Molecular detection, phylogenetic analysis, and genetic diversity of Babesia gibsoni isolates from dogs in India based on the 18S rRNA gene

Introduction

Canine babesiosis is a globally significant tick-borne haemoprotozoan disease caused by intraerythrocytic protozoa belonging to the genus Babesia resulting in subclinical infections to severe multisystemic illness, thereby posing a major challenge to canine health worldwide. [14] . Among the small Babesia spp., B. gibsoni is considered one of the most clinically important pathogen infecting domestic dogs. It is a small pleomorphic intraerythrocytic protozoan that predominantly appears as ring-shaped organisms, although oval and elongated forms spanning the erythrocyte may occasionally be observed (Conrad et al., 1991; Casapulla et al., 1998) [6,4] . Based on genetic diversity, B. gibsoni has been classified into three major genotypes: the Asian genotype, the Californian strain, and the Spanish isolate (Karasova et al., 2022) [14] . The parasite has a worldwide distribution and has been reported from all continents except Antarctica (Conrad et al., 1991; Casapulla et al., 1998) [6,4] . In India, Rhipicephalus sanguineus is the principal vector of Babesia gibsoni, with confirmed transstadial transmission (Chao et al., 2017) [5] . The parasite can also be transmitted through blood transfusion, dog bites, contaminated needles, and transplacental infection (Fukumoto et al., 2005; Jefferies et al., 2007) [8,13] . Clinical manifestations range from anorexia, lethargy, weakness, pale mucous membranes, fever, hypoalbuminemia, and hyperbilirubinemia to severe complications including haemolytic anaemia, thrombocytopenia, renal and hepatic dysfunction, pancreatitis, neurological and cardiac disorders, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), and septic shock (Irwin, 2009; Solano-Gallego and Baneth, 2011; Solano-Gallego et al., 2016) [11,15,16] [10,12] . The correlation between these molecular markers and clinical severity remains critical, as distinct genotypes may influence the pathogenesis and disease progression observed in both domestic and wild canids (Guo et al., 2020) [9] . Although some investigations have highlighted clinical signs such as depression, anorexia, and lymphadenopathy, the potential for zoonotic transmission remains poorly substantiated in current literature (Alobaidii et al., 2024; Badawi & Yousif, 2020) [1,2] .

Consequently, rigorous molecular characterization targeting highly conserved 18S rRNA sequences gene will be useful to elucidate the phylogenetic relationship of B. gibsoni isolates as suggested by (Birkenheuer et al., 2003) [3] . The present study aimed to characterize B. gibsoni isolates by analyzing their genetic diversity and phylogenetic relationships with globally reported isolates, thereby providing molecular insights into the epidemiology of the parasite.

Materials and Methods

Sample collection, microscopy and DNA extraction

A total of 180 whole blood samples were aseptically collected from dogs clinically suspected of babesiosis presented to veterinary clinics and hospitals in Uttar Pradesh, Delhi, Haryana, and Tamil Nadu, India. Thin blood smears were prepared from each sample, stained with Giemsa, and examined microscopically under the oil immersion lens (1000X) for the presence of intraerythrocytic B. gibsoni piroplasms. Genomic DNA was extracted from 200 µL of whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was quantified and stored at -20 °C until further analysis.

PCR amplification of the 18S rRNA gene

Molecular detection of B. gibsoni was performed by PCR targeting the 18S rRNA gene using the primer pair: forward 5′-CCTTTTTGGCGGCGTTTATTA-3′ and reverse 5′-CAATCCCTAGTCGGCATGGTTTA-3′. PCR amplification was carried out in a final reaction volume of 25 µL containing 2X PCR Master Mix, 10 pmol of each primer, template DNA, and nuclease-free water. The thermal cycling conditions consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 50 °C for 45 s, and extension at 72 °C for 45 s, with a final extension at 72 °C for 10 min. Amplified products were resolved by electrophoresis on 1.5% agarose gel stained with RedSafe™ nucleic acid stain (Intron Biotechnology, South Korea) and visualized using a Gel Documentation System (Syngene, USA).

DNA sequencing and molecular characterization

Representative PCR-positive amplicons were purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol and subjected to bidirectional Sanger sequencing. The obtained chromatograms were manually inspected, edited, and assembled to generate consensus sequences. Newly generated sequences were submitted to the National Center for Biotechnology Information (NCBI) GenBank database, and accession numbers were assigned. Sequence similarity was determined using the Basic Local Alignment Search Tool (BLAST), and homologous reference sequences were retrieved from the GenBank database for comparative sequence analysis.

Phylogenetic analysis

Phylogenetic analysis was performed using seven newly generated partial 18S rRNA gene sequences (787 bp) together with 32 representative B. gibsoni sequences retrieved from the NCBI GenBank database (Supplementary table 1). Multiple sequence alignment was carried out prior to phylogenetic reconstruction. The evolutionary relationships among the isolates were inferred using the Maximum Likelihood (ML) method and Tamura model implemented in MEGA version 11. The robustness of the inferred phylogeny was evaluated by bootstrap analysis using 1,000 replicates.

Results

Detection of Babesia gibsoni

Of the 180 canine blood samples examined, 42 (23.3%) were positive for Babesia gibsoni by Giemsa-stained blood smear examination, whereas PCR targeting the 18S rRNA gene detected 58 (32.2%) positive samples. PCR identified 16 additional infections that were negative by microscopy, indicating its superior sensitivity for detecting low parasitaemia.

Sequence analysis

Seven representative PCR amplicons were successfully sequenced, generating partial 18S rRNA gene sequences of 787 bp. The sequences were submitted to the NCBI GenBank database and assigned accession numbers PV018514-PV018520.

Phylogenetic analysis

Phylogenetic analysis of the 18S rRNA gene revealed that all B. gibsoni isolates formed a well-supported monophyletic group with bootstrap value 76 that was clearly separated from the outgroup, Babesia canis (KT844899, Poland) (Fig. 2). Within the B. gibsoni clade, isolates obtained from different geographical regions showed a high degree of genetic similarity, with relatively short branch lengths throughout the tree. The Indian isolates were distributed among sequences originating from several Asian countries, including China, Korea, Japan, Taiwan, Nepal, Myanmar, and Sri Lanka, rather than forming a completely independent lineage. The newly generated Indian sequences (PV018514-PV018520) clustered within the principal B. gibsoni lineage. Most of these isolates grouped closely with previously reported Indian isolates from Uttar Pradesh, Delhi, Haryana (Hisar), Tamil Nadu, West Bengal, Kerala, Assam, Maharashtra, and Odisha, demonstrating close genetic relatedness among Indian populations. Several international isolates, including those from Taiwan, China, Korea, Japan, Nepal, Myanmar, and Sri Lanka, were interspersed among Indian isolates, further supporting the presence of a genetically homogeneous Asian population. In contrast, isolates from Brazil, Malawi (Africa), Italy, and Texas (USA) occupied nearby branches within the same major lineage without showing extensive divergence, reflecting the conserved nature of the 18S rRNA gene. The pairwise genetic distance matrix demonstrated very low nucleotide divergence among B. gibsoni isolates. The overall mean genetic distance was approximately 0.004, indicating high conservation of the 18S rRNA gene across geographically diverse isolates.

Discussion

The present study provides molecular evidence of B. gibsoni infection in dogs across India, employing a PCR based diagnostic approach that demonstrated significantly higher sensitivity than conventional microscopic examination. While 42 samples were identified as positive via Giemsa stained blood smears, PCR targeting the 18S rRNA gene successfully detected 58 positive samples. These results corroborate findings from previous studies (Birkenheuer et al., 2003; Fukumoto et al., 2001; Jain et al., 2017; Liu et al., 2019) [3,7,12,17] . which consistently show that PCR assays overcome the inherent limitations of microscopic examination, specifically, the difficulty of detecting low level parasitemia common in subclinical or chronic infections. [9,10,12,17,18] . Our findings demonstrate that Indian isolates group closely with those reported from other Asian countries, including China, Korea, Japan, Nepal, Myanmar, and Sri Lanka. This clustering pattern, often referred to as the Asian genotype (Birkenheuer et al., 2003; Jain et al., 2017) [3,12] , supports the existence of a genetically homogeneous population, likely facilitated by the widespread circulation of related strains. In contrast, while sequences from other regions (e.g., Brazil, Italy, Texas) may show minor divergence, they remain within the same major lineage, reinforcing the high conservation of the 18S rRNA gene. Although this gene is highly effective for species identification, the observed lack of extensive differentiation suggests that additional, more polymorphic markers may be required for finer-resolution strain differentiation and epidemiological tracing of B. gibsoni isolates in future studies.

Acknowledgement

AcknowledgementThe authors are thankful to Director, ICAR-IVRI, Izatnagar for providing necessary facilities for completing this research work.

References

1. Alobaidii WA, Al-Baroodi SY, Almashhadany DA. Molecular and phylogenetic analysis of Babesia gibsoni in dogs and its infested ticks in Nineveh province, Iraq. Iraqi Journal of Veterinary Sciences. 2024;38(Suppl IIV):27-33.
2. Badawi NM, Yousif AA. Survey and molecular study of Babesia gibsoni in dogs of Baghdad province, Iraq. The Iraqi Journal of Veterinary Medicine. 2020;44(E0):34-41.
3. Birkenheuer AJ, Levy MG, Breitschwerdt EB. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples. Journal of Clinical Microbiology. 2003;41(9):41724177.
4. Casapulla R, Baldi L, Avallone V, Sannino R, Pazzanese L, Mizzoni V. Canine piroplasmosis due to Babesia gibsoni: Clinical and morphological aspects. Veterinary Record. 1998;142:168-169.
5. Chao LL, Liao HT, Ho TY, Shih CM. First detection and molecular identification of Babesia gibsoni from Rhipicephalus sanguineus ticks. Acta Tropica. 2017;166:356-362.
6. Conrad P, Thomford J, Yamane I, Whiting J, Bosma L, Uno T, et al. Hemolytic anemia caused by Babesia gibsoni infection in dogs. Journal of the American Veterinary Medical Association. 1991;199(5):601-605.
7. Fukumoto S, Xuan X, Nishikawa Y, Inoue N, Igarashi I, Nagasawa H, et al. Identification and expression of a 50-kilodalton surface antigen of Babesia gibsoni and evaluation of its diagnostic potential in an enzymelinked immunosorbent assay. Journal of Clinical Microbiology. 2001;39(7):2603-2609.
8. Fukumoto S, Suzuki H, Igarashi I, Xuan X. Fatal experimental transplacental Babesia gibsoni infections in dogs. International Journal for Parasitology. 2005;35(9):1031-1035.
9. Guo WP, Xie GC, Li D, Su M, Jian R, Du LY. Molecular detection and genetic characteristics of Babesia gibsoni in dogs in Shaanxi Province, China. Parasites & Vectors. 2020;13(1):366.
10. He L, Miao X, Hu J, Huang Y, He P, He J, et al. First molecular detection of Babesia gibsoni in dogs from Wuhan, China. Frontiers in Microbiology. 2017;8:1577.
11. Irwin PJ. Canine babesiosis: From molecular taxonomy to control. Parasites & Vectors. 2009;2(Suppl 1):S4.
12. Jain KJ, Lakshmanan B, Syamala K, Praveena JE, Aravindakshan T. High prevalence of small Babesia species in canines of Kerala, South India. Veterinary World. 2017;10(11):1319-1323.
13. Jefferies R, Ryan UM, Jardine J, Broughton DK, Robertson ID, Irwin PJ. Blood, bull terriers and babesiosis: Further evidence for direct transmission of Babesia gibsoni in dogs. Australian Veterinary Journal. 2007;85(11):459-463.
14. Karasová M, Tóthová C, Grelová S, Fialkovičová M. The etiology, incidence, pathogenesis, diagnostics, and treatment of canine babesiosis caused by Babesia gibsoni infection. Animals. 2022;12(6):739.
15. Solano-Gallego L, Baneth G. Babesiosis in dogs and cats: Expanding parasitological and clinical spectra. Veterinary Parasitology. 2011;181(1):48-60.
16. Solano-Gallego L, Sainz Á, Roura X, Estrada-Peña A, Miró G. A review of canine babesiosis: The European perspective. Parasites & Vectors. 2016;9:336.
17. Yin F, Guo C, Tian Z, Li D, Mu D, Liu H, et al. Analysis of genetic diversity and population structure of Babesia gibsoni. Frontiers in Veterinary Science. 2023;10:1147958.
18. Zahler M, Rinder H, Zweygarth E, Fukata T, Maede Y, Schein E, et al. Babesia gibsoni of dogs from North America and Asia belong to different species. Parasitology. 2000;120(4):365-369.