Syst Parasitol (2014) 89:83–89 DOI 10.1007/s11230-014-9510-7

Coccidial dispersion across New World marsupials: tejerai Scorza, Torrealba & Dagert, 1957 (: ) 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 & recovered from urine samples were ellipsoidal, Dagert, 1957 is a primitive coccidian parasite reported 20.4 9 12.7 lm, with sporocyst residuum composed from the New World marsupials Didelphis marsupialis of scattered spherules and c.13 sporozoites per sporo- (Linnaeus) and Marmosa demerarae (Thomas). The cyst, with refractile bodies and nucleus. Macrogametes, current work describes K. tejerai from the Brazilian microgametes, sporonts, sporoblasts/sporocysts were common opossum Didelphis aurita (Wied-Neuwied) in identified within parasitophorous vacuoles of epithelial Southeastern Brazil, evidencing the coccidial dispersion cells located near the renal corticomedullary junction. across opossums of the same family. The sporocysts Didelphis marsupialis should not have transmitted K. tejerai to D. aurita because they are not sympatric; however M. demerarae is sympatric with D. marsupialis C. S. dos Santos M. D. Cordeiro and D. aurita. Therefore, D. aurita becomes the third Curso de Po´s-Graduac¸a˜o em Cieˆncias Veterina´rias, host species for K. tejerai in South America. Universidade Federal Rural do Rio de Janeiro (UFRRJ), BR-465 km 7, 23897-970 Serope´dica, RJ, Brazil

& B. P. Berto ( ) Introduction Departamento de Biologia Animal, Instituto de Biologia, UFRRJ, BR-465 km 7, 23897-970 Serope´dica, RJ, Brazil e-mail: [email protected] Opossums in the New World represent 99 different species. The vast majority of these (95 species, 96%) B. do Bomfim Lopes inhabits South America. Didelphis spp. are common in Programa de Po´s-graduac¸a˜o em Cieˆncia, Tecnologia e Inovac¸a˜o em Agropecua´ria, UFRRJ, BR-465 km 7, South America; however, one of the six species, 23897-970 Serope´dica, RJ, Brazil Didelphis virginiana (Kerr) has distribution in North and Central Americas (IUCN, 2014). A. H. da Fonseca Didelphis spp. became epidemiologically relevant Departamento de Epidemiologia e Sau´de Pu´blica, Instituto de Veterina´ria, UFRRJ, BR-465 km 7, in the New World when they were identified as 23897-970 Serope´dica, RJ, Brazil definitive hosts for some coccidian parasites of the genus Lankester, 1882. Among these W. L. T. Filho C. W. G. Lopes Sarcocystis spp., Sarcocystis neurona Dubey, Davis, Departamento de Parasitologia Animal, Instituto de Veterina´ria, UFRRJ, BR-465 km 7, Speer, Bowman, Lahunta, Granstrom, Topper, Hamir, 23897-970 Serope´dica, RJ, Brazil Cummings & Suter, 1991 is recognised as the 123 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

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 Serope´dica (22°440S, 43°420W), state of Rio de Janeiro, Brazil. The opossums were transported to the Veterinary Institute (Instituto de Veterina´ria – IV) at the UFRRJ, and were reared and fed in small enclo- sures approximately 1 9 1 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 author- ised by Brazilian Institute of Environment and Natural Renewable Resources (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renova´veis – IBAMA) under protocol # 34701-2. Sample pro- cessing and data analysis were conducted at the Laboratory of and Coccidiosis (Laborato´rio Fig. 1 Urinary sporocysts of Klossiella tejerai from the de Coccı´dios e Coccidioses – LCC) located at UFRRJ. Brazilian common opossum Didelphis aurita. A, Composite Urine samples were collected and placed in plastic line drawing; B–C, Photomicrographs. Scale-bars:10lm vials. Sporocysts of Klossiella spp. were recovered by centrifugal sedimentation and examined microscopi- ethiological agent of equine protozoal myeloenceph- cally using the technique described by Duszynski & alitis (Dubey & Lindsay, 1998; Monteiro et al., 2013). Wilber (1997). The opossums positive for sporocysts Besides Sarcocystis spp., other coccidia infect of Klossiella spp. in urine were necropsied. Kidneys Didelphis spp., including spp. (Teixeira were examined grossly and representative samples of et al., 2007) and Klossiella tejerai Scorza, Torrealba kidney tissue were collected into 10% neutral buffered & Dagert, 1957 (see Scorza et al., 1957). Klossiella formalin. Once fixed, these tissues were embedded in spp. have been reported from various marsupials, paraffin, sectioned at 4 lm, and stained routinely with primarily from Australian peramelids, petaurids and hematoxylin and eosin. Morphological observations, macropodids (Barker et al., 1975, 1985; Bennett et al., line drawing and photomicrographs were made using 2007). However, in the New World, only K. tejerai an Olympus BX binocular microscope coupled to a was described from Didelphis marsupialis (Linnaeus) digital camera Eurocam 5.0. All measurements are in in Venezuela (Scorza et al., 1957), and subsequently micrometres and are presented as the range followed reported from this host species in Panama (Edgcomb by the mean.

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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, Oo¨cyst with free mature sporoblasts/sporocysts; I, Macrogamete (right) and early sporont (middle) within parasitophorous vacuoles and oo¨cyst with free mature sporoblasts/sporocysts (left). Scale-bars:10lm

Results Locality: Brazil, State of Rio de Janeiro, Municipality of Serope´dica (22°440S, 43°420W). Twenty Brazilian common opossums were examined; Material studied: One-half of the sporocysts from one of them (5%) shed Klossiella-like sporocysts in urine samples are kept in 10% aqueous buffered the urine. The current description follows the guide- formalin (v/v) and the other half in 70% ethanol for lines of Duszynski & Wilber (1997) and Berto et al. future molecular studies, according Duszynski & (2014a) for the urinary sporocysts and the examples of Gardner (1991). Both samples and the renal tissue Scorza et al. (1957), Barker et al. (1975; 1985), slides were deposited in the Parasitology Collection of Gardiner et al. (1998) and Bennett et al. (2007) for the the Laborato´rio de Coccı´dios e Coccidioses, at nomenclature of tissue stages of Klossiella. UFRRJ, located at the Municipality of Seropedica in the State of Rio Janeiro, Brazil. Photovouchers and Klossiella tejerai Scorza, Torrealba & Dagert, 1957 line drawings are deposited and available (http://r1. ufrrj.br/lcc) as well. Photographs of the host specimen Host: Didelphis aurita Wied-Neuwied (Mammalia: are deposited in the same collection. The repository Didelphimorphia: Didelphidae). number is 53/2014. 123 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 7–11 9 5–9 (8.6 9 7.2) (8 9 6) (12) 4–14 (9) Parasitophorous vacuole 14–29 9 14–22 –– – size (22.6 9 18.3) Microgamete Shape Subspheroidal to ovoidal – Ovoidal – Size 5–6 9 3–5 (5.3 9 3.8) (6 9 2) (9 9 6) – Sporont Shape Subspheroidal to irregular – – – Size 15–31 9 14–21 up to 27 – 25–39 (28) (21.3 9 16.4) Number of nuclei 8–13 (10) – – – Sporoblast/Sporocyst Shape Ellipsoidal – – – Size 10–13 9 6–9 (11.4 9 6.8) (12 9 9) (13.7 9 9) 14–17 (16) Number per oo¨cyst 12–30 (18) (18) 16–22 – Oo¨cyst Shape Irregular – – – Size 57–103 9 36–57 – (80 9 40) – (71.6 9 47.2) Urinary sporocysts/ sporozoites Shape Ellipsoidal – – – Size 19–22 9 12–14 –– – (20.4 9 12.7) Length/width ratio 1.5–1.8 (1.6) – – – Sporocyst residuum Granular and diffuse – Present – Number of sporozoites 12–14 (13) (12) 14–22 – Refractile body 2, refringent, in both ends – – – Nucleus Refringent and central – – –

Site of infection: Epithelium of the renal tubules. Endogenous stages Endogenous stages in parasitophorous vacuoles within Description (Figs. 1, 2) renal epithelial cells located near the corticomedullary junction. Macrogametes subspheroidal to ovoidal, Exogenous stages 7–11 9 5–9 (8.6 9 7.2), with basophilic nucleus and Sporocysts ellipsoidal, 19–22 9 12–14 (20.4 9 12.7); contained within a subspheroidal to irregular parasitoph- length/width (L/W) ratio 1.5–1.8 (1.6) (Fig. 1). Spo- orous vacuole, 14–29 9 14–22 (22.6 9 18.3) (Fig. 2A– rocyst residuum present, composed of scattered B, I). Macrogamete in syzygy with microgametes in spherules. Sporozoites 12–14 (13), with anterior and some cases (Fig. 2C). Microgametes subspheroidal to posterior refractile bodies and central nucleus. ovoidal, 5–6 9 3–5 (5.3 9 3.8), with basophilic

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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, 15–31 9 of Scorza et al. (1957) merogonic stages in the renal 14–21 (21.3 9 16.4), dotted circumferentially with 8–13 histology were not observed. The photomicrographs (10) basophilic nuclei (Fig. 2D–E, I). Immature sporo- of Edgcomb et al. (1976) are confusing and may have blasts ellipsoidal, 5–6 9 3–4 (5.4 9 3.5) (Fig. 2F–G). been misinterpreted. In contrast, the meronts and Mature sporoblasts/sporocysts 12–30 (18), ellipsoidal, merozoites observed by Boulard (1975) are evident. 10–13 9 6–9 (11.4 9 6.8), with multiple dotted baso- Scorza et al. (1957) suggested that the merogonic philic nuclei (Fig. 2H). Oo¨cysts irregular, 57–103 9 stage should occur in other organs of the host, such as 36–57 (71.6 9 47.2) (Fig. 2H–I). 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 Discussion Boulard (1975) and Edgcomb et al. (1976) are K. tejerai or another species. The description of K. tejerai of the current work confers In general, coccidiosis is an important disease that with the original description of Scorza et al. (1957)in affects the health, physiology and behavior of the all characteristic features which were compared. The hosts. The immunity against coccidia develops descriptions of Edgcomb et al. (1976) and Boulard depending on the number of oo¨cysts/sporocysts (1975) had some divergences such as the measures of ingested; however, generally this immunity does not the microgamete and sporoblast/sporocyst and the prevent re-infection. In adult animals balance is number of sporozoites per sporocyst (Table 1). maintained between the constant re-infection and the Additionally, Edgcomb et al. (1976) and Boulard degree of immunity (Hunsaker, 1977; Aguilar et al., (1975) observed merogonic stages (schizonts). How- 2008). In this sense, coccidiosis in wildlife in a habitat ever, in the current work and in the original description without environmental impacts is rarely a significant

123 88 Syst Parasitol (2014) 89:83–89 problem; on the other hand, epizootics should occur References when environmental disturbances and/or anthropo- genic factors contribute to change the behavior and/or Aguilar, T. M., Maia, R., Santos, E. S., & Macedo, R. H. (2008). mainly stressing the wild animals, leading to immun- Parasite levels in blue-black grassquits correlate with male displays but not female mate preference. Behavioral odepression. In this context, coccidia and coccidiosis Ecology, 19, 292–301. in wildlife, such as K. tejerai in D. aurita, assume role Barker, I. K., Munday, B. L., & Harrigan, K. E. (1975). Klos- of biomarkers of anthropization and/or environmental siella spp. in the kidneys of peramelid, petaurid, and disturbance (Giraudeau et al., 2014). In the current macropodid marsupials. Zeitschrift fu¨r Parasitenkunde, 46, 35–41. work, the low prevalence, besides of the positive Barker, I. K., Munday, B. L., & Hartley, W. J. (1985). Klossiella opossum to be apparently healthy, demonstrate that the (Apicomplexa, Klossiellidae) in Petaurid and Macropodid location of capture, although anthropized, is favorable Marsupials in Australia. Journal of Protozoology, 32, to the demands of its ecological niche. On another point 520–522. Bennett, M. D., Woolford, L., O’Hara, A. J., Nicholls, P. K., of view, the generalist habit of the opossums may have Warren, K. S., Friend, J. A., & Swan, R. A.. (2007). allowed its adaptation in an anthropized environment. Klossiella quimrensis (Apicomplexa: Klossiellidae) causes In another aspect, the coccidia are biomarkers of renal coccidiosis in western barred bandicoots Perameles bougainville transmission and, therefore, dispersion (Berto et al., (Marsupialia: Peramelidae) in Western Aus- tralia. Journal of Parasitology, 93, 89–92. 2014b). Klossiella tejerai have been reported from only Berto, B. P., McIntosh, D., & Lopes, C. W. G. (2014a). Studies two species of New World opossums. The route of on coccidian oocysts (Apicomplexa: ). Re- infection of this coccidian species is urine-oral; therefore, vista Brasileira de Parasitologia Veterina´ria, 23, 1–15. the coccidial transmission should be usual among Berto, B. P., Lopes, B. doB., Melinski, R. D., Souza, A., Ribas, C., Abreu, F., Ferreira, I., & Lopes, C. W. G. (2014b). sympatric opossums which have close ecological niches Coccidial dispersion across trans- and cis-Andean antbirds favouring rapid transmission, since the sporocysts are not (Passeriformes: Thamnophilidae): sagittulae resistant to desiccation, solar radiation, and other envi- McQuistion and Capparella, 1992 (Apicomplexa: Eimeri- ronmental factors, due to thin sporocyst wall. Figure 3 idae) from non-sympatric hosts. Canadian Journal of Zoology, 92, 383–388. shows the geographic ranges of these opossums to assist Boulard, Y. (1975). Etude morphologique des coccidies (Ade- in understanding the dynamics of dispersal of K. tejerai in leidae) Klossiella killicki n. sp. chez des microchiropterae the New World. The direct transmission of K. tejerai Africans et Klossiella tejerai Scorza, 1957, chez un mar- Bulletin of the Museum of Natural from D. marsupialis to D. aurita is unlikely, because they supial sud-americain. History, 284, 83–89. are not sympatric. In contrast, M. demerarae is sympatric Dubey, J. P., & Lindsay, D. S. (1998). Isolation in immunode- with both D. marsupialis and D. aurita (Fig. 3A); ficient mice of Sarcocystis neurona from opossum therefore, M. demerarae may potentially transmit K. (Didelphis virginiana) faeces, and its differentiation from tejerai for these two hosts, and other sympatric suscep- Sarcocystis falcatula. International Journal for Parasitol- ogy, 28, 1823–1828. tible hosts. In this sense, Didelphis albiventris (Lund) Duszynski, D. W., & Gardner, S. L. (1991). Fixing coccidian may be a potential susceptible host, which has wide oocysts is not an adequate solution to the problem of pre- geographic range in South America and could disperse K. serving protozoan type material. Journal of Parasitology, tejerai for Didelphis spp. and other New World opossums 77, 52–57. Duszynski, D. W., & Wilber, P. G. (1997). A guideline for the (Fig. 3B). preparation of species descriptions in the Eimeridae. Finally, the current work is based on the concept of Journal of Parasitology, 83, 333–336. intra-family specificity proposed by Duszynski & Edgcomb, J. H., Walker, D. H., & Johnson, C. M. (1976). Klossiella Veterinary Pathology, 13 Wilber (1997) which allows new hosts of the same in the opossum. , 315–318. family. Therefore, considering Didelphidae as host- Gardiner, C. H., Fayer, R., & Dubey, J. P. (1998). An atlas of family for K. tejerai with only two host species, D. protozoan parasites in animal tissues. Washington: Armed aurita becomes the third host species. Forces Institute of Pathology, 84 pp. Giraudeau, M., Mousel, M., Earl, S., & McGraw, K. (2014). Parasites in the city: degree of urbanization predicts pox- Acknowledgements This study was supported by grants from virus and coccidian infections in house finches (Haemor- the Fundac¸a˜o Carlos Chagas Filho de Amparo a` Pesquisa do hous mexicanus). PLoS One, 9, e86747. Estado do Rio de Janeiro (FAPERJ) to B. P. Berto (E-26/ Hunsaker, D. (1977). The Biology of Marsupials. New York: 110.987/2013). Academic Press, 537 pp.

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IUCN. (2014). International Union for Conservation of Nature Scorza, J. V., Torrealba, J. F., & Dagert, C. (1957). Klossiella and Natural Resources. http://www.iucnredlist.org. Cited tejerai nov. sp. y Sarcocystis didelphidis nov. sp. parasitos 18 May, 2014. de un Didelphis marsupialis de Venezuela. Acta Biologica Monteiro, R. M., Keid, L. B., Richtzenhain, L. J., Valadas, S. Venezuelica, 2, 97–108. Y., Muller, G., & Soares, R. M. (2013). Extensively Teixeira, M., Rauta, P. D., Albuquerque, G. R., & Lopes, C. variable surface antigens of Sarcocystis spp. infecting W. G. (2007). Eimeria auritanensis n. sp. and E. gambai Brazilian marsupials in the genus Didelphis occur in Carini, 1938 (Apicomplexa: ) from the opossum myriad allelic combinations, suggesting sexual recom- Didelphis aurita Wied-Newied, 1826 (Marsupialia: Di- bination has aided their diversification. Veterinary Par- delphidae) from southeastern Brazil. Revista Brasileira de asitology, 196, 64–70. Parasitologia Veterina´ria, 16, 83–86.

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