First detection and molecular identification of Babesia gibsoni and Hepatozoon canis in an Asiatic wild dog (Cuon alpinus) from Thailand

Babesia spp. and Hepatozoon spp. are apicomplexan parasites that infect a wide range of domestic and wild animals. The life cycle of these parasites requires a tick vector as a definitive host and various vertebrates as reservoir hosts. The objective of this study was to detect and characterize Babesia spp. and Hepatozoon spp. in an Asiatic wild dog (Cuon alpinus) from a natural habitat in northeastern Thailand. Heart and spleen samples of an adult male wild dog were screened for Babesia spp. and Hepatozoon spp. by polymerase chain reaction (PCR) of the partial fragment of 18S ribosomal RNA gene sequence. Both Babesia sp. and Hepatozoon sp. were detected in the spleen of the wild dog. Nucleotide sequence and phylogenetic analyses showed that the detected parasites were Babesia gibsoni and Hepatozoon canis. This is the first report of B. gibsoni and H. canis in the Asiatic wild dog from Thailand using PCR. Our results indicate that wild dogs can serve as a potential reservoir of the protozoan parasites and that they may play an important role in the transmission of these parasites to other wild or domestic canids.


Introduction
The Asiatic wild dog or dhole (Cuon alpinus) is a medium-sized mammal in the family Canidae occurring in southern and southeastern Asia. It is a social, pack-living species that prefers to hunt medium to large-sized ungulate prey (Jenks et al., 2014). This species is mainly found in several protected areas in Thailand but can sometimes be found outside parks or sanctuaries (Jenks et al., 2012). Owing to a decline in its population from habitat loss, hunting, and invasion by domestic animals and their related pathogens, C. alpinus has been classified as an endangered species (Kamler et al., 2015).
Protozoan parasites of the genera Babesia and Hepatozoon are tickborne pathogens that infect a wide variety of mammals, birds, reptiles and amphibians (Smith, 1996;Baneth et al., 2003;Peirce and Parsons, 2012;Schnittger et al., 2012). These parasites are associated with negative worldwide economic, clinical and epidemiological impacts, mainly in farm and pet animals (Smith, 1996;Schnittger et al., 2012). Babesia spp., which cause babesiosis, are transmitted by ixodid ticks. The clinical signs of babesiosis are mainly hemolysis and anemia (Margalit Levi et al., 2018). The severity of infection by Babesia spp. can range from subclinical or chronic to severe or fatal, depending on the virulence of the species and the immune response of the hosts (Decaro et al., 2013;Alvarado-Rybak et al., 2016;Solano-Gallego et al., 2016). Hepatozoon spp., which cause hepatozoonosis, are transmitted by ingestion of an infected tick. The infection by Hepatozoon spp. often presents subclinical infection, but can occasionally present clinical signs such as weight loss, lethargy, fever, anemia and lymphadenopathy (Baneth and Weigler, 1997;Baneth et al., 2003;Alvarado-Rybak et al., 2016). The same species of Babesia and Hepatozoon have been reported in both domestic dogs and wild carnivores. From an evolutionary perspective, the parasites can be transferred from wild canines to domestic dogs living in the same geographical areas. In addition, the transmission of the parasitic diseases between wild and domestic canines also depends on other factors such as the canine host species-specificity, the different habitats of domestic and wild canines, and the exposure to a different spectrum of tick-borne vectors (Baneth, 2001;Penzhorn, 2011;Hodžić et al., 2018;Margalit Levi et al., 2018).
In Thailand, Babesia and Hepatozoon infections have been reported in domestic and wild animals and also in tick vectors (Jittapalapong et al., 2006;Salakij et al., 2008Salakij et al., , 2010Laummaunwai et al., 2014;Piratae et al., 2015;Sumrandee et al., 2015;Bhusri et al., 2017). However, there are not previous records of Babesia and Hepatozoon infections in the Asiatic wild dog. Therefore, the objective of this study was to detect and characterize Babesia spp. and Hepatozoon spp. in the Asiatic wild dog from a natural habitat in northeastern Thailand using the partial 18S ribosomal RNA (18S rRNA) gene sequence.

Sample collection
In August 2018, heart and spleen samples of an Asiatic wild dog from a natural habitat in northeastern Thailand were collected and sent to the Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MoZWE), Faculty of Veterinary Science, Mahidol University to screen for parasitic infections. The heart and spleen samples were collected by wildlife authorities from a wild dog found dead in a forested area. Our study protocol was approved by the Faculty of Veterinary Science, Mahidol University Animal Care and Use Committee (Ref. MUVS-2019-02-07).

DNA extraction and 18S rRNA amplification
Genomic DNA was extracted from the heart and spleen samples using a DNeasy Blood & Tissue Kit (QIAGEN, Germany) according to the manufacturer's protocol. The polymerase chain reaction (PCR) assays were run to detect Babesia spp. and Hepatozoon spp. infections. A partial fragment of 18S rRNA gene was amplified using multiplex-PCR. For Babesia spp., a 619 base pair (bp) fragment was amplified by a primer pair consisting of a forward primer Ba-103F (5 ′ -CCAATCCTGACA-CAGGGAGGTAGTGACA-3 ′ ) and a reverse primer Ba-721R (5 ′ -CCCCA-GAACCCAAAGACTTTGATTTCTCTCAAG-3 ′ ), whereas Hepatozoon spp, a 737 bp fragment was amplified by a primer pair consisting of a forward primer Hep-001F (5 ′ -CCTGGCTATACATGAGCAAAATCTCAACTT-3 ′ ) and a reverse primer Hep-737R (5 ′ -CCAACTGTCCCTATCAATCAT-TAAAGC-3 ′ ) (Kledmanee et al., 2009). The multiplex-PCR reaction mixture consisted of 12.5 μl of QIAGEN Multiplex-PCR Master Mix, 10.25 μl of nuclease-free water, 0.125 μl of each primer (100 μM), and 2 μl of DNA template in a total reaction volume of 25 μl. The PCR thermocycling conditions consisted of one step of 95 • C for 15 min, followed by 35 cycles of 94 • C for 45 s, 65 • C for 45 s, and 72 • C for 90 s, with a final extension step at 72 • C for 10 min. Amplified PCR products were separated by gel electrophoresis on a 2% agarose gel, visualized with GelRed™ (Biotium, USA) under UV light. The GeneRuler™ 100 bp DNA ladder (Thermo Scientific) was used to estimate the size of DNA fragments on the gel.

DNA cloning
The PCR products were cloned using the pGEM®-T Easy Vector System (Promega) according to the manufacturer's protocol. Briefly, each PCR product was ligated into the vector, before the ligated vector was transformed into the competent E. coli DH5α. The competent cells were screened for recombinant vector by blue/white selection technique in a Luria-Bertani (LB) plate supplemented with ampicillin/IPTG/X-Gal. The white colony carrying the PCR product was cultured in LB medium with the addition of an antibiotic before the plasmids (the ligation vectors) were extracted using the QIAprep Spin Miniprep Kit for the next step DNA sequencing.

DNA sequencing and phylogenetic analysis
DNA sequencing of PCR products was performed using an ABI 3730 XL DNA analyzer at Bioneer (Daejeon, Korea) with T7 universal primer. The nucleotide sequences were edited and analyzed using MEGA 11 (Molecular Evolutionary genetic analysis) software (Tamura et al., 2021). Multiple alignments of all nucleotide sequences were done using ClustalW within MEGA 11. The nucleotide sequence result was compared to sequences deposited in the GenBank database using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree based on neighbor-joining (NJ) method with bootstrapping (1000 replicates) was constructed by MEGA 11 software (Tamura et al., 2021).

Results and discussion
Both Babesia sp. and Hepatozoon sp. were detected in the spleen of the adult male wild dog. The nucleotide sequences of both parasites were compared with available sequences in the GenBank database by BLAST. The sequence of Babesia sp. showed 99.64% identity to B. gibsoni sequences (GenBank: MN134517), whereas the nucleotide sequence of Hepatozoon sp. showed 98.51% identity to H. canis (GenBank: MN393911). The sequences of B. gibsoni (GenBank: MK144331) and H. canis (GenBank: MK144332) were used in the phylogenetic analysis. The phylogenetic tree of the 18S rRNA of B. gibsoni and H. canis and other related species was established using an NJ method ( Fig. 1 and 2). Babesia gibsoni isolated from the wild dog in this study was included in the cluster with B. gibsoni isolated from the domestic dog and other wild animals; i.e. masked palm civet in Taiwan  This study reports for the first time the occurrence of B. gibsoni and H. canis in the Asiatic wild dog. The post-mortem examination by veterinarians revealed that the wild dog was killed by another wild dog in a fight. Although blood parasite infections in many wildlife species are typically asymptomatic, the hosts can develop clinical signs under certain circumstances such as unnatural hosts, stress, habitat degradation, climate fluctuation, or immunosuppression (Williams et al., 2014). In such cases, a wild dog may present subclinical or mild infections according to the clinical manifestation of Babesia and Hepatozoon infections in wild canids which are typically subclinical (McCully et al., 1975;Schoeman, 2009;Penzhorn, 2011). Babesia infection can cause severe illness, especially in young and immunosuppressed dogs. Colly and Nesbit (1992) reported a fatal acute babesiosis in a juvenile African wild dog (Lycaon pictus). Additionally, the stress in captive wild animals can enhance the severity of babesiosis, since in many cases infected wild animals died soon after capture or translocation (Penzhorn, 2006). Canine babesiosis, caused by B. canis vogeli, is one of the most important tick-borne diseases of dogs in Thailand (Niwetpathomwat et al., 2006;Piratae et al., 2015;Juasook et al., 2016). However, there is no previous report of canine babesiosis in wild canids in the country. In this study, the nucleotide sequence of Babesia sp, was highly similar to that of B. gibsoni corresponding with the result from phylogenetic analysis. The infection by B. gibsoni has been found worldwide, especially from domestic dogs in Asian countries (Groves and Yap, 1968;He et al., 2017;Do et al., 2021). Furthermore, B. gibsoni has also been reported in wild carnivores such as golden jackal and red fox (Penzhorn, 2006).
Canine hepatozoonosis is also an important tick-borne disease of dogs in Thailand (Jittapalapong et al., 2006). In this study, the nucleotide sequence of Hepatozoon sp. from the wild dog was highly similar to that of H. canis. The result from the phylogenetic analysis revealed that H. canis (MK144332) was included in the group with other isolated H. canis. The infection by H. canis has been reported in domestic dogs from many countries of Asia, Europe, Africa, and America (Oyamada et al., 2005;Allen et al., 2011;Cimpan et al., 2020;Guo et al., 2020). Additionally, infection with H. canis have been reported in many wild carnivores such as African wild dog (Netherlands et al., 2021), crab-eating fox (Criado-Fornelio et al., 2006), golden jackal (Farkas et al., 2014), and red fox (Farkas et al., 2014;Imre et al., 2015;Ebani et al., 2017).
The transmission of B. gibsoni and H. canis is usually attributed to hard tick species. Rhipicephalus sanguineus, the brown dog tick (spread worldwide with domestic dogs) is the main vector of H. canis (Baneth, 2001) and B. canis vogeli (Penzhorn, 2011), but B. gibsoni in Asia is mainly transmitted by the tick Haemaphysalis longicornis . Two tick species (Haemaphysalis lagrangei and H. shimoga) and one tick genus (Amblyomma) were recorded from Asiatic wild dogs in Thailand (Unpublished data). These ticks might be playing an important role in transmitting those parasites in the forest. In Japan, B. gibsoni was detected in Amblyomma testudinarium collected from wild boar, while H. canis was detected in H. longicornis collected from a dog (Masatani et al., 2017). The source of both parasites in the wild dog and the responsible tick species could not be established with certainty in this study.
Based on their genetic relationship, it is important to determine the transmission pathway between domestic and wild dogs because such transmission can be devastating to endangered species as reported elsewhere (Woodroffe et al., 2012).
The co-infection by B. canis and H. canis in domestic dogs has been reported in several countries such as Costa Rica (Rojas et al., 2014), Philippines (Adao et al., 2017) and Thailand (Piratae et al., 2015;Do et al., 2021). However, a few reports of co-infection by B. gibsoni and H. canis are available. Therefore, the molecular detection of B. gibsoni and H. canis co-infection in a free-ranging Asiatic wild dog in this study is very important as it provides complementary information for monitoring disease outbreaks in other wild and domestic canids, and also for preventing wildlife extinction. To provide more efficient recommendations on the management plan for this vulnerable species, it is important that wildlife authorities should understand the extent of infection, distribution and dynamics of these parasites and their vectors in wildlife-domestic animals interface areas. The control measures focusing on prevention of wild and domestic animal interactions including restriction of domestic animal access to the protected area may reduce the risk of cross-species transmission of these parasites as well as other pathogens. Further epidemiological studies are required to clarify the relationships among parasites, ticks and reservoir hosts. More transdisciplinary engagements between conservation biologists, wildlife veterinarians and epidemiologists are important for establishing the comprehensive management plans aimed at endangered wild canid conservation.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence