the parasites. Under oil immersion (1000 ×) examination, the small form appeared as typical annular or oval shapes (signet rings) and measured no more than one-eighth the diameter of the host erythrocyte in a Giemsa-stained peripheral blood smear (Fig. 1c, d). Similarly, the large form appeared oval/pyriform in shape (Figs. 1a, b, 2a). Upon micrometry, the sizes of the small and large forms varied from 1.0–2.0 × 0.5–2.6 μm and 2.8–3 × 3.5–7 μm, respectively (Fig. 1). In the case of the large form, multiple piroplasms within a single RBC were observed (Fig. 2a), and due to parasite-induced RBC lysis, extracellular piroplasms were evident in some of the blood smears (Fig. 2b).
Fig. 1.
Micrometric images of large (a, b) and small forms (c, d) of Babesia in Giemsa-stained peripheral blood smears of infected dogs
Fig. 2.
Microscopic images of a large form of canine Babesia. Four typical piroplasms inside an erythrocyte (a) and two extracellular piroplasms (b) of B. vogeli in an infected dog in a Giemsa-stained peripheral blood smear
Isolation of genomic DNA and estimation of its integrity, concentration and purity
Genomic DNA was successfully isolated from 216 blood samples exhibiting high integrity (> 95%) and concentrations ranging from 10–850 ng/μL. Spectrophotometric analysis (NanoDrop® ND- 2000 UV, Thermo Fisher Scientific, USA) revealed purity ratios (A260/A280) between 1.65 and 1.85, indicating suitable DNA quality for downstream applications.
Molecular detection using cPCR assays
PCR amplification yielded amplicons of ~ 600 bp (Supplementary Fig. 2) and 693 bp (Supplementary Fig. 3) for the B. vogeli internal transcribed spacer regions (ITS1–5.8S–ITS2) and cytb gene, respectively. A total of 48 out of 216 samples (22.22%) tested positive for B. vogeli infection based on cytb gene, with results consistent with those obtained from ITS region analysis. Conventional PCR targeting the BgTRAP and coxI genes of B. gibsoni produced amplicons of ~ 855 bp (Supplementary Fig. 4) and 649 bp (Supplementary Fig. 5), respectively. A total of 12 out of 216 (5.55%) samples were found positive for B. gibsoni infection with each gene.
Comparison of conventional (microscopy) and molecular (conventional PCR) techniques for the detection of Babesia spp. infection
The overall prevalence of canine babesiosis by microscopy and conventional PCR was found to be 27/216 (12.5%) and 60/216 (27.78%), respectively (Table 1). The PCR-based detection, using one nuclear and one mitochondrial marker each for B. vogeli and B. gibsoni, showed consistent prevalence rates for B. vogeli (22.22%) and B. gibsoni (5.55%), indicating similar sensitivity of the PCR assays for both species.
Table 1.
Comparative detection of Babesia spp. infection by microscopy and conventional PCR
| Total number of cases | Microscopy (%) | Pearson Chi-Square (χ2) | P value | cPCR (%) | Pearson Chi-Square (χ2) | P value | Total number of positive cases | Molecular prevalence (%) | |
|---|---|---|---|---|---|---|---|---|---|
| B. vogeli | 216 | 23 (10.64%) | 98.953 | 0.000 | 48 (22.22%) | 216.000 | 0.000 | 48 | 22.22 |
| B. gibsoni | 216 | 04 (1.85%) | 69.283 | 0.000 | 12 (5.55%) | 216.000 | 0.000 | 12 | 5.55 |
| Total | 216 | 27 (12.5%) | 60 (27.78%) | 60 | 27.78 |
Analysis and interpretation of risk factors and clinical variables
Risk factors and clinical variables associated with B. vogeli infection in dogs
The analysis of various risk factors and clinical variables for B. vogeli infection in dogs (Table 2) revealed significant associations with breed and lymph node enlargement (P < 0.05). Specifically, non-descript dogs were found to be significantly more susceptible to B. vogeli infection compared to other breeds (OR: 27.491; 95% CI: 1.092–692.018; P < 0.05). Additionally, dogs presented with enlarged lymph nodes were three times more likely to test positive for B. vogeli infection than those without lymphadenopathy (OR = 3.338; 95% CI: 1.345–8.287; P < 0.05). Seventy-nine percent of B. vogeli infected dogs exhibited lymphadenopathy. Dogs less than one year of age showed higher odds of infection (OR = 1.765; 95% CI: 0.663–4.703), although this association was not statistically significant. No significant associations were found between B. vogeli infection and other evaluated risk factors and vital clinical parameters, including body temperature, pulse rate, and respiration rate.
Table 2.
Risk factors and clinical variables associated with B. vogeli infection in dogs
| Factor | Variable | N | Positive | % Positive | Pearson Chi-Square (χ2) | P value | OR | 95% CI (OR) | P value |
|---|---|---|---|---|---|---|---|---|---|
| Overall | 216 | 48 | 22.2 | ||||||
| Place | 0.192 | 0.661 | |||||||
| Hisar | 152 | 35 | 23.0 | 1.107 | 0.462—2.657 | 0.819 | |||
| Others | 64 | 13 | 20.3 | 1.0 | |||||
| Breed | 6.523 | 0.089 | |||||||
| Labrador | 38 | 12 | 31.6 | 2.546 | 0.801—8.099 | 0.113 | |||
| Non-descript | 55 | 16 | 29.1 | 27.491 | 1.092—692.018 | 0.044 | |||
| German Shepherd | 57 | 11 | 19.3 | 1.704 | 0.578—5.025 | 0.334 | |||
| Others | 66 | 09 | 13.6 | 1.0 | |||||
| Age | 4.197 | 0.123 | |||||||
| Up to 1 year | 85 | 25 | 29.4 | 1.765 | 0.663—4.703 | 0.256 | |||
| 1–4 years | 73 | 13 | 17.8 | 1.024 | 0.353—2.968 | 0.966 | |||
| Above 4 years | 58 | 10 | 17.2 | 1.0 | |||||
| Sex | 0.520 | 0.471 | |||||||
| Female | 77 | 15 | 19.5 | 0.738 | 0.330—1.648 | 0.458 | |||
| Male | 139 | 33 | 23.7 | 1.0 | |||||
| Season | 0.785 | 0.675 | |||||||
| Monsoon | 78 | 17 | 21.8 | 0.587 | 0.191—1.801 | 0.351 | |||
| Summer | 106 | 22 | 20.8 | 0.516 | 0.181—1.474 | 0.217 | |||
| Autumn and winter | 32 | 09 | 28.1 | 1.0 | |||||
| Raised as | 1.089 | 0.297 | |||||||
| Pet | 161 | 33 | 20.5 | 34.690 | 1.168—1030.084 | 0.040 | |||
| Stray and semi-captive | 55 | 15 | 27.3 | 1.0 | |||||
| Setting | 0.109 | 0.741 | |||||||
| Rural | 11 | 02 | 18.2 | 1.081 | 0.178—6.565 | 0.932 | |||
| Urban | 205 | 46 | 22.4 | 1.0 | |||||
| Tick infestation | 1.082 | 0.298 | |||||||
| No | 86 | 16 | 18.6 | 1.890 | 0.593—6.028 | 0.282 | |||
| Yes | 130 | 32 | 24.6 | 1.0 | |||||
| History of tick infestation | 0.714 | 0.398 | |||||||
| No | 53 | 14 | 26.4 | 1.226 | 0.405—3.708 | 0.718 | |||
| Yes | 163 | 34 | 20.9 | 1.0 | |||||
| Products used against ticks | 4.279 | 0.233 | |||||||
| Injectable | 30 | 04 | 13.3 | 0.784 | 0.174—3.531 | 0.751 | |||
| None | 108 | 30 | 27.8 | 1.865 | 0.518—6.710 | 0.340 | |||
| Shampoo | 35 | 07 | 20.0 | 1.218 | 0.314—4.719 | 0.776 | |||
| Others | 43 | 07 | 16.3 | 1.0 | |||||
| Was there any exposure to other animals? | 3.625 | 0.057 | |||||||
| No | 146 | 27 | 18.5 | 0.236 | 0.055—1.004 | 0.051 | |||
| Yes | 70 | 21 | 30.0 | 1.0 | |||||
| If yes, did they have tick infestation? | 8.221 | 0.066 | |||||||
| No | 13 | 04 | 30.7 | 0.095 | 0.006—1.435 | 0.089 | |||
| Yes | 57 | 20 | 35.1 | 1.0 | |||||
| Body temperature (°F) | 0.640 | 0.726 | |||||||
| 99.5–102.5 | 42 | 10 | 23.8 | 0.526 | 0.152—1.819 | 0.310 | |||
| 102.6–105 | 136 | 28 | 20.6 | 0.566 | 0.207—1.544 | 0.266 | |||
| 105.1 and above | 38 | 10 | 26.3 | 1.0 | |||||
| Pulse rate | 0.245 | 0.621 | |||||||
| 70–120 | 189 | 43 | 22.8 | 1.256 | 0.371—4.255 | 0.715 | |||
| Above 120 | 27 | 05 | 18.5 | 1.0 | |||||
| Respiration rate | 5.245 | 0.073 | |||||||
| 18–34 | 36 | 12 | 33.3 | 1.418 | 0.438—4.592 | 0.561 | |||
| 35–68 | 133 | 23 | 17.3 | 0.665 | 0.255—1.737 | 0.405 | |||
| Above 68 | 47 | 13 | 27.7 | 1.0 | |||||
| Colour of mucous membranes | 1.873 | 0.392 | |||||||
| Congested | 44 | 08 | 18.2 | 0.642 | 0.155—2.664 | 0.541 | |||
| Pale | 135 | 34 | 25.2 | 0.989 | 0.303—3.225 | 0.985 | |||
| Others | 37 | 06 | 16.2 | 1.0 | |||||
| Lymph node | 8.073 | 0.004 | |||||||
| Enlarged | 133 | 38 | 28.6 | 3.338 | 1.345—8.287 | 0.009 | |||
| Normal | 83 | 10 | 12.0 | 1.0 |
N Number of samples examined, OR Odds Ratio, CI Confidence interval
Risk factors and clinical variables associated with B. gibsoni infection in dogs
Similar to B. vogeli, the association of several risk factors and clinical variables with B. gibsoni infection was analyzed, with results presented in Table 3. Age and sex were found to be significantly associated with the occurrence of B. gibsoni infection (P < 0.05). Dogs over four years of age were found to be more susceptible to B. gibsoni infection. Similarly, male dogs, in comparison to females, were more likely to contract B. gibsoni infection. No significant associations were found between B. gibsoni infection in dogs and the other risk factors. Similarly, vital parameters (temperature, pulse rate, and respiration rate) showed no significant association with B. gibsoni infection (Table 3).
Table 3.
Risk factors and clinical variables associated with B. gibsoni infection in dogs
| Factor | Variable | N | Positive | % Positive | Pearson Chi-Square (χ2) | P value | OR | 95% CI (OR) | P value |
|---|---|---|---|---|---|---|---|---|---|
| Overall | 216 | 12 | 5.6 | ||||||
| Place | 12.544 | 0.000 | |||||||
| Hisar | 152 | 03 | 2.0 | 0.235 | 0.047—1.167 | 0.077 | |||
| Others | 64 | 09 | 14.1 | 1.0 | |||||
| Breed | 6.444 | 0.092 | |||||||
| Labrador | 38 | 03 | 7.9 | 0.561 | 0.061—5.171 | 0.610 | |||
| Non-descript | 55 | Nil | 0.0 | 1.159 | 0.000—6587.159 | 0.973 | |||
| German Shepherd | 57 | 06 | 10.5 | 0.829 | 0.109—6.298 | 0.856 | |||
| Others | 66 | 03 | 4.5 | 1.0 | |||||
| Age | 16.260 | 0.000 | |||||||
| Up to 1 year | 85 | Nil | 0.0 | 0.317 | 0.044—2.300 | 0.256 | |||
| 1–4 years | 73 | 03 | 4.1 | 0.305 | 0.046—2.043 | 0.221 | |||
| Above 4 years | 58 | 09 | 15.5 | 1.0 | |||||
| Sex | 4.132 | 0.042 | |||||||
| Female | 77 | 01 | 1.3 | 0.581 | 0.106—3.179 | 0.531 | |||
| Male | 139 | 11 | 7.9 | 1.0 | |||||
| Season | 0.686 | 0.710 | |||||||
| Monsoon | 78 | 03 | 3.8 | 1.078 | 0.120—9.702 | 0.946 | |||
| Summer | 106 | 07 | 6.6 | 0.908 | 0.119—6.941 | 0.926 | |||
| Autumn and winter | 32 | 02 | 6.3 | 1.0 | |||||
| Raised as | 4.341 | 0.067 | |||||||
| Pet | 161 | 12 | 7.5 | 0.971 | 0.000—6563.401 | 0.995 | |||
| Stray and semi-captive | 55 | Nil | 0.0 | 1.0 | |||||
| Setting | 0.276 | 0.599 | |||||||
| Rural | 11 | 01 | 9.1 | 0.870 | 0.016—46.061 | 0.945 | |||
| Urban | 205 | 11 | 5.4 | 1.0 | |||||
| Tick infestation | 0.010 | 0.920 | |||||||
| No | 86 | 05 | 5.8 | 1.044 | 0.090—12.053 | 0.972 | |||
| Yes | 130 | 07 | 5.4 | 1.0 | |||||
| History of tick infestation | 0.415 | 0.520 | |||||||
| No | 53 | 04 | 7.5 | 1.562 | 0.175—13.968 | 0.690 | |||
| Yes | 163 | 08 | 4.9 | 1.0 | |||||
| Products used against ticks | 1.398 | 0.706 | |||||||
| Injectable | 30 | 03 | 10.0 | 0.884 | 0.041—19.165 | 0.937 | |||
| None | 108 | 05 | 4.6 | 1.445 | 0.098—21.299 | 0.788 | |||
| Shampoo | 35 | 02 | 5.7 | 1.007 | 0.067—15.210 | 0.996 | |||
| Others | 43 | 02 | 4.7 | 1.0 | |||||
| Was there any exposure to other animals? | 5.838 | 0.056 | |||||||
| No | 146 | 12 | 8.2 | 2.496 | 0.064—97.962 | 0.625 | |||
| Yes | 70 | Nil | 0.0 | 1.0 | |||||
| If yes, did they have tick infestation? | 5.838 | 0.054 | |||||||
| No | 13 | Nil | 0.0 | 1.104 | 0.025—49.599 | 0.959 | |||
| Yes | 57 | Nil | 0.0 | 1.0 | |||||
| Body temperature (°F) | 2.572 | 0.276 | |||||||
| 99.5–102.5 | 42 | 04 | 9.5 | 1.044 | 0.082—13.349 | 0.973 | |||
| 102.6–105 | 136 | 05 | 3.7 | 0.546 | 0.064—4.654 | 0.580 | |||
| 105.1 and above | 38 | 03 | 7.9 | 1.0 | |||||
| Pulse rate | 1.739 | 0.187 | |||||||
| 70–120 | 189 | 12 | 6.3 | 1.426 | 0.095—21.490 | 0.798 | |||
| Above 120 | 27 | Nil | 0.0 | 1.0 | |||||
| Respiration rate | 4.930 | 0.085 | |||||||
| 18–34 | 36 | 05 | 13.9 | 0.795 | 0.080—7.926 | 0.845 | |||
| 35–68 | 133 | 06 | 4.5 | 0.668 | 0.098—4.547 | 0.680 | |||
| Above 68 | 47 | 01 | 2.1 | 1.0 | |||||
| Colour of mucous membranes | 0.499 | 0.779 | |||||||
| Congested | 44 | 02 | 4.5 | 1.792 | 0.069—46.314 | 0.725 | |||
| Pale | 135 | 07 | 5.2 | 2.705 | 0.183—39.887 | 0.469 | |||
| Others | 37 | 03 | 8.1 | 1.0 | |||||
| Lymph node | 0.734 | 0.392 | |||||||
| Enlarged | 133 | 09 | 6.8 | 1.034 | 0.206—5.192 | 0.967 | |||
| Normal | 83 | 03 | 3.6 | 1.0 |
N Number of samples examined, OR Odds Ratio, CI Confidence interval
Hematological parameters
Significant decreases were observed in B. vogeli-infected dogs' Hb (8.05 ± 0.35 g/dL), TEC (4.38 ± 0.19 million/µL), PCV (28.26 ± 1.25%), MCH (18.50 ± 0.40 pg), MCHC (28.63 ± 0.73 g/dL), and platelet count (97.17 ± 7.24 103/µL) compared to healthy controls. Similarly, B. gibsoni-infected dogs showed significant decreases in Hb (6.71 ± 0.64 g/dL), TEC (3.52 ± 0.38 million/µL), PCV (22.44 ± 2.06%), and platelet count (71.50 ± 8.26 103/µL) compared to healthy controls. No significant differences were observed in MCV, MCHC, TLC, neutrophils, lymphocytes, and monocytes between B. vogeli- and B. gibsoni-infected dogs and healthy controls (Supplementary Table 3). After treatment, these parameters significantly improved and returned to normal ranges in dogs recovered from both B. vogeli and B. gibsoni infections (Supplementary Table 7, 8).
Comparison of hematological parameters between B. vogeli- and B. gibsoni-infected dogs before treatment revealed significant differences in TEC, PCV, and platelet count. Babesia gibsoni-infected dogs had lower TEC (3.52 ± 0.38 million/µL), PCV (22.44 ± 2.06%), and platelet count (71.50 ± 8.26 × 103/µL) compared to B. vogeli-infected dogs (TEC: 4.38 ± 0.19 million/µL, PCV: 28.26 ± 1.25%, and platelets: 97.17 ± 7.24 × 103/µL). No significant differences were observed in Hb, MCV, MCH, MCHC, TLC, neutrophils, lymphocytes, and monocytes between the two groups (Supplementary Table 4).
Serum biochemical parameters
Babesia vogeli-infected dogs showed significant increases in serum ALT (80.54 ± 8.23 U/L), TB (1.35 ± 0.14 mg/dL), DB (0.52 ± 0.05 mg/dL), IDB (0.83 ± 0.09 mg/dL), and BUN (34.74 ± 2.69 mg/dL) before treatment compared to healthy controls (Supplementary Table 5). After treatment, these parameters significantly decreased and returned to normal ranges in recovered dogs (Supplementary Table 9). No significant differences were observed in TP, albumin, blood glucose, and creatinine before treatment between infected and healthy dogs (Supplementary Table 5). However, albumin levels increased significantly after treatment (Supplementary Table 9).
Babesia gibsoni-infected dogs showed significant increases in serum ALT (118.17 ± 24.08 U/L), TB (3.44 ± 0.11 mg/dL), DB (1.07 ± 0.06 mg/dL), IDB (2.37 ± 0.05 mg/dL), and BUN (59.45 ± 5.07 mg/dL) before treatment compared to healthy controls (Supplementary Table 5). Significant decreases were observed in TP (5.48 ± 0.29 g/dL) and albumin (2.2 ± 0.20 g/dL). Post-treatment, most parameters normalized in convalescent dogs, except for BUN, which decreased non-significantly (Supplementary Table 10). No significant differences were observed in blood glucose and creatinine before treatment between infected and healthy dogs (Supplementary Table 5).
A comparative analysis of serum biochemical parameters in dogs infected with B. vogeli and B. gibsoni revealed distinct differences, with B. gibsoni-infected dogs exhibiting significantly elevated levels of TB, IDB, and BUN. Parameters such as ALT, TP, albumin, DB, blood glucose, and creatinine showed no notable variation between the two groups, indicating differing effects of these infections on canine liver and kidney function (Supplementary Table 6).
Therapeutic management of clinical cases of canine babesiosis
Treatment of the infected cases was done according to the Babesia spp. diagnosed, as the treatment protocols for B. vogeli and B. gibsoni are completely disparate. All the recovered animals tested negative for B. vogeli and B. gibsoni infections by cPCR on day 15 and day 45 post-treatment, respectively. This negative test result served as additional confirmation of the effectiveness of the treatment regimen employed for both B. vogeli and B. gibsoni infections.
Treatment of B. vogeli
A single dose of imidocarb dipropionate @ 6.6 mg/kg was administered subcutaneously to treat B. vogeli infection. Atropine sulphate was administered intramuscularly @ 0.04 mg/kg, 30 min before imidocarb dipropionate, to mitigate its cholinergic side effects. Furthermore, symptomatic and supportive treatments were administered as needed. Liver tonics were given based on body weight, with dosages of 2.5 mL (up to 5 kg), 5.0 mL (5–15 kg), 7.5 mL (15–25 kg), 10–15 mL (25–50 kg), and 30 mL (above 50 kg), given two to three times daily for 15 days. Antioxidant therapy included Vitamin E (10–15 IU/kg orally for 5 days) and N-acetyl cysteine (70 mg/kg IV or orally for 5 days). Choleretic support involved ursodeoxycholic acid (20–25 mg/kg orally once daily for 15 days). Symptomatic treatment included ondansetron as an anti-emetic @ 0.1–0.5 mg/kg body weight intramuscularly or intravenously, administered twice daily as needed; ranitidine as an H2 blocker @ 0.5–2 mg/kg body weight intramuscularly as needed; and ethamsylate as a hemostat @ 5–10 mg/kg body weight intramuscularly as needed. Additionally, ectoparasite control was achieved using tick treatments such as Fipronil 9.8% spot-on, 12.5% Amitraz dip, or 12% Selamectin spot-on.
Treatment of B. gibsoni
Triple therapy was administered to dogs positive for B. gibsoni infection, comprising diminazene aceturate (3.5 mg/kg, intramuscularly, once on day 1), imidocarb dipropionate (6.6 mg/kg subcutaneously once on day 2), and clindamycin (30 mg/kg orally, every 12 h for 45 days). Furthermore, symptomatic and supportive treatments were provided as needed, similar to the approach for B. vogeli cases.
Discussion
Canine babesiosis, a tick-borne disease caused by Babesia species, is a significant concern globally. In India, B. vogeli and B. gibsoni are the documented species [3, 4, 13, 31, 40]. Despite sporadic reports based on conventional diagnostic methods, the true prevalence and epidemiological status of the disease in India remain poorly understood. This study aimed to bridge this knowledge gap by employing conventional PCR assays to detect Babesia species, examining hematobiochemical alterations, determining associated risk factors, and evaluating therapeutic management.
This study examined 216 dogs suspected of canine babesiosis, observing varied clinical signs consistent with previous research [14, 27, 41–43]. An overall prevalence of 12.5% by microscopy and 27.78% by conventional PCR, with higher PCR detection likely due to greater sensitivity, chronic infection, and low or intermittent parasitaemia at the time of presentation of dogs in microscopy-negative cases. The prevalence rates for B. vogeli by microscopy and conventional PCR were 10.64% and 22.22%, respectively, whereas the corresponding rates for B. gibsoni were 1.85% and 5.55%, respectively. Similar findings were recently reported in Haryana [12, 40], though many earlier studies identified B. gibsoni as predominant in India [15, 18, 44–47]. The underlying reasons for the low percent positivity of B. gibsoni in the current study are difficult to pinpoint. The higher detection of canine babesiosis by PCR-based assays compared to microscopy, as observed in the present study, has also been reported by several authors [13, 18, 45, 47, 48]. The prevalence of canine babesiosis in various parts of northern India has been reported to range from 0.66% to 8.9% [20, 49], whereas in southern India, Kumar et al. [50] recorded prevalence rates of 3.9% and 84.9% for B. canis (now B. vogeli) and B. gibsoni, respectively. The wide variation in climatic conditions across different regions of India may contribute to the differing prevalence rates of tick-borne infections.
Among all the risk factors analyzed, non-descript dogs were 27 times more likely to contract B. vogeli infection compared to other breeds. This could be due to the high number of stray or unowned dogs in India, which often serve as reservoirs for tick vectors due to poor grooming, lack of veterinary care, and frequent exposure to infested environments [17]. The absence of regular screening and treatment in these animals, combined with their potential for greater mobility and interaction with other dogs, likely increases the risk of contact-based transmission of infected ticks [19, 51]. In the present study, over 50% of B. vogeli cases occurred in dogs under one year, consistent with earlier reports [1, 52, 53]. The absence of significant associations between B. vogeli infection and other evaluated risk factors or vital clinical parameters may be attributed to the relatively low pathogenicity of B. vogeli and the non-specific nature of physiological variables, which can be influenced by factors including stress, disease stage, host immune status, and prior treatment. For B. gibsoni, males were more frequently infected, which may be attributed to their more aggressive and socializing behaviour, while dogs over four years had higher infection rates, possibly due to increased exposure to infected dogs and ticks [26, 49]. Similarly, the absence of significant associations between B. gibsoni infection and other evaluated variables may be due to the variable clinical presentation of the disease and the non-specific nature of these parameters, which are influenced by factors such as disease stage, parasite burden, host immune status, and prior treatment. Additionally, the limited number of infected dogs may have reduced the statistical power to detect significant associations.
Dogs infected with B. vogeli and B. gibsoni exhibited hematological changes, with anemia being a common finding in both, though more severe in B. gibsoni-infected dogs. Both infections caused marked reductions in Hb, TEC, and PCV compared to healthy dogs, with B. gibsoni showing greater declines [14, 16, 27, 40, 54–56]. Hemolytic anemia in canine babesiosis is multifactorial and is attributed to parasite-induced RBC damage, immune-mediated destruction, oxidative injury, increased osmotic fragility of erythrocytes, splenic erythrophagocytosis, complement activation, and the production of parasite-derived hemolytic factors [1, 10, 57]. In both infections, MCH and MCHC were significantly lower than those of healthy controls, with a more pronounced decline in B. vogeli-infected dogs, likely reflecting erythrocyte destruction associated with parasitic invasion. The decreased MCHC observed in the present study was consistent with the rare occurrence of hemoglobinuria, suggesting limited intravascular hemolysis. In contrast, increased MCHC has been reported in conditions characterized by marked intravascular hemolysis, including canine babesiosis and human malaria [27, 58–60].
Significant thrombocytopenia was also observed in dogs infected with both B. vogeli and B. gibsoni, which may have resulted from immune-mediated platelet destruction, consumptive coagulopathy secondary to hemolytic or vascular injury, splenic sequestration, and platelet involvement in host defense mechanisms against the parasite [10, 61, 62]. This decrease in platelet count, TEC, and PCV was more pronounced in B. gibsoni infections, which may be attributed to the higher virulence, chronic nature of the infection, and greater treatment challenges associated with this species. Similar trends have been reported in previous studies [14, 27, 56, 63].
Dogs with babesiosis exhibited significant biochemical alterations compared to healthy controls, including elevated ALT, TB, DB, IDB, and BUN, along with decreased albumin levels. Elevated ALT likely indicated hepatocellular damage, hemorrhage, and hemolysis [14, 41, 59, 64]. Acute fulminant hepatic failure (FHF) is a rare but life-threatening complication of canine babesiosis. While liver damage is common in severe babesiosis, the progression to acute FHF typically occurs only in complicated, virulent cases [42, 59]. Both B. vogeli and B. gibsoni-infected dogs had lower albumin levels, possibly due to increased acute-phase proteins from inflammation, liver damage, vasculitis, or protein-losing gastropathy and nephropathy [54, 59, 65, 66]. Hyperbilirubinemia was linked to hemolysis and hepatic damage [14, 54]. Elevated BUN levels likely reflected azotemia associated with pre-renal hypoperfusion or renal dysfunction, although contributions from hemolysis and gastrointestinal hemorrhage could not be excluded [28, 67]. Babesia gibsoni-infected dogs had significantly higher BUN, TB, and IDB levels than B. vogeli-infected dogs, possibly due to its greater pathogenicity, chronic infection, and treatment challenges.
Various drugs and combinations have been used to treat canine babesiosis [7, 68–70], although their mechanisms and comparative efficacy remain incompletely understood [38, 61, 70, 71]. Drug susceptibility varies among Babesia species, necessitating species-specific therapeutic approaches [1, 7]. Imidocarb dipropionate remains the treatment of choice for large Babesia, particularly B. vogeli [37, 72]. In contrast, treatment of B. gibsoni is more challenging, as single-drug therapies often fail to eliminate infection [73–77]. The triple-therapy protocol comprising diminazene aceturate, imidocarb dipropionate, and clindamycin used in the present study was adopted from Lin et al. [38] and resulted in PCR negativity in all treated dogs by day 45 post-treatment. Although atovaquone combined with azithromycin has shown good efficacy, particularly against the Asian genotype of B. gibsoni [61], complete parasite clearance remains challenging. While the PCR-negative status observed in the present study suggests a favorable therapeutic response, the 45-day follow-up period may not be sufficient to definitively exclude chronic carrier states, and longer-term monitoring would be required to confirm sustained parasite clearance.
Treatment must be tailored to Babesia species, drug resistance, and patient factors (e.g., age, immunity, and clinical status). Limited drug options, species-specific responses, and chronic carrier states, especially in small Babesia, complicate therapy. Further studies on new drugs and synergistic regimens are essential for improved management.
The study's limitations include a relatively small control group, which may have influenced the hematobiochemical comparisons, and a regional study population that may limit the generalizability of the findings to other geographical areas. In addition, the relatively small number of B. gibsoni-positive dogs (n = 12) may have reduced the statistical power of the risk factor analyses for this species and contributed to imprecise odds ratio estimates. Furthermore, because the study employed a cross-sectional design, the observed associations between risk factors and infection should not be interpreted as causal relationships. Despite these limitations, the study has important translational relevance, as its findings on molecular detection and therapeutic management can directly inform clinical practice and improve treatment outcomes in dogs with babesiosis.
Conclusions
This study revealed a significant molecular prevalence of canine babesiosis in northern India, with B. vogeli being the predominant species. Conventional PCR was more sensitive than microscopy for detecting Babesia species infection in dogs. Key risk factors and clinical variables identified included non-descript breed dogs being highly susceptible to B. vogeli infection and dogs with enlarged lymph nodes being more likely to test positive for B. vogeli. Similarly, B. gibsoni infection was notably associated with older dogs and males. The study also highlighted significant hematological and biochemical alterations in infected dogs, with B. gibsoni infections causing more severe changes. The treatment protocols used, specifically imidocarb dipropionate for B. vogeli and a combination therapy (diminazene aceturate, imidocarb dipropionate, and clindamycin) for B. gibsoni, demonstrated promising efficacy. These findings contribute to the understanding of Babesia epidemiology in the region and highlight the need for further studies to inform the development of targeted control strategies.
Supplementary Information
Acknowledgements
The authors are thankful to the Director of Research and the Dean, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, for providing the necessary facilities to carry out this work.
Authors’ contributions
Conceptualization, AKN; Data curation, AKN; Formal analysis, AK, AKN and ADM; Investigation, AK, DA, AKN and ADM; Methodology, AK, AKN and ADM; Project administration, AKN; Software, AK and AKN; Supervision, AKN; Writing – original draft, AK and AKN; Writing – review & editing, DA, AKN and ADM.
Funding
Authors are thankful to the RKVY- RAFTAAR (Scheme No. 4067(PFMS)-C(g)-VPS-01-OA (RKVY)) for enriching the Department with the facilities, which were utilized during the research. No funds were received for publication of this research.
Data availability
The datasets generated and/or analyzed during the current study are provided within the supplementary files.
Declarations
Ethics approval and consent to participate
The study protocol involving the collection of blood samples was approved by the Institutional Animal Ethics Committee of Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, ensuring compliance with ethical standards for animal research (Permission No. VCC/IAEC/2022/1679–1705). Informed consent was obtained from the animal owner(s) to utilize the animals in this study.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Ansu Kumari and Anil Kumar Nehra contributed equally to this work.
Contributor Information
Ansu Kumari, Email: anshushehrawat1096@gmail.com.
Anil Kumar Nehra, Email: anilnehra15@gmail.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated and/or analyzed during the current study are provided within the supplementary files.