Coccidial dispersion across New World marsupials:Klossiella tejerai Scorza, Torrealba & Dagert, 1957(Apicomplexa: Adeleorina) from the Brazilian commonopossum 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

� 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

recovered from urine samples were ellipsoidal,

20.4 9 12.7 lm, with sporocyst residuum composed

of scattered spherules and c.13 sporozoites per sporo-

cyst, 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, 1882. Among these

Sarcocystis spp., Sarcocystis neurona Dubey, Davis,

Speer, Bowman, Lahunta, Granstrom, Topper, Hamir,

Cummings & Suter, 1991 is recognised as the

C. S. dos Santos � M. D. Cordeiro

Curso de Pos-Graduacao em Ciencias Veterinarias,

Universidade Federal Rural do Rio de Janeiro (UFRRJ),

BR-465 km 7, 23897-970 Seropedica, RJ, Brazil

B. P. Berto (&)

Departamento de Biologia Animal, Instituto de Biologia,

UFRRJ, BR-465 km 7, 23897-970 Seropedica, RJ, Brazil

e-mail: bertobp@ufrrj.br

B. do Bomfim Lopes

Programa de Pos-graduacao em Ciencia, Tecnologia e

Inovacao em Agropecuaria, UFRRJ, BR-465 km 7,

23897-970 Seropedica, RJ, Brazil

A. H. da Fonseca

Departamento de Epidemiologia e Saude Publica,

Instituto de Veterinaria, UFRRJ, BR-465 km 7,

23897-970 Seropedica, RJ, Brazil

W. L. T. Filho � C. W. G. Lopes

Departamento de Parasitologia Animal, Instituto de

Veterinaria, UFRRJ, BR-465 km 7,

23897-970 Seropedica, RJ, Brazil

123

Syst Parasitol (2014) 89:83–89

DOI 10.1007/s11230-014-9510-7

ethiological agent of equine protozoal myeloenceph-

alitis (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

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

Seropedica (22�440S, 43�420W), state of Rio de

Janeiro, Brazil. The opossums were transported to the

Veterinary Institute (Instituto de Veterinaria – 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 Renovaveis

– IBAMA) under protocol # 34701-2. Sample pro-

cessing and data analysis were conducted at the

Laboratory of Coccidia and Coccidiosis (Laboratorio

de Coccıdios 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 microscopi-

cally 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.

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

84 Syst Parasitol (2014) 89:83–89

123

Results

Twenty Brazilian common opossums were examined;

one of them (5%) shed Klossiella-like sporocysts in

the urine. The current description follows the guide-

lines 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 Seropedica (22�440S, 43�420W).

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 Laboratorio de Coccıdios 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 (http://r1.

ufrrj.br/lcc) as well. Photographs of the host specimen

are deposited in the same collection. The repository

number is 53/2014.

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, Oocyst

with free mature sporoblasts/sporocysts; I, Macrogamete (right) and early sporont (middle) within parasitophorous vacuoles and oocyst

with free mature sporoblasts/sporocysts (left). Scale-bars: 10 lm

Syst Parasitol (2014) 89:83–89 85

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Site of infection: Epithelium of the renal tubules.

Description (Figs. 1, 2)

Exogenous stages

Sporocysts ellipsoidal, 19–22 9 12–14 (20.4 9 12.7);

length/width (L/W) ratio 1.5–1.8 (1.6) (Fig. 1). Spo-

rocyst residuum present, composed of scattered

spherules. Sporozoites 12–14 (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,

7–11 9 5–9 (8.6 9 7.2), with basophilic nucleus and

contained within a subspheroidal to irregular parasitoph-

orous vacuole, 14–29 9 14–22 (22.6 9 18.3) (Fig. 2A–

B, I). Macrogamete in syzygy with microgametes in

some cases (Fig. 2C). Microgametes subspheroidal to

ovoidal, 5–6 9 3–5 (5.3 9 3.8), with basophilic

Table 1 Comparative morphology of Klossiella tejerai recovered from New World opossums

Host Didelphis aurita (Wied-

Neuwied)

Didelphis marsupialis

(L.)

Marmosa demerarae

(Thomas)

D. marsupialis (L.)

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

size

14–29 9 14–22

(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

(21.3 9 16.4)

up to 27 – 25–39 (28)

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 oocyst 12–30 (18) (18) 16–22 –

Oocyst

Shape Irregular – – –

Size 57–103 9 36–57

(71.6 9 47.2)

– (80 9 40) –

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 – – –

86 Syst Parasitol (2014) 89:83–89

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nucleus. Sporonts subspheroidal to irregular, 15–31 9

14–21 (21.3 9 16.4), dotted circumferentially with 8–13

(10) basophilic nuclei (Fig. 2D–E, I). Immature sporo-

blasts ellipsoidal, 5–6 9 3–4 (5.4 9 3.5) (Fig. 2F–G).

Mature sporoblasts/sporocysts 12–30 (18), ellipsoidal,

10–13 9 6–9 (11.4 9 6.8), with multiple dotted baso-

philic nuclei (Fig. 2H). Oocysts irregular, 57–103 9

36–57 (71.6 9 47.2) (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). How-

ever, 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 oocysts/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

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

Syst Parasitol (2014) 89:83–89 87

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problem; on the other hand, epizootics should occur

when environmental disturbances and/or anthropo-

genic factors contribute to change the behavior and/or

mainly stressing the wild animals, leading to immun-

odepression. 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 envi-

ronmental 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 suscep-

tible 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 host-

family for K. tejerai with only two host species, D.

aurita becomes the third host species.

Acknowledgements This study was supported by grants from

the Fundacao Carlos Chagas Filho de Amparo a Pesquisa do

Estado do Rio de Janeiro (FAPERJ) to B. P. Berto (E-26/

110.987/2013).

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