Molecular identification of the Hepatozoan canis in Rhipicephalus sanguineus ticks in Tamil Nadu

Introduction

Rhipicephalus sanguineus sensu lato ticks can transmit a variety of pathogens to dogs (Dantas-Torres et al., 2012) [5] , including bacteria such as Ehrlichia canis, Rickettsia conorii, Rickettsia rickettsii and protozoa (Hepatozooncanis, Babesia vogeli). Rhipicephalus sanguineus s.l. ticks are found worldwide, primarily in tropical and subtropical regions, due to their high adaptability to different environmental conditions (Dantas-Torres et al., 2013; Giannelli et al., 2013) [7] . Dogs, foxes and jackals can become infected by H. canis (Eucoccidiorida, Hepatozoidae) by ingestion of nymphs or adult ticks that harbour infective oocysts (Baneth, 2011; Giannelli et al., 2013) [7] . Hepatozoon canis is a tick-borne apicomplexan parasite that causes a variety of symptoms in dogs from mild infection to severe illness characterized by cachexia, fever, weight loss and anaemia when parasitic infection is severe. Canine hepatozoonosis has been reported from America, Africa, Asia, and Europe, with some areas (e.g., southern Italy) being particularly suitable for the occurrence of this infection (Baneth, 2011; Dantas-Torres et al., 2012; Giannelli et al., 2013) [2,5,7] . This study aimed to detect H. canis infection by molecular analysis in R sanguineus sensu Lato ticks collected from different places in Tamil Nadu state, India.

Materials and Methods

Tick collection and Morphological identification

A total of 324 semi engorged ticks from naturally infested dogs were collected from Blue Cross Society in Chennai, Veterinary Poly Clinic in Madurai, and Namakkal. From each location, a minimum of five semi-engorged ticks attached to dogs were collected. The collected ticks were identified morphologically and stored in 70% ethanol based on Barker and Walker (2014) [3] .

Tick dissection

Each semi-engorged tick was externally sterilized for five to ten minutes in a 70% ethanol solution before being dissected, and then it was rinsed in sterilized distilled water. Every midgut dissection was carried out under a microscope. With a few minor adjustments, the ticks were dissected by removing the upper cuticle using a sterile blade in order to separate the midgut, following the procedure used by Lalzar et al. (2014) [10] .

Extraction of genomic DNA

The genomic DNA extraction from dissected midgut using the DNeasy Blood and Tissue kit (Qiagen) following the instructions from the manufacturer. The concentration of genomic DNA was evaluated with a Nano Drop TM 2000 (Thermo Scientific, USA), and stored at -20 °C until further use.

PCR Amplification

The 18S rDNA was amplified using PCR with the Red dye Taq 2x Mastermix, Amplicon. It was aimed to obtain a 666 bp fragment of the 3´ half of the 18S rRNA genes. PCR conditions included an initial denaturation step at 94 °C for 5 min followed by 30 cycles for 30 s at 94 °C, 30 s for annealing at 55 °C and 60 s for extension at 72 °C. A final extension step was carried out for 5 min at 72 °C. The primers details used for the amplification of the 666 bp fragment were: forward, 5´ ATACATGAGCAAAAT CTCAAC-3´; reverse, 5´-CTTATTATTCCATGCTGCAG-3´ (Inokuma, et al. 2002) [9] . PCR was done by based on the protocols followed by Szabo et al. (2005). All PCR reactions were carried out with a thermocycler (T100 thermocycler TM , BioRad) at a final volume of 20 µl. Following PCR, 1% agarose-gel electrophoresis was used to verify for amplified fragments by comparing them to a molecular ladder (MX-0917-01 100bp DNA Ladder TM , Medox).

Data submission in GenBank

The 18S rRNA sequence of tick samples were submitted in GenBank (http://www.ncbi.nlm.nih.gov/genbank) to record each sequence with accession number.

Results

Molecular identification of H. canis by 18S rRNA gene

The genomic DNA samples of R. sanguineus ticks were screened by PCR using specific primers of 18S rRNA in accordance with documented length of amplification fragments in tick species the expected length for the 18S rRNA fragment was approximately 666 bp (Figure 1). [4,1,7,1] . This study confirmed the presence of H. canis infections in R. sanguineus ticks from three agro climatical zones of Tamil Nadu. Further research is required to gain more insights into the global transmission of H. canis by R. sanguineus ticks.

Conclusion

This study confirmed the presence of Hepatozoon canis in Rhipicephalus sanguineus sensu lato ticks collected from three major districts of Tamil Nadu, with infection rates of 15%, 25%, and 20% in Chennai, Madurai, and Namakkal, respectively. Molecular detection using 18S rRNA PCR and sequencing revealed a 666 bp amplicon showing 98.10% homology with known H. canis isolates, validating the reliability of the assay. These findings highlight the significant role of R. sanguineus ticks as vectors in the regional transmission of canine hepatozoonosis. Understanding tick-borne pathogen distribution is essential for improving diagnostic, preventive, and control strategies. Future studies should explore seasonal dynamics, vector competence, and broader epidemiological patterns across India.

References

1. Aktas M, Ozubek S, Ipek ND. Molecular investigations of Hepatozoon species in dogs and developmental stages of Rhipicephalus sanguineus. Parasitol Res. 2013;112:2381-2385.
2. Baneth G. Perspectives on canine and feline hepatozoonosis. Vet Parasitol. 2011;181:3-11.
3. Barker SC, Walker AR. Ticks of Australia: The species that infest domestic animals and humans. Zootaxa. 2014;3816:1-144.
4. Torres DF. The brown dog tick, Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae): From taxonomy to control. Vet Parasitol. 2008;152:173-185.
5. Torres DF, Latrofa MS, Weigl S, Tarallo VD, Lia RP, Otranto D. Hepatozoon canis infection in ticks during spring and summer in Italy. Parasitol Res. 2012;110:695698.
6. Giannelli A, Ramos RA, Paola GD, Mencke N, Torres DF, Baneth G, et al. Transstadial transmission of Hepatozoon canis from larvae to nymphs of Rhipicephalus sanguineus. Vet Parasitol. 2013;196:1-5.
7. Helm CS, Himmelstjerna VSG, Liesner JM, Kohn B, Müller E, Schaper R. Identical 18S rRNA haplotypes of Hepatozoon canis in dogs and foxes in Brandenburg, Germany. Ticks Tick Borne Dis. 2020;11:101520.
8. Inokuma H, Okuda M, Ohno K, Shimoda K, Onishi T. Analysis of the 18S rRNA gene sequence of a Hepatozoon detected in two Japanese dogs. Vet Parasitol. 2002;106:265-271.
9. Lalzar I, Friedmann Y, Gottlieb Y. Tissue tropism and vertical transmission of Coxiella in Rhipicephalus sanguineus and Rhipicephalus turanicus ticks. Environ Microbiol. 2014;16:3657-3668.