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Molecular characterization of Hepatozoon spp. infection in endangered Indian wild felids and canids
Veterinary Parasitology 186 (2012) 475–479
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Molecular characterization of Hepatozoon spp. infection in endangered Indian wild felids and canids Rahul Mohanchandra Pawar a , Anantula Poornachandar a , Pasham Srinivas b , Kancharapu Ramachandra Rao a , Uthandaraman Lakshmikantan a , Sisinthy Shivaji a,∗ a b
Laboratory for Conservation of Endangered Species, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India Nehru Zoological Park (NZP), Hyderabad, India
a r t i c l e
i n f o
Article history: Received 24 August 2011 Received in revised form 8 November 2011 Accepted 14 November 2011 Keywords: Hepatozoon felis Hepatozoon canis Asiatic lion Bengal tiger Indian leopard and Indian wild dog
a b s t r a c t Hepatozoon species are parasites that infect a wide variety of domestic and wild animals. The objective of this study was to perform the molecular detection and characterization of Hepatozoon spp. in Asiatic lion, Indian tiger, Indian leopard, Indian wild dog, Indian domestic dog and cat based on partial 18S rRNA gene sequences from Hepatozoon spp. in the naturally infected animals. Hepatozoon spp. could be detected in blood samples of 5 out of 9 Asiatic lions, 2 out of 5 Indian tigers, 2 out of 4 Indian leopards and 2 out of 2 Indian wild dogs and, 2 out of 4 domestic cats and 2 out of 3 domestic dog samples by PCR. Sequencing of PCR amplicon and BLAST analysis of partial 18S rRNA gene sequences indicated that the Hepatozoon spp. in Asiatic lion, Bengal tiger, Indian leopard and domestic cat was Hepatozoon felis (98–99% similarity) and in the Indian wild and domestic dog the phylogenetic neighbour was Hepatozoon canis (97–100% similarity). Presence of H. felis and H. canis in both domestic and wild animals suggested that they are not host specific and the same parasite causes infection in domestic and wild felids and canids in India and from different parts of the world. To our knowledge, this is the first report on detection and molecular characterization of H. felis infection in Asiatic lions, Indian tigers, Indian leopards and H. canis in Indian wild dog. Hepatozoon spp. may be a potential pathogen and an opportunistic parasite in immuno-compromised animals and could thus represent a threat to endangered Indian wild felids and canids. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The Asiatic lion (Panthera leo persica), Indian tiger (Panthera tigris tigris) and Indian leopard (Panthera pardus fusca) are major big cats found in India, the others being the snow leopard (Panthera uncia) and clouded leopard (Neofelis nebulosa) (Pandit et al., 2007). All these cats are considered endangered in India and habitat destruction and poaching are the two anthropogenic factors responsible for the decline of these animals. The Asiatic lion which exists as a single wild population in the Gir forest, Gujarat, India, is
∗ Corresponding author. Tel.: +91 40 27192504; fax: +91 40 27160311. E-mail address: [email protected] (S. Shivaji). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.11.036
considered to be the most endangered of the big cats followed by the Indian tiger and then the Indian leopard. The International Union for Conservation of Nature (IUCN) has classified the Asiatic lion and the Indian tiger as endangered and the Indian leopard as near threatened. The Indian wild dog or Dhole (Cuon alpinus) is also categorised by IUCN as endangered and the reasons attributed for their decline are habitat loss, depletion of prey base, competition from other predators, persecution and possibly diseases from domestic and feral dogs (Durbin et al., 2008). In fact disease could also be responsible for the decline in the population of big cats (Roelke-Parker et al., 1996; Smith et al., 2006). Hepatozoonosis is a parasitic disease caused by species of Hepatozoon, a genus in the phylum Apicomplexa, suborder Adeleorina and family Hepatozoidae. Hepatozoon spp.
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infects a wide variety of mammals, birds, reptiles, and amphibian hosts (Smith, 1996). Hepatozoonosis is characterized by symptoms such as anorexia, pale mucous membranes, weight loss, pain, diarrhoea, vomiting, gait abnormalities, fever, polyuria and polydipsia and under severe conditions it could be fatal (Gondim et al., 1998; Kocan et al., 2000; Garret et al., 2005; East et al., 2008). The life cycle of a Hepatozoon is divided between a vertebrate, the intermediate host, which gets infected through ingestion of Hepatozoon infected arthropods, such as ticks, which are the definitive hosts of the parasite and contain sporulated oocysts (Smith, 1996; Baneth et al., 2007). Schizogony occurs in various organs of the intermediate vertebrate hosts, and merozoites invade leukocytes (Smith, 1996). Infection may also occur by predation (Smith, 1996). The objectives of the study were to investigate whether the Asiatic lion, Indian tiger, Indian leopard and Indian wild dog are infected with Hepatozoon species, to identify the species based on phylogenetic analysis of the partial 18S rRNA gene sequence and to ascertain whether Hepatozoon spp. causing infection in Indian wild felids and wild dog are phylogenetically identical or different from the Hepatozoon spp. causing infection in domestic felids and canids in India and from different parts of the world. This is the first report of Hepatozoon felis and Hepatozoon canis infection in endangered Asiatic lion, Indian tiger, Indian leopard and endangered Indian wild dog. 2. Material and methods Blood samples were collected from captive Asiatic lions, tigers, leopards and wild dogs housed at the Nehru Zoological Park (NZP), Hyderabad, India, between 2007 and 2010 (Table 1). Asiatic lions, tigers and wild dogs were captiveraised whereas Indian leopards were wild-caught. Blood samples were collected when animals were anesthetized for routine health check-up, or therapeutic intervention. Blood samples from domestic cats and dogs were collected from a veterinary polyclinic, Hyderabad. Samples were collected directly into EDTA containing vacuette vials (BD, Franklin Lakes, NJ, USA) and frozen at −20 ◦ C until use. DNA was extracted from the frozen blood samples using the standard proteinase K/phenol: chloroform protocol of Sambrook et al. (1989), dissolved in TE (10 mM Tris–HCL, 1.0 mM EDTA, pH 8.0) buffer and stored at −20 ◦ C until use. As described by Criado-Fornelio et al. (2003) universal Babesia–Theileria–Hepatozoon primers (BTH-1F, 5 - CCTGMGARACGGCTACCACATCT and BTH-1R, 5 TTGCGACCATACTCCCCCCA-3 ) for 18S rRNA gene were used for the detection of Hepatozoon spp. in the DNA samples by PCR. PCR amplification was performed essentially as described by Criado-Fornelio et al. (2003) using Expand High Fidelity PCR enzyme mix (2.5 U for 25 l; Roche, Basel, Switzerland). The PCR products were gel purified using a PureLink quick gel extraction kit (Invitrogen, Carlsbad, CA, USA) and sequenced using BigDye Terminator and ABI 3700 (Applied Biosystem, Foster, CA, USA) genetic analyser. For accuracy both the strands were sequenced using primers BTH-F1 and BTH-1R, aligned using Autoassembler software (Applied Biosystem, Foster, CA, USA) and subjected to the basic local alignment
search tool (BLAST) sequence similarity search (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the nearest phylogenetic neighbour. Subsequently, the partial 18S rRNA gene sequences from this study and similar sequences from the GenBank database were aligned using CLUSTAL X (Thompson et al., 1997) and used to construct phylogenetic trees by Neighbour-joining method, Maximum parsimony and UPGMA (Kumar et al., 2008) with Plasmodium vivax as an out-group. For evaluation of the robustness of tree topologies bootstrap analysis was performed based on 1000 resamplings using MEGA 4.1 program (Kumar et al., 2008). 3. Results and discussion Earlier reports demonstrated that Hepatozoon infections in wild animals are normally subclinical (McCully et al., 1975; Mercer et al., 1988; Averbeck et al., 1990; Kocan et al., 2000; Metzger et al., 2008; East et al., 2008; André et al., 2010) though infection with Hepatozoon spp. in young wild canids has been occasionally associated with the presence of clinical disease (Kocan et al., 2000; Garret et al., 2005) and mortality in hyenas (East et al., 2008). Hepatozoon infected animals in this study were also asymptomatic. DNA from the Hepatozoon spp. infected (suspected) Asiatic lion, Indian tiger, Indian leopard and Indian wild dog following PCR yielded a single amplified product of about 730 bp using universal PCR primers (BTH-F1 and BTH-R1). In the blood of 5 out of 9 Asiatic lions, 2 out of 5 Indian tigers, 2 out of 4 Indian leopards, 2 out of 2 Indian wild dogs, 2 out of 4 domestic cats and 2 out of 3 domestic dog samples the expected amplified product of about 730 bp was detected (Table 1). Sequencing and BLAST analysis of the amplicon indicated that it was similar to a partial 18S rRNA gene sequence of H. felis (98–99%) when the source DNA was from the Asiatic lion, Indian tiger and Indian leopard and domestic cat. All the 18S rRNA gene sequences of H. felis from this study showed 100% nucleotide identity with sequences from Asiatic lions, Bengal tigers, Indian leopards and domestic cats. When 18S rRNA gene sequences of H. felis from the different hosts were compared the similarity varied from 98.5% to 99.8%. But, when the source DNA was from the Indian wild and domestic dog the nearest phylogenetic neighbour was H. canis with which it exhibited 97–100% similarity (Table 1). All the 18S rRNA gene sequences of H. canis from Indian wild and domestic dogs were identical (100% nucleotide identity) between themselves and between host species. 18S rRNA gene sequence of H. felis and H. canis showed 95% similarity. Phylogenetic trees constructed using either Neighbourjoining, Maximum parsimony or UPGMA methods using the partial 18S rRNA gene sequences (625 nucleotides) from this study along with sequences of 29 Hepatozoon isolates from 9 different species of animals downloaded from the GenBank database (Fig. 1) exhibited three major clades (A, B, and C) representing Hepatozoon ursi (clade A), H. felis (clade B), and H. canis (clade C), with a robust bootstrap value in all the three phylogenetic trees (Fig. 1). The phylogenetic relationship within the genus Hepatozoon as observed in the present study was similar to that reported earlier (Criado-Fornelio et al., 2006; Rubini et al., 2005;
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Table 1 PCR detection of Hepatozoon spp. in the blood of Asiatic lion, Indian tiger, Indian leopard, Indian wild dog and domestic cat and dog based on partial 18S rRNA gene sequences. Host species
Number of blood samples
Number of samples positive for Hepatozoon spp.
Nearest phylogenetic neighbour
18S rRNA gene sequence similarity (%)
Asiatic lion Bengal tiger Indian leopard Indian wild dog Domestic cat Domestic dog
9 5 4 2 4 3
5 2 2 2 2 2
H. felis from a leopard cat from Thailand (GQ926901) H. felis from a leopard cat from Korea (GQ377216) H. felis from a leopard cat from Korea (GQ377218) H. canis from a dog from USA (AF176835) H. felis from a domestic cat from Spain (AY628681) H. canis from a domestic dog from Taiwan (EU289222)
99 99 99 97 98 100
Fig. 1. Phylogenetic analysis of the Hepatozoon spp. partial 18S rRNA gene sequence amplified from the blood of Asiatic lion, Indian tiger, Indian leopard Indian wild dog, Indian domestic dog and cat using primers BTH-F1 and BTH-R1. The phylogenetic tree was constructed using partial 18S rRNA gene sequence corresponding to a stretch of 625 nucleotides common to all the sequences obtained in the study and retrieved from the database. 18S rRNA gene sequence of Plasmodium vivax was used as the out-group. Bootstrap values are shown at the nodes. Jukes-cantor algorithms were utilized, and phylogenetic trees were constructed by Neighbour-joining method using MEGA 4.1 program. *Indicates sequences from this study.
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Metzger et al., 2008; Salakij et al., 2010; Kubo et al., 2010; Pawar et al., 2011). All the isolates of H. ursi from Indian sloth bear and black bear from Japan formed a very compact clade A. In contrast clade B included isolates of H. felis from Indian tiger, Asiatic lion, leopard from India, leopard cat from Korea and Thailand, domestic cat from India, Spain and Brazil. The isolates in this clade showed 99–100% similarity, with the isolates from Asiatic lion and Indian leopard showing 100% similarity to each other. These results would indicate that H. felis infects wild and domestic felids across countries. The Hepatozoon spp. found in wild and domestic felines in our study formed two genotypes (I and II) within the major clade B (Fig. 1). Hepatozoon genotype I caused infection in Bengal tiger and domestic cat whereas genotype II caused infection in Asiatic lion and Indian leopard. It was also observed that in a single zoo both the genotypes were present in different host species indicating that wild and domestic animals in India are parasitized by more than one species, or genotype, of H. felis. Further studies are needed to confirm if any correlation exists between the genotype and the host species. Clade C included sequences of 18S rRNA gene of H. canis from Indian wild dog, domestic dog from USA, Spain, Brazil, Taiwan, Thailand and India, fox from Croatia, crab-eating and Pampas fox from Brazil. This confirms the presence of H. canis infection in wild and domestic dogs, and fox from across various countries. Additional information on how the Hepatozoon sp. was transmitted to the wild and domestic animals, in the present study, is not known. Unlike other vector borne protozoon infections, Hepatozoon infection in canines or felines occurs by ingestion of vector containing mature oocytes of Hepatozoon in the haemocoel. However, ticks were not found parasitizing the wild and domestic animals included in our study. Earlier study showed that H. canis is transmitted by Rhipicephalus sanguineus (Baneth et al., 2001) and Amblyomma ovale ticks (Forlano et al., 2005; Rubini et al., 2009). Furthermore, there is also the possibility that infection could occur by predation of prey with Hepatozoon cysts (McCully et al., 1975; Smith, 1996). Vertical intra-uterine transmission of the parasite may also occur, as shown for H. canis infection in domestic dogs (Murata et al., 1993). The grooming behavior in these wild animals to remove ticks is also known to facilitate transmission of Hepatozoon spp. (East et al., 2008). Infection with Hepatozoon spp. in young coyotes has been occasionally associated with the presence of clinical disease (Kocan et al., 2000; Garret et al., 2005) and mortality in hyenas (East et al., 2008). The concomitant infection with pathogens of viral or bacterial origin potentially weakens the animal immune system and in such immunocompromised animals Hepatozoon spp. may be a potential pathogen and an opportunistic parasite (Davis et al., 1978; Baneth et al., 1998; Kubo et al., 2006). Very few reports are available regarding the feline and canine hepatozoonosis in India. Earlier, canine hepatozoonosis was reported in domestic dogs in Mumbai, associated with clinical signs of anaemia, thrombocytopenia, hepatitis, hyperglobulinaemia and elevate blood urea and nitrogen (Pawar and Gatne, 2005). Information with regard to the immune
status of these dogs and co-infection with other vectorborne diseases was not available. In most cases however, subclinical infections occurs with a prevalence ranging from 3% to 9% (Gupta et al., 1994; Sharma et al., 1997). However, earlier reports on the feline and canine hepatozoonosis in Indian wildlife is totally lacking. This is the first report of H. felis infection in endangered Asiatic lion, Indian tiger and Indian leopard and H. canis infection in endangered Indian wild dog. In our study, H. felis was found in cat family while H. canis in dog family in both domestic and wild animals suggest that they are not host specific. More studies on the prevalence, transmission, and the pathogenesis of the infection in Indian wild felids and canids should be conducted since Hepatozoon infection may represent a threat to the survival of the endangered Indian wild felids and canids. It is also possible that these carnivores may serve as a reservoir of the parasite (Davis et al., 1978; Mercer et al., 1988; Kocan et al., 2000) for subsequent transmission to other animals. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements We would like to thank the CSIR, Ministry of Science and Technology, Govt. of India and Central Zoo Authority (CZA) of India, Ministry of Environment and Forests, Govt. of India, for financial assistance. We would like to thank Director, Nehru Zoological Park (NZP), Hyderabad, for providing permission to collect samples and Dr. Hakeem for his support during sample collection. We would like to acknowledge B.V.N. Naveen Kumar for formatting the manuscript. References André, M.R., Adania, C.H., Teixeira, R.H.F., Vargas, G.H., Falcade, M., Sousae, L., Salles, A.R., Allegretti, S.M., Felippe, P.A.N., Machado, R.Z., 2010. Molecular detection of Hepatozoon spp. In Brazilian and exotic wild carnivores. Vet. Parasitol. 173, 134–138. Averbeck, G.A., Bjjork, K.E., Packer, C., Herbst, L., 1990. Prevalence of hematozoans in lions (Panthera onca) and cheetah (Acinonyx jubatus) in Serengeti National Park and Ngorongoro Crater, Tanzania. J. Wildl. Dis. 26, 392–394. Baneth, G., Aroch, I., Tal, N., Harrus, S., 1998. Hepatozoon species infection in domestic cats: a retrospective study. Vet. Parasitol. 79, 123–133. Baneth, G., Samish, M., Alekseev, E., Aroch, I., Shkap, V., 2001. Transmission of Hepatozoon canis to dogs by naturally-fed or percutaneously injected Rhipicephalus sanguineus ticks. J. Parasitol. 87, 606–611. Baneth, G., Samish, M., Shkap, V., 2007. Life cycle of Hepatozoon canis (Apicomplexa: Adeleorina: Hepatozoidae) in the tick Rhipicephalus sanguineus and domestic dog (Canis familiaris). J. Parasitol. 93, 283–299. Criado-Fornelio, A., Martinez-Marcos, A., Buling-Sarana, A., BarbaCarretero, J.C., 2003. Molecular studies on Babesia, Theileria and Hepatozoon in southern Europe. Part I. Epizootiological aspects. Vet. Parasitol. 113, 189–201. Criado-Fornelio, A., Ruas, J.L., Casado, N., Farias, N.A.R., Soares, M.P., Müller, G., Brum, J.G.W., Berne, M.E.A., Buling-Saranã, A., Barba-Carretero, J.C., 2006. New molecular data on mammalian Hepatozoon species (Apicomplexa: Adeleorina) from Brazil and Spain. J. Parasitol. 92, 93–99. Davis, D.S., Robinson, R.M., Craig, T.M., 1978. Naturally occurring hepatozoonosis in a coyote. J. Wildl. Dis. 14, 244–246.
R.M. Pawar et al. / Veterinary Parasitology 186 (2012) 475–479 Durbin, L.S., Hedges, S., Duckworth, J.W., Tyson, M., Lyenga, A., Venkataraman, A., 2008. Cuon alpines. In: IUCN 2008. IUCN Red List of Threatened Species. East, M.L., Wibbelt, G., Lieckfeldt, D., Ludwig, A., Goller, K., Wilhelm, K., Schares, G., Thierer, D., Hofer, H., 2008. A Hepatozoon species genetically distinct from H. canis infecting spotted hyenas in the Serengeti ecosystem, Tanzania. J. Wildl. Dis. 44, 45–52. Forlano, M., Scofield, R., Elisei, C., Fernandes, K.R., Ewingii, S.A., Massard, C.L., 2005. Diagnosis of Hepatozoon spp. in Amblyomma ovale and its experimental transmission in domestic dogs in Brazil. Vet. Parasitol. 134, 1–7. Garret, J.J., Kocan, A.A., Reichard, M.V., Panciera, R.J., Bahr, R.J., 2005. Experimental infection of adult and juvenile coyotes with domestic dog and wild coyote isolates of Hepatozoon americanum (Apicomplexa: Adeleorina). J. Wildl. Dis. 41, 588–592. Gondim, L.F.P., Kohayagawa, A., Alencar, N.X., Biondo, A.W., Takahira, R.K.E., Franco, S.R.V., 1998. Canine hepatozoonosis in Brazil: description of eight naturally occurring cases. Vet. Parasitol. 74, 319–323. Gupta, M.P., Nauriyal, D.C., Juyal, P.D., Kalra, I.S., Khahra, S.S., Mohan, R., 1994. Therapeutic trials of Hepatozoon canis infection in dogs. Indian Vet. J. 71, 1221–1221. Kocan, A.A., Cummings, C.A., Panciera, R.J., Mathew, J.S., Ewingii, S.A., Barker, R.W., 2000. Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma. J. Wildl. Dis. 36, 149–153. Kubo, M., Miyoshi, N., Yasuda, N., 2006. Hepatozoonosis in two species of Japanese wild cats. J. Vet. Med. Sci. 68, 833–837. Kubo, M., Jeong, A., Kim, S.I., Kim, Y.J., Lee, H., Kimura, J., Agatsuma, T., Sakai, H., Yanai, T., 2010. The first report of Hepatozoon species infection in leopard cats (Prionailurus bengalensis) in Korea. J. Parasitol. 96, 437–439. Kumar, S., Dudley, J., Nei, M., Tamura, K., 2008. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief. Bioinform. 9, 299–306. McCully, R.M., Basson, P.A., Bigalke, R.D., De-Vos, V., Young, E., 1975. Observations on naturally acquired hepatozoonosis of wild carnivores and dogs in the Republic of South Africa. Onderstepoort J. Res. 42, 117–134. Mercer, S.H., Jones, L.P., Rappole, J.H., Twed, D., Laack, L.L., Craig, T.M., 1988. Hepatozoon sp. in wild carnivores in Texas. J. Wildl. Dis. 24, 574–576.
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Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Molecular characterization of Hepatozoon spp. infection in endangered Indian wild felids and canids Rahul Mohanchandra Pawar a , Anantula Poornachandar a , Pasham Srinivas b , Kancharapu Ramachandra Rao a , Uthandaraman Lakshmikantan a , Sisinthy Shivaji a,∗ a b
Laboratory for Conservation of Endangered Species, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India Nehru Zoological Park (NZP), Hyderabad, India
a r t i c l e
i n f o
Article history: Received 24 August 2011 Received in revised form 8 November 2011 Accepted 14 November 2011 Keywords: Hepatozoon felis Hepatozoon canis Asiatic lion Bengal tiger Indian leopard and Indian wild dog
a b s t r a c t Hepatozoon species are parasites that infect a wide variety of domestic and wild animals. The objective of this study was to perform the molecular detection and characterization of Hepatozoon spp. in Asiatic lion, Indian tiger, Indian leopard, Indian wild dog, Indian domestic dog and cat based on partial 18S rRNA gene sequences from Hepatozoon spp. in the naturally infected animals. Hepatozoon spp. could be detected in blood samples of 5 out of 9 Asiatic lions, 2 out of 5 Indian tigers, 2 out of 4 Indian leopards and 2 out of 2 Indian wild dogs and, 2 out of 4 domestic cats and 2 out of 3 domestic dog samples by PCR. Sequencing of PCR amplicon and BLAST analysis of partial 18S rRNA gene sequences indicated that the Hepatozoon spp. in Asiatic lion, Bengal tiger, Indian leopard and domestic cat was Hepatozoon felis (98–99% similarity) and in the Indian wild and domestic dog the phylogenetic neighbour was Hepatozoon canis (97–100% similarity). Presence of H. felis and H. canis in both domestic and wild animals suggested that they are not host specific and the same parasite causes infection in domestic and wild felids and canids in India and from different parts of the world. To our knowledge, this is the first report on detection and molecular characterization of H. felis infection in Asiatic lions, Indian tigers, Indian leopards and H. canis in Indian wild dog. Hepatozoon spp. may be a potential pathogen and an opportunistic parasite in immuno-compromised animals and could thus represent a threat to endangered Indian wild felids and canids. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The Asiatic lion (Panthera leo persica), Indian tiger (Panthera tigris tigris) and Indian leopard (Panthera pardus fusca) are major big cats found in India, the others being the snow leopard (Panthera uncia) and clouded leopard (Neofelis nebulosa) (Pandit et al., 2007). All these cats are considered endangered in India and habitat destruction and poaching are the two anthropogenic factors responsible for the decline of these animals. The Asiatic lion which exists as a single wild population in the Gir forest, Gujarat, India, is
∗ Corresponding author. Tel.: +91 40 27192504; fax: +91 40 27160311. E-mail address: [email protected] (S. Shivaji). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.11.036
considered to be the most endangered of the big cats followed by the Indian tiger and then the Indian leopard. The International Union for Conservation of Nature (IUCN) has classified the Asiatic lion and the Indian tiger as endangered and the Indian leopard as near threatened. The Indian wild dog or Dhole (Cuon alpinus) is also categorised by IUCN as endangered and the reasons attributed for their decline are habitat loss, depletion of prey base, competition from other predators, persecution and possibly diseases from domestic and feral dogs (Durbin et al., 2008). In fact disease could also be responsible for the decline in the population of big cats (Roelke-Parker et al., 1996; Smith et al., 2006). Hepatozoonosis is a parasitic disease caused by species of Hepatozoon, a genus in the phylum Apicomplexa, suborder Adeleorina and family Hepatozoidae. Hepatozoon spp.
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infects a wide variety of mammals, birds, reptiles, and amphibian hosts (Smith, 1996). Hepatozoonosis is characterized by symptoms such as anorexia, pale mucous membranes, weight loss, pain, diarrhoea, vomiting, gait abnormalities, fever, polyuria and polydipsia and under severe conditions it could be fatal (Gondim et al., 1998; Kocan et al., 2000; Garret et al., 2005; East et al., 2008). The life cycle of a Hepatozoon is divided between a vertebrate, the intermediate host, which gets infected through ingestion of Hepatozoon infected arthropods, such as ticks, which are the definitive hosts of the parasite and contain sporulated oocysts (Smith, 1996; Baneth et al., 2007). Schizogony occurs in various organs of the intermediate vertebrate hosts, and merozoites invade leukocytes (Smith, 1996). Infection may also occur by predation (Smith, 1996). The objectives of the study were to investigate whether the Asiatic lion, Indian tiger, Indian leopard and Indian wild dog are infected with Hepatozoon species, to identify the species based on phylogenetic analysis of the partial 18S rRNA gene sequence and to ascertain whether Hepatozoon spp. causing infection in Indian wild felids and wild dog are phylogenetically identical or different from the Hepatozoon spp. causing infection in domestic felids and canids in India and from different parts of the world. This is the first report of Hepatozoon felis and Hepatozoon canis infection in endangered Asiatic lion, Indian tiger, Indian leopard and endangered Indian wild dog. 2. Material and methods Blood samples were collected from captive Asiatic lions, tigers, leopards and wild dogs housed at the Nehru Zoological Park (NZP), Hyderabad, India, between 2007 and 2010 (Table 1). Asiatic lions, tigers and wild dogs were captiveraised whereas Indian leopards were wild-caught. Blood samples were collected when animals were anesthetized for routine health check-up, or therapeutic intervention. Blood samples from domestic cats and dogs were collected from a veterinary polyclinic, Hyderabad. Samples were collected directly into EDTA containing vacuette vials (BD, Franklin Lakes, NJ, USA) and frozen at −20 ◦ C until use. DNA was extracted from the frozen blood samples using the standard proteinase K/phenol: chloroform protocol of Sambrook et al. (1989), dissolved in TE (10 mM Tris–HCL, 1.0 mM EDTA, pH 8.0) buffer and stored at −20 ◦ C until use. As described by Criado-Fornelio et al. (2003) universal Babesia–Theileria–Hepatozoon primers (BTH-1F, 5 - CCTGMGARACGGCTACCACATCT and BTH-1R, 5 TTGCGACCATACTCCCCCCA-3 ) for 18S rRNA gene were used for the detection of Hepatozoon spp. in the DNA samples by PCR. PCR amplification was performed essentially as described by Criado-Fornelio et al. (2003) using Expand High Fidelity PCR enzyme mix (2.5 U for 25 l; Roche, Basel, Switzerland). The PCR products were gel purified using a PureLink quick gel extraction kit (Invitrogen, Carlsbad, CA, USA) and sequenced using BigDye Terminator and ABI 3700 (Applied Biosystem, Foster, CA, USA) genetic analyser. For accuracy both the strands were sequenced using primers BTH-F1 and BTH-1R, aligned using Autoassembler software (Applied Biosystem, Foster, CA, USA) and subjected to the basic local alignment
search tool (BLAST) sequence similarity search (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the nearest phylogenetic neighbour. Subsequently, the partial 18S rRNA gene sequences from this study and similar sequences from the GenBank database were aligned using CLUSTAL X (Thompson et al., 1997) and used to construct phylogenetic trees by Neighbour-joining method, Maximum parsimony and UPGMA (Kumar et al., 2008) with Plasmodium vivax as an out-group. For evaluation of the robustness of tree topologies bootstrap analysis was performed based on 1000 resamplings using MEGA 4.1 program (Kumar et al., 2008). 3. Results and discussion Earlier reports demonstrated that Hepatozoon infections in wild animals are normally subclinical (McCully et al., 1975; Mercer et al., 1988; Averbeck et al., 1990; Kocan et al., 2000; Metzger et al., 2008; East et al., 2008; André et al., 2010) though infection with Hepatozoon spp. in young wild canids has been occasionally associated with the presence of clinical disease (Kocan et al., 2000; Garret et al., 2005) and mortality in hyenas (East et al., 2008). Hepatozoon infected animals in this study were also asymptomatic. DNA from the Hepatozoon spp. infected (suspected) Asiatic lion, Indian tiger, Indian leopard and Indian wild dog following PCR yielded a single amplified product of about 730 bp using universal PCR primers (BTH-F1 and BTH-R1). In the blood of 5 out of 9 Asiatic lions, 2 out of 5 Indian tigers, 2 out of 4 Indian leopards, 2 out of 2 Indian wild dogs, 2 out of 4 domestic cats and 2 out of 3 domestic dog samples the expected amplified product of about 730 bp was detected (Table 1). Sequencing and BLAST analysis of the amplicon indicated that it was similar to a partial 18S rRNA gene sequence of H. felis (98–99%) when the source DNA was from the Asiatic lion, Indian tiger and Indian leopard and domestic cat. All the 18S rRNA gene sequences of H. felis from this study showed 100% nucleotide identity with sequences from Asiatic lions, Bengal tigers, Indian leopards and domestic cats. When 18S rRNA gene sequences of H. felis from the different hosts were compared the similarity varied from 98.5% to 99.8%. But, when the source DNA was from the Indian wild and domestic dog the nearest phylogenetic neighbour was H. canis with which it exhibited 97–100% similarity (Table 1). All the 18S rRNA gene sequences of H. canis from Indian wild and domestic dogs were identical (100% nucleotide identity) between themselves and between host species. 18S rRNA gene sequence of H. felis and H. canis showed 95% similarity. Phylogenetic trees constructed using either Neighbourjoining, Maximum parsimony or UPGMA methods using the partial 18S rRNA gene sequences (625 nucleotides) from this study along with sequences of 29 Hepatozoon isolates from 9 different species of animals downloaded from the GenBank database (Fig. 1) exhibited three major clades (A, B, and C) representing Hepatozoon ursi (clade A), H. felis (clade B), and H. canis (clade C), with a robust bootstrap value in all the three phylogenetic trees (Fig. 1). The phylogenetic relationship within the genus Hepatozoon as observed in the present study was similar to that reported earlier (Criado-Fornelio et al., 2006; Rubini et al., 2005;
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Table 1 PCR detection of Hepatozoon spp. in the blood of Asiatic lion, Indian tiger, Indian leopard, Indian wild dog and domestic cat and dog based on partial 18S rRNA gene sequences. Host species
Number of blood samples
Number of samples positive for Hepatozoon spp.
Nearest phylogenetic neighbour
18S rRNA gene sequence similarity (%)
Asiatic lion Bengal tiger Indian leopard Indian wild dog Domestic cat Domestic dog
9 5 4 2 4 3
5 2 2 2 2 2
H. felis from a leopard cat from Thailand (GQ926901) H. felis from a leopard cat from Korea (GQ377216) H. felis from a leopard cat from Korea (GQ377218) H. canis from a dog from USA (AF176835) H. felis from a domestic cat from Spain (AY628681) H. canis from a domestic dog from Taiwan (EU289222)
99 99 99 97 98 100
Fig. 1. Phylogenetic analysis of the Hepatozoon spp. partial 18S rRNA gene sequence amplified from the blood of Asiatic lion, Indian tiger, Indian leopard Indian wild dog, Indian domestic dog and cat using primers BTH-F1 and BTH-R1. The phylogenetic tree was constructed using partial 18S rRNA gene sequence corresponding to a stretch of 625 nucleotides common to all the sequences obtained in the study and retrieved from the database. 18S rRNA gene sequence of Plasmodium vivax was used as the out-group. Bootstrap values are shown at the nodes. Jukes-cantor algorithms were utilized, and phylogenetic trees were constructed by Neighbour-joining method using MEGA 4.1 program. *Indicates sequences from this study.
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Metzger et al., 2008; Salakij et al., 2010; Kubo et al., 2010; Pawar et al., 2011). All the isolates of H. ursi from Indian sloth bear and black bear from Japan formed a very compact clade A. In contrast clade B included isolates of H. felis from Indian tiger, Asiatic lion, leopard from India, leopard cat from Korea and Thailand, domestic cat from India, Spain and Brazil. The isolates in this clade showed 99–100% similarity, with the isolates from Asiatic lion and Indian leopard showing 100% similarity to each other. These results would indicate that H. felis infects wild and domestic felids across countries. The Hepatozoon spp. found in wild and domestic felines in our study formed two genotypes (I and II) within the major clade B (Fig. 1). Hepatozoon genotype I caused infection in Bengal tiger and domestic cat whereas genotype II caused infection in Asiatic lion and Indian leopard. It was also observed that in a single zoo both the genotypes were present in different host species indicating that wild and domestic animals in India are parasitized by more than one species, or genotype, of H. felis. Further studies are needed to confirm if any correlation exists between the genotype and the host species. Clade C included sequences of 18S rRNA gene of H. canis from Indian wild dog, domestic dog from USA, Spain, Brazil, Taiwan, Thailand and India, fox from Croatia, crab-eating and Pampas fox from Brazil. This confirms the presence of H. canis infection in wild and domestic dogs, and fox from across various countries. Additional information on how the Hepatozoon sp. was transmitted to the wild and domestic animals, in the present study, is not known. Unlike other vector borne protozoon infections, Hepatozoon infection in canines or felines occurs by ingestion of vector containing mature oocytes of Hepatozoon in the haemocoel. However, ticks were not found parasitizing the wild and domestic animals included in our study. Earlier study showed that H. canis is transmitted by Rhipicephalus sanguineus (Baneth et al., 2001) and Amblyomma ovale ticks (Forlano et al., 2005; Rubini et al., 2009). Furthermore, there is also the possibility that infection could occur by predation of prey with Hepatozoon cysts (McCully et al., 1975; Smith, 1996). Vertical intra-uterine transmission of the parasite may also occur, as shown for H. canis infection in domestic dogs (Murata et al., 1993). The grooming behavior in these wild animals to remove ticks is also known to facilitate transmission of Hepatozoon spp. (East et al., 2008). Infection with Hepatozoon spp. in young coyotes has been occasionally associated with the presence of clinical disease (Kocan et al., 2000; Garret et al., 2005) and mortality in hyenas (East et al., 2008). The concomitant infection with pathogens of viral or bacterial origin potentially weakens the animal immune system and in such immunocompromised animals Hepatozoon spp. may be a potential pathogen and an opportunistic parasite (Davis et al., 1978; Baneth et al., 1998; Kubo et al., 2006). Very few reports are available regarding the feline and canine hepatozoonosis in India. Earlier, canine hepatozoonosis was reported in domestic dogs in Mumbai, associated with clinical signs of anaemia, thrombocytopenia, hepatitis, hyperglobulinaemia and elevate blood urea and nitrogen (Pawar and Gatne, 2005). Information with regard to the immune
status of these dogs and co-infection with other vectorborne diseases was not available. In most cases however, subclinical infections occurs with a prevalence ranging from 3% to 9% (Gupta et al., 1994; Sharma et al., 1997). However, earlier reports on the feline and canine hepatozoonosis in Indian wildlife is totally lacking. This is the first report of H. felis infection in endangered Asiatic lion, Indian tiger and Indian leopard and H. canis infection in endangered Indian wild dog. In our study, H. felis was found in cat family while H. canis in dog family in both domestic and wild animals suggest that they are not host specific. More studies on the prevalence, transmission, and the pathogenesis of the infection in Indian wild felids and canids should be conducted since Hepatozoon infection may represent a threat to the survival of the endangered Indian wild felids and canids. It is also possible that these carnivores may serve as a reservoir of the parasite (Davis et al., 1978; Mercer et al., 1988; Kocan et al., 2000) for subsequent transmission to other animals. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements We would like to thank the CSIR, Ministry of Science and Technology, Govt. of India and Central Zoo Authority (CZA) of India, Ministry of Environment and Forests, Govt. of India, for financial assistance. We would like to thank Director, Nehru Zoological Park (NZP), Hyderabad, for providing permission to collect samples and Dr. Hakeem for his support during sample collection. We would like to acknowledge B.V.N. Naveen Kumar for formatting the manuscript. References André, M.R., Adania, C.H., Teixeira, R.H.F., Vargas, G.H., Falcade, M., Sousae, L., Salles, A.R., Allegretti, S.M., Felippe, P.A.N., Machado, R.Z., 2010. Molecular detection of Hepatozoon spp. In Brazilian and exotic wild carnivores. Vet. Parasitol. 173, 134–138. Averbeck, G.A., Bjjork, K.E., Packer, C., Herbst, L., 1990. Prevalence of hematozoans in lions (Panthera onca) and cheetah (Acinonyx jubatus) in Serengeti National Park and Ngorongoro Crater, Tanzania. J. Wildl. Dis. 26, 392–394. Baneth, G., Aroch, I., Tal, N., Harrus, S., 1998. Hepatozoon species infection in domestic cats: a retrospective study. Vet. Parasitol. 79, 123–133. Baneth, G., Samish, M., Alekseev, E., Aroch, I., Shkap, V., 2001. Transmission of Hepatozoon canis to dogs by naturally-fed or percutaneously injected Rhipicephalus sanguineus ticks. J. Parasitol. 87, 606–611. Baneth, G., Samish, M., Shkap, V., 2007. Life cycle of Hepatozoon canis (Apicomplexa: Adeleorina: Hepatozoidae) in the tick Rhipicephalus sanguineus and domestic dog (Canis familiaris). J. Parasitol. 93, 283–299. Criado-Fornelio, A., Martinez-Marcos, A., Buling-Sarana, A., BarbaCarretero, J.C., 2003. Molecular studies on Babesia, Theileria and Hepatozoon in southern Europe. Part I. Epizootiological aspects. Vet. Parasitol. 113, 189–201. Criado-Fornelio, A., Ruas, J.L., Casado, N., Farias, N.A.R., Soares, M.P., Müller, G., Brum, J.G.W., Berne, M.E.A., Buling-Saranã, A., Barba-Carretero, J.C., 2006. New molecular data on mammalian Hepatozoon species (Apicomplexa: Adeleorina) from Brazil and Spain. J. Parasitol. 92, 93–99. Davis, D.S., Robinson, R.M., Craig, T.M., 1978. Naturally occurring hepatozoonosis in a coyote. J. Wildl. Dis. 14, 244–246.
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