Toxocara canis: Effect of inoculum size on pulmonary pathology and cytokine expression in BALB/c mice

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Abstract

Infection of mice with Toxocara canis results in pulmonary inflammation and the induction of a Th2 type of immune response. The aim of this study was to determine whether the effect of infection with this nematode depends on the inoculum size. Results indicate that mice infected with either a high or a low inoculum size showed, in a dose-dependent manner, pulmonary inflammation with eosinophil infiltration, increased levels of total IgE, and Toxocara-specific IgG1 that persisted up to 60 days p.i. Relative quantification of cytokine expression in lungs of mice infected with different doses showed proportional increased expression of the IL-4, IL-5, and IL-10 transcripts, whereas the expression of the IFN-γ transcript was not different from that of uninfected controls. These results indicate that infection of BALB/c mice with T. canis results in chronic pulmonary inflammation and a dominant Th2 type of immune response, independent of the inoculum size.

Introduction

Toxocara canis is a roundworm of dogs and the causative agent of toxocariasis (Kayes, 1997). Humans and mice can also become infected with T. canis after ingestion of embryonated eggs present in soil contaminated with dog feces. In these paratenic hosts, the larvae do not develop to the adult stage but persist in tissues as the larval stage for many years (Schantz, 1989). Once the eggs are ingested, the larvae hatch, penetrate the small intestine and migrate to different tissues in the body inducing inflammatory responses. Migration of T. canis larvae can lead to a syndrome known as visceral larva migrans (VLM). Symptoms of VLM include fever, hepatosplenomegaly, and respiratory distress such as wheezing, coughing, and episodic airflow obstruction (Feldman and Parker, 1992, Taylor et al., 1988). Other symptoms include eosinophilic pneumonia (Loeffler’s pneumonia) that bears a clinical resemblance to the pulmonary inflammatory responses observed in asthmatic patients. Immunological features of toxocariasis include eosinophilia and increased serum IgE levels (Obwaller et al., 1998, Sugane and Ohshima, 1984).

Using murine models, several groups have shown that infection with T. canis results in pulmonary inflammation characterized by the infiltration of eosinophils, neutrophils, and lymphocytes (Buijs et al., 1994, Kayes et al., 1987, Pinelli et al., 2001). Recently, we reported on airway hyper-responsiveness, pulmonary inflammation, and increased levels of IgE that persist in BALB/c mice for months after a single T. canis infection (Pinelli et al., 2005).

Early studies have shown that inoculum size is a significant factor determining the proportional recovery of larvae from different organs (Kayes and Oaks, 1976). Studies on cerebral toxocariasis have shown that the infective dose influences not only the larval burden in brain but also the behavior of Toxocara-infected mice (Cox and Holland, 1998). Information regarding the effect of T. canis inoculum size on pulmonary inflammation and type of immune response induced is limited. In the present study, we monitored the effect of different T. canis inoculum size on antibody production, pulmonary pathology, and cytokine expression in lungs of BALB/c mice.

Section snippets

Parasites and experimental infection

Toxocara canis adult worms were recovered from naturally infected dogs, after routine deworming using antihelminthic treatment. Eggs were collected from the uteri of female worms and were allowed to embryonate in 0.1 M H2SO4 in the dark at room temperature for 4–6 weeks. Embryonated eggs were stored in 0.1 M H2SO4 at 4 °C until use.

Specified pathogen-free female BALB/c mice (5–6 weeks) were obtained from Harlan Netherlands BV (Horst, The Netherlands). Mice were housed in Macrolon III cages with

Histology

Infection of BALB/c mice with 100 embryonated eggs leads to moderate perivascular infiltration, mainly consisting of esosinophils and lymphocytes, slight peribronchiolitis with the same pattern of inflammatory cells, and minimal alveolitis containing large macrophages, eosinophils, and microhaemorrhages (Table 2). We also observed moderate hypertrophy of the bronchiolar goblet cells with PAS-positive staining indicating mucus production (data not shown). The observed changes persisted up to 60

Discussion

We have previously shown that infection of BALB/c mice with 1000 T. canis embryonated eggs results after 7 days in marked histological changes in the lungs and increased levels of total IgE in serum (Pinelli et al., 2001). The observed pulmonary inflammation has been shown by us and others to persist for months after infection (Buijs et al., 1995, Pinelli et al., 2005). In the present study, we aim at determining whether the T. canis inoculum size has an effect on the type of immune response

Acknowledgments

The authors thank H. Strootman, P.J. van Schaaik, and D. Elberts for their technical assistance.

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