Crithidia deanei infection in normal and dexamethasone–immunosuppressed Balb/c mice

doi:10.4236/health.2010.26087

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Vol.2, No.6, 589-594 (2010) Health

doi:10.4236/health.2010.26087

Crithidia deanei infection in normal and

dexamethasone–immunosuppressed Balb/c mice

Dilvani Oliveira Santos1*, Saulo C. Bourguignon1, Helena Carla Castro1, Alice Miranda2,3,

Rodrigo Tonioni Vieira1, Suzana Corte-Real4, Otílio Machado Pereira Bastos5

1Department of Cellular and Molecular Biology, Federal Fluminense University (UFF), Rio de Janeiro, Brazil;

*Corresponding Author: santosdilvani@gmail.com; lacelauff@yahoo.com.br

2Department of Mycobacteriosis, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil

3Department of Pathology, FCM, University of State of Rio de Janeiro, Rio de Janeiro, Brazil

4Laboratory of Biology Structural, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil

5Department of Microbiology and Parasitology, Federal Fluminense University (UFF), Rio de Janeiro, Brazil

Received 18 October 2009; revised 21 December 2009; accepted 24 December 2009.

ABSTRACT

Monoxenous trypanossomatids protozoa are

not believed to cause in vivo infection in verte-

brate hosts throughout their life cycle. However,

there are reports mentioning some cases of HIV-

positive patients who have presented oppor-

tunistic infections caused by these protozoa.

Recently, we have demonstrated the in vitro in-

fection of mouse dermal fibroblasts by these

protozoa. The aim of the present work is to in-

vestigate the possibility of Crithidia deanei, a

endosymbiont-bearing monoxenous trypanos-

somatid, infect BALB/c mice under or not

Dexamethasone treatment. To attend it, distinct

gro- ups of adult BALB/c mice were immuno-

suppressed with 50 mg/kg of Dexamethasone.

This immunosuppressor was administered 24

hours before infection and daily, for 15 days

after C. deanei inoculation. Control groups: C.

deanei–inoculated animals but non-immuno-

suppressed and non-inoculated animals but

immunosuppressed were also used. Light Mi-

croscopy analysis revealed an infection process

characterized by the presence of the trypanos-

somatid inside dermal cells in the groups stud-

ied. The experimental inoculation resulted in a

non-lethal infection characterized by the pres-

ence of the trypanossomatid inside dermal cells

in the normal BALB/c mice, but notably, in the C.

deanei–inoculated immunosuppressed group.

These preliminary results lead to the following

conclusions: 1) C. deanei is able to infect nor-

mal BALB/c mice; 2) the immunosupressed

mice seemed to be more susceptible to the C.

deanei infection compared to the control group.

Besides C. deanei in dexamethasone-immuno-

suppressed mice provides a useful model for

studies of monoxenous trypanosomatids ‘in

vivo’ infection, resembling that one presumably

occurring in imunodeficient individuals with

AIDS.

Keywords: Monoxenous Trypanossomatid;

‘In Vivo’ Infection; Immunosuppression

1. INTRODUCTION

Trypanossomatids parasitize a diverse range of hosts

including animals, plants and protists [1]. Some of them,

such as Trypanosoma and Leishmania, are heteroxenous

and are ethiological agents of serious diseases in humans

and experimental animals. Others are a monoxenous and

are mostly found in insects [2]. Monoxenous trypano-

somatids had never been confirmed as pathogenic in

vertebrate host. However, there is one report of trypano-

somatid, other than Trypanosoma and Leishmania, in

some opportunistic cutaneous infections in immuno-

compromised individuals [3] or those without any pre-

vious history of immunodepression [4]. In addition, our

group was pioneer in proving the infection of mouse

dermal fibroblasts by two different monoxenous try-

panosomatid species—Crithidia deanei and Herpetomo-

nas roitmani [5]. Although some of these trypanosomat-

ids were classified as a divergent member of the

Leishmania genus [6], a visceral leishmaniasis—like in-

fection was described in an HIV-positive patient as

caused by Leptomonas pulexsimulantis, a monoxenous

trypanosomatid found in dog’s flea [3], suggesting that

monoxenous protozoa can be considered opportunistic

agents in immunocompromised individuals. Therefore,

D. O. Santos et al. / HEALTH 2 (2010) 589-594

590

we investigated the ability of C. deanei to infect verte-

brate host. For that purpose, we have used BALB/c mice

under or not Dexamethasone treatment as an experi-

menttal model, based on a previous report of mouse

dermal fibroblasts infection by C. deanei and H. roit-

mani [5].

2. MATERIALS AND METHODS

Parasite culture. Crithidia deanei was kindly provided

by Dr. M. Auxiliadora de Souza (Trypanosomatids Colll-

etion of the Oswaldo Cruz Institute, Rio de Janeiro,

Brasil). The monoxenous were kept at 28°C with serial

passages at 48 h intervals in Warrens’ medium [7] con-

taining 10% fetal calf serum.

Experimental animal infection. Female 8-week old

BALB/c mice (Nau, Instituto de Biologia /UFF) were

used. Animals housed in standard conditions were

treated with Dexamethasone (Aziume) [8] 24 hours be-

fore infection with C. deanei. After infection with 107

2-day-old promastigotes C. deanei by subcutaneous

route (hind foot pad) —day 0, dexameth-asone 50 mg/kg

was administered daily, for 15 days. Four BALB/c mice

group were used: control without dexamethasone; con-

trol with dexamethasone; C. deanei–inoculated with

dexamethasone and C. deanei–inoculated without dexa-

methasone (Table 1). A determined number of mice

from each group were euthanasiated at 6 h, 1 d, 2 d, 3 d,

d 7 and d 15 after C. deanei inoculation. At each control

point, mice were weighted and parasite burdens were

determined in foot pad by histological analysis.

Histological analysis. Specimens of foot pad were

fixed in 10% buffered formalin. After dehydration in

graded ethanol, the tissues were embedded in paraffin

and, then, processed routinely as previously reported [9].

5 μm thick sections were obtained with a Leica micro-

tome. After that, they were collected on glass slides for

Hematoxilin-Eosin (HE) staining. The tissues samples

infected or not were observed at least 400 randomly se-

lected cells at 1000 × magnification, using a Zeiss

photomicroscope.

3. RESULTS

Clinical finding’s. No mortality, weight loss or clinical

signs were observed in mice infected with either dexam-

ethasone or not.

Macroscopy findings. Both groups C.deanei—inocu-

lated immunosuppressed mice and not inoculated immu-

nosuppressed mice displayed splenomegaly and heap-

tomegaly.

Histological analysis. Through light microscopy the

morphological analysis just of the foot pad was done. At

necropsy, parasites were found in the foot pad from the

mice inoculated with C. deanei, regardless immunosup-

ressed or not. In the Dexamethasone treated-controls

groups (in the absence of C. deanei inoculation), no hist-

ological and inflammatory reations of the foot pad were

observed until d15 (Figure 2(e1)).

Surprisingly, in both experimental design-in the pres-

ence or not of dexamethasone, C. deanei was infective to

BALB/c mice (Figure 1 and 2), but, notably, in the imm-

unosupressed BALB/c mice (Figure 2).

Using light microscopy, it observed C.

deanei–infected mouse dermal cells after 24 h infection

(Figures 1(a1) and (a2)). On the 2nd post infection day,

C. deanei was also observed within mice dermal cells

(Figures 1(b1) and (b2)). and, between whiles, ex-

tracellular parasites were seen (Figure 1(b1)). A large

numbers of parasites were clearly present in the dermal

cells after the third post-infection day (Figures 1(c1) and

(c2)). Although it was possible to observe C. deanei

within the dermal cells, their mechanism of entrance is

still not clear as it can involve phagocytosis, penetration

in the cell or inducing membrane invagination. Anyway,

one mechanism of the C. deanei–infection might be

through sincicious formation from the host cells as the

image of the Figures 1(c1) and (c2) suggest. After 7

days of infection it still can observe parasites present in

dermal cells C. deanei–infected mice (Figures 1(d1) and

(d2)). At this time, some extracellular parasites were still

seen (Figures 1(d1) and (d2)).

After 15 days of infection, the light microscopy still

revealed intracellular forms of C. dean ei as well as some

extracellular forms of this parasite attached to the dermal

cells surface (Figures 1(e1) and (e2)).

In the controls groups (in the absence of C. deanei in-

oculation and presence of dexamethasone) no histology-

cal and inflammatory reactions of the foot pat were obs-

erved until day 15 (Figure 2(e1)).

Interestingly, the kinetics of infection in foot pad from

C. deanei–inoculated Dexamethasone immunosuppressed

mice showed parasites as early as 6h in the subcutaneous

tissues (Figures 2(a1) and (a2)). Notably, the most exu-

berant C. deanei–infection was observed in the presence

of Dexamethasone on the first day of infection (Figures

2(b1) and (b2). In the meanwhile, it can clearly observe

a C. deanei within a vacuole (Figure 2(b2)). On the foll-

owing day, it can still observe a large numbers of C.

deanei inside the cells (Figures 2(c1) and (c2)). In this

time of infection, similar to the findings on the previous

day, it can see that each parasite occupies its own vacu-

ole (Figures 2(c1) and (c2)). The image of C. deanei inside

D. O. Santos et al. / HEALTH 2 (2010) 589-594

591

Table 1. Distribution of the experimental groups according to the animals number e the respective data of necropsy.

Animals number / time of necropsy

Groups

6 h 24 h 48 h 72 h day 7 day 15

I (C. deanei–inoculated DMT treated mice) 2 2 2 2 2 2

II (C. deanei–inoculated mice) 2 2 2 2 2 2

III (DMT treated mice) 1 1 1 1 1 1

IV (not DMT treated and not C. deanei inoculated mice)–Control Groups 1 1 1 1 1 1

Total 6 6 6 6 6 6

 DMT-Dexamethasone 50 mg/kg.

 In a total 36 animals were used.

 The animals were euthanazed according to the rules of ethical comitê (Comissão de ética no uso de animais (CEUA- FIOCRUZ) P8317 ação:

1201 no. P024705).

Figure 1. Analysis by light microscopy showing C. deanei interaction with Balb/c mouse. Pictures were taken in a two different

plans from the same field (e.g. a1 and a2, etc…) in order to show whole extension of cell infection. Representative sections from skin

samples of 24h (a1 and a2); 48h (b1 and b2); 72h (c1 and c2); 7 days (d1 and d2) and 15 days (e1 and e2) C. deanei post-infection

(original magnification x 100). Grey Arrow shows some free C. deanei extracellular forms (b1; d1 and d2) as well as C. deanei ex-

tracellular forms attached to the dermal cells surface (e1 and e2). Dark arrow show multiple C. deanei-infected dermal cells. Note the

presence of sincicious formation in c1 and c2. N = Dermal cells nucleous.

a1 b1

d1 d2 e1

D. O. Santos et al. / HEALTH 2 (2010) 589-594

592

Figure 2. Analysis by light microscopy showing C. deanei interaction with dexamethasone immunosuppressed Balb/c mouse. Pic-

tures were taken in a two different plans from the same field (e.g. a1 and a2, etc…) in order to show whole extension of cell infection.

Representative sections from skin samples of 6h (a1 and a2); 24h (b1 and b2); 48h (c1 and c2); 72 h(d1 and d2) C. deanei

post-infection (original magnification x 100). Dark arrow show multiple strongly C. deanei–infected dermal cells. Occasionaly, some

parasites seemed to be clearly envolved by a vacuole (a2, b2, c1, c2 and d2). Controls groups 15 days after dexamethasone treatment

or not (e1: dexamethasone-immunosuppressed Balb/c mouse; and e2: normal Balb/c mouse; original magnification (e1) × 50 and (e2)

× 10). N = Dermal cell nucleous.

vacuoles continues to be seen in the third day of inf-

ection (Figures 2(d1) and (d2)). Here some tissue degra-

dation was also observed (Figures 2(d1) and (d2)). Int-

erestingly, at the last days of infection time (7 and 15

days) in immunosupressed BALB/c mice, no parasites

were found contrasting to some tissue alterations which

were observed (data not shown).

4. DISCUSSION

Several clinical cases suggesting that monoxenous try-

panosomatids could be implicated in human infections

have been described in the last years. They have been

emerging as possible opportunistic pathogens in imuno-

compromised individuals. An unusual Leishmania-like

parasite was found in a HIV-positive patient with symp-

toms of Leishmania infection [10]. Despite the previ-

ous-mentioned data, genotypic and phenotypic charac-

terization showed that a flagellate parasite, found in the

bone marrow of a Brazilian HIV-positive patient pre-

senting a visceral leishamniasis-like reaction, was indeed

a monoxenous trypanosomatid, although no tissue inva-

sion could be detected [3]. Surprinsingly, a new case of

cutaneous infection by a presumed monoxenous try-

panosomatid was reported in the island of Martinique;

however, the individual had no history of immunosup-

pression, particularly HIV infection [4].

As stated earlier, Santos et al. (2004) first reported

that endosymbiont-bearing trypanosomatid C. deanei

and Herpetomonas roitmani are able to infect mouse

dermal-derived fibroblasts while Crithidia fasciculate

and Herpetomonas samuelpessoai (trypanossomatid

a1 b1 c1

a2 b2 c2

d1 d2 e1 e2

D. O. Santos et al. / HEALTH 2 (2010) 589-594

593

endosimbiont free) did not infect. It is also of interest to

observe that both C. deanei and H. roitmani can be res-

istant to lysis mediated by the complement system. In

contrast, H. samuelpessoai and C. fasciculate displayed

100% of lysis after incubation with the complement sys-

tem [5]. The symbionts of C. deanei can influence the

phagocytosis of these parasites by macrophages as have

been presented by [11]. And, most recently, [12], re-

ported the infection of HIV-1-infected prim- ary human

macrophages by Blastocrithidia culicis (another endo-

symbiont-bearing monoxenous trypanosomatid). Our

present data further emphasize the large capacity of C.

deanei to infect vertebrate host and reinforce the idea

that monoxenous trypanosomatids present low host

specifity [2,13,14]. As demonstrated by our work, C.

deanei can readily infect normal BALB/c mice by sub-

cutaneous route and infection persist in the dermal cells

for 15 days. These are very interestingly results, since

we have previously reported the “in vitro” C. deanei–

infection of dermal cells obtained from a different spe-

cie of mouse-the Swiss mouse [5]. Besides, as observed

in our present work, extracellular forms of C. deanei are

displayed in dermal tissue of the BALB/c mice (Figures

1(b1), (d1) and (d2)). This fact is interesting to be men-

tioned since it might suggest that, after intracellular C.

deanei cycle, these parasites leave the host cell and, after

that, appear in the extracellular medium (in a flagellate

form) to re-infect others dermal cells. Taken together,

these evidences reinforce the idea that monoxenous try-

panosomatids are able to infect and to survive once

reaching the vertebrate host. Over and again, we demon-

strated the infection of BALB/c mice, but, a much more

pronounced C. deanei–infection in a different experi-

mental design: in Dexamethasone-immunodepressed

mice (Figure 2). Through its limphopenic activity, spe-

cially about T cell production [15], the dexamethasone

can reduce the mechanisms of anti-parasite effect of

immune system and it might explain the increase of sus-

ceptibily to C. deanei infection observed in all immuno-

suppressed animals. The important survival of the para-

site in the murine experimental host contrast strikingly

with the weak clinical-pathological effects observed with

absence of lymphocytic infiltrates in parasitized foot pad.

This can be paralleled to that observed during human

visceral leishmaniasis where patent infections with para-

site dissemination are frequently associated with T cell

unresponsiveness to Leishmania antigen [16], while cure

is accompanied with restoration of the cellular response

[17,18]. Although monoxenous trypanosomatids in hu-

mans are more correlated to opportunistic parasites, our

work is pioneer in demonstrating that C. deanei is able

to infect normal mice (whithout dexamethasone treat-

ment). Our findings corroborate to the reports of [4],

who also found monoxenous tripanosomatids in a non-

immunocompromised individual though in a localized

skin lesion. Besides, our previous report demonstrated

the monoxenous trypanosomatid infection by dermal

cells isolated from skin of normal Swiss mice [5]. Nev-

erthless, our data shows that the infection of C. deanei

by dexamethasone-treated mice, although earlier proemi-

nent at the beginning of the time of infection (Figures

2(a),(b)), could not be followed longer, since the dermal

cells seemed to be degenerated (data not shown). These

results suggest that C. deanei might induce dermal cells

degeneration. Most recently, [19] reported that C. deanei

was able to induce fibroblasts lysis.

Besides the interaction of monoxenous trypanossom-

atids with vertebrate cells, the literature have also men-

tioned some results obtained from the interaction of

these trypanossomatids with invertebrate cells. Then,

[20,21], reported the colonization of Aedes aegypti mid-

gut by the endosymbiont-bearing trypanosomatid Blas-

tocrithidia culicis and C. deanei respectively.

Considering the colonization of hematophagous in-

sects by monoxenous trypanosomatids and their low host

specificity, human cases of infection with lower tryp-

anosomatids could have been largely underestimated

until now due to their morphological similarity with

Leishmania species. This emphasizes the relevance of

enzymatic characterization, whenever possible, of all

Leishmania-like parasites isolated from skin or visceral

lesions of patients with or not immunosuppression his-

tory. Taken together, these reports reinforce the idea of

the urgent need of elucidating the epidemiology of these

lower trypanosomatids that so far remains poorly known.

5. ACKNOWLEDGEMENTS

We thank FAPERJ, CNPq, UFF and FIOCRUZ for the finantial sup-

port.

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