Introduction. Syst Parasitol (2014) 89:83 89 DOI /s

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1 Syst Parasitol (2014) 89:83 89 DOI /s Coccidial dispersion across New World marsupials: Klossiella tejerai Scorza, Torrealba & Dagert, 1957 (Apicomplexa: Adeleorina) from the Brazilian common opossum Didelphis aurita (Wied-Neuwied) (Mammalia: Didelphimorphia) Caroline Spitz dos Santos Bruno Pereira Berto Bruno do Bomfim Lopes Matheus Dias Cordeiro Adivaldo Henrique da Fonseca Walter Leira Teixeira Filho Carlos Wilson Gomes Lopes Received: 11 June 2014 / Accepted: 14 July 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Klossiella tejerai Scorza, Torrealba & Dagert, 1957 is a primitive coccidian parasite reported from the New World marsupials Didelphis marsupialis (Linnaeus) and Marmosa demerarae (Thomas). The current work describes K. tejerai from the Brazilian common opossum Didelphis aurita (Wied-Neuwied) in Southeastern Brazil, evidencing the coccidial dispersion across opossums of the same family. The sporocysts C. S. dos Santos M. D. Cordeiro Curso de Pós-Graduação em Ciências Veterinárias, Universidade Federal Rural do Rio de Janeiro (UFRRJ), BR-465 km 7, Seropédica, RJ, Brazil B. P. Berto (&) Departamento de Biologia Animal, Instituto de Biologia, UFRRJ, BR-465 km 7, Seropédica, RJ, Brazil bertobp@ufrrj.br B. do Bomfim Lopes Programa de Pós-graduação em Ciência, Tecnologia e Inovação em Agropecuária, UFRRJ, BR-465 km 7, Seropédica, RJ, Brazil A. H. da Fonseca Departamento de Epidemiologia e Saúde Pública, Instituto de Veterinária, UFRRJ, BR-465 km 7, Seropédica, RJ, Brazil W. L. T. Filho C. W. G. Lopes Departamento de Parasitologia Animal, Instituto de Veterinária, UFRRJ, BR-465 km 7, Seropédica, RJ, Brazil recovered from urine samples were ellipsoidal, lm, with sporocyst residuum composed of scattered spherules and c.13 sporozoites per sporocyst, with refractile bodies and nucleus. Macrogametes, microgametes, sporonts, sporoblasts/sporocysts were identified within parasitophorous vacuoles of epithelial cells located near the renal corticomedullary junction. Didelphis marsupialis should not have transmitted K. tejerai to D. aurita because they are not sympatric; however M. demerarae is sympatric with D. marsupialis and D. aurita. Therefore, D. aurita becomes the third host species for K. tejerai in South America. Introduction Opossums in the New World represent 99 different species. The vast majority of these (95 species, 96%) inhabits South America. Didelphis spp. are common in South America; however, one of the six species, Didelphis virginiana (Kerr) has distribution in North and Central Americas (IUCN, 2014). Didelphis spp. became epidemiologically relevant in the New World when they were identified as definitive hosts for some coccidian parasites of the genus Sarcocystis Lankester, Among these Sarcocystis spp., Sarcocystis neurona Dubey, Davis, Speer, Bowman, Lahunta, Granstrom, Topper, Hamir, Cummings & Suter, 1991 is recognised as the

2 84 Syst Parasitol (2014) 89:83 89 et al., 1976) and from the woolly mouse opossum Marmosa demerarae (Thomas) in Guyana (Boulard, 1975). The present study describes K. tejerai infecting a Brazilian common opossum Didelphis aurita (Wied- Neuwied) in Southeastern Brazil, evidencing the coccidial dispersion across opossums of the same family. Materials and methods Fig. 1 Urinary sporocysts of Klossiella tejerai from the Brazilian common opossum Didelphis aurita. A, Composite line drawing; B C, Photomicrographs. Scale-bars: 10 lm ethiological agent of equine protozoal myeloencephalitis (Dubey & Lindsay, 1998; Monteiro et al., 2013). Besides Sarcocystis spp., other coccidia infect Didelphis spp., including Eimeria spp. (Teixeira et al., 2007) and Klossiella tejerai Scorza, Torrealba & Dagert, 1957 (see Scorza et al., 1957). Klossiella spp. have been reported from various marsupials, primarily from Australian peramelids, petaurids and macropodids (Barker et al., 1975, 1985; Bennett et al., 2007). However, in the New World, only K. tejerai was described from Didelphis marsupialis (Linnaeus) in Venezuela (Scorza et al., 1957), and subsequently reported from this host species in Panama (Edgcomb Twenty opossums D. aurita were captured on and around the Campus of the Federal Rural University of Rio de Janeiro (Universidade Federal Rural do Rio de Janeiro UFRRJ), located in the municipality of Seropédica ( S, W), state of Rio de Janeiro, Brazil. The opossums were transported to the Veterinary Institute (Instituto de Veterinária IV) at the UFRRJ, and were reared and fed in small enclosures approximately m. Feed and water were administered ad libitum. The capture, maintenance and collection of samples was approved by UFRRJ Ethics Committee under protocol No. 255/2012 and authorised by Brazilian Institute of Environment and Natural Renewable Resources (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis IBAMA) under protocol # Sample processing and data analysis were conducted at the Laboratory of Coccidia and Coccidiosis (Laboratório de Coccídios e Coccidioses LCC) located at UFRRJ. Urine samples were collected and placed in plastic vials. Sporocysts of Klossiella spp. were recovered by centrifugal sedimentation and examined microscopically using the technique described by Duszynski & Wilber (1997). The opossums positive for sporocysts of Klossiella spp. in urine were necropsied. Kidneys were examined grossly and representative samples of kidney tissue were collected into 10% neutral buffered formalin. Once fixed, these tissues were embedded in paraffin, sectioned at 4 lm, and stained routinely with hematoxylin and eosin. Morphological observations, line drawing and photomicrographs were made using an Olympus BX binocular microscope coupled to a digital camera Eurocam 5.0. All measurements are in micrometres and are presented as the range followed by the mean.

3 Syst Parasitol (2014) 89: Fig. 2 Photomicrographs of life-cycle stages of Klossiella tejerai in renal tissue from the Brazilian common opossum Didelphis aurita. A B, Macrogametes contained within parasitophorous vacuoles; C, Macrogamete and microgamete in syzygy within parasitophorous vacuole; D E, Early sporonts within parasitophorous vacuoles; F G, Late budding sporonts within parasitophorous vacuoles; H, Oöcyst with free mature sporoblasts/sporocysts; I, Macrogamete (right) and early sporont (middle) within parasitophorous vacuoles and oöcyst with free mature sporoblasts/sporocysts (left). Scale-bars: 10 lm Results Twenty Brazilian common opossums were examined; one of them (5%) shed Klossiella-like sporocysts in the urine. The current description follows the guidelines of Duszynski & Wilber (1997) and Berto et al. (2014a) for the urinary sporocysts and the examples of Scorza et al. (1957), Barker et al. (1975; 1985), Gardiner et al. (1998) and Bennett et al. (2007) for the nomenclature of tissue stages of Klossiella. Klossiella tejerai Scorza, Torrealba & Dagert, 1957 Host: Didelphis aurita Wied-Neuwied (Mammalia: Didelphimorphia: Didelphidae). Locality: Brazil, State of Rio de Janeiro, Municipality of Seropédica ( S, W). Material studied: One-half of the sporocysts from urine samples are kept in 10% aqueous buffered formalin (v/v) and the other half in 70% ethanol for future molecular studies, according Duszynski & Gardner (1991). Both samples and the renal tissue slides were deposited in the Parasitology Collection of the Laboratório de Coccídios e Coccidioses, at UFRRJ, located at the Municipality of Seropedica in the State of Rio Janeiro, Brazil. Photovouchers and line drawings are deposited and available ( ufrrj.br/lcc) as well. Photographs of the host specimen are deposited in the same collection. The repository number is 53/2014.

4 86 Syst Parasitol (2014) 89:83 89 Table 1 Comparative morphology of Klossiella tejerai recovered from New World opossums Host Didelphis aurita (Wied- Didelphis marsupialis Marmosa demerarae D. marsupialis (L.) Neuwied) (L.) (Thomas) Reference Present study Scorza et al. (1957) Boulard (1975) Edgcomb et al. (1976) Macrogamete Shape Subspherical to ovoidal Subspherical to ovoidal Size ( ) (8 9 6) (12) 4 14 (9) Parasitophorous vacuole size Microgamete ( ) Shape Subspheroidal to ovoidal Ovoidal Size ( ) (6 9 2) (9 9 6) Sporont Shape Subspheroidal to irregular Size up to (28) ( ) Number of nuclei 8 13 (10) Sporoblast/Sporocyst Shape Ellipsoidal Size ( ) (12 9 9) ( ) (16) Number per oöcyst (18) (18) Oöcyst Shape Irregular Size ( ) ( ) Urinary sporocysts/ sporozoites Shape Ellipsoidal Size ( ) Length/width ratio (1.6) Sporocyst residuum Granular and diffuse Present Number of sporozoites (13) (12) Refractile body 2, refringent, in both ends Nucleus Refringent and central Site of infection: Epithelium of the renal tubules. Description (Figs. 1, 2) Exogenous stages Sporocysts ellipsoidal, ( ); length/width (L/W) ratio (1.6) (Fig. 1). Sporocyst residuum present, composed of scattered spherules. Sporozoites (13), with anterior and posterior refractile bodies and central nucleus. Endogenous stages Endogenous stages in parasitophorous vacuoles within renal epithelial cells located near the corticomedullary junction. Macrogametes subspheroidal to ovoidal, ( ), with basophilic nucleus and contained within a subspheroidal to irregular parasitophorous vacuole, ( ) (Fig. 2A B, I). Macrogamete in syzygy with microgametes in some cases (Fig. 2C). Microgametes subspheroidal to ovoidal, ( ), with basophilic

5 Syst Parasitol (2014) 89: Fig. 3 Geographic ranges of some New World opossums, according IUCN (2014). A, Hosts for Klossiella tejerai are the didelphid opossums Didelphis marsupialis, Marmosa demerarae and Didelphis aurita. Didelphis marsupialis is not sympatric with D. aurita; however, M. demerarae is sympatric with D. marsupialis and D. aurita; B, Didelphis albiventris is another didelphid opossum with wide geographic range sympatric with D. marsupialis and D. aurita which could disperse K. tejerai for Didelphis spp. and other New World opossums nucleus. Sporonts subspheroidal to irregular, ( ), dotted circumferentially with 8 13 (10) basophilic nuclei (Fig. 2D E, I). Immature sporoblasts ellipsoidal, ( ) (Fig. 2F G). Mature sporoblasts/sporocysts (18), ellipsoidal, ( ), with multiple dotted basophilic nuclei (Fig. 2H). Oöcysts irregular, ( ) (Fig. 2H I). Discussion The description of K. tejerai of the current work confers with the original description of Scorza et al. (1957) in all characteristic features which were compared. The descriptions of Edgcomb et al. (1976) and Boulard (1975) had some divergences such as the measures of the microgamete and sporoblast/sporocyst and the number of sporozoites per sporocyst (Table 1). Additionally, Edgcomb et al. (1976) and Boulard (1975) observed merogonic stages (schizonts). However, in the current work and in the original description of Scorza et al. (1957) merogonic stages in the renal histology were not observed. The photomicrographs of Edgcomb et al. (1976) are confusing and may have been misinterpreted. In contrast, the meronts and merozoites observed by Boulard (1975) are evident. Scorza et al. (1957) suggested that the merogonic stage should occur in other organs of the host, such as the lungs, spleen, pancreas, testicles, etc. Thus, further studies are needed to detail the merogonic stage of K. tejerai and/or confirm that the specimens observed by Boulard (1975) and Edgcomb et al. (1976) are K. tejerai or another species. In general, coccidiosis is an important disease that affects the health, physiology and behavior of the hosts. The immunity against coccidia develops depending on the number of oöcysts/sporocysts ingested; however, generally this immunity does not prevent re-infection. In adult animals balance is maintained between the constant re-infection and the degree of immunity (Hunsaker, 1977; Aguilar et al., 2008). In this sense, coccidiosis in wildlife in a habitat without environmental impacts is rarely a significant

6 88 Syst Parasitol (2014) 89:83 89 problem; on the other hand, epizootics should occur when environmental disturbances and/or anthropogenic factors contribute to change the behavior and/or mainly stressing the wild animals, leading to immunodepression. In this context, coccidia and coccidiosis in wildlife, such as K. tejerai in D. aurita, assume role of biomarkers of anthropization and/or environmental disturbance (Giraudeau et al., 2014). In the current work, the low prevalence, besides of the positive opossum to be apparently healthy, demonstrate that the location of capture, although anthropized, is favorable to the demands of its ecological niche. On another point of view, the generalist habit of the opossums may have allowed its adaptation in an anthropized environment. In another aspect, the coccidia are biomarkers of transmission and, therefore, dispersion (Berto et al., 2014b). Klossiella tejerai have been reported from only two species of New World opossums. The route of infection of this coccidian species is urine-oral; therefore, the coccidial transmission should be usual among sympatric opossums which have close ecological niches favouring rapid transmission, since the sporocysts are not resistant to desiccation, solar radiation, and other environmental factors, due to thin sporocyst wall. Figure 3 shows the geographic ranges of these opossums to assist in understanding the dynamics of dispersal of K. tejerai in the New World. The direct transmission of K. tejerai from D. marsupialis to D. aurita is unlikely, because they are not sympatric. In contrast, M. demerarae is sympatric with both D. marsupialis and D. aurita (Fig. 3A); therefore, M. demerarae may potentially transmit K. tejerai for these two hosts, and other sympatric susceptible hosts. In this sense, Didelphis albiventris (Lund) may be a potential susceptible host, which has wide geographic range in South America and could disperse K. tejerai for Didelphis spp. and other New World opossums (Fig. 3B). Finally, the current work is based on the concept of intra-family specificity proposed by Duszynski & Wilber (1997) which allows new hosts of the same family. Therefore, considering Didelphidae as hostfamily for K. tejerai with only two host species, D. aurita becomes the third host species. Acknowledgements This study was supported by grants from the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) to B. P. Berto (E-26/ /2013). References Aguilar, T. M., Maia, R., Santos, E. S., & Macedo, R. H. (2008). Parasite levels in blue-black grassquits correlate with male displays but not female mate preference. Behavioral Ecology, 19, Barker, I. K., Munday, B. L., & Harrigan, K. E. (1975). Klossiella spp. in the kidneys of peramelid, petaurid, and macropodid marsupials. Zeitschrift für Parasitenkunde, 46, Barker, I. K., Munday, B. L., & Hartley, W. J. (1985). Klossiella (Apicomplexa, Klossiellidae) in Petaurid and Macropodid Marsupials in Australia. Journal of Protozoology, 32, Bennett, M. D., Woolford, L., O Hara, A. J., Nicholls, P. K., Warren, K. S., Friend, J. A., & Swan, R. A.. (2007). Klossiella quimrensis (Apicomplexa: Klossiellidae) causes renal coccidiosis in western barred bandicoots Perameles bougainville (Marsupialia: Peramelidae) in Western Australia. Journal of Parasitology, 93, Berto, B. P., McIntosh, D., & Lopes, C. W. G. (2014a). Studies on coccidian oocysts (Apicomplexa: Eucoccidiorida). Revista Brasileira de Parasitologia Veterinária, 23, Berto, B. P., Lopes, B. dob., Melinski, R. D., Souza, A., Ribas, C., Abreu, F., Ferreira, I., & Lopes, C. W. G. (2014b). Coccidial dispersion across trans- and cis-andean antbirds (Passeriformes: Thamnophilidae): Isospora sagittulae McQuistion and Capparella, 1992 (Apicomplexa: Eimeriidae) from non-sympatric hosts. Canadian Journal of Zoology, 92, Boulard, Y. (1975). Etude morphologique des coccidies (Adeleidae) Klossiella killicki n. sp. chez des microchiropterae Africans et Klossiella tejerai Scorza, 1957, chez un marsupial sud-americain. Bulletin of the Museum of Natural History, 284, Dubey, J. P., & Lindsay, D. S. (1998). Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula. International Journal for Parasitology, 28, Duszynski, D. W., & Gardner, S. L. (1991). Fixing coccidian oocysts is not an adequate solution to the problem of preserving protozoan type material. Journal of Parasitology, 77, Duszynski, D. W., & Wilber, P. G. (1997). A guideline for the preparation of species descriptions in the Eimeridae. Journal of Parasitology, 83, Edgcomb, J. H., Walker, D. H., & Johnson, C. M. (1976). Klossiella in the opossum. Veterinary Pathology, 13, Gardiner, C. H., Fayer, R., & Dubey, J. P. (1998). An atlas of protozoan parasites in animal tissues. Washington: Armed Forces Institute of Pathology, 84 pp. Giraudeau, M., Mousel, M., Earl, S., & McGraw, K. (2014). Parasites in the city: degree of urbanization predicts poxvirus and coccidian infections in house finches (Haemorhous mexicanus). PLoS One, 9, e Hunsaker, D. (1977). The Biology of Marsupials. New York: Academic Press, 537 pp.

7 Syst Parasitol (2014) 89: IUCN. (2014). International Union for Conservation of Nature and Natural Resources. Cited 18 May, Monteiro, R. M., Keid, L. B., Richtzenhain, L. J., Valadas, S. Y., Muller, G., & Soares, R. M. (2013). Extensively variable surface antigens of Sarcocystis spp. infecting Brazilian marsupials in the genus Didelphis occur in myriad allelic combinations, suggesting sexual recombination has aided their diversification. Veterinary Parasitology, 196, Scorza, J. V., Torrealba, J. F., & Dagert, C. (1957). Klossiella tejerai nov. sp. y Sarcocystis didelphidis nov. sp. parasitos de un Didelphis marsupialis de Venezuela. Acta Biologica Venezuelica, 2, Teixeira, M., Rauta, P. D., Albuquerque, G. R., & Lopes, C. W. G. (2007). Eimeria auritanensis n. sp. and E. gambai Carini, 1938 (Apicomplexa: Eimeriidae) from the opossum Didelphis aurita Wied-Newied, 1826 (Marsupialia: Didelphidae) from southeastern Brazil. Revista Brasileira de Parasitologia Veterinária, 16,