Resumen

Palabras clave: inmunosupresión - ratas - Strongyloides venezuelensis - estrongiloidiasis - transmisión transmamaria.

Con el fin de investigar los niveles de anticuerpos y antígenos en muestras de lavado

broncoalveolar (LBA) y suero, ratas hembras inmunosuprimidas o no inmunosuprimidas e

infectadas experimentalmente con Strongyloides venezuelensis y sus crías fueron utilizadas. Para

ello, el ensayo inmunoabsorbente ligado a enzimas (ELISA) se realizó usando extractos alcalinos

de larvas y anti-L3 anticuerpo policlonal producido en conejos. Los resultados mostraron que las

crías de las hembras inmunosuprimidas fueron significativamente mayores para la detección de

IgG en el suero y LBA. Por otra parte, la detección de antígeno en muestras de suero también fue

más evidente en las crias de ratas inmunosuprimidas. Así pues, aquí reforzamos la ruta

transmamaria de la infección por Strongyloides en el modelo experimental de infección.

ORIGINAL ARTICLE / ARTÍCULO ORIGINAL

TRANSMAMMARY TRANSMISSION OF STRONGYLOIDIASIS IN

IMMUNOSUPPRESSED RATS

TRANSMISIÓN TRANSMAMARIA DE ESTRONGILOIDIASIS EN RATAS

INMUNOSUPRIMIDAS

1* 1 1

Ana Lúcia Ribeiro Gonçalves , Claudio Vieira Silva , Edson Fernando Goulart Carvalho ,

2 1

Marlene Tiduko Ueta & Julia Maria Costa-Cruz

Abstract

In order to investigate the levels of antibodies and antigen in sera and in broncho-alveolar lavage

fluid (BALF) samples, female rats immunosuppressed or non-immunosuppressed female rats and

rats experimentally infected with Strongyloides venezuelensis and their young offspring were

used. For this, enzyme-linked immune-sorbent assay (ELISA) was conducted using alkaline

parasite extracts and anti-L3 polyclonal antibody produced in rabbits. Findings revealed that

young offspring from immunosuppressed females presented significantly higher positivity for

IgG detection in serum samples and in BALF. In addition, antigen detection in serum samples was

also more evident in offspring from immunosuppressed rats. Thus, we reinforce the trans-

mammalian route of Strongyloides infection in experimental model infection.

Keywords: immunosuppression - rats - Strongyloides venezuelensis - strongyloidiasis - transmammary transmission.

Suggested citation: Gonçalves, ALR, Silva, CV, Carvalho, EFG, Ueta, MT & Costa-Cruz, JM. 2013. Transmammary

transmission of Strongyloidiasis in immunosuppressed rats. Neotropical Helminthology, vol. 7, n°2, jul-dec, pp. 195 - 200.

1 2

Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, MG; Laboratório de Parasitologia, Universidade Estadual de Campinas, SP, Brazil.

E-mail: analuciabio@yahoo.com.br

Neotrop. Helminthol., 7(2), 2013

2013 Asociación Peruana de Helmintología e Invertebrados Afines (APHIA)

ISSN: 2218-6425 impreso / ISSN: 1995-1043 on line

195

INTRODUCTION

Strongyloides venezuelensis Brumpt, 1934 is a

rodent parasite that has been extensively used as

an experimental model to study human infection

(Baek et al., 1998; Gonçalves et al., 2010). In

experimental infections, infective larvae from S.

venezuelensis migrate to lungs before

establishing in duodenal mucosa. Thus, its

migration in the rodent host is similar to that of

Strongyloides stercoralis Bavay, 1876 in

humans (Negrão-Corrêa et al., 2003).

Although transplacental transmission of larvae

has not been reported, the transmammary

transmission can occur if the female acquire

st rd

infection between the 1 and 3 day post-birth.

Vertical transmission of larvae is a major

pathway in the life cycle of several species of

Strongyloides, including Strongyloides ratti

Sandground, 1925 and S. venezuelensis in rats

(Katz, 1969; Nolan & Katz, 1981). On the other

hand, evidence of vertical transmission in

human with S. stercoralis is absent (Shoop et al.,

2002).

In addition, in murine models Strongyloides sp.

induces the production of cytokines such as IL-

3, IL-4 and IL-5, with subsequent secretion of

specific IgM, IgG, IgG1, IgA and IgE, essential

to the elimination of the parasite (Onah & Nawa,

2000; Rodrigues et al., 2009). However, little

attention has been given to immune response in

immunosuppressed rats and infected with S.

venezuelensis. For this reason, IgG antibodies

and the antigen detection at vertical transmission

can influence the immune response to the

parasite during its implantation and/or expulsion

from the host.

The present study investigated the levels of

antibodies and antigen in sera and

bronchoalveolar lavage fluid (BALF) of female

rats immunosupressed or not and experimentally

infected with S. venezuelensis and their

offspring.

Animals

Two female Wistar rats weighing 100-120g,

bred at the Instituto de Ciências Biomédicas,

Universidade Federal de Uberlândia (ICBIM-

UFU), were used in the experiments. During this

study, the rats were maintained at the animal

facilities of the Centro de Experimentação e

Utilização de Animais, UFU, MG, Brazil, fed

with laboratory ration, and tap water to drink ad

libitum. All experiments were conducted in

accordance with animal ethics guidelines and

were approved by the Comitê de Ética na

Utilização de Animais of the Universidade

Federal de Uberlândia (CEUA-UFU 096/10).

Parasites

Strongyloides venezuelensis third-stage

infective larvae (L ) were obtained from

charcoal cultures of infected rat faeces. The

cultures were stored at 28°C for 48 h, and the

infective larvae were collected and concentrated

using the Rugai method (Rugai et al., 1954). The

pellet (3mL) from the conical cup were diluted

10 times in distilled water and larvae were

counted using stereomicroscopy. Larvae were

quantified according to the following formula: N

x 10 x 60 (N = the number of larvae counted, 10 =

dilution factor and 60 = correction factor when

using pipettes of 50 uL). For each infection,

1500 S. venezuelensis L larvae were inoculated

subcutaneously in rat abdominal cavity.

Immunosuppression and infection of female rats

before conception

Before the infection, the immunosuppressed

-1

female rat received 5 ug·mL of dexamethasone

disodium phosphate, in water, for 5 days as

described previously (Romand et al., 1998). For

the infection the two female rats were inoculated

subcutaneously in the abdominal cavity with

1500 S. venezuelensis L , after conception.

Serum and brochoalveolar lavage fluid samples

On the 76 day post-infection, two female rats

and their offspring (six of female non

immunosuppressed and six of female

immunossuppressed) were anesthetized with 60

Gonçalveset al.

Transmammary Transmission of Strongyloidiasis

196

MATERIALS AND METHODS

IgG (Sigma, Chemical Co., St Louis, MO) at the

ideal dilution of 1:8000 for 45 min at 37ºC. The

reaction was revealed by adding the enzyme

substrate (0.03% H O and o-phenylenediamine

2 2

[OPD] in 0.1M citrate-phosphate buffer, pH 5.0)

and incubated for 15 min at room temperature.

The reaction was stopped by adding 2N H SO

2 4

and the optical density (OD) was determined at

492 nm in a plate reader (Titertek Multiskan;

Flow Laboratories, McLean VA). BALF with EI

> 1.0 were considered positive.

Measurement of antigen in serum and BALF

samples

For serum samples, polystyrene microplates

were coated overnight, as previously described

(Gonçalves et al., 2012). Sera with EI > 1.0 were

considered positive.

For BALF samples, high-binding microtitre

plates (Corning-Costar; Laboratory Sciences

Company, New York, NY) were coated

-1

overnight at 4ºC with 50µL·well of IgG anti-S.

-1

venezuelensis (40µg·mL ) in 0.06 M carbonate-

bicarbonate buffer (pH 9.6). Plates were washed

three times for 5 minutes with PBS containing

0.05% Tween 20 (PBS-T). After washing, plates

were incubated with undiluted bronchoalveolar

lavage fluid samples for 2 h at 37ºC and

subsequently with the secondary antibody

consisting of peroxidase-labeled rabbit anti-S.

venezuelensis IgG at the optimal dilution of 1:40

for 45 min at 37ºC. The reaction was revealed as

described above. BALF with EI > 1.0 were

considered positive.

Statistical analysis

Each experiment was performed twice.

Statistical variations were analyzed using

Student't test followed by Mann-Whitney test.

The criterion for statistical significance was set

at P < 0.05.

Parasite-specific IgG was measured in serum

samples from infected immunosuppressed

female rats and offspring on the 76 day post-

infection, and these values were then compared

-1 -1

mg·Kg ketamine and 7 mg·Kg xilazine s.c, and

blood samples were collected by cardiac

puncture. Blood was then centrifuged, and the

serum was stored at -20°C. Subsequently, the

chest cavity of each animal was carefully

opened, and the trachea was exposed and

catheterized. The catheter was tied in place, and

sterile phosphate buffered saline (PBS)/sodium

citrate (0.5%) was infused in three 1 mL

aliquots. The BALF was collected and placed on

ice, and then the aliquots were stored at -20°C.

Alkaline parasite extracts

Alkaline extracts were prepared using 300,000

S. venezuelensis larvae, as previously described

(Machado et al., 2003). The protein content of

the supernatant was determined by the Lowry

method (Lowry et al.,1951).

Production of immune serum and conjugate

Two rabbits were immunized for anti-S.

venezuelensis immune serum production.

Immunization and anti-S. venezuelensis specific

IgG purification and horseradish peroxidase

conjugation was carried out as previously

described (Gonçalves et al., 2010).

Measurement of specific IgG in serum and

BALF samples

For serum samples, polystyrene microplates

were coated overnight, as previously described

(Gonçalves et al., 2012). Results were arbitrarily

expressed as ELISA index (EI), previously

reported in research on human strongyloidiasis

according to the following formula: EI = OD

sample/cut off, where the cut off was established

as the mean OD of three negative control sera

plus two standard deviations. Sera with EI > 1.0

were considered positive.

For BALF samples, high-binding microtitre

plates (Corning-Costar) were coated overnight

-1

at 4 oC with 50 µL·well of S. venezuelensis

-1

alkaline extract (10 µg·mL ) in 0.06 M

carbonate-bicarbonate buffer (pH 9.6). Plates

were washed three times with PBS-T. After

washing, plates were incubated with undiluted

BALF samples for 45 min at 37ºC and

subsequently with the secondary antibody

consisting of peroxidase-labeled goat anti-rat

197

Neotrop. Helminthol., 7(2), 2013

RESULTS

with those found for infected non

immunosuppressed female rats and offspring

(Fig. 1A). Serological assays demonstrated that

2 / 6 ( 3 3 . 3 % ) o f f s p r i n g o f a n o n

immunosuppressed female rat and 5/6 (83.3%)

offspring of an immunosuppressed female rat

were seropositive to S. venezuelensis. Levels of

IgG antibodies to S. venezuelensis were higher in

immunosuppressed female rat (Fig. 1A). Levels

of IgG antibodies to S. venezuelensis in BALF

samples demonstrated that 1/6 (16.7%)

offspring of a non immunosuppressed female rat

and 6/6 (100%) pups of immunosuppressed

female rat were positive. These results were

significantly higher in the offspring of an

immunosuppressed female rat (P < 0.01) (Fig.

1B).

When ELISA was used for the detection of S.

venezuelensis antigen in serum samples (Fig.

1C), 3/6 (50%) offspring of a non

immunosuppressed female rat and 3/6 (50%)

offspring of an immunosuppressed female rat

were seropositive. In BALF samples, levels of

a n t i g e n w e r e d e t e c t e d o n l y i n

immunosuppressed female rat and in an

offspring from a non immunosuppressed female

(Fig 1D).

198

Figure 1. Levels of S. venezuelensis specific IgG detection in serum (A) and BALF (B) samples, using alkaline parasite extract

and levels of S. venezuelensis antigen detection in serum (C) and BALF (D) samples from non immunosuppressed or

immunosuppressed female rats and their offspring. Dashed lines indicate the detection limit for the antibody or antigen. **

p<0.01.

Gonçalveset al.

Transmammary Transmission of Strongyloidiasis

199

DISCUSSION

Vertical transmission of larvae, especially

through the transmammary route, is a major

pathway in the life cycle of several species of

Strongyloides, including Strongyloides ransomi

Schwartz & Alicata,1930; in swine (Moncol &

Batte, 1966; Gomes, 2009), Strongyloides

papillosus Wedl,1856 in cattle and sheep (Lyons

et al., 1970; Andrade, 2010), Strongyloides

westeri Ihle, 1917 in horses (Lyons et al., 1969),

and S. ratti and S. venezuelensis in rats (Katz,

1969; Nolan & Katz, 1981; Pereira, 2008).

Moreover, many studies verified that vertical

transmission through prenatal pathways does

not occur in dogs infected with S. stercoralis. On

the other hand, vertical transmission of this

parasite through transmammary routes is

possible in dogs (Shoop et al., 2002).

Our approach was meant to disclose the

possibility of S. venezuelensis vertical

transmission through transmammary route in

non immunossupressed or immunosuppressed

rats. Our findings revealed that the offspring

from immunosuppressed females presented

significantly higher positivity for IgG detection

in serum samples. In addition, antigen detection

in serum samples was also more evident in

offspring from immunossupressed rats.

Previous studies showed that antigen detection

in serum samples may not be a good approach

for strongyloidiasis diagnosis, because the

detection rates were below the cut-off limits for

normal and immunocompromised rats during

the kinetics of infection (Gonçalves et al., 2012).

When BALF samples were used, it was observed

that the levels of specific IgG may be an

alternative to study vertical transmission

through transmammary route in rats. However,

the antigen detection may not be a good assay for

these analyses.

Previous studies showed that continued cycling

of larvae during disseminated strongyloidiasis

could have the same effect as those parasitic

species that use arrested development as a tactic

to keep larvae available for the time period when

the host becomes gravid (Shoop et al., 2002).

The limitation of the work focuses on control of

clutches making it difficult to extend the results

to different hosts. Commonly the dissemination

occurs in immunosuppressed host that reinforce

the approach of the present study. In conclusion,

we reinforce the importance of transmammarian

route of Strongyloides infection in experimental

animal infection.

We are grateful to the Brazilian Research

Council (CAPES), Fundação de Amparo à

Pesquisa de Minas Gerais (FAPEMIG) and

Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq).

ACKOWLEDGEMENTS

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Correspondence to author/ Autor para

correspondencia:

Ana Lúcia R. Gonçalves

Av. Pará 1720, Uberlândia, 38400-902, Minas

Gerais, Brazil. Tel.: +55 34 3218 2600.

E-mail / Correo electrónico:

analuciabio@yahoo.com.br

Received April 23, 2013.

Accepted June 23, 2013.

Gonçalveset al.

Transmammary Transmission of Strongyloidiasis