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Full Research Article

Molecular Characterization of Canine Babesiosis in Coastal Odisha

A
Anita Dora1
M
Manaswini Dehuri1,*
B
Bijayendranath Mohanty1
A
Ananta Hembram1
P
Prasanta Kumar Koustasa Mishra2
A
Adhikari Sahu1
B
Biswaranjan Maharana3
S
Satyapragyan Mishra1
1Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.
2Department of Veterinary Biochemistry, Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.
3Referral Laboratory (Parasitology Division), HPKV, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.

ABSTRACT

Background: Babesiosis poses a significant threat to canine health, contributing substantially to morbidity and mortality. Therefore, the present study was undertaken to molecularly characterize Babesia spp. isolates obtained from dogs in coastal Odisha.

Methods: A total of 102 blood samples presented to Veterinary Clinical Complex Odisha University of Agriculture and Technology, Bhubaneswar belonging to both, male and female dogs of different age group were screened for presence of Babesia spp. Blood smears from the collected samples were perfectly prepared and duly stained by Giemsa stain. Primers targeting 18S rRNA for Babesia genus identification (PIRO1/PIRO B) was done for Primary PCR, while for nested PCR of B. vogeli (BAB1/BAB4) and B. gibsoni (Gib 599 and Gib 1270) was carried out using species specific primer. Also Duplex PCR was carried out for simultaneous detection of both Babesia isolates. The sequence alignment of 18S rRNA sequences of Babesia isolate from Odisha with other geographical isolates was done by Clustal Omega and two phylogenetic trees were constructed using Mega XI software tool.

Result: The overall prevalence of canine babesiosis based on microscopy was 15.68% (16/102) while the primary PCR assay detected Babesia spp was 26.47% (27/102). The nested PCR assay amplified 600 bp and 670 bp partial 18S rRNA of B. vogeli and B. gibsoni, respectively and the overall prevalence of B. gibsoni and B. vogeli in coastal Odisha was 23.52% (24/102) and 3.92% (4/102) respectively. Pimary PCR assay from 38 tick samples (Rhipicephalus sanguineus) yielded 9 samples positive (23.68%) for Babesia spp.  There was statistically (p<0.01) greater presence of canine babesiosis in age group of 2-4 years while higher presence was revealed for male dogs though statistically non-significant (p>0.05). The partial sequences were assigned GenBank Accession no of PQ505093.1 and PQ504910.1 for B. gibsoni and PQ580773.1 for B.vogeli isolate. The evolutionary history analysis of both isolates revealed genetic similarity with Indian and well as foreign isolates. A higher presence of Babesia gibsoni was detected, with age group of 2-4 years and males being more vulnerable to canine babesiosis in the present study.

INTRODUCTION

Babesiosis is a severe, globally occurring vector-borne disease incriminating various Babesia species and affecting both domestic and wild animals, transmitted by certain Ixodid ticks. In 1895, the disease was first detected in dogs in northern Italy while Smith and Kilborne associated ixodid ticks as vectors in the transmission of Babesia infection. Canine babesiosis is a tick-borne illness affecting dogs worldwide, caused by intra-erythrocytic protozoa belonging to order Piroplasmida whose prevalence and geographical distribution are influenced by the types of ticks like Rhipicephalus sanguineus found in different regions (Solano-Gallego et al., 2016). Canine babesiosis is caused by both large and small forms of Babesia spp. These species are found worldwide due to international travel with pets and climate change, which has led to the expansion of tick habitats. The rise in temperature and humidity, along with the growing population of stray dogs, have contributed to increased transmission of Babesia spp. among dogs in India (Abd Rani et al., 2011). Canine babesiosis does not present with specific symptoms; but common signs include fever, loss of appetite, nosebleeds, petechiae and an enlarged spleen along with haemoglobinuria, anaemia and low platelet count (Sudhakara et al., 2016). Most infected dogs display anaemia and all cases involve thrombocytopenia.
       
Diagnosis of babesiosis is primarily based upon crucial analysis of exhibited clinical signs and and detection of Babesia spp. in stained blood smear under microscope. But in this method, sometimes it is difficult to detect piroplasms even in acute infection and also in higher level of parasitemia. In this circumstances, relevant immunological or molecular method using PCR is more useful for detection of babesiosis (Kushwaha et al., 2017). But immunological methods also exhibit certain drawbacks particularly in acute and long standing chronic cases, where false negative results are very often found due to lack of optimum antibody to show the results of the tests. Therefore, molecular method like PCR is the best technique for detection of babesiosis as it is highly sensitive and accurate, even in acute or chronic cases or in cases which was found negative in microscopic examination. After consistent research and development, nested PCR has been considered perfect and this test is currently in use to detect Babesia up to species level.
       
Babesiosis, an emerging disease in many parts of the world, requires accurate diagnosis to support effective treatment. In India, data on the molecular prevalence and characterization of Babesia in dogs are limited, with some recent studies using molecular techniques to estimate infection rates (Singh et al., 2016 in Punjab; Jain et al., 2017 in Kerala; Jalajakshi et al., 2021 in Andhra Pradesh). As there is a requisite to identify the genotype of the parasite during clinical illness and assess the strains and genetic diversity present in specific geographical area, the present study was undertaken to carry out molecular characterisation of Babesia species in the coastal districts of Odisha, Eastern India.

MATERIALS AND METHODS

Collection of blood sample and ticks
 
The canine blood samples routinely presented for diagnostic purpose to Veterinary Clinical Complex, Odisha University of Agriculture and Technology, Bhubaneswar, from March to December 2024 were collected, taking note of details of the animals.
 
Microscopic examination
 
The present investigation was carried out on 102 blood samples which were subjected to microscopic examination by making peripheral thin blood smears and staining with Giemsa stain (diluted with 1:9). The stained smears were washed, air dried and examined under oil immersion lens of microscope for detection of Babesia piroplasm. The blood samples were further stored at -20°C until DNA extraction and PCR analysis.
 
Primary, nested and duplex PCR
 
Genomic DNA was isolated from 200 μl of whole blood by using QIAamp®DNA blood mini kit (QIAGEN, GmbH, Germany) following the manufacturer’s recommendations. DNA from ticks were isolated using DNEasy blood and tissue kit (QIAGEN, GmbH, Germany) by trichurating the tick samples in buffer saline and subsequent steps were followed as per the manufacture’s protocol. DNA was quantified by using Nano drop (M/S Qiagen). PCR amplification of 18S rRNA gene was done using appropriate primers (Table 1) and using a gradient thermal cycler (M/S Prima, Himedia) in a final reaction volume of 25 μl with contents (Table 2) in a 200 μl PCR tube. The PCR  was carried with different cycling conditions (Table 3). In addition to the test DNA samples, a positive control (DNA of known sequence), a negative control constituting ultra-pure water in substitution of DNA in equal volume were included in each of the PCR amplification. The DNA samples from suspected canine blood were subjected to primary PCR amplification and then 2 μl of PCR product obtained from primary PCR were used as a template in nested PCR using species specific primer to produce the amplicon of desired size. Duplex PCR was attempted for simultaneous detection of B. vogeli and B. gibsoni from the blood samples of suspected dogs. The expected PCR product for Babesia genus was 410 bp DNA fragment while for B. vogeli and B. gibsoni were 600 bp and 670 bp respectively for nested PCR. In case of Duplex PCR, the expected product size for B. vogeli  and B. gibsoni were 600 bp and 488 bp respectively.

Table 1: List of primers for different PCR.



Table 2: Constituents of PCR tube used for PCR amplification.



Table 3: Thermocycling conditions for amplification in different PCR types.


 
Agarose gel electrophoresis, purification and sequencing of PCR product
 
The agarose gel electrophoresis was carried out for checking DNA as well as evaluation of PCR products. The DNA/ amplified PCR products were analyzed on 1.5% agarose gel. The horizontal gel electrophoresis was carried out at 80V for about 60 minutes or till the dye migrated to two-third distance down the gel. The bands were visualised using an UV transilluminator (Genei, Bangalore). PCR products of 18S rRNA gene of Babesia vogeli and Babesia gibsoni were commercially purified using Purification kit and sequenced by using Sanger dideoxy sequencing in an automated DNA sequencer at Barcode Biosciences and Eurofinns Genomics, Bengaluru.
 
Sequence analysis
 
The sequences were visualised and manually edited with the help of the program BioEdit. Consensus sequence was obtained by DNA star software program. The sequence similarity was then checked against sequences deposited in the GenBank using the BLAST software. Reference sequences of 18S rRNA genes of Babesia vogeli and Babesia gibsoni were retrieved from NCBI GenBank and were used in the multiple sequence alignment of each gene using Clustal Omega software.
 
Phylogenetic analysis
 
The phylogenetic tree was based on a Clustal alignment of the sequences using the Neighbor-Joining method conducted in MEGA XI software. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analysed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. Bootstrap replications (1000) were performed, which were expressed on each node as a percentage The evolutionary distances were computed using the Maximum Composite Likelihood method. Two phylogenetic trees were constructed by taking sequences from the present study (Babesia gibsoni and Babesia vogeli) and other sequences of 18S rRNA gene from different countries (China, Japan, USA, Myanmar, West Indies, Spain, Australia, Bangladesh, Malaysia, Brazil,) as well as Indian cities (Junagarh, Kerala, Kolkata, Delhi, Chennai) retrieved from GenBank.
 
Statistical analysis
 
Statistical analysis with regards to prevalence analysis of age and sex was done using chi square method by SPSS 27.0 software.

RESULTS AND DISCUSSION

Microscopic examination
 
During the present investigation, 102 canine blood samples were examined by conventional microscopy and PCR. In microscopical examination, 16 (15.68%) samples were found positive for Babesia organisms in Giemsa stained blood smears by observing typical piroplasms in RBC.
 
Molecular detection
 
The examination of blood sample (n=102) by primary PCR assay revealed that 27 samples (26.47%) were positive for Babesia spp.  based on amplification of 18S rRNA gene yielding 410bp product (Fig 1). Nested PCR assay on the positive Babesia spp. yielded 4 samples (3.92%) were positive for Babesia vogeli yielding 600 bp product (Fig 2). while 24 samples were found (23.52%) positive for Babesia gibsoni amplifying 670 bp product (Fig 3) The oligonucleotide primers for each set of PCR reactions did not yield any PCR product with the negative control. Duplex PCR for simultaneous detection of Babesia vogeli and Babesia gibsoni was performed using the two primers at the annealing temperature of 54°C. The samples which were used in PCR assay were the similar samples which we found positive for Babesia vogeli and Babesia gibsoni by singleplex PCR assay. The results of duplex PCR confirmed that amplification of genomic DNA of Babesia vogeli and Babesia gibsoni yielded a PCR product of 600 bp and 488 bp respectively (Fig 4).

Fig 1: Agarose gel electrophoresis of Primary PCR product for detection of Babesia spp from canine blood smear.



Fig 2: Agarose gel electrophoresis of nested PCR product for detection of Babesia vogeli.



Fig 3: Agarose gel electrophoresis of Nested PCR product for detection of Babesia gibsoni.



Fig 4: Agarose gel electrophoresis of duplex PCR product for detection of Babesia gibsoni and Babesia vogeli


       
Pimary PCR assay from 38 tick samples (Rhipicephalus sanguineus) yielded 9 samples positive (23.68%) for Babesia spp. yielding 410 bp. In the present study there was no occurrence of any other tick species from the suspected dogs.
 
Age and sex wise prevalence
 
The highest (13.72%) prevalence of Babesia infections was found in canines of age group 2-4 years followed by 7.84% corresponding to age group 0-2 years  and the lowest value (4.9%)was recorded in the age group of 4 years (Table 4). There was significant difference (p<0.01) in the prevalence of different age groups. Sex-wise analysis revealed a higher prevalence of babesiosis in male dogs (16.67%) compared with female dogs (9.80%). The overall prevalence did not vary significantly (p>0.05) between male and female canines.

Table 4: Age and sex wise prevalence of canine babesiosis.


 
Sequencing and phylogenetic analysis
 
Three representative samples for the haemo-protozoan parasites were subjected to sequencing. The partial sequences were assigned GenBank Accession no of PQ505093.1 and PQ504910.1 for B. gibsoni and PQ580773.1 for B. vogeli isolate. When compared to those18SrRNA gene sequences published in GenBank, the identity rates of the isolates under study ranged from 98 to 100%.
       
The phylogenetic tree constructed showed that Babesia vogeli (Fig 5) isolated from dogs of coastal Odisha was most closely related with B, vogeli isolate of Chennai (MN165666.1), Junagarh (MZ646048.1) and Delhi (MN165667.1) in India, while 18S rRNA of Babesia vogeli in the present study showed close genetic relationship with isolate from Brazil (JX535812.1) with a high bootstrap value. A lower genetic relationship was observed among the present B. vogeli isolate with that of isolates from Kerala (MN190268.1), Punjab (MZ413886.1), China (MK881129.1) and Columbia (KT946902.1). The phylogenetic tree for Babesia gibsoni (Fig 6) constructed showed that one Babesia gibsoni isolated from dogs of coastal Odisha was most closely related with B. gibsoni isolate of Junagarh (MG675030.1) Kolkata (KJ142323.1) in India and Malaysia (KU500919.1) being in the same clade while another B. gibsoni isolate from our investigation was genetically similar to Australia (AY102164), Bangladesh (LC006968.1) and USA (EU084679.1) isolates. There was lesser genetic resemblance with isolate from Japan (AB478319.1) for both isolates obtained in the present study.

Fig 5: Phylogenetic analysis of Babesia vogeli isolates by neighbor joining method.



Fig 6: Phylogenetic analysis of Babesia gibsoni isolates by neighbor joining method.


       
Tick-borne diseases, particularly those caused by haemoprotozoan parasites, are common in tropical and subtropical regions. The severity of these infections often hinges on the extent of host erythrocyte destruction due to the parasite’s rapid proliferation. In India, Babesiosis in dog was first identified in 1910 (Patton, 1910) and since its discovery, it has posed considerable challenges to veterinarians, particularly in clinical, diagnostic and therapeutic management. The PCR assay for Babesia spp. began in 2000s and the diagnosis through molecular means was widely followed by researchers abroad. In the present study, there was detection of Babesia spp. in 26.47% dogs by Primary PCR by amplification of 18S rRNA. An overall prevalence ranging from 20-30% has been reported earlier from Haryana (Bhagwan et al., 2024) Bengaluru (Mahalingaiah et al., 2017), Maharashtra (Debbarma et al., 2022) and Andhra Pradesh (Jalajakshi et al., 2021). A higher range of prevalence ranging from about 50-60% has also been recorded from North eastern regions of India (Laha et al., 2014; Mittal et al., 2019) and South India (Arthi et al., 2017).
       
Further, species specific primers were used in nested PCR, which revealed that 23.53% dogs were positive for Babesia gibsoni while only 3.92% cases were positive for Babesia vogeli. A higher presence of Babesia gibsoni in comparison to Babesia vogeli was also documented from other parts of India (Mittal et al., 2019; Jalajakshi et al., 2021). However, a higher presence of Babesia vogeli has also been documented from Haryana (Bhagwan et al., 2024); Maharashtra (Debbarma et al., 2022) Assam (Laha et al., 2014) and Rajasthan (Choudhary et al., 2025).
       
B. gibsoni has been identified across five continents, including Asia, Africa, North America, Europe and Australia and is known to cause highly severe disease (Irwin, 2010). Also, chances of possible transmission of this small form of Babesia by ingestion of blood, contact with wounds or saliva have been contemplated (Irizarry-Rovira et al., 2001).

Interestingly, a previous study conducted in the same geographical area reported a predominance of the large form of Babesia, identified as B. vogeli, with only a minor occurrence of the small form, B.gibsoni (Panda et al., 2021). In contrast, the present investigation revealed a different pattern of occurrence, indicating a shift in the predominant species/form detected in dogs from the coastal Odisha region. As far as detection of canine babesiosis outside India is concerned, Babesia vogeli has been widely documented in Africa, (Mghirbi and Bouattour, 2008; Selim et al., 2022), Europe (Solano-Gallego et al., 2008), Both B. gibsoni and B.vogeli have been recorded from canines in European countries (Schafer et al., 2023)  and Thailand (Do et al., 2021).
       
The lower prevalence of B.vogeli during the present investigation is quite consistent with earlier reports from Karnataka (Lavanya et al., 2019) depicting a prevalence lesser than 10% and also in Egypt (Selim et al., 2022) and Brazil (de Oliveira et al., 2016). There is disparity between these prevalence rates due to variation in study, design and duration, environment parameters, presence of vectors and reservoir hosts and kind of diagnostic tests employed. The genes used for diagnosis through PCR assay also varies. There is also differences with regards to veterinary health care like the frequency and mode of treatment.
       
During this present research, there was no instance of coinfection with B. vogeli or B. gibsoni. However, concurrent infections of larger and smaller forms of Babesia have been reported from dogs elsewhere (Vairamuthu et al., 2014; Mahalingaiah et al., 2017).
       
A higher detection of canine babesiosis by PCR-based assays in comparison to conventional microscopic examination as noticed in the present investigation, has also been reported by several researchers in India and abroad pointing at higher sensitivity of molecular assay (Laha et al., 2014; Birkenheuer et al., 2003).
       
As per our investigation, there was higher presence of Babesiosis in male dogs as compared to females which corroborates the earlier observations elsewhere (Mahalingaiah et al., 2017, Jalajakshi et al., 2021). The higher prevalence of babesiosis in male dogs may be associated with sex-related differences in immune function, as testosterone has been reported to modulate immune responses and may increase susceptibility to parasitic infections (Roberts et al., 2001). Nevertheless, this association should be considered a possible contributing factor rather than a direct causal explanation for the higher prevalence observed in males. Also it has been postulated that the hormonal influence and temperament of males could result in limited attention from owners (Mellanby et al., 2011). Whereas in female dogs, estrogen improves the immune reactivity and produces resistance to invading infection (Taneja, 2018). However, in contrast to the present study, previous reports from other states (Arthi, 2017, Lavanya et al., 2019) and Madhya Pradesh (Shrivastava et al., 2014) have depicted a higher presence of Babesia infection in females over males. The reasoning being a sedentary lifestyle in females especially during pregnancy and nursing, making it more prone to tick infestation and lowering of immunity.
       
The study presented that dogs in the age group 2-4 years showed a higher presence of canine babesiosis. Earlier reports have noted higher prevalence in dogs above one year of age (Singh et al. 2014 and Kumar et al. 2015). Also reports from India (Shrivastava et al., 2014) and Nigeria (Obeta et al., 2020) have revealed higher age wise prevalence in the age group of 1 to 3 years. Younger dogs, due to maternal immunity have lesser chance to gain infection and lack of past exposure in comparison to older dogs makes it less prone to babesiosis. There has been evidence of older dogs being prone to Babesia infection as reported by higher presence of B. gibsoni in adults (Kumar et al., 2009). As per earlier research, babesiosis in dog’s increases with age, with the infection reaching a peak between 3-5 years’ age group after which it starts declining (Hornok et al., 2016). However, young pups have a compromised immune status and are more active and agile making it more prone to tick exposure and thereby infection (Obeta et al., 2020).
       
The haemoprotozoan infection due to Babesia vogeli and Babesia gibsoni was confirmed by sequencing of the portion of 18S rRNA gene for randomly selected positive dog samples. There was construction of two phylogenetic tree by utilising the Neighbor-Joining method in MEGA 11. The tree was built through sequences of 18S rRNA gene obtained in this study and additional sequences available in GenBank. Phylogenetic analysis of Babesia vogeli 18S rRNA partial sequences presented a close similarity with Indian isolates as well as with isolate from Brazil. The B. gibsoni isolates from the present study formed a group with other geographical isolates outside India including Australia, Bangladesh, America and Australia. Studies conducted in different parts of India (Roopesh et al., 2018; Deepa, 2023) also indicated that Indian B. gibsoni strains clustered into a major group with isolates from other parts of the country.

CONCLUSION

The current study suggests that babesiosis is an emerging or re-emerging tick-borne hemoprotozoan disease affecting dogs in coastal Odisha with Babesia gibsoni showing a greater presence over Babesia vogeli. PCR proved to be a highly sensitive, rapid and reliable method for detecting Babesia infection in both carrier and clinically suspected animals. Accurate differentiation of Babesia species is clinically important because the infecting species can differ considerably in pathogenicity, disease severity, prognosis and response to treatment. Therefore, species-level identification is essential for appropriate treatment selection and effective clinical management of canine babesiosis. Future studies regarding interaction of tick vectors and the parasite could yield a better epidemiological understanding of canine babesiosis in the area.

ACKNOWLEDGEMENT

The present study was supported by College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
Not applicable.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Molecular Characterization of Canine Babesiosis in Coastal Odisha

    A
    Anita Dora1
    M
    Manaswini Dehuri1,*
    B
    Bijayendranath Mohanty1
    A
    Ananta Hembram1
    P
    Prasanta Kumar Koustasa Mishra2
    A
    Adhikari Sahu1
    B
    Biswaranjan Maharana3
    S
    Satyapragyan Mishra1
    1Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.
    2Department of Veterinary Biochemistry, Odisha University of Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.
    3Referral Laboratory (Parasitology Division), HPKV, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.

    ABSTRACT

    Background: Babesiosis poses a significant threat to canine health, contributing substantially to morbidity and mortality. Therefore, the present study was undertaken to molecularly characterize Babesia spp. isolates obtained from dogs in coastal Odisha.

    Methods: A total of 102 blood samples presented to Veterinary Clinical Complex Odisha University of Agriculture and Technology, Bhubaneswar belonging to both, male and female dogs of different age group were screened for presence of Babesia spp. Blood smears from the collected samples were perfectly prepared and duly stained by Giemsa stain. Primers targeting 18S rRNA for Babesia genus identification (PIRO1/PIRO B) was done for Primary PCR, while for nested PCR of B. vogeli (BAB1/BAB4) and B. gibsoni (Gib 599 and Gib 1270) was carried out using species specific primer. Also Duplex PCR was carried out for simultaneous detection of both Babesia isolates. The sequence alignment of 18S rRNA sequences of Babesia isolate from Odisha with other geographical isolates was done by Clustal Omega and two phylogenetic trees were constructed using Mega XI software tool.

    Result: The overall prevalence of canine babesiosis based on microscopy was 15.68% (16/102) while the primary PCR assay detected Babesia spp was 26.47% (27/102). The nested PCR assay amplified 600 bp and 670 bp partial 18S rRNA of B. vogeli and B. gibsoni, respectively and the overall prevalence of B. gibsoni and B. vogeli in coastal Odisha was 23.52% (24/102) and 3.92% (4/102) respectively. Pimary PCR assay from 38 tick samples (Rhipicephalus sanguineus) yielded 9 samples positive (23.68%) for Babesia spp.  There was statistically (p<0.01) greater presence of canine babesiosis in age group of 2-4 years while higher presence was revealed for male dogs though statistically non-significant (p>0.05). The partial sequences were assigned GenBank Accession no of PQ505093.1 and PQ504910.1 for B. gibsoni and PQ580773.1 for B.vogeli isolate. The evolutionary history analysis of both isolates revealed genetic similarity with Indian and well as foreign isolates. A higher presence of Babesia gibsoni was detected, with age group of 2-4 years and males being more vulnerable to canine babesiosis in the present study.

    INTRODUCTION

    Babesiosis is a severe, globally occurring vector-borne disease incriminating various Babesia species and affecting both domestic and wild animals, transmitted by certain Ixodid ticks. In 1895, the disease was first detected in dogs in northern Italy while Smith and Kilborne associated ixodid ticks as vectors in the transmission of Babesia infection. Canine babesiosis is a tick-borne illness affecting dogs worldwide, caused by intra-erythrocytic protozoa belonging to order Piroplasmida whose prevalence and geographical distribution are influenced by the types of ticks like Rhipicephalus sanguineus found in different regions (Solano-Gallego et al., 2016). Canine babesiosis is caused by both large and small forms of Babesia spp. These species are found worldwide due to international travel with pets and climate change, which has led to the expansion of tick habitats. The rise in temperature and humidity, along with the growing population of stray dogs, have contributed to increased transmission of Babesia spp. among dogs in India (Abd Rani et al., 2011). Canine babesiosis does not present with specific symptoms; but common signs include fever, loss of appetite, nosebleeds, petechiae and an enlarged spleen along with haemoglobinuria, anaemia and low platelet count (Sudhakara et al., 2016). Most infected dogs display anaemia and all cases involve thrombocytopenia.
           
    Diagnosis of babesiosis is primarily based upon crucial analysis of exhibited clinical signs and and detection of Babesia spp. in stained blood smear under microscope. But in this method, sometimes it is difficult to detect piroplasms even in acute infection and also in higher level of parasitemia. In this circumstances, relevant immunological or molecular method using PCR is more useful for detection of babesiosis (Kushwaha et al., 2017). But immunological methods also exhibit certain drawbacks particularly in acute and long standing chronic cases, where false negative results are very often found due to lack of optimum antibody to show the results of the tests. Therefore, molecular method like PCR is the best technique for detection of babesiosis as it is highly sensitive and accurate, even in acute or chronic cases or in cases which was found negative in microscopic examination. After consistent research and development, nested PCR has been considered perfect and this test is currently in use to detect Babesia up to species level.
           
    Babesiosis, an emerging disease in many parts of the world, requires accurate diagnosis to support effective treatment. In India, data on the molecular prevalence and characterization of Babesia in dogs are limited, with some recent studies using molecular techniques to estimate infection rates (Singh et al., 2016 in Punjab; Jain et al., 2017 in Kerala; Jalajakshi et al., 2021 in Andhra Pradesh). As there is a requisite to identify the genotype of the parasite during clinical illness and assess the strains and genetic diversity present in specific geographical area, the present study was undertaken to carry out molecular characterisation of Babesia species in the coastal districts of Odisha, Eastern India.

    MATERIALS AND METHODS

    Collection of blood sample and ticks
     
    The canine blood samples routinely presented for diagnostic purpose to Veterinary Clinical Complex, Odisha University of Agriculture and Technology, Bhubaneswar, from March to December 2024 were collected, taking note of details of the animals.
     
    Microscopic examination
     
    The present investigation was carried out on 102 blood samples which were subjected to microscopic examination by making peripheral thin blood smears and staining with Giemsa stain (diluted with 1:9). The stained smears were washed, air dried and examined under oil immersion lens of microscope for detection of Babesia piroplasm. The blood samples were further stored at -20°C until DNA extraction and PCR analysis.
     
    Primary, nested and duplex PCR
     
    Genomic DNA was isolated from 200 μl of whole blood by using QIAamp®DNA blood mini kit (QIAGEN, GmbH, Germany) following the manufacturer’s recommendations. DNA from ticks were isolated using DNEasy blood and tissue kit (QIAGEN, GmbH, Germany) by trichurating the tick samples in buffer saline and subsequent steps were followed as per the manufacture’s protocol. DNA was quantified by using Nano drop (M/S Qiagen). PCR amplification of 18S rRNA gene was done using appropriate primers (Table 1) and using a gradient thermal cycler (M/S Prima, Himedia) in a final reaction volume of 25 μl with contents (Table 2) in a 200 μl PCR tube. The PCR  was carried with different cycling conditions (Table 3). In addition to the test DNA samples, a positive control (DNA of known sequence), a negative control constituting ultra-pure water in substitution of DNA in equal volume were included in each of the PCR amplification. The DNA samples from suspected canine blood were subjected to primary PCR amplification and then 2 μl of PCR product obtained from primary PCR were used as a template in nested PCR using species specific primer to produce the amplicon of desired size. Duplex PCR was attempted for simultaneous detection of B. vogeli and B. gibsoni from the blood samples of suspected dogs. The expected PCR product for Babesia genus was 410 bp DNA fragment while for B. vogeli and B. gibsoni were 600 bp and 670 bp respectively for nested PCR. In case of Duplex PCR, the expected product size for B. vogeli  and B. gibsoni were 600 bp and 488 bp respectively.

    Table 1: List of primers for different PCR.



    Table 2: Constituents of PCR tube used for PCR amplification.



    Table 3: Thermocycling conditions for amplification in different PCR types.


     
    Agarose gel electrophoresis, purification and sequencing of PCR product
     
    The agarose gel electrophoresis was carried out for checking DNA as well as evaluation of PCR products. The DNA/ amplified PCR products were analyzed on 1.5% agarose gel. The horizontal gel electrophoresis was carried out at 80V for about 60 minutes or till the dye migrated to two-third distance down the gel. The bands were visualised using an UV transilluminator (Genei, Bangalore). PCR products of 18S rRNA gene of Babesia vogeli and Babesia gibsoni were commercially purified using Purification kit and sequenced by using Sanger dideoxy sequencing in an automated DNA sequencer at Barcode Biosciences and Eurofinns Genomics, Bengaluru.
     
    Sequence analysis
     
    The sequences were visualised and manually edited with the help of the program BioEdit. Consensus sequence was obtained by DNA star software program. The sequence similarity was then checked against sequences deposited in the GenBank using the BLAST software. Reference sequences of 18S rRNA genes of Babesia vogeli and Babesia gibsoni were retrieved from NCBI GenBank and were used in the multiple sequence alignment of each gene using Clustal Omega software.
     
    Phylogenetic analysis
     
    The phylogenetic tree was based on a Clustal alignment of the sequences using the Neighbor-Joining method conducted in MEGA XI software. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analysed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. Bootstrap replications (1000) were performed, which were expressed on each node as a percentage The evolutionary distances were computed using the Maximum Composite Likelihood method. Two phylogenetic trees were constructed by taking sequences from the present study (Babesia gibsoni and Babesia vogeli) and other sequences of 18S rRNA gene from different countries (China, Japan, USA, Myanmar, West Indies, Spain, Australia, Bangladesh, Malaysia, Brazil,) as well as Indian cities (Junagarh, Kerala, Kolkata, Delhi, Chennai) retrieved from GenBank.
     
    Statistical analysis
     
    Statistical analysis with regards to prevalence analysis of age and sex was done using chi square method by SPSS 27.0 software.

    RESULTS AND DISCUSSION

    Microscopic examination
     
    During the present investigation, 102 canine blood samples were examined by conventional microscopy and PCR. In microscopical examination, 16 (15.68%) samples were found positive for Babesia organisms in Giemsa stained blood smears by observing typical piroplasms in RBC.
     
    Molecular detection
     
    The examination of blood sample (n=102) by primary PCR assay revealed that 27 samples (26.47%) were positive for Babesia spp.  based on amplification of 18S rRNA gene yielding 410bp product (Fig 1). Nested PCR assay on the positive Babesia spp. yielded 4 samples (3.92%) were positive for Babesia vogeli yielding 600 bp product (Fig 2). while 24 samples were found (23.52%) positive for Babesia gibsoni amplifying 670 bp product (Fig 3) The oligonucleotide primers for each set of PCR reactions did not yield any PCR product with the negative control. Duplex PCR for simultaneous detection of Babesia vogeli and Babesia gibsoni was performed using the two primers at the annealing temperature of 54°C. The samples which were used in PCR assay were the similar samples which we found positive for Babesia vogeli and Babesia gibsoni by singleplex PCR assay. The results of duplex PCR confirmed that amplification of genomic DNA of Babesia vogeli and Babesia gibsoni yielded a PCR product of 600 bp and 488 bp respectively (Fig 4).

    Fig 1: Agarose gel electrophoresis of Primary PCR product for detection of Babesia spp from canine blood smear.



    Fig 2: Agarose gel electrophoresis of nested PCR product for detection of Babesia vogeli.



    Fig 3: Agarose gel electrophoresis of Nested PCR product for detection of Babesia gibsoni.



    Fig 4: Agarose gel electrophoresis of duplex PCR product for detection of Babesia gibsoni and Babesia vogeli


           
    Pimary PCR assay from 38 tick samples (Rhipicephalus sanguineus) yielded 9 samples positive (23.68%) for Babesia spp. yielding 410 bp. In the present study there was no occurrence of any other tick species from the suspected dogs.
     
    Age and sex wise prevalence
     
    The highest (13.72%) prevalence of Babesia infections was found in canines of age group 2-4 years followed by 7.84% corresponding to age group 0-2 years  and the lowest value (4.9%)was recorded in the age group of 4 years (Table 4). There was significant difference (p<0.01) in the prevalence of different age groups. Sex-wise analysis revealed a higher prevalence of babesiosis in male dogs (16.67%) compared with female dogs (9.80%). The overall prevalence did not vary significantly (p>0.05) between male and female canines.

    Table 4: Age and sex wise prevalence of canine babesiosis.


     
    Sequencing and phylogenetic analysis
     
    Three representative samples for the haemo-protozoan parasites were subjected to sequencing. The partial sequences were assigned GenBank Accession no of PQ505093.1 and PQ504910.1 for B. gibsoni and PQ580773.1 for B. vogeli isolate. When compared to those18SrRNA gene sequences published in GenBank, the identity rates of the isolates under study ranged from 98 to 100%.
           
    The phylogenetic tree constructed showed that Babesia vogeli (Fig 5) isolated from dogs of coastal Odisha was most closely related with B, vogeli isolate of Chennai (MN165666.1), Junagarh (MZ646048.1) and Delhi (MN165667.1) in India, while 18S rRNA of Babesia vogeli in the present study showed close genetic relationship with isolate from Brazil (JX535812.1) with a high bootstrap value. A lower genetic relationship was observed among the present B. vogeli isolate with that of isolates from Kerala (MN190268.1), Punjab (MZ413886.1), China (MK881129.1) and Columbia (KT946902.1). The phylogenetic tree for Babesia gibsoni (Fig 6) constructed showed that one Babesia gibsoni isolated from dogs of coastal Odisha was most closely related with B. gibsoni isolate of Junagarh (MG675030.1) Kolkata (KJ142323.1) in India and Malaysia (KU500919.1) being in the same clade while another B. gibsoni isolate from our investigation was genetically similar to Australia (AY102164), Bangladesh (LC006968.1) and USA (EU084679.1) isolates. There was lesser genetic resemblance with isolate from Japan (AB478319.1) for both isolates obtained in the present study.

    Fig 5: Phylogenetic analysis of Babesia vogeli isolates by neighbor joining method.



    Fig 6: Phylogenetic analysis of Babesia gibsoni isolates by neighbor joining method.


           
    Tick-borne diseases, particularly those caused by haemoprotozoan parasites, are common in tropical and subtropical regions. The severity of these infections often hinges on the extent of host erythrocyte destruction due to the parasite’s rapid proliferation. In India, Babesiosis in dog was first identified in 1910 (Patton, 1910) and since its discovery, it has posed considerable challenges to veterinarians, particularly in clinical, diagnostic and therapeutic management. The PCR assay for Babesia spp. began in 2000s and the diagnosis through molecular means was widely followed by researchers abroad. In the present study, there was detection of Babesia spp. in 26.47% dogs by Primary PCR by amplification of 18S rRNA. An overall prevalence ranging from 20-30% has been reported earlier from Haryana (Bhagwan et al., 2024) Bengaluru (Mahalingaiah et al., 2017), Maharashtra (Debbarma et al., 2022) and Andhra Pradesh (Jalajakshi et al., 2021). A higher range of prevalence ranging from about 50-60% has also been recorded from North eastern regions of India (Laha et al., 2014; Mittal et al., 2019) and South India (Arthi et al., 2017).
           
    Further, species specific primers were used in nested PCR, which revealed that 23.53% dogs were positive for Babesia gibsoni while only 3.92% cases were positive for Babesia vogeli. A higher presence of Babesia gibsoni in comparison to Babesia vogeli was also documented from other parts of India (Mittal et al., 2019; Jalajakshi et al., 2021). However, a higher presence of Babesia vogeli has also been documented from Haryana (Bhagwan et al., 2024); Maharashtra (Debbarma et al., 2022) Assam (Laha et al., 2014) and Rajasthan (Choudhary et al., 2025).
           
    B. gibsoni     has been identified across five continents, including Asia, Africa, North America, Europe and Australia and is known to cause highly severe disease (Irwin, 2010). Also, chances of possible transmission of this small form of Babesia by ingestion of blood, contact with wounds or saliva have been contemplated (Irizarry-Rovira et al., 2001).

    Interestingly, a previous study conducted in the same geographical area reported a predominance of the large form of Babesia, identified as B. vogeli, with only a minor occurrence of the small form, B.gibsoni (Panda et al., 2021). In contrast, the present investigation revealed a different pattern of occurrence, indicating a shift in the predominant species/form detected in dogs from the coastal Odisha region. As far as detection of canine babesiosis outside India is concerned, Babesia vogeli has been widely documented in Africa, (Mghirbi and Bouattour, 2008; Selim et al., 2022), Europe (Solano-Gallego et al., 2008), Both B. gibsoni and B.vogeli have been recorded from canines in European countries (Schafer et al., 2023)  and Thailand (Do et al., 2021).
           
    The lower prevalence of B.vogeli during the present investigation is quite consistent with earlier reports from Karnataka (Lavanya et al., 2019) depicting a prevalence lesser than 10% and also in Egypt (Selim et al., 2022) and Brazil (de Oliveira et al., 2016). There is disparity between these prevalence rates due to variation in study, design and duration, environment parameters, presence of vectors and reservoir hosts and kind of diagnostic tests employed. The genes used for diagnosis through PCR assay also varies. There is also differences with regards to veterinary health care like the frequency and mode of treatment.
           
    During this present research, there was no instance of coinfection with B. vogeli or B. gibsoni. However, concurrent infections of larger and smaller forms of Babesia have been reported from dogs elsewhere (Vairamuthu et al., 2014; Mahalingaiah et al., 2017).
           
    A higher detection of canine babesiosis by PCR-based assays in comparison to conventional microscopic examination as noticed in the present investigation, has also been reported by several researchers in India and abroad pointing at higher sensitivity of molecular assay (Laha et al., 2014; Birkenheuer et al., 2003).
           
    As per our investigation, there was higher presence of Babesiosis in male dogs as compared to females which corroborates the earlier observations elsewhere (Mahalingaiah et al., 2017, Jalajakshi et al., 2021). The higher prevalence of babesiosis in male dogs may be associated with sex-related differences in immune function, as testosterone has been reported to modulate immune responses and may increase susceptibility to parasitic infections (Roberts et al., 2001). Nevertheless, this association should be considered a possible contributing factor rather than a direct causal explanation for the higher prevalence observed in males. Also it has been postulated that the hormonal influence and temperament of males could result in limited attention from owners (Mellanby et al., 2011). Whereas in female dogs, estrogen improves the immune reactivity and produces resistance to invading infection (Taneja, 2018). However, in contrast to the present study, previous reports from other states (Arthi, 2017, Lavanya et al., 2019) and Madhya Pradesh (Shrivastava et al., 2014) have depicted a higher presence of Babesia infection in females over males. The reasoning being a sedentary lifestyle in females especially during pregnancy and nursing, making it more prone to tick infestation and lowering of immunity.
           
    The study presented that dogs in the age group 2-4 years showed a higher presence of canine babesiosis. Earlier reports have noted higher prevalence in dogs above one year of age (Singh et al. 2014 and Kumar et al. 2015). Also reports from India (Shrivastava et al., 2014) and Nigeria (Obeta et al., 2020) have revealed higher age wise prevalence in the age group of 1 to 3 years. Younger dogs, due to maternal immunity have lesser chance to gain infection and lack of past exposure in comparison to older dogs makes it less prone to babesiosis. There has been evidence of older dogs being prone to Babesia infection as reported by higher presence of B. gibsoni in adults (Kumar et al., 2009). As per earlier research, babesiosis in dog’s increases with age, with the infection reaching a peak between 3-5 years’ age group after which it starts declining (Hornok et al., 2016). However, young pups have a compromised immune status and are more active and agile making it more prone to tick exposure and thereby infection (Obeta et al., 2020).
           
    The haemoprotozoan infection due to Babesia vogeli and Babesia gibsoni was confirmed by sequencing of the portion of 18S rRNA gene for randomly selected positive dog samples. There was construction of two phylogenetic tree by utilising the Neighbor-Joining method in MEGA 11. The tree was built through sequences of 18S rRNA gene obtained in this study and additional sequences available in GenBank. Phylogenetic analysis of Babesia vogeli 18S rRNA partial sequences presented a close similarity with Indian isolates as well as with isolate from Brazil. The B. gibsoni isolates from the present study formed a group with other geographical isolates outside India including Australia, Bangladesh, America and Australia. Studies conducted in different parts of India (Roopesh et al., 2018; Deepa, 2023) also indicated that Indian B. gibsoni strains clustered into a major group with isolates from other parts of the country.

    CONCLUSION

    The current study suggests that babesiosis is an emerging or re-emerging tick-borne hemoprotozoan disease affecting dogs in coastal Odisha with Babesia gibsoni showing a greater presence over Babesia vogeli. PCR proved to be a highly sensitive, rapid and reliable method for detecting Babesia infection in both carrier and clinically suspected animals. Accurate differentiation of Babesia species is clinically important because the infecting species can differ considerably in pathogenicity, disease severity, prognosis and response to treatment. Therefore, species-level identification is essential for appropriate treatment selection and effective clinical management of canine babesiosis. Future studies regarding interaction of tick vectors and the parasite could yield a better epidemiological understanding of canine babesiosis in the area.

    ACKNOWLEDGEMENT

    The present study was supported by College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    Not applicable.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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