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Experimental transmission of Hepatozoon americanum to rodents
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Veterinary Parasitology 151 (2008) 164–169 www.elsevier.com/locate/vetpar
Experimental transmission of Hepatozoon americanum to rodents Eileen M. Johnson *, Kelly E. Allen, Melanie A. Breshears, Roger J. Panciera, Susan E. Little, Sidney A. Ewing Oklahoma State University, College of Veterinary Health Sciences, Department of Veterinary Pathobiology, Room 250 McElroy Hall, Stillwater, Ok 74078-2007, United States Received 25 May 2007; received in revised form 25 October 2007; accepted 26 October 2007
Abstract Laboratory-raised cotton rats (Sigmodon hispidus), outbred white mice (Mus musculus), and C57BL/6J-Lystbg-J/J mice (M. musculus) that were administered approximately 50 sporulated oocysts of Hepatozoon americanum (AF176836) by gavage developed inflammatory lesions containing parasitic cystozoites in cardiac and skeletal muscle, kidney, and lung. Sprague–Dawley rats (Rattus norvegicus) similarly exposed showed no evidence of infection. Cystozoites were first detected by histopathologic examination four weeks after exposure to oocysts. Globular, PAS-positive material accumulated around the cystozoites as the duration of infection lengthened. Nested PCR analysis of tissues collected 16 weeks post-exposure was positive for the 18S rRNA Hepatozoon sp. gene and the DNA sequence of the fragment amplified was 99.6% and 99.8% identical to H. americanum sequences previously reported from naturally-infected dogs (AF176836 and AY864676, respectively). Merogonous and gamontogonous stages of the parasite were not detected in any of the cystozoite-infected rodents. # 2007 Elsevier B.V. All rights reserved. Keywords: Cystozoite; Hepatozoon americanum; Paratenic host; Peromyscus leucopus; Sigmodon hispidus
1. Introduction Amblyomma maculatum has been shown to acquire Hepatozoon americanum by feeding experimentally on dogs and coyotes (Mathew et al., 1998; Ewing et al., 2002a; Garrett et al., 2005). However, numerous field surveys for vertebrate hosts of the tick indicate that neither dogs nor wild carnivores are preferred hosts for any of the three feeding stages of this tick (Semtner and Hair, 1973; Greiner et al., 1984; Apperson et al., 1990; Wehinger et al., 1995; Forrester et al., 1996; Clark et al., 1998; Teel et al., 1998; Kinsey et al., 2000; Allan et al.,
* Corresponding author. Tel.: +1 405 744 2473; fax: +1 405 744 5275. E-mail address: [email protected] (E.M. Johnson). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.10.017
2001; Clark et al., 2001; Durden and Kinsey, 2001; Goldberg et al., 2002; Barker et al., 2004). Tick host preference findings raise questions about the reservoir of infection for immature A. maculatum in nature. Investigators have postulated that the natural reservoir of H. americanum might be some as-yet-unidentified wild vertebrate species that harbors developmental stages, is a host and source of infection for larval and/or nymphal A. maculatum, and is normal prey for carnivores (Ewing et al., 2002b). Ewing and Panciera (2003) proposed that predation was likely involved in the transmission of H. americanum to dogs and coyotes. Transmission of H. americanum from ticks to dogs and coyotes has been studied fairly extensively (Mathew et al., 1998; Ewing et al., 2002a,b; Garrett et al., 2005) but other possible host-parasite interactions have not been examined.
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Predation of prey that might host cystozoite stages of H. americanum has been suggested as a possible means of transmission to canids and an important area for study (Craig et al., 1978; Kocan and Kocan, 1991). Cystozoites, a ‘‘quiescent tissue stage’’, have been described for H. canis (Baneth and Shkap, 2003; Baneth et al., 2007) but have not been detected in H. americanum-infected dog or coyote tissues (Kocan et al., 1999; Panciera et al., 1999, 2001). Attempted transmission of H. americanum (Texas strain of H. canis) by a method other than feeding sporulated oocysts involved feeding tissues of infected dogs to susceptible ones (Nordgren and Craig, 1984); transmission was not demonstrated. Transmission to laboratory mice by administering merozoites collected from dog muscle was also unsuccessful. Exposure of common prey of dogs and coyotes by oral administration of sporulated oocysts collected from infected A. maculatum has not been studied. The purpose of the present study was to determine the susceptibility of laboratory-raised rodents to infection with H. americanum. One objective was to determine if S. hispidus, a preferred host for immature stages of A. maculatum (Barker et al., 2004), could support development of H. americanum and act as a ‘‘natural’’ reservoir. The second objective was to find a laboratory animal model for American canine hepatozoonosis (ACH). 2. Material and methods 2.1. Rodents Rodent species tested were laboratory-raised cotton rats (S. hispidus), Sprague–Dawley rats (Rattus norvegicus.), outbred white mice (Mus musculus), and C57BL/6J-Lystbg–J/J mice (M. musculus). Six, young, sexually mature female rodents of each species/strain were acquired from commercial vendors and maintained in IACUC-approved facilities through Oklahoma State University (OSU) Laboratory Animal Resources (LAR). One rodent from each species was housed individually in a separate cage and served as a nonexposed control. Cotton rat principals were housed in individual cages. Sprague–Dawley rat principals were housed with two animals per cage; and outbred and C57BL/6J-Lystbg-J/J mice principals were each housed as groups in individual cages.
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Agricultural Experiment Station Tick Laboratory, were fed on a dog experimentally infected with H. americanum (AF176836) according to the methods of Mathew et al. (1998). Twenty aliquots of 50 sporulated oocysts each were aspirated from the hemocoel of dissected ticks and stored in Eppendorf tubes with 1 ml of phosphate-buffered saline (PBS) at room temperature. 2.3. Rodent exposure Principals for each species were dosed with 50 oocysts of H. americanum by gavage under anesthesia with isofluorane. Gavage was performed less than 6 h after oocysts were collected. 2.4. Monitoring methods Prior to gavage and euthanasia, whole blood for blood smear preparation and PCR analysis was obtained by cardiac or retro-orbital puncture or by tail snip conducted under isofluorane anesthesia. One principal from each of the four groups of rodents was euthanatized at 4-week intervals; at necropsy, tissues were collected for histopathologic examination, PCR analysis and DNA sequencing. Controls were euthanatized for necropsy and histopathologic examination at the end of the 16-week observation period. Tissues routinely collected included lung, liver, kidney, heart, skeletal muscle, bone marrow and spleen. 2.5. PCR and DNA sequencing DNA extractions were performed using the GFXTM Genomic Blood Purification Kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s instructions. The 18S rRNA Hepatozoon gene was amplified by nested PCR and sequenced as previously described (Johnson et al., 2007). 2.6. Histopathology and immunohistopathology Five-micrometer sections of paraffin-embedded formalin-fixed tissues were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff-diastase (PAS). Selected sections were subjected to immunohistochemical assay (Panciera et al., 2001) to verify identity of parasite stages observed in principals by routine histopathology.
2.2. H. americanum oocysts
3. Results
Four hundred laboratory-raised, specific-pathogenfree A. maculatum nymphs, acquired from the Oklahoma
Pre-exposure PCR results on blood from all rodents were negative. Clinical illness attributable to exposure
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to H. americanum oocysts was not recognized among rodents during the 16-week course of the experiment. However, one C57BL/6J-Lystbg-J/J mouse died 1 week post-gavage due to aspiration pneumonia and one outbred mouse died 9 weeks post-exposure of unknown causes. The cotton rat control died during anesthesia and bleeding at 4-week. No evidence of infection was found in the control or principals that died prematurely. Neither Hepatozoon gamonts nor gross necropsy lesions were observed in either principals or controls at any time during the study. Histopathologic examination revealed multifocal and confluent non-suppurative inflammatory lesions in heart of cotton rats, C57BL/6J-Lystbg-J/J mice and, less consistently, in outbred mice as early as 4 weeks post-exposure. Lesions initially consisted of loose aggregates of lymphocytes and monocytes; they later became larger, more compactly cellular and predominantly monocytic. Myofiber degeneration, atrophy, and necrosis existed in areas of inflammation (Fig. 1). Similar but fewer lesions were found in renal cortex, skeletal muscle, and lung of cotton rats; such lesions were present less frequently in C57BL/6J-Lystbg-J/J mice and rarely in outbred mouse principals. Lesions suggestive of Hepatozoon infection were not observed in any rodent controls or in Sprague–Dawley rat principals. Histologic evidence of merogony was not observed in any subjects. Immunohistochemical positive, comma-shaped parasites (cystozoites) were within cytoplasmic vacuoles of rodent host cells located proximate to or within inflammatory foci detected in heart, renal cortex, skeletal muscle, and lung. Parasites were approximately
2 12 mm with a central or paracentral, denselystained nucleus (Fig. 2). Single parasites were embedded in lightly basophilic material (Fig. 3) composed of a dense aggregate of globules that stained positively in PAS-diastase treated sections. This globular material was more voluminous in infections of longer duration. The monozoic cysts containing cystozoites were morphologically similar to those described by Desser (1990) in H. griseisciuri-infected grey squirrels. Monozoic cysts were not observed in any rodent controls or in Sprague–Dawley rat principals. The 18S rRNA Hepatozoon gene was detected by PCR amplification and analysis of tissues from the cotton rats, outbred mouse and C57BL/6J-Lystbg-J/J
Fig. 1. Inflammatory focus in myocardium of cotton rat 16 weeks after experimental exposure to sporulated oocysts of H. americanum. There is atrophy, degeneration, and necrosis of myofibers within a focus of moncytic inflammatory infiltrate. HE, Bar = 50 mm.
Fig. 3. Myocardium of cotton rat. Crescentic, encysted zoite within rodent macrophage whose nucleus is displaced and compressed to margin of the host cell and whose cytoplasmic volume is expanded by globular mucopolysaccharide material. HE, Bar = 15 mm.
Fig. 2. Cystozoite in minimally expanded host cell from cotton rat myocardium 4 weeks post-exposure to sporulated oocysts of H. americanum. Zoite (arrow) is banana-shaped with densely basophilic nucleus. Host cell cytoplasm is vaguely perceptible. HE, Bar = 10 mm.
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mice principals necropsied at week-16 post-exposure. The sequence of the 18S rDNA fragment from the cotton rat tissues containing cystozoites (EU249993) was identical to the sequence of the fragment amplified from the dog experimentally infected with H. americanum (EU249992) on which the ticks fed. The sequence from cotton rat tissues containing cystozoites was 99.8% or 99.6% identical to H. americanum sequences previously reported from naturally-infected dogs (AY864676 and AF176836, respectively), although the two previously reported sequences and the sequence reported here did differ from one another by at least one base in the variable region considered. 4. Discussion Exposure of cotton rats, outbred mice, and C57BL/ 6J-Lystbg-J/J mice to oocysts of H. americanum resulted in successful transmission of infection characterized by development of cystozoites in heart, kidney, skeletal muscle and lung; Sprague–Dawley rats were resistant. A previously reported attempt to transmit a North American isolate of Hepatozoon sp. to rodents was unsuccessful (Nordgren and Craig, 1984); in that study mice were fed skeletal muscle of dogs infected with H. americanum (Texas strain of H. canis). Cystozoites have been demonstrated in the tissues of facultative intermediate (paratenic) hosts in the life cycles of some apicomplexan parasites of predators such as dogs, cats and birds of prey (Gardiner et al., 1988). Latent dizoic cystic forms have been demonstrated in the life cycles of Hepatozoon species that infect snakes, lizards, and anurans (Smith et al., 1994, 1996; Stehbens and Johnston, 1968; Wozniak et al., 1996; Kim et al., 1998; Lainson et al., 2003; Paperna and Lainson, 2004). In such cycles, transmission of Hepatozoon spp. to an obligate vertebrate intermediate host can occur not only through ingestion of haematophagous arthropod definitive hosts containing sporulated oocysts, but also through ingestion of cystozoites in an obligate or facultative host (paratenic), or through both methods (Smith, 1996). Mammalian intermediate hosts have been found to harbor cystozoites in their tissues as well (Baneth and Shkap, 2003; Desser, 1990; Laakkonen et al., 2001; Uni et al., 2003). Desser (1990) found cystic forms in grey squirrels infected with H. griseisciuri; he suggested that cystozoites could be an important mode of transmission for Hepatozoon spp. in mammals. However, attempts to transmit Hepatozoon spp. of mammals by oral exposure to cystozoite stages have not been reported. In spite of extensive histologic examination of many canids
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infected with H. americanum, cystozoites have not been observed in their tissues (Panciera et al., 1999; Kocan et al., 1999; Panciera et al., 2001; Cummings et al., 2005; Garrett et al., 2005). The recognition of H. americanum cystozoites in experimentally infected rodents in the present study is unique, and lends support to Desser’s hypothesis concerning possible transmission of Hepatozoon spp. among mammals by cystozoites. Smith (1996) proposed that the Hepatozoon group of haemogregarines has evolved alternate modes of transmission that exploit the predator-prey relationships of the vertebrate intermediate hosts of these parasites. Development of cystozoites in rodents experimentally challenged with sporulated H. americanum oocysts suggests that paratenic hosts may be involved in the natural transmission cycle of this parasite, a finding that may be of use in better understanding the epidemiology of ACH. Frequently, in parasite life cycles that involve arthropod intermediate host(s), vertebrate paratenic host(s), and definitive vertebrate host(s), the paratenic host is prey for the definitive host. In many such life cycles, the paratenic host is more likely to feed on the arthropod intermediate host than is the definitive host. This host-parasite interaction is well exemplified by the life cycle of Halipegus occidualis, a trematode of the green frog, Rana clamitans. The dragonfly nymph is a required paratenic host in the trematode life cycle because of the feeding gap between the microscopic ostracod second intermediate host and the frog definitive host (Esch, 2004). Similar host feeding habits come into play in life cycles of Hepatozoon spp. In the life cycle of H. sipedon, the culicine mosquito definitive hosts are a natural food source for the first intermediate host, the northern leopard frog, but are not ordinarily fed upon by the second intermediate host, the northern water snake. The frog is a natural food source for the snake and, when harboring cystozoites, is the source of infection for the snake (Smith et al., 1994). Mosquitoes that contain sporulated oocysts are not infectious for the snake. These types of relationships between hosts and parasites that evolved over time enhance chances of survival of the parasite. Cotton rats were chosen as experimental subjects in this study because they are known to be preferred hosts for the larvae and nymphs of A. maculatum, they are abundant in ACH-endemic sites in Oklahoma, and they are easy prey for dogs and coyotes throughout the known geographic distribution of ACH. However, transmission of ACH to dogs with cystozoites contained in experimentally infected rodents and detection of
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cystozoites in wild-trapped rodents with subsequent experimental transmission to dogs are necessary to confirm the role of these rodents as paratenic hosts for H. americanum. In an effort to find a laboratory animal model for ACH, laboratory rats and mice strains were incorporated into the study because of their susceptibility to infection with known Hepatozoon spp., including the type species for the genus, H. muris. Usefulness of the selected rodents as laboratory animal models for ACH is limited because neither merogony nor gamontogony was observed during the 16-week observation period. However, laboratory rodents harboring cystozoites of H. americanum may offer a method for maintenance of the organism which could be a useful and efficient alternative to current methods involving long-term maintenance of infected dogs and experimental infection of ticks. The findings of the present study emphasize the need for further investigations into the potential role that paratenic host(s) may play in the epizootology of ACH. To understand the epidemiology of ACH, investigations directed toward identification of natural reservoirs of H. americanum are critical. Vertebrates, such as birds, present in the geographic range of ACH that feed on nymphal and adult A. maculatum or that may be predators of the preferred hosts of this tick would be good subjects for investigation. It is apparent from findings in this study that molecular analysis of gamonts and cystozoites found in vertebrate hosts is essential for accurate speciation and delineation of hepatozoa. 5. Conclusion Cystozoite stages of H. americanum developed in three of four rodent species/strains exposed to sporulated oocysts; merogony and gamontogony were not observed. This is not only the first report of the existence of a cystozoite stage of H. americanum but also the first report of infection of vertebrate hosts other than carnivores with H. americanum. Although a role for rodents in the epizootiology of American canine hepatozoonosis has not been definitively established, findings of the present study indicate that rodents could serve as paratenic hosts of H. americanum and be a potential source of infection for canids. Future epidemiologic investigations should include this alternative mode of transmission as a means for spread of ACH, which previously was believed to be acquired exclusively by ingestion of infected A. maculatum.
Acknowledgements This research was supported by an OSU CVHS Research Advisory Committee seed Grant, the Wendell H. and Nellie G. Krull Endowment Fund, and funds from the OSU CVHS Department of Pathobiology. References Allan, S.A., Simmons, L.A., Burridge, M.J., 2001. Ixodid ticks on white-tailed deer and feral swine in Florida. J. Vector Ecol. 26, 93–102. Apperson, C.S., Levine, J.F., Nicholson, W.L., 1990. Geographic occurrence of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) infesting white-tailed deer in North Carolina. J. Wildlife Dis. 24, 550–553. Baneth, G., Shkap, V., 2003. Monozoic cysts of Hepatozoon canis. J. Parasitol. 89, 379–381. Baneth, G., Samish, M., Shrap, 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. Barker, R.W., Kocan, A.A., Ewing, S.A., Wetteman, R.P., Payton, M.E., 2004. Occurrence of the Gulf coast tick (Acari: Ixodidae) on wild and domestic mammals in North-Central Oklahoma. J. Med. Entomol. 41, 170–178. Clark, K.L., Oliver Jr., J.H., McKechnie, D.B., Williams, D.C., 1998. Distribution, abundance, and seasonal activities of ticks collected from rodents and vegetation in South Carolina. J. Vector Ecol. 23, 89–105. Clark, K.L., Oliver Jr., J.H., Grego, J.M., James, A.M., Durden, L.A., Banks, C.W., 2001. Host associations of ticks parasitizing rodents at Borrelia burgdorferi enzootic sites in South Carolina. J. Parasitol. 87, 1379–1386. Craig, T.M., Smallwood, J.E., Knauer, K.W., McGrath, J.P., 1978. Hepatozoon canis infection in dogs: clinical, radiographic, and hemotologic findings. J. Am. Vet. Med. Assoc. 173, 967–972. Cummings, C.A., Panciera, R.J., Kocan, K.M., Ewing, S.A., 2005. Characterization of stages of Hepatozoon americanum and of parasitized canine host cells. Vet. Parasitol. 42, 788–796. Desser, S.S., 1990. Tissue ‘‘cysts’’ of Hepatozoon griseisciuri in the grey squirrel, Sciurus carolinensis: the significance of these cysts in species of Hepatozoon. J. Parasitol. 76, 257–259. Durden, L.A., Kinsey, A.A., 2001. Ticks (Acari: Ixodidae) and spirochetes (spirochaetaceae: spirochaetales) recovered from birds on a Georgia barrier island. J. Med. Entomol. 38, 231–236. Esch, G.W., 2004. Ecological studies in Charlie’s pond: ‘‘A stream of consciousness’’. In: Parasites, People, and Places: Essays on Field Parasitology, Cambridge University Press, Cambridge, United Kingdom, pp. 69–96. Ewing, S.A., Mathew, J.S., Panciera, R.J., 2002a. Transmission of Hepatozoon americanum (Apicomplex: Adeleorina) by ixodids (Acari: Ixodidae). J. Med. Entomol. 39, 631–634. Ewing, S.A., Dubois, J.G., Mathew, J.S., Panciera, R.J., 2002b. Larval Gulf coast ticks (Amblyomma americanum) [Acari: Ixodidae] as host for Hepatozoon americanum [Apicomplexa: Adeleorina]. Vet. Parasitol. 1031, 43–51. Ewing, S.A., Panciera, R.J., 2003. American canine hepatozoonosis. Clin. Microbiol. Rev. 16, 688–697.
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Veterinary Parasitology 151 (2008) 164–169 www.elsevier.com/locate/vetpar
Experimental transmission of Hepatozoon americanum to rodents Eileen M. Johnson *, Kelly E. Allen, Melanie A. Breshears, Roger J. Panciera, Susan E. Little, Sidney A. Ewing Oklahoma State University, College of Veterinary Health Sciences, Department of Veterinary Pathobiology, Room 250 McElroy Hall, Stillwater, Ok 74078-2007, United States Received 25 May 2007; received in revised form 25 October 2007; accepted 26 October 2007
Abstract Laboratory-raised cotton rats (Sigmodon hispidus), outbred white mice (Mus musculus), and C57BL/6J-Lystbg-J/J mice (M. musculus) that were administered approximately 50 sporulated oocysts of Hepatozoon americanum (AF176836) by gavage developed inflammatory lesions containing parasitic cystozoites in cardiac and skeletal muscle, kidney, and lung. Sprague–Dawley rats (Rattus norvegicus) similarly exposed showed no evidence of infection. Cystozoites were first detected by histopathologic examination four weeks after exposure to oocysts. Globular, PAS-positive material accumulated around the cystozoites as the duration of infection lengthened. Nested PCR analysis of tissues collected 16 weeks post-exposure was positive for the 18S rRNA Hepatozoon sp. gene and the DNA sequence of the fragment amplified was 99.6% and 99.8% identical to H. americanum sequences previously reported from naturally-infected dogs (AF176836 and AY864676, respectively). Merogonous and gamontogonous stages of the parasite were not detected in any of the cystozoite-infected rodents. # 2007 Elsevier B.V. All rights reserved. Keywords: Cystozoite; Hepatozoon americanum; Paratenic host; Peromyscus leucopus; Sigmodon hispidus
1. Introduction Amblyomma maculatum has been shown to acquire Hepatozoon americanum by feeding experimentally on dogs and coyotes (Mathew et al., 1998; Ewing et al., 2002a; Garrett et al., 2005). However, numerous field surveys for vertebrate hosts of the tick indicate that neither dogs nor wild carnivores are preferred hosts for any of the three feeding stages of this tick (Semtner and Hair, 1973; Greiner et al., 1984; Apperson et al., 1990; Wehinger et al., 1995; Forrester et al., 1996; Clark et al., 1998; Teel et al., 1998; Kinsey et al., 2000; Allan et al.,
* Corresponding author. Tel.: +1 405 744 2473; fax: +1 405 744 5275. E-mail address: [email protected] (E.M. Johnson). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.10.017
2001; Clark et al., 2001; Durden and Kinsey, 2001; Goldberg et al., 2002; Barker et al., 2004). Tick host preference findings raise questions about the reservoir of infection for immature A. maculatum in nature. Investigators have postulated that the natural reservoir of H. americanum might be some as-yet-unidentified wild vertebrate species that harbors developmental stages, is a host and source of infection for larval and/or nymphal A. maculatum, and is normal prey for carnivores (Ewing et al., 2002b). Ewing and Panciera (2003) proposed that predation was likely involved in the transmission of H. americanum to dogs and coyotes. Transmission of H. americanum from ticks to dogs and coyotes has been studied fairly extensively (Mathew et al., 1998; Ewing et al., 2002a,b; Garrett et al., 2005) but other possible host-parasite interactions have not been examined.
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Predation of prey that might host cystozoite stages of H. americanum has been suggested as a possible means of transmission to canids and an important area for study (Craig et al., 1978; Kocan and Kocan, 1991). Cystozoites, a ‘‘quiescent tissue stage’’, have been described for H. canis (Baneth and Shkap, 2003; Baneth et al., 2007) but have not been detected in H. americanum-infected dog or coyote tissues (Kocan et al., 1999; Panciera et al., 1999, 2001). Attempted transmission of H. americanum (Texas strain of H. canis) by a method other than feeding sporulated oocysts involved feeding tissues of infected dogs to susceptible ones (Nordgren and Craig, 1984); transmission was not demonstrated. Transmission to laboratory mice by administering merozoites collected from dog muscle was also unsuccessful. Exposure of common prey of dogs and coyotes by oral administration of sporulated oocysts collected from infected A. maculatum has not been studied. The purpose of the present study was to determine the susceptibility of laboratory-raised rodents to infection with H. americanum. One objective was to determine if S. hispidus, a preferred host for immature stages of A. maculatum (Barker et al., 2004), could support development of H. americanum and act as a ‘‘natural’’ reservoir. The second objective was to find a laboratory animal model for American canine hepatozoonosis (ACH). 2. Material and methods 2.1. Rodents Rodent species tested were laboratory-raised cotton rats (S. hispidus), Sprague–Dawley rats (Rattus norvegicus.), outbred white mice (Mus musculus), and C57BL/6J-Lystbg–J/J mice (M. musculus). Six, young, sexually mature female rodents of each species/strain were acquired from commercial vendors and maintained in IACUC-approved facilities through Oklahoma State University (OSU) Laboratory Animal Resources (LAR). One rodent from each species was housed individually in a separate cage and served as a nonexposed control. Cotton rat principals were housed in individual cages. Sprague–Dawley rat principals were housed with two animals per cage; and outbred and C57BL/6J-Lystbg-J/J mice principals were each housed as groups in individual cages.
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Agricultural Experiment Station Tick Laboratory, were fed on a dog experimentally infected with H. americanum (AF176836) according to the methods of Mathew et al. (1998). Twenty aliquots of 50 sporulated oocysts each were aspirated from the hemocoel of dissected ticks and stored in Eppendorf tubes with 1 ml of phosphate-buffered saline (PBS) at room temperature. 2.3. Rodent exposure Principals for each species were dosed with 50 oocysts of H. americanum by gavage under anesthesia with isofluorane. Gavage was performed less than 6 h after oocysts were collected. 2.4. Monitoring methods Prior to gavage and euthanasia, whole blood for blood smear preparation and PCR analysis was obtained by cardiac or retro-orbital puncture or by tail snip conducted under isofluorane anesthesia. One principal from each of the four groups of rodents was euthanatized at 4-week intervals; at necropsy, tissues were collected for histopathologic examination, PCR analysis and DNA sequencing. Controls were euthanatized for necropsy and histopathologic examination at the end of the 16-week observation period. Tissues routinely collected included lung, liver, kidney, heart, skeletal muscle, bone marrow and spleen. 2.5. PCR and DNA sequencing DNA extractions were performed using the GFXTM Genomic Blood Purification Kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s instructions. The 18S rRNA Hepatozoon gene was amplified by nested PCR and sequenced as previously described (Johnson et al., 2007). 2.6. Histopathology and immunohistopathology Five-micrometer sections of paraffin-embedded formalin-fixed tissues were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff-diastase (PAS). Selected sections were subjected to immunohistochemical assay (Panciera et al., 2001) to verify identity of parasite stages observed in principals by routine histopathology.
2.2. H. americanum oocysts
3. Results
Four hundred laboratory-raised, specific-pathogenfree A. maculatum nymphs, acquired from the Oklahoma
Pre-exposure PCR results on blood from all rodents were negative. Clinical illness attributable to exposure
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to H. americanum oocysts was not recognized among rodents during the 16-week course of the experiment. However, one C57BL/6J-Lystbg-J/J mouse died 1 week post-gavage due to aspiration pneumonia and one outbred mouse died 9 weeks post-exposure of unknown causes. The cotton rat control died during anesthesia and bleeding at 4-week. No evidence of infection was found in the control or principals that died prematurely. Neither Hepatozoon gamonts nor gross necropsy lesions were observed in either principals or controls at any time during the study. Histopathologic examination revealed multifocal and confluent non-suppurative inflammatory lesions in heart of cotton rats, C57BL/6J-Lystbg-J/J mice and, less consistently, in outbred mice as early as 4 weeks post-exposure. Lesions initially consisted of loose aggregates of lymphocytes and monocytes; they later became larger, more compactly cellular and predominantly monocytic. Myofiber degeneration, atrophy, and necrosis existed in areas of inflammation (Fig. 1). Similar but fewer lesions were found in renal cortex, skeletal muscle, and lung of cotton rats; such lesions were present less frequently in C57BL/6J-Lystbg-J/J mice and rarely in outbred mouse principals. Lesions suggestive of Hepatozoon infection were not observed in any rodent controls or in Sprague–Dawley rat principals. Histologic evidence of merogony was not observed in any subjects. Immunohistochemical positive, comma-shaped parasites (cystozoites) were within cytoplasmic vacuoles of rodent host cells located proximate to or within inflammatory foci detected in heart, renal cortex, skeletal muscle, and lung. Parasites were approximately
2 12 mm with a central or paracentral, denselystained nucleus (Fig. 2). Single parasites were embedded in lightly basophilic material (Fig. 3) composed of a dense aggregate of globules that stained positively in PAS-diastase treated sections. This globular material was more voluminous in infections of longer duration. The monozoic cysts containing cystozoites were morphologically similar to those described by Desser (1990) in H. griseisciuri-infected grey squirrels. Monozoic cysts were not observed in any rodent controls or in Sprague–Dawley rat principals. The 18S rRNA Hepatozoon gene was detected by PCR amplification and analysis of tissues from the cotton rats, outbred mouse and C57BL/6J-Lystbg-J/J
Fig. 1. Inflammatory focus in myocardium of cotton rat 16 weeks after experimental exposure to sporulated oocysts of H. americanum. There is atrophy, degeneration, and necrosis of myofibers within a focus of moncytic inflammatory infiltrate. HE, Bar = 50 mm.
Fig. 3. Myocardium of cotton rat. Crescentic, encysted zoite within rodent macrophage whose nucleus is displaced and compressed to margin of the host cell and whose cytoplasmic volume is expanded by globular mucopolysaccharide material. HE, Bar = 15 mm.
Fig. 2. Cystozoite in minimally expanded host cell from cotton rat myocardium 4 weeks post-exposure to sporulated oocysts of H. americanum. Zoite (arrow) is banana-shaped with densely basophilic nucleus. Host cell cytoplasm is vaguely perceptible. HE, Bar = 10 mm.
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mice principals necropsied at week-16 post-exposure. The sequence of the 18S rDNA fragment from the cotton rat tissues containing cystozoites (EU249993) was identical to the sequence of the fragment amplified from the dog experimentally infected with H. americanum (EU249992) on which the ticks fed. The sequence from cotton rat tissues containing cystozoites was 99.8% or 99.6% identical to H. americanum sequences previously reported from naturally-infected dogs (AY864676 and AF176836, respectively), although the two previously reported sequences and the sequence reported here did differ from one another by at least one base in the variable region considered. 4. Discussion Exposure of cotton rats, outbred mice, and C57BL/ 6J-Lystbg-J/J mice to oocysts of H. americanum resulted in successful transmission of infection characterized by development of cystozoites in heart, kidney, skeletal muscle and lung; Sprague–Dawley rats were resistant. A previously reported attempt to transmit a North American isolate of Hepatozoon sp. to rodents was unsuccessful (Nordgren and Craig, 1984); in that study mice were fed skeletal muscle of dogs infected with H. americanum (Texas strain of H. canis). Cystozoites have been demonstrated in the tissues of facultative intermediate (paratenic) hosts in the life cycles of some apicomplexan parasites of predators such as dogs, cats and birds of prey (Gardiner et al., 1988). Latent dizoic cystic forms have been demonstrated in the life cycles of Hepatozoon species that infect snakes, lizards, and anurans (Smith et al., 1994, 1996; Stehbens and Johnston, 1968; Wozniak et al., 1996; Kim et al., 1998; Lainson et al., 2003; Paperna and Lainson, 2004). In such cycles, transmission of Hepatozoon spp. to an obligate vertebrate intermediate host can occur not only through ingestion of haematophagous arthropod definitive hosts containing sporulated oocysts, but also through ingestion of cystozoites in an obligate or facultative host (paratenic), or through both methods (Smith, 1996). Mammalian intermediate hosts have been found to harbor cystozoites in their tissues as well (Baneth and Shkap, 2003; Desser, 1990; Laakkonen et al., 2001; Uni et al., 2003). Desser (1990) found cystic forms in grey squirrels infected with H. griseisciuri; he suggested that cystozoites could be an important mode of transmission for Hepatozoon spp. in mammals. However, attempts to transmit Hepatozoon spp. of mammals by oral exposure to cystozoite stages have not been reported. In spite of extensive histologic examination of many canids
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infected with H. americanum, cystozoites have not been observed in their tissues (Panciera et al., 1999; Kocan et al., 1999; Panciera et al., 2001; Cummings et al., 2005; Garrett et al., 2005). The recognition of H. americanum cystozoites in experimentally infected rodents in the present study is unique, and lends support to Desser’s hypothesis concerning possible transmission of Hepatozoon spp. among mammals by cystozoites. Smith (1996) proposed that the Hepatozoon group of haemogregarines has evolved alternate modes of transmission that exploit the predator-prey relationships of the vertebrate intermediate hosts of these parasites. Development of cystozoites in rodents experimentally challenged with sporulated H. americanum oocysts suggests that paratenic hosts may be involved in the natural transmission cycle of this parasite, a finding that may be of use in better understanding the epidemiology of ACH. Frequently, in parasite life cycles that involve arthropod intermediate host(s), vertebrate paratenic host(s), and definitive vertebrate host(s), the paratenic host is prey for the definitive host. In many such life cycles, the paratenic host is more likely to feed on the arthropod intermediate host than is the definitive host. This host-parasite interaction is well exemplified by the life cycle of Halipegus occidualis, a trematode of the green frog, Rana clamitans. The dragonfly nymph is a required paratenic host in the trematode life cycle because of the feeding gap between the microscopic ostracod second intermediate host and the frog definitive host (Esch, 2004). Similar host feeding habits come into play in life cycles of Hepatozoon spp. In the life cycle of H. sipedon, the culicine mosquito definitive hosts are a natural food source for the first intermediate host, the northern leopard frog, but are not ordinarily fed upon by the second intermediate host, the northern water snake. The frog is a natural food source for the snake and, when harboring cystozoites, is the source of infection for the snake (Smith et al., 1994). Mosquitoes that contain sporulated oocysts are not infectious for the snake. These types of relationships between hosts and parasites that evolved over time enhance chances of survival of the parasite. Cotton rats were chosen as experimental subjects in this study because they are known to be preferred hosts for the larvae and nymphs of A. maculatum, they are abundant in ACH-endemic sites in Oklahoma, and they are easy prey for dogs and coyotes throughout the known geographic distribution of ACH. However, transmission of ACH to dogs with cystozoites contained in experimentally infected rodents and detection of
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cystozoites in wild-trapped rodents with subsequent experimental transmission to dogs are necessary to confirm the role of these rodents as paratenic hosts for H. americanum. In an effort to find a laboratory animal model for ACH, laboratory rats and mice strains were incorporated into the study because of their susceptibility to infection with known Hepatozoon spp., including the type species for the genus, H. muris. Usefulness of the selected rodents as laboratory animal models for ACH is limited because neither merogony nor gamontogony was observed during the 16-week observation period. However, laboratory rodents harboring cystozoites of H. americanum may offer a method for maintenance of the organism which could be a useful and efficient alternative to current methods involving long-term maintenance of infected dogs and experimental infection of ticks. The findings of the present study emphasize the need for further investigations into the potential role that paratenic host(s) may play in the epizootology of ACH. To understand the epidemiology of ACH, investigations directed toward identification of natural reservoirs of H. americanum are critical. Vertebrates, such as birds, present in the geographic range of ACH that feed on nymphal and adult A. maculatum or that may be predators of the preferred hosts of this tick would be good subjects for investigation. It is apparent from findings in this study that molecular analysis of gamonts and cystozoites found in vertebrate hosts is essential for accurate speciation and delineation of hepatozoa. 5. Conclusion Cystozoite stages of H. americanum developed in three of four rodent species/strains exposed to sporulated oocysts; merogony and gamontogony were not observed. This is not only the first report of the existence of a cystozoite stage of H. americanum but also the first report of infection of vertebrate hosts other than carnivores with H. americanum. Although a role for rodents in the epizootiology of American canine hepatozoonosis has not been definitively established, findings of the present study indicate that rodents could serve as paratenic hosts of H. americanum and be a potential source of infection for canids. Future epidemiologic investigations should include this alternative mode of transmission as a means for spread of ACH, which previously was believed to be acquired exclusively by ingestion of infected A. maculatum.
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