Molecular Investigation of Canine Babesiosis in and Around Bhubaneswar, India

Canine babesiosis, a hemolytic protozoan disease represents an important veterinary problem caused primarily by large and small forms of piroplasms of Babesia spp. A molecular-based survey on the overall occurrence of natural Babesia infection in stray(n=98 ) and pet dogs(n=100) from Bhubaneswar and nearby areas using PCR technique targeting 18s RNA gene fragment along with genetic sequence analysis was carried out. A total of 38 (pet:22, stray:16) samples (19.19%) were found positive for babesiosis based on the amplication of 450 bp amplicon region of the gene while 4 samples (0.02%) showed co-infection with Hepatozoon canis. The sequenced PCR products were submitted to NCBI ,and on BLAST analysis the isolates with accession no KT246303, KT246306, KT246307 showing similarity with Babesia vogeli, while KT246305 was identical to B.gibsoni isolates and KT246304 was identical to Hepatozoon canis. This is the rst report on the molecular diagnosis of canine babesiosis in the state. PCR assay was found to be more precise over microscopic diagnosis, the use of more specic and sensitive tests along with more samples could aid in a better understanding of the epidemiology of canine babesiosis in this region.


Introduction
Canine babesiosis is an important tick-borne life threatening haemo-protozoan disease caused by the intra-erythrocytic protozoan parasites belonging to the genus Babesia. The Babesia species mainly incriminated for causing disease in canids are the small form (Babesia gibsoni, Babesia conrade, Babesia vulpes) and large form (Babesia canis, Babesia vogeli. Babesia rossi ) in different parts of the world (Solano-Gallego et al., 2016). The transmission occurs by Dermacentor reticulatus in Europe, Rhipicephalus sanguineus in tropical and subtropical regions, and Haemaphysalis eliptica in South Africa (Uilenberg, 2006). In Indian subcontinent, canids are infested majorly with Rhipicephalus sanguineus and Detection of Babesia spp. is usually achieved using microscopic examination of stained blood smears, but this technique is limited because of low sensitivity, chronic evolution of the disease, and the di culty of distinguishing morphologically similar strains and species (Irwin, 2005). The serological test such as the immuno uorescent antibody test is useful but has poor speci city as a result of antigen cross-reactivity (Rani et al., 2011) and fails to identify current infection. The employment of recent biotechnological techniques like PCR, nested & semi-nested PCR, PCR-RFLP, and multiplex PCR has lead to advancement in the detection of this protozoan parasite. The true status of canine babesiosis is still not clear in India barring a few reports (Rani et al., 2011;Sarma et al.,2019) and in the region under study (Sahu et al.,2014) which was mostly based on microscopic examination of blood smears. The present study is a molecular-based investigation of canine babesiosis in Bhubaneswar, Odisha.
The study was conducted in and around Bhubaneswar (Odisha) located between 20° 14' 0" North, 85° 50' 0" East, and having an average altitude of 45 m (148 ft) above sea level. The average relative humidity and annual rainfall recorded in the region are 70% and 1,542 mm respectively. Blood samples from 198 dogs presented to Teaching Veterinary Clinical Complex and Animal Birth Control Programme,Bhubaneswar (stray:98, pet:100) belonging to either sex and different age groups, showing tick infestation and clinical signs like pyrexia, anorexia or lethargy were selected for this study. Blood smear examination using Giemsa stain was conducted and the blood samples which were found positive or negative for Babesia piroplasms were preserved at -20 ° C for further study using molecular techniques. DNA was isolated from 200 µl of blood ( with anticoagulant, EDTA) sampled from each dog Then the rest volume was adjusted to 25 µl by addition of nuclease free water (Genei, Bangalore).The ampli cation was performed in a thermal cycler (Gene Amp PCR System 9700, Applied Biosystem) with cyclic conditions comprising of an initial denaturation at 94 degrees C for 5 min with 35 rounds of denaturation, primer annealing, and polymerization at 94 °C, 62 °C, and 72 °C, respectively for 30 seconds each. A nal chain extension at 72 °C for 6 minutes completed the cycle program. Then the ampli ed PCR products were analyzed on 1.5% agarose gel (Lonza, 0.5 µg/ml)(120 V, 40 min) and visualized under UV transilluminator (Alpha Innotech, New Delhi).PCR products of the expected nucleotide size were puri ed with QIAquick PCR Puri cation Kit according to the manufacturer's instruction (Qiagen, Germany).The puri ed PCR products were sequenced by the automated DNA sequencer (310 Genetic Analyser, Applied Biosystems) using ABI Prism Dye Terminator kit and both forward and reverse primers, and the sequences obtained were subjected to BLAST analysis. Phylogenetic analysis was done by MEGA X using the Neighbour-joining tree method based on the partial gene sequences of 18S rRNA obtained in the study and reference sequences obtained from the NCBI GenBank database. The number at the node was the proportion of 500 bootstraps.

Results
The most consistent clinical signs observed during the examination were elevated rectal temperature above 39.6°C, anorexia, pale mucous membrane , tick infestation, coughing , and vomition in 85.71%, 68.57%, 65.71%, 57.14%, 37.14% and 8.57% of dogs under study respectively. On microscopical examination of stained blood smears piroplasms of Babesia spp. were detected in 8.08% (16/198) samples. The overall detection of babesiosis by conventional PCR technique was 19.19% (38/198) on the basis of presence of 450 bp amplicon (Fig 1). Four samples showed double band after gel puri cation having 450 and 520 bp amplicon size indicating mixed infection with Hepatozoon canis (Fig  2). The infection in pet dogs (22%) was higher than stray dogs(16.23%).
The nucleotide sequence obtained after sequencing of PCR product was submitted to GenBank and accession numbers KT246303, KT246304, KT246305, KT246306,KT246307 were assigned. BLAST analysis of GenBank revealed that the obtained nucleotide sequences of KT246303, KT246306 and KT246307 were 100% homologous to previously deposited 18S rRNA gene sequences of B. vogeli. The sequence of KT246305 showed the best matches (100%) with Babesia gibsoni sequences from different regions available in NCBI database. The sequence KT246304 showed 99% similarity with the previously deposited 18S rRNA gene sequences of H.canis.
On phylogenetic analysis (Fig 3), the sequences clustered into three distinct clades. B. vogeli isolates of the study (KT246303 and KT246307) were clustered together to form a well-de ned group Based on sequencing results and BLAST analysis, it was also observed that one sequence (KT246305) showed maximum homogeneity with sequences of B.gibsoni with a stray match with B.canis. In India, B.canis is yet to be reported in any molecular diagnosis possibly due to absence of potential vector. B.gibsoni infection in dogs has been reported earlier in blood smear examination from Bhubaneswar (Sahu et al.,2014) while molecular detection have been reported in different regions of India (Singh et al.,2014;Jain et al.,2018;Sarma et al.,2019). The molecular identi cation of B. gibsoni, B. vogeli and H.canis from canines of Punjab has been previously described (Singla et al., 2016). On phylogenetic analysis the present three isolates showed an a liation with other B. vogeli isolates from different geographical regions. But one of the isolates showed no a liation with other B. vogeli isolates though it was con rmed to be B. vogeli from Blast analysis, which might have originated from a different strain. B.gibsoni infected dogs exhibit varying clinical manifestation ranging from subclinical to fatal depending on the host body condition. Though B.vogeli is less pathogenic exhibiting moderate symptoms in adult dogs, they cause severe condition in puppies and splenectomised dogs (Wang et al., 2018).The study clearly re-established reliability of PCR as a technique over microscopy. Therefore, molecular diagnosis can facilitate pertinent treatment and control regimen.

Conclusion
Traditionally, the presumptive diagnosis is based on fever, anemia and thrombocytopenia, while the microscopic examination still remains the most rapid con rmatory method for diagnosis of canine babesiosis under eld condition. PCR assay has been found to be more speci c method, could detect even in carrier state where there were no clinical signs and symptoms while co-infection with other vectorborne agents were also recorded. Further studies utilising more sensitive tests and species-speci c primers along with a larger number of clinical samples needs to be analyzed to get insight into the epidemiological patterns of canine babesiosis as well as devising an effective control program. Phylogenetic Analysis using MEGA X by Neighbour-joining tree method based on the partial gene sequences of 18S rRNA