Babesiosis is a clinically significant, tick-borne disease caused by intraerythrocytic apicomplexan parasites of the genus Babesia, affecting a broad spectrum of vertebrate hosts and often resulting in considerable morbidity and mortality in both domestic and wild animals (Kuttler and Ristic, 1988). In canines, babesiosis is primarily attributed to two morphologically distinct groups, namely the large-form Babesia canis and B. vogeli and the small-form B. gibsoni, which differ not only in size but also in pathogenicity, epidemiology and clinical progression.
       
The life cycle of Babesia gibsoni involves a complex interplay between the vertebrate host and the tick vector, with asexual replication occurring within erythrocytes and sexual stages developing within the tick (Uilenberg, 2006). In Giemsa-stained peripheral blood smears, the parasite is typically identified as small (1-3 µm), pleomorphic intraerythrocytic piroplasms exhibiting ring, oval or comma-shaped morphology, which serve as key diagnostic features. Although tick transmission remains the principal route of infection, increasing evidence highlights the role of alternative transmission pathways, including blood transfusion and transplacental spread, thereby enhancing the epidemiological complexity of the disease (Jefferies et al., 2007).
       
Infections with B. gibsoni may be less acute than those caused by some large-form Babesia species; however, they can follow a persistent and debilitating course. The disease spectrum ranges from subclinical infection to severe, life-threatening illness culminating in multi-organ dysfunction and death (Suarez et al., 2001; Irwin, 2010). The acute phase is typically marked by pyrexia, lethargy, hemolytic anaemia and pronounced thrombocytopenia (Meinkoth et al., 2002). Chronic carrier status may also occur, with recovered dogs harbouring the parasite for prolonged periods and potentially serving as reservoirs of infection (Groves and Dennis, 1972). Furthermore, investigations of disease-associated biomarkers, such as serum hepcidin, have highlighted the potential value of biochemical indicators for assessing disease severity and predicting clinical outcomes in canine babesiosis (Rajamanickam et al., 2021).
       
The clinical severity of infection is influenced not merely by parasitemia but is largely governed by host immune responses, with younger animals, particularly those below two years of age, exhibiting heightened susceptibility and more severe manifestations. In addition to haematological derangements, affected dogs may develop a spectrum of biochemical and systemic abnormalities including hemoglobinuria, hypoglycemia, acid-base imbalances, azotemia, proteinuria and elevated hepatic enzymes, often accompanied by lymphadenopathy and splenomegaly. In complicated cases, the disease may precipitate coagulopathies, immune-mediated haemolytic anaemia and systemic inflammatory responses leading to multiple organ dysfunction syndromes such as acute renal failure, hepatic insufficiency, jaundice and pancreatitis (Karasová et al., 2022). Consistent with the clinical complexity of canine babesiosis, naturally infected dogs have also been reported to exhibit significant haematological, biochemical and oxidative alterations, highlighting the potential utility of laboratory parameters in assessing disease severity and clinical status (Shil et al., 2022).
       
Recent molecular epidemiological investigations have demonstrated that Babesia gibsoni is the predominant small-form Babesia infecting dogs in Kerala, with PCR-based prevalence substantially exceeding that detected by microscopy (Jain et al., 2017; Augustine et al., 2017; Ajith et al., 2024; Venugopal et al., 2024). Similar observations have been reported from different regions of India including Punjab, Gujarat, Tamil Nadu and the north-eastern states (Singla et al., 2016; Bilwal et al., 2017; Gonmei et al., 2020; Jaisree et al., 2025). Molecular investigations in naturally infected dogs from other parts of India have also documented the occurrence of canine babesiosis and emphasized the importance of epidemiological and risk-associated factors in disease occurrence (Kopparthi et al., 2021; Choudhary et al., 2025). Worldwide, B. gibsoni is increasingly recognized as an important emerging canine tick-borne pathogen (Karasová et al., 2022; Chan et al., 2025).
       
Despite its recognized clinical importance, there remains a need for region-specific data on the epidemiology, risk determinants and integrated haemato-biochemical alterations associated with small-form canine babesiosis, particularly in tropical regions such as Southern Kerala where tick-borne diseases are endemic. Most available studies either focus on prevalence alone or do not comprehensively correlate clinical presentation, risk factors and laboratory alterations.
       
Furthermore, limited emphasis has been placed on systematically evaluating naturally occurring infections under field conditions, which are important for understanding real-world disease dynamics and guiding evidence-based clinical management.
       
Accordingly, the present study aimed to determine the microscopic prevalence of small-form Babesia infection, evaluate selected epidemiological and clinical risk determinants and characterize associated haematological and biochemical alterations in naturally infected dogs presented to a veterinary clinic in Southern Kerala. We hypothesized that infection would be associated with identifiable epidemiological or clinical risk factors and with measurable alterations in haematological and biochemical parameters compared with clinically healthy, non-infected dogs.

Study area and animals
 
The present study was conducted on client-owned dogs presented to a private veterinary clinic in Varkala, Kerala, India during January 2024 to March 2025. A total of 152 dogs exhibiting clinical signs suggestive of haemoprotozoan infection, including anorexia, pyrexia, anaemia, pale or icteric mucous membranes, haemoglobinuria, tick infestation and general weakness, were included in the investigation. Dogs were categorized into two age groups: less than one year of age (puppies and young dogs) and more than one year of age (adult dogs) for epidemiological analysis.
 
Sample collection and processing
 
Approximately 4 mL of blood was aseptically collected from the cephalic vein of each animal. Of this, 2 mL was transferred into ethylenediaminetetraacetic acid (EDTA)-coated vacutainer tubes (HiMedia) for hematological analysis, while the remaining 2 mL was placed into serum vacutainers containing clot activator (Nasmed Diagnostics) for biochemical investigations.
       
Thin blood smears were prepared immediately after sample collection, air-dried, fixed and stained with Giemsa stain. The stained smears were examined under oil immersion (100×) using light microscopy for the detection of small-form Babesia spp. based on characteristic intraerythrocytic morphology. Because microscopy has limited sensitivity, particularly in low-parasitaemia infections and cannot reliably distinguish Babesia species, the diagnosis in the present study should be regarded as microscopy-based detection of small-form Babesia-like organisms rather than definitive species identification. This diagnostic limitation may have resulted in an underestimation of prevalence.
 
Assessment of risk factors
 
A detailed case history was recorded for each animal. Epidemiological and clinical variables evaluated as potential risk factors included age, sex, breed, presence of fever, tick infestation, haemoglobinuria, reduced appetite, pale mucous membranes, dyspnoea and epistaxis. For the haematological and biochemical comparisons, the control group comprised clinically healthy, non-infected dogs that were negative for small-form Babesia on peripheral blood smear examination.
       
Season was not included as a variable because uneven sample collection across seasons could affect the precision of estimates and potentially introduce bias. The presence of ticks was assessed based on owner history and clinical examination; therefore, formal tick species identification was not performed systematically for every dog. However, ticks collected and identified by the attending clinician in most cases were morphologically consistent with Rhipicephalus sanguineus. This observation should be interpreted cautiously because molecular confirmation of tick species and vector competence was not performed.
 
Hematological analysis
 
Hematological parameters were analyzed using an automated hematology analyzer (IDEXX ProCyte Dx™). The parameters evaluated included total erythrocyte count (TEC), haemoglobin concentration, haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), total leukocyte count (TLC), differential leukocyte count and platelet count.
 
Biochemical analysis
 
Serum was separated by centrifugation and subjected to biochemical analysis using an automated veterinary biochemistry analyzer (SMT-120V Vet, Seamaty). The parameters assessed included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin, blood urea nitrogen (BUN), creatinine, total protein, albumin, globulin and albumin-to-globulin (A:G) ratio.
 
Statistical analysis
 
Associations between categorical risk factors and microscopy-based detection of small-form Babesia spp. were assessed using the chi-square test, with Odds Ratios (ORs) and 95% Confidence Intervals (CIs) calculated where appropriate. Hematological and biochemical parameters were compared between infected and control groups using an unpaired t-test, with statistical significance set at p<0.05.
 
Ethical considerations
 
The study involved client-owned animals presented for clinical examination and all procedures were performed with the consent of the owners. Blood collection was carried out following standard veterinary clinical practices, ensuring minimal discomfort to the animals.

Out of 152 dogs examined, 23 dogs were positive for small-form Babesia spp. on peripheral blood smear examination, morphologically consistent with Babesia gibsoni, yielding an overall microscopic prevalence of 15.13% (Fig 1, 2). The occurrence of canine babesiosis across diverse agro-climatic regions of India, including Sikkim (Rani et al., 2011), Gujarat (Bilwal et al., 2017), Tamil Nadu (Jaisree et al., 2025) and Punjab (Singla et al., 2016), underscores its widespread distribution.




       
During microscopic examination, only small-form intraerythrocytic piroplasms consistent with Babesia gibsoni morphology were detected. No large-form Babesia organisms suggestive of Babesia canis or Babesia vogeli were observed. No microscopic evidence of other haemoprotozoan infections including Ehrlichia canis, Hepatozoon canis or mixed haemoparasitic infections was observed in the examined blood smears.
       
Reports from Kerala indicate substantially higher prevalence rates when molecular tools are employed, with Babesia gibsoni detected at 47.3% by PCR compared to 26.67% by microscopy (Jain et al., 2017) and similarly 50% versus 25.86% (Augustine et al., 2017). Recent studies have further documented molecular prevalence ranging from 40.89% to 58.33% (Ajith et al., 2024; Venugopal et al., 2024), supported by earlier reports (Karunakaran et al., 2011; Tresamol et al., 2013). These findings highlight the limited sensitivity of microscopy and suggest that the 15.13% prevalence observed in the present study may underestimate the true burden of infection. The estimate should therefore be interpreted as a microscopy-based prevalence among clinically suspected dogs rather than a definitive population prevalence.
       
Although the present investigation relied on conventional microscopy because of its affordability and routine applicability in field veterinary practice, previous molecular studies from Kerala have consistently demonstrated considerably higher detection rates using PCR (Ajith et al., 2024; Venugopal et al., 2024). Therefore, the prevalence reported in the present study likely represents the detectable microscopic burden rather than the true prevalence. Future investigations combining microscopy with molecular assays would improve species confirmation and detection of mixed haemoparasitic infections.
 
Risk factors
 
The association of various epidemiological and clinical variables with babesiosis is presented in Table 1. A higher prevalence was observed in dogs older than one year, females and non-descript breeds; however, only age showed a statistically significant association (p<0.05), corroborating the findings of Singh et al., (2014). The increased susceptibility observed in older animals may be related to cumulative exposure to tick vectors, although the cross-sectional design does not permit causal inference.


       
A highly significant association was observed between babesiosis and haemoglobinuria (p<0.01), with all four dogs presenting haemoglobinuria testing positive (4/4). However, the OR estimate of 297.00 is based on very sparse data and should therefore be interpreted with considerable caution; the extremely wide 95% CI (13.806-6389.141) further indicates substantial uncertainty around the estimate. Haemoglobinuria may reflect severe intravascular haemolysis in some canine cases. Fever was also significantly associated (p<0.01), with 34.48% positivity among febrile dogs, in agreement with Bilwal et al., (2017).
       
Tick infestation emerged as a major risk factor, with significantly higher infection rates in infested dogs (51.49%) compared to non-infested animals (4.27%) (p<0.01), consistent with Godara et al., (2010).Ticks were morphologically identified as Rhipicephalus sanguineus in many cases presented with ticks, a known vector of Babesia spp. in India (Jose et al., 2018). As the study was also conducted in Varkala, a tropical humid coastal region with year-round warmth, the favorable environmental conditions for tick survival may have contributed to the observed occurrence of babesiosis. Nevertheless, because tick species were not systematically identified in every dog and vector competence was not assessed, the observed association should not be interpreted as definitive evidence of transmission by a particular tick species.
       
Notably, the present study highlights that commonly perceived clinical indicators such as mucous membrane pallor and dyspnea may lack statistical reliability as standalone predictors, emphasizing the need for laboratory confirmation. These findings should, however, be interpreted in the context of the study population, which comprised clinically suspected dogs presenting to a veterinary clinic and should not be generalized directly to the wider dog population.
 
Hematological alterations
 
Hematological analysis (Table 2) revealed a highly significant reduction (p<0.01) in total erythrocyte count, hemoglobin concentration and hematocrit in infected dogs, indicating marked anaemia. These findings are consistent with Reddy et al. (2014); Nalubamba et al. (2015) and Anju et al., (2022). The pathogenesis of anaemia in babesiosis is multifactorial, involving erythrocyte destruction by piroplasms, immune-mediated hemolysis, oxidative damage and splenic sequestration (Meinkoth et al., 2002; Reddy et al., 2016).

Considering red cell indices, MCH showed a significant reduction, while MCHC was significantly elevated; MCV was increased but not significantly, in agreement with Gonmei et al., (2020). Leukogram analysis demonstrated a significant increase (p<0.05) in total leukocyte count, along with marked neutrophilia (p<0.01), indicating an active inflammatory response, as also reported by Bilwal et al., (2017) and Shah et al., (2011). Lymphocyte counts were elevated but non-significant, possibly reflecting chronic antigenic stimulation (Yogeshpriya et al., 2018; Vishnurahav et al., 2014).


       
Significant monocytosis and eosinopaenia were observed, aligning with Gryshchenko et al., (2023). Platelet counts were markedly reduced (p<0.01), consistent with Gonmei et al., (2020) and Anju et al., (2022), likely due to immune-mediated destruction, splenic sequestration and consumptive coagulopathy (Boozer and Macintire, 2005). These findings collectively indicate substantial haematological disturbance in infected dogs, although the cross-sectional design precludes determining whether individual abnormalities preceded infection or resulted from disease progression.
 
Biochemical alterations
 
Biochemical parameters (Table 3) revealed significant elevations in AST and ALT (p<0.01), indicative of hepatocellular damage, in agreement with Wadhwa et al., (2011), Bilwal et al., (2017) and Gryshchenko et al. (2023). Increased enzyme activity may result from hepatocellular necrosis or increased membrane permeability.


       
Although ALP levels were elevated, the increase was not statistically significant, contrasting with reports by Gonmei et al. (2020) and Anju et al., (2022). Total bilirubin levels were significantly increased (p<0.05), reflecting hemolysis and possible cholestatic dysfunction (Chan et al., 2025), consistent with Shah et al., (2011) and Bilwal et al., (2017), but contrary to some reports (Gonmei et al., 2020; Gryshchenko et al., 2023).
       
Blood urea nitrogen levels were significantly elevated (p<0.05), likely due to increased protein catabolism and hemolysis-related nitrogen load (Reddy et al., 2014; Gryshchenko et al., 2023). Creatinine levels showed a non-significant increase, suggesting limited renal compromise in most cases, although contrasting findings have been reported (Vishnurahav et al., 2014; Reddy et al., 2024).
       
Total protein and globulin levels were significantly elevated (p<0.01), reflecting enhanced acute-phase and immunoglobulin synthesis during systemic inflammation (Tóthová et al., 2020). In contrast, albumin levels and A:G ratio was significantly reduced (p<0.01), possibly due to increased vascular permeability and redistribution and altered hepatic protein synthesis, supporting Gonmei et al., (2020).
       
Molecular methods such as PCR generally provide greater analytical sensitivity and enable species-level identification, but may be less accessible in routine clinical settings. Microscopy remains inexpensive, widely available and rapid; however, it is less sensitive in low-parasitaemia infections, may miss small-form Babesia and cannot confirm species.  As reported in previous studies from Kerala, Babesia gibsoni is among the most commonly identified small-form Babesia species and the morphological features observed in this study are consistent with findings suggestive of B. gibsoni (Deepa, 2021; Anju et al., 2022).  However, in the absence of molecular confirmation, species-level identification remains presumptive. The microscopy-based diagnostic approach may therefore have led to underestimation of prevalence and may also have preferentially detected dogs with higher parasitaemia.
       
The present study integrates epidemiological risk profiling with detailed haematological and biochemical assessment under field conditions in Southern Kerala. This integrated approach provides a useful clinicopathological framework for small-form canine babesiosis. However, the findings should be interpreted in light of the study's single-clinic setting, microscopy-based diagnosis and lack of molecular species confirmation.
       
Additionally, the identification of statistically significant associations involving age, fever, tick infestation and haemoglobinuria, alongside non-significant associations for several other clinical indicators, underscores the limitations of symptom-based diagnosis and reinforces the importance of laboratory-supported decision-making. A limitation of the present investigation is that species confirmation was not performed using molecular techniques; therefore, parasite identification was based solely on characteristic microscopic morphology. In addition, co-infections with other tick-borne pathogens, including Ehrlichia and Hepatozoon species, may occur in endemic regions and could contribute to or modify the observed haematological and biochemical abnormalities. As the present study did not include molecular or serological testing for other vector-borne pathogens, their potential contribution cannot be excluded. These limitations should be considered when interpreting the findings and highlight the need for future studies incorporating molecular species confirmation and comprehensive screening for concurrent vector-borne infections.

The present study demonstrated that small-form canine babesiosis is endemic in Southern Kerala with a microscopic prevalence of 15.13%. Age, fever, tick infestation and haemoglobinuria were identified as significant risk determinants. Infection was associated with marked anaemia, thrombocytopenia and significant hepatic and biochemical alterations. These findings provide valuable baseline epidemiological and clinicopathological information for improving diagnosis, clinical management and future molecular epidemiological investigations on canine babesiosis in Southern India.

The study was conducted in Dr. Tails Veterinary Clinic, Varkala, Kerala, India. The authors are thankful to all the staffs of the clinic and pet owners for their valuable co-operation. 

The authors declare that there are no conflicts of interest regarding the publication of this paper.