establishment of besnoitia darlingi from opossums (didelphis virginiana) in experimental...

Establishment of Besnoitia darlingi from opossums (Didelphis virginiana)in experimental intermediate and definitive hosts, propagation in cellculture, and description of ultrastructural and genetic characteristicsq

J.P. Dubeya,*, D.S. Lindsayb, B.M. Rosenthala, C. Sreekumara, D.E. Hilla, S.K. Shena,O.C.H. Kwoka, L.G. Rickardc, S.S. Blackc,d,1, A. Rashmir-Ravenc

aParasite Biology, Epidemiology and Systematics Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville Agricultural

Research Center, Animal and Natural Resources Institute, Building 1001, BARC-East, Beltsville, MD 20705-2350, USAbDepartment of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of

Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USAcCollege of Veterinary Medicine, Mississippi State University, P.O. Box 9825, Mississippi State, MS 39762, USA

dMississippi Veterinary Diagnostic Laboratory, 2531 North West Street, Jackson, MS 39216, USA

Received 30 January 2002; received in revised form 11 March 2002; accepted 11 March 2002

Abstract

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in

mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts

from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and

interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African

green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats

fed experimentally-infected mouse tissues. These schizonts measured up to 45 £ 25 mm and contained many merozoites. A few schizonts

were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were

similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B.

darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA. Published by Elsevier Science Ltd. on behalf of

Australian Society for Parasitology Inc.

Keywords: Besnoitia darlingi; Opossum; Didelphis virginiana; Cat; Felis catus; Ultrastructure; Molecular; Schizont; Cell culture

1. Introduction

Besnoitia darlingi is a coccidian parasite for which cats

can serve as definitive hosts and for which opossums serve

as intermediate hosts (Frenkel, 1977; Smith and Frenkel,

1977, 1984). Opossums may become infected with B.

darlingi by ingesting infected tissues or by ingesting food

or water contaminated with oocysts excreted by cats (Smith

and Frenkel, 1977). Macroscopic B. darlingi tissue cysts

may be found in many opossum tissues, especially in the

adrenal glands (Smith and Frenkel, 1977). In the United

States, B. darlingi has been reported from opossums in

Kentucky (Conti-Diaz et al., 1970), Missouri and Illinois

(Flatt et al., 1971),Texas (Stabler and Welch, 1961) and

Kansas City (Smith and Frenkel, 1977).

In the United States, natural B. darlingi infections have not

been identified in hosts other than opossums, and oocysts have

not been identified in the faeces of naturally-infected cats.

Thus, little is known of the epidemiology of B. darlingi.

A B. darlingi-like parasite also occurs in another species

of opossum, Didelphis marsupialis in Panama (Darling,

1910; Schneider, 1967a,b,c). A similar parasite has also

been reported from lizards (Basiliscus basiliscus, Ameiva

ameiva, Ameiva leptophrys, Ameiva festiva) from Panama

(Schneider, 1967b) and probably from other hosts (Smith

and Frenkel, 1977). Recently, Paperna and Lainson (2001)

studied the biology of a Besnoitia sp. from naturally-

infected lizards (A. ameiva) from Brazil but they were

unable to transmit the parasite to domestic cats. Thus, it is

uncertain whether the parasite in lizards is B. darlingi.

International Journal for Parasitology 32 (2002) 1053–1064

0020-7519/02/$20.00. Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc.

PII: S0020-7519(02)00060-7

www.parasitology-online.com

q GenBank accession numbers: AF489696, AF489697.

* Corresponding author. Tel.: 11-301-504-8128; fax: 11-301-504-9222.

E-mail address: [email protected] (J.P. Dubey).1 Present address: 898 East Road, Starkville, MS 39759, USA.

Because there are no cultures or DNA of B. darlingi from

different hosts available for comparison, there is no way to

critically evaluate the taxonomy of B. darlingi. Here, biolo-

gical, ultrastructural, and genetic characteristics of B.

darlingi isolated from opossums from Mississippi are

described.

2. Materials and methods

2.1. Naturally-infected opossums

Two naturally-infected opossums (nos. 1, 2) were the

source of the materials used in the present investigation

(Table 1). Both opossums were trapped on horse farms in

Mississippi in February–March, 2001 as part of a study on

the epidemiology of Sarcocystis neurona infections in

Mississippi (Dubey et al., 2001). Portions of naturally-

infected tissues with macroscopic tissue cysts (ears and

kidneys of opossum no. 1 and liver of opossum no. 2)

were transported by air on cold pack from Mississippi to

Beltsville and received in the laboratory 4 days after

necropsy of the opossums.

Infected tissues with grossly visible tissue cysts were

fixed in buffered neutral 10% formalin for histological

studies, and in Karnovsky fixative for TEM, frozen at

270 8C for genetic characterisation, and bioassayed in

mice and cats. The experiments in animals were performed

according to United States Department of Agriculture’s

approved guidelines for Animal Care.

2.2. Infection in mice

Infected tissues were inoculated s.c. or i.p. into inter-

feron-gamma gene knockout (KO) mice (Dubey and Lind-

say, 1998) or outbred Swiss Webster female albino mice

(Taconic Farms) (Table 1). In one trial, Swiss Webster

mice were given dexamethasone in drinking water (1 mg/

ml, Sigma). For mouse inoculation, infected tissues were

homogenized in an aqueous 0.9% NaCl (saline), mixed

with antibiotics (penicillin 1000 units, streptomycin 100

mg per ml of saline), and inoculated into mice (Table 1).

2.3. Infection in cats

Five laboratory-raised, parasite-free cats (Dubey, 1995)

were used (Table 2). Two cats were fed opossum tissues

containing tissue cysts (opossum no. 1 to cat 500, opossum

no. 2 to cat 496). Three cats were fed tissue cysts from

experimentally-infected mice and euthanised 4, 8, and 13

days p.i. Faeces of cats were examined for oocyst shedding

(Smith and Frenkel, 1977). Oocysts were separated from cat

faeces by floatation in sugar solution, washed in water,

incubated at 22 8C in 2% H2SO4 for at least 1 week. Sporu-

lated oocysts from cat 496 were fed to two Swiss Webster

mice (Table 1).

2.4. In vitro cultivation

Tachyzoites obtained from the peritoneal exudate of a KO

mouse (no. 7094, Table 1) were inoculated onto bovine

monocytes as described (Dubey et al., 1999). After two

J.P. Dubey et al. / International Journal for Parasitology 32 (2002) 1053–10641054

Table 1

Transmission of Besnoitia darlingi from opossums to mice

Opossum no. Type of mouse Inoculum Route Day of necropsyd Passage no. Parsitised tissue(s)f

1 KO 8096 Bradyzoites i.p. D11 Original Pex

KO 8094 Bradyzoites s.c. D12 Original Li, Lu

KO 8095 Bradyzoites s.c. D12 Original Li, Lu

SW 8100 Tachyzoites i.p. DK4 Ist Pex

KO 6298 Tachyzoites i.p. DK6 IInd Pex

KO 7094a Tachyzoites i.p. DK5 IInd Pex

KO A Tachyzoites s.c. DK9 IInd Li, Lu

KO 7144 Tachyzoites s.c. DK11 IInd Li, Lu

SW 1–4b Tachyzoites s.c. K154 IInd Neg.

2 3 SWc 8360–8362b Bradyzoites s.c. K153 Original He, Te

4 SW 8364–8368 Bradyzoites s.c. K154 Original Neg.

3 SW 8369–8374 Bradyzoites i.p. DK11–14 Original Pex

4 SW 8216–8219b Bradyzoites s.c. K142 Ist He

4 SW 8252–8255 Oocysts Oral K121 Cat 496 oocysts He

2 SW 9963 Oocysts Oral D11 Cat 570 oocysts H, Li, Lu, S

2 SW 9964 Oocysts Oral D11 Cat 570 oocysts H, Li, Lu

a Tachyzoites used for cell culture and for cryopreservation.b Tissues fed to cats for oocyst shedding.c Given dexamethasone from day 0 to 11.d D, died; DK, killed when ill; K, killed.e Tissue cyst.f H, heart; Li, liver; Lu, lung; Pex, peritoneal exudate; S, spleen; T, tongue.

subcultures, the organisms were stored in liquid nitrogen for

18 months prior to thawing for growth in bovine monocytes.

The B. darlingi isolate was sent to the laboratory in Virginia

for descriptive studies and TEM. The tachyzoites were

grown in African green monkey (Cercopithecus aethiops)

kidney cells (CV-1 cells, ATCC CCL-70). Tachyzoites were

inoculated onto glass coverslips containing a monolayer of

CV-1 cells in multi-welled tissue culture plates. Coverslips

of cells to be examined 1 and 2 days p.i. were inoculated

with 2 £ 105 tachyzoites and those examined at 3 and 4 days

were inoculated with 1 £ 105 merozoites. Coverslips of

infected CV-1 cells were fixed in 10% phosphate buffered

formalin for 30 min, placed in 100% methanol for 10 min

and stained with a rapid staining technique (Diff-Quick,

Dade Behring Inc.). Coverslips were also fixed in Bouin’s

fixative and stained with Giemsa. Coverslips were examined

with light microscopy.

2.5. Histologic and TEM examinations

Tissues of opossums, mice and cats were fixed in 10%

buffered neutral formalin. Paraffin-embedded sections were

cut at 5 mm, and examined after staining with H&E. To

search for enteroepithelial stages the entire small intestine

J.P. Dubey et al. / International Journal for Parasitology 32 (2002) 1053–1064 1055

Table 2

Infectivity of Besnoitia darlingi tissue cysts to cats

Cat no. Tissue cyst source Oocyst shedding

Observation period (days) Oocysts shed Schizonts in

Intestine Other organs

500 Opossum 1 39 No No No

496 Opossum 2 21 Yes No M.L.c

572 Experimentala 4 No Yesb No

571 Experimental 8 No Yes M.L., liver

570 Experimental 13 Yes Yes M.L.

a See Table 1.b Individual zoites.c M.L., mesenteric lymph nodes.

Fig. 1. Stages of Besnoitia darlingi. (A) Section of a tissue cyst from the liver of a naturally-infected opossum. Note thick cyst wall (arrow) with myriads of

bradyzoites and host cell nuclei (arrowheads). H&E stain. (B) Sections of bradyzoites from the tissue cyst from the opossum in (A). Note longitudinally cut

bradyzoites (arrowheads on opposite ends). Toluidine blue stain. (C) A tachyzoite (arrow) in smear from the lung of an experimentally-infected mouse. Giemsa

stain. (D) An unsporulated oocyst (arrow) form the faeces of a cat fed naturally-infected opossum liver. Unstained.

of cats 570, 571, and 572 (Table 2) was fixed in formalin and

40 sections from each cat were examined. In addition,

sections of mesenteric lymph nodes, colon, lung, liver,

spleen, heart, kidneys and adrenals of cats were examined

for B. darlingi stages. Virtually all mouse tissues were fixed

for histologic studies.

For TEM of tachyzoites, a monolayer of CV-1 cells in a

25-cm2 cell-culture flask was inoculated with B. darlingi

tachyzoites. Six days p.i. the infected monolayer was

removed from the plastic growth surface with a cell scraper,

place in a 15-ml tube and pelleted by centrifugation. The

cell pellet was fixed in 13% (v/v) glutaraldehyde in PBS (pH

7.4) for 3 days. Cell pellets were post-fixed in 1% (w/v)

osmium tetroxide in 0.1 M phosphate buffer, dehydrated

in a series of ethanols, passed through two changes of propy-

lene oxide, and embedded in Poly/Bed 812 resin (Poly-

sciences Inc.). Thin sections were stained with uranyl

acetate and lead citrate and examined with a JOEL 100

CX II TEM operating at 80 kV. Tissue cysts of B. darlingi

collected from naturally infected opossum liver which had


Fig. 2. TEM of a tissue cyst of Besnoitia darlingi from the liver of a naturally-infected opossum. (A) Note a thick host connective tissue (ct) overlying the cyst

wall (cw) and bradyzoites butted with the cyst wall. (B) Longitudinal section of a bradyzoite (arrows). Note numerous micronemes located mostly anterior to

the nucleus (n), rhoptries (r) extending up to the posterior end, amylopectin (a), and enigmatic bodies (e).

been fixed in Karnovsky fixative were processed and exam-

ined using identical methods.

2.6. Genetic characterisation

DNA was extracted from Besnoitia tissue cysts of the

naturally-infected opossum no. 1 by proteinase K digestion

and subsequent purification on Qiagen DNAeasy columns

according to the manufacturer’s instructions. Half of the

large subunit nuclear ribosomal RNA was amplified by

PCR using primers KL2 and K6A of Mugridge et al.

(1999). The more variable ITS-1 portion of nuclear rDNA

was amplified with primers 69 and 70 of Tanhauser et al.

(1999). Amplification products were directly sequenced

using these and internal primers using an ABI 3100 auto-

mated DNA sequencer and compared with homologues

previously reported from related parasite species.

3. Results

Besnoitia darlingi tissue cysts from opossum were glis-

tening white and up to 1 mm in diameter. The tissue cysts

contained hundreds of slender, approximately 10 £ 1:5 mm-

sized bradyzoites (Fig. 1A,B). Ultrastructurally, a thick

layer of host connective tissue surrounded the tissue cyst

proper (Fig. 2). The bradyzoites were curved and it was rare

to find longitudinally cut bradyzoites (Fig. 2B). Ultrastruc-

turally, bradyzoites contained numerous prominent micro-

nemes located mostly towards the conoidal end; however, a

few were located posterior to the nucleus. The micronemes


Fig. 3. TEM of bradyzoites of Besnoitia darlingi from Fig. 2. Note numerous micronemes (m) anterior to the nucleus (n), up to three rhoptries (r) in one plane of

section, amylopectin (a), enigmatic bodies (e) and unidentified membrane-bound bodies (x).

were not arranged in any particular fashion. The nucleus

was located centrally or towards the posterior (non-conoi-

dal) half of the bradyzoite. Rhoptries were elongated and

some extended even towards the posterior end (Fig. 3A). A

maximum of three rhoptries were seen in any bradyzoite.

Other organelles noted were amylopectin granules, dense

granules, unidentified membrane-bound bodies towards

the conoidal end (Fig. 3C), and enigmatic bodies, character-

istic of Besnoitia bradyzoites. The enigmatic bodies were

about twice the length of micronemes and were located

mostly towards the posterior end of the bradyzoite (Figs. 2

and 3).

Tachyzoites were found in mice (Fig. 4A,B) inoculated

with bradyzoites obtained from tissue cysts from opossums

and the tachyzoites were infective to other mice by subino-

culation (Table 1). Tissue cysts (Fig. 4C,D) formed in mice

inoculated with tachyzoites (Table 1).

The cat (no. 496) fed infected liver tissue from opossum

no. 2 shed a few oocysts (Fig. 1D) with a prepatent period of

11 days. The cat (no. 500) fed tissues from opossum no. 1

did not shed oocysts. The cat (no. 571) fed experimentally-

infected mouse tissues shed oocysts with a prepatent period

of 9 days. The oocysts were approximately 11 mm in

diameter and were shed unsporulated in faeces (Fig. 1D).

Schizogonic stages were found in the small intestine of

cats killed 4–13 days after being fed infected tissues (Fig. 5).

Only individual zoites were seen in the lamina propria of

small intestine of the cat euthanised 4 days p.i. Developing

and mature schizonts were located in the lamina propria

throughout the small intestine of cats euthanised 8 and 13

days p.i. (Fig. 5). The host cell parasitised was not defini-

tively identified definitively but some schizonts appeared to

be in the capillary endothelium (Fig. 5D). The host cell

nucleus was sometimes hypertrophied and indented (Fig.


Fig. 4. Lesions and Besnoitia darlingi stages in sections of tissues of experimentally-infected mice. H&E stain. (A,B) Heart of a KO mouse fed oocysts. Eleven

days p.i. Note necrosis (large arrow) of myocardium associated with tachyzoites (small arrows). (C) Heart of a Swiss Webster mouse 153 days after s.c.

inoculation with bradyzoites. Note granulomatous inflammation around a degenerating tissue cyst (arrowheads) and an intact tissue cyst (arrow) without any

host reaction. (D) Brain of a Swiss Webster mouse 121 days after feeding oocysts. Note granuloma around a degenerating tissue cyst (arrow).

5C). Merozoites divided by schizogony. Small schizonts

were about 10 mm in diameter and were difficult to distin-

guish from host cells (Fig. 5A). The multinucleated stage

consisted of nuclei (Fig. 5D) that were not distinctly sepa-

rated from each other. The largest schizont seen was 45 £ 25

mm in size (Fig. 5G). The merozoites were tiny (approxi-

mately 3 mm long), slender, and often arranged in groups or

whorls (Fig. 5F,G). Gamonts were not seen.

Few schizonts were seen in sections of mesenteric lymph

nodes and liver of cats fed tissue cysts (Table 2). The largest

schizont observed in the mesenteric lymph node was 55 £

25 mm (Fig. 5H). Schizonts in liver and mesenteric lymph

nodes appeared structurally identical to intestinal schizonts.

The two KO mice fed oocysts from cat no. 496 died of

acute besnoitiosis 11 days p.i. The predominant lesion was

myocardial necrosis with tachyzoites in myofibres (Fig.

4A,B). Granulomatous inflammation was associated in

several organs with ruptured tissue cysts (Fig. 4C,D). Tissue

cysts in experimentally-infected mice remained micro-

scopic even 5 months p.i.

Tachyzoites were visible intracellularly on the second

day after inoculation and the cells in the original flasks

survived until day 30 (opossum no. 1) and day 48 (opossum

no. 2), respectively. Organisms were successfully passaged

into bovine monocytes or equine kidney cells and were

cryopreserved for future studies.

Besnoitia darlingi tachyzoites penetrated CV-1 cells and

underwent development by endodyogeny by day 1 p.i. (Fig.


Fig. 5. Schizonts of Besnoitia darlingi in the small intestine (A–G) and mesenteric lymph node (H) of cats fed tissue cysts. H&E stain. Bar applies to all parts.

All schizonts are located in the lamina propria; the intestinal lumen in each picture is oriented on the top. The host cell nucleus is indented (arrowheads) and

occasionally hypertrophied. Schizonts are arranged in presumed stages of development, (A) being the youngest and (G) being the most mature. (A–C)

Schizonts with nucleus in early stages of division. (D) Schizont in a capillary with an erythrocyte (arrow). (E) Multinucleated schizont. (F,G) Schizonts with

merozoites arranged in groups. (H) The largest schizont seen with merozoites arranged in whorls.

6). In coverslip preparations fixed in Bouin’s and stained

with Giemsa, intracellular tachyzoites were 4–5 £ 1:5 mm in

size and they were located in a vacuole usually near the host

cell nucleus (Fig. 6A). The nucleus was vesicular and

located centrally or towards the anterior half of the parasite.

Occasionally the tachyzoites were located in more than one

parasitophorous vacuole within the cell (Fig. 6A). The para-

site divided, often with simultaneous endodyogeny, within a

given vacuole (Fig. 6B). Some host cells contained rosettes

(Fig. 6C). More than 30 tachyzoites could be seen within

one parasitophorous vacuole (Fig. 6D). Extracellular forms

were more crescentic and the nucleus appeared darker than

the intracellular tachyzoites (Fig. 6E). In formalin–metha-

nol-fixed smears, tachyzoites at 1 day p.i. were 5:3 £ 2:2 mm

(range 4:5–6:3 £ 1:8–2:7 mm; n ¼ 10), at 2 days p.i. they

were 5:6 £ 2:1 mm (range 5:4–7:2 £ 1:8–2:7 mm; n ¼ 10), at

3 days p.i. they were 6:0 £ 2:1 mm (range 5:4–7:2 £ 1:8–2:7

mm; n ¼ 10) and at 4 days p.i. they were 5:5 £ 2:2 mm

(range 4:5–6:3 £ 1:8–2:7 mm; n ¼ 10).

Ultrastructurally, tachyzoites were located in a parasito-

phorous vacuole in the host cell cytoplasm (Figs. 7 and 8).

The parasitophorous vacuole contained tubular network and

amorphous to granular material (Fig. 8). Before division,

tachyzoites contained a posteriorly located nucleus, several

micronemes, rhoptries, and dense granules (Fig. 7A). The

micronemes were arranged in rows and located mostly

towards the conoidal end (Fig. 7A). Rhoptry contents

were electron-dense and up to 4 rhoptries were seen in a

given section (Fig. 8). An electron dense material was seen

lining the conoidal end of the subpellicle (Fig. 8). Tachy-

zoites divided into 2 progeny by endodyogeny (Fig. 7B).

Schizonts and tissue cysts were not seen in cell cultures.

Direct sequencing of portions of the ribosomal RNA

array amplified by PCR was employed in order to facilitate


Fig. 6. Tachyzoites of Besnoitia darlingi in African green monkey cells 70 h p.i. Arrowheads point to host cell nucleus. Bouin’s fixed, Giemsa stained. Bar

applies to all parts. (A) Parasites in 15 vacuoles. Some organisms are dividing (arrows). (B) Parasites in four or more vacuoles. All eight organisms in one

vacuole are dividing (arrows). (C) Parasites with two groups in rosettes (arrows). (D) Thirty-four zoites, presumably in one vacuole. (E) Groups of tachyzoites,

with one in division (arrow). (F) Extracellular tachyzoites (arrows) which are slender and longer than intracellular tachyzoites.

comparison of the present parasite isolate with others which

have been, or which will be in the future, characterised

genetically. Over an 1153 bp portion of the large subunit

ribosomal RNA, B. darlingi opossum isolate shared over

99% identity with that reported from B. besnoiti of cattle.

However, many distinguishing substitutions have accrued in

the more variable ITS-1 portion of the ribosomal RNA of

these two taxa. A pairwise BLAST search identified

conserved, homologous residues (81% identity) in the first

124 bp (representing a portion of the small-subunit rDNA)

and the final 46 bp (incorporating a portion of the 5.8S

rDNA). In the intervening spacer region, however, such

positional homology was harder to establish. Far more simi-

larity, however, could be established between the ITS-1 of

B. darlingi and that of B. jellisoni. Whereas these two

differed from each other at merely 4.5% of 279 aligned

bases, they each differed from B. besnoiti at over 19% of

these. These B. darlingi sequences have been deposited in

GenBank as accession numbers AF489696 and AF489697.

Live culture of Besnoitia darlingi (designated strain OP1)

deposited in ATCC (No. 50978).

4. Discussion

This study confirms shedding of B. darlingi oocysts by

cats reported by Smith and Frenkel (1977). In addition, we

describe the ultrastructure of tachyzoites and bradyzoites

and the development of B. darlingi schizonts in the lamina

propria of cats. Confirming cats as a definitive host, impor-

tantly, differentiates B. darlingi from the lizard parasites

studied by Paperna and Lainson (2001) which did not

produce oocysts in the faeces of any of the three cats fed

infected tissues. Additionally, neither B. jellisoni (of

rodents) nor a Besnoitia sp. of reindeer were transmitted

to cats experimentally (Frenkel, 1977; Ayroud et al.,

1995). Although Peteshev et al. (1974) reported shedding

of Besnoitia-like oocysts from cats after feeding tissues

naturally-infected with B. besnoiti from cattle, these experi-

ments were uncontrolled and repeated attempts to find a

definitive host for B. besnoiti have failed (Diesing et al.,

1988). Thus, differences exist in the definitive host range

of Besnoitia species, and these differences can be used to aid

in their diagnosis.

The paucity of oocysts shed by cats fed naturally or

experimentally-infected tissues suggests that cats may not

play a central role in the natural epidemiology of B.

darlingi. The observed scarcity of oocysts was probably

not limited by the inoculum size because even a single

macroscopic tissue cyst from these naturally-infected opos-

sums contained thousands of bradyzoites. Why cat no. 500,

fed many tissue cysts, did not shed oocysts is unknown but

may be related to cats being a poor host for B. darlingi. It is

possible that most transmission of B. darlingi, which has not

been reported in any other hosts, occurs when an opossum

feeds upon the remains of others.

Finding schizonts in the intestines of cats fed Besnoitia-

infected tissues in the present study is interesting because B.


Fig. 7. TEM of culture-derived tachyzoites of Besnoitia darlingi. (A) A zoite, probably before start of nuclear division. Note micronemes (mi) arranged in rows,

mitochondria (mt), rhoptry (r), dense granules (g) and a nucleus (n). A few micronemes are present even posterior to the nucleus (B) Mother tachyzoite

containing two daughter organisms. The pellicle (arrowheads) of one progeny is visible. Each daughter organism has a nucleus (n) and rhoptries (rp). A rhoptry

(rm) of the mother zoite is also visible.

darlingi schizonts were previously unknown (Smith and

Frenkel, 1977). In location, B. darlingi schizonts are some-

what similar to those of Besnoitia wallacei, which also colo-

nize the vascular endothelium in the lamina propria of the

ileum (Wallace and Frenkel, 1975; Frenkel, 1977). Despite

their common location in the lamina propria, the tenfold

difference in size between B. wallacei and B. darlingi schi-

zonts make them readily distinguishable. Besnoitia wallacei

schizonts may reach 800 mm in length, whereas typical B.

darlingi schizonts measure only 45 £ 25 mm. The B.

darlingi schizonts occur throughout the small intestine,

particularly in the jejunum, whereas B. wallacei was

found in the ileum of cats (Frenkel, 1977).

Unlike B. wallacei, B. darlingi was successfully trans-

mitted to mice by inoculation with tachyzoites, bradyzoites,

and sporozoites. However, in mice, tissue cysts were few

and remained microscopic, whereas in naturally-infected

opossums tissue cysts were macroscopic. Thus, mice may

not be a natural intermediate host for B. darlingi.

Besnoitia darlingi was easily maintained in cell culture

by subpassage. Live tachyzoites were recovered in cell

cultures inoculated with B. darlingi cultures stored in liquid

nitrogen for 18 months. Besnoitia besnoiti and B. jellisoni

have also been cultivated in vitro (Bigalke, 1962; Frenkel,

1965; Fayer et al., 1969). Thus, cell culture-derived tachy-

zoites may be useful to differentiate species belonging to the

genus Besnoitia.

The ultrastructure of B. darlingi tissue cysts and tachy-

zoites described for the first time in the present report was

essentially similar to that of tissue cysts of the Besnoitia


Fig. 8. TEM of a group of Besnoitia darlingi tachyzoites in parasitophorous vacuole (PV). Note conoid (C), rhoptries (R), micronemes (M) and dense granules

(G), and electron-dense layer covering the conoidal end of tachyzoites (opposing arrowheads).

species (Sheffield, 1968; Scholtyseck et al., 1968; Heydorn

et al., 1984; Paperna and Lainson, 2001).

Tachyzoites of B. darlingi and Toxoplasma gondii cannot

be distinguished by light microscopy. However, they can be

distinguished ultrastructurally by their rhoptries and micro-

nemes. Rhoptries of B. darlingi are electron-dense whereas

those of T. gondii tachyzoites are electron-lucent (Dubey,

1993). Micronemes in T. gondii tachyzoites are few and

arranged haphazardly whereas micronemes of B. darlingi

are more numerous and arranged in rows. In addition, elec-

tron dense material lines the anterior part of the innermost

layer of the pellicle of B. darlingi tachyzoites which is

absent in T. gondii tachyzoites. The electron dense material

was first reported by Paperna and Lainson (2001) in tachy-

zoites from the peritoneal exudate of mice infected with the

lizard Besnoitia from Brazil. This electron-dense material

was not reported from in vivo or in vitro derived tachyzoites

of B. jellisoni (Sheffield, 1966; Senaud et al., 1974; Senaud

and Mehlhorn, 1978) or other species of Besnoitia (Gobel et

al., 1985).

The life cycle of B. darlingi resembles that of T. gondii in

the experimental intermediate host (mouse). Tachyzoites

and tissue cysts are formed in many organs. However, the

life cycle is different in the definitive host, the cat. Although

the oocysts of T. gondii and B. darlingi are morphologically

similar, their endogenous stages are distinct. Unlike T.

gondii, B. darlingi schizonts are formed in the lamina

propria, whereas T. gondii schizonts occur in the enterocytes

(Dubey and Beattie, 1988). In addition, B. darlingi schizonts

are found in extra-intestinal organs (liver and mesenteric

lymph nodes), whereas in T. gondii, schizonts are confined

to the intestines.

The close relationship between B. darlingi and other

members of the genus was confirmed by nearly identical

large subunit ribosomal RNA genes. Shared genetic attri-

butes unique to Besnoitia spp. provide an additional means

to recognize species belonging to this genus. Although para-

sites in their intermediate hosts may be readily and reliably

designated as belonging to the genus on the basis of their

macroscopic tissue cysts, genetic means may prove particu-

larly useful in defining the natural definitive host range of B.

darlingi and its congeners. Coupled with the greater inter-

specific variation evident in the ITS-1 molecule, these find-

ings provide a needed tool to further characterize the natural

transmission and evolutionary relationships among the

members of this large but poorly understood parasite

group. The present study provides the most complete

morphologic, biologic, ultrastructural and genetic character-

ization available for any species of Besnoitia.

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