Toxocara canis and Toxocara cati whose definitive hosts belong to the Canidae and Felidae respectively. After eating eggs containing infective T. canis larvae, ...
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Identification of excretory/secretory antigens produced by L2 stage larvae of Toxocara canis involving in induction of IgG response in mice by proteomics approach D. T. Vo1, K. P. U Le2, T. T. H. Nguyen2 and H. H. Nguyen3 1
Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam International University, Vietnam National University of Ho Chi Minh City 3 Institute of Tropical Biology, Vietnam Academy of Science and technology (VAST) 2
Abstract— Toxocara canis is a kind of round worms that parasitise Canidae. Human and other animals are their paratenic hosts in which the worm is limited in the second larval stage and therefore they can’t complete their life cycle. In these paratenic hosts, excretory/secretory antigens (E/S-Ags) produced by T. canis larvae were shown involve in regulating immune response and inducing IgG production that in turn becomes protecting molecules and biomarkers for toxocariasis sero-diagnosis. In this study, a proteomics aprroach of 2D-gel electrophoresis and a western blotting assay were applied for determination of biomarkers derived from E/S-Ags of T. canis larvae that are regconized by IgG in infected mice. Our results showed that the majority constituents of E/S-Ags have pI at pH 9-10 and the minority one have pI at pH 4-8. Western blotting analysis showed that 33.1kDa and 49.4kDa E/S-Ags were strongly recognized by specific IgG in infected mice. We conclude that these 33.1kDa and 49.4kDa constituents in E/SAgs might be potential targets for protection of the host immune system as well as biomarkers for developing diagnosis products used in veterinary and human medicine. Keywords— Toxocara canis, IgG, sero-diagnosis, paratenic host, excretory/secretory antigens. I. INTRODUCTION
Toxocariasis is a zoonosis disease found in several countries in the world. The disease is caused by infection with Toxocara canis and Toxocara cati whose definitive hosts belong to the Canidae and Felidae respectively. After eating eggs containing infective T. canis larvae, human and many other animals (defined as paratenic host) become infected. However, in these paratenic hosts, the development cycle of the nematodes is limited to the second larval stage which remains in the tissues for a long period of time [1] causing different syndromes: visceral larva migrans (VLM), ocular larva migrans (OLM), neural larva migrans (NLM) or covert or common toxocariasis (CT). In many countries including Vietnam, toxocariasis is considered as a neglected disease. However, published works showed that 38.8% of school children were infected in Brazil [2] and in Iran the infection level was 15.8% [3]. In the USA 13.9% of people >6 years old age were infected
[4]. In Vietnam around 30% of patients who visited hospitals were infected [5]. T. canis larvae produce antigens so called E/S-Ags that have been intensively studied previously [6-8]. However, how E/S-Ags involve in interaction between the worms and the defense system of infected hosts is still unclear. Some recent studies have shown that the E/S-Ags contribute to and influence on regulation of the host immune response. An in vitro study showed that mucins in E/S-Ags regulate immune system by inducing TH2 cytokine production (IL-4, IL-5, IL-6, IL-10 and TGF-b) and inhibiting TH1 and TH17 responses in mouse spleenocytes [9]. Foxp3 expressing regulatory T cells (Treg cells), which play critical role in dampens immune response, were shown increase in liver and spleen of infected mice [10]. In contrast, IgG antibodies produced by host immune plasma cells after infection are likely important in term of neutralizing these E/S-Ags to prevent their activities. In addition, the specific IgG antibodies have been be considered as useful biomarkers for sero-diagnosis of toxocariasis in both animals and human [11]. Nevertheless identification of constituents in E/S-Ags that are regconized by IgG in infected host might help to understand inside into mechanisms that the hosts govern to protect themselve against T. canis infection and biomarkers for sero-diagnosis development. In this study, by using a proteomics approach of 2D-gel electrophoresis and a western blotting assay we attempt address this issue. II. MATERIALS AND METHODS
A. Animals Swiss outbred mice (albino) (20g in weight) were purchased from the Pasteur Institute in Ho Chi Minh City, Vietnam. Pups (2-3 months old) were purchased in Ninh Thuan Province, Vietnam. All animals were then kept in our laboratory during experiments.
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B. Preparation of T. canis larvae and E/S-Ags T. canis larvae were prepared as shown elsewhere [12] in our group. The larvae were cultured at a density of 1000 larvae/ml in DMEM medium in the presence of penicillin (100U/ml) and streptomycin (100ug/ml) at 37oC in a 5% CO2 atmosphere. Culture medium was harvested every four weeks. E/S antigens in the culture medium were concentrated by centrifugation with the Amicon centrifugal filter units (10kDa cut off). A cocktail of protease inhibitors (2mM AEBSF, 0.3 µM Aprotinin, 130 µM Bestatin, 1 mM EDTA, 14 µM E-64, 1 µM Leupeptin) (Sigma) was added to E/SAgs which were then stored at -20oC until use. The protein concentration was determined by Bradford assay. C. Infection of mice with T. canis larvae Mice were intraperitoneally injected with 1000 larvae of T. canis [13]. Four weeks later, Blood from these animals was collected and kept at 2-8oC overnight. Sera were then harvested by centrifugation and kept at -20oC until use. Sera obtained from untreated animals were used as negative control. Toxocariasis animals were confirmed by using a dot blot assay with E/S antigens produced by T. canis larvae [12]. D. SDS-PAGE E/S-Ags (1µg and 10µg/well) were subjected to SDSPAGE under reducing condition on a 10% gel. Molecular weight standards were purchased from Pierce Biotechnology PageRuler broad range unstained protein ladder. For protein detection, coomassie blue staining was used. Protein molecular weight was estimated by using Quantity One software v4.6.3 (Bio-Rad Laboratories).
1hr with T. canis infected mouse sera (pooled form 3 infected mice) diluted 1:1000 in TBST and then washed with TBST buffer. The membrane was incubated with a 1:5000 diluted goat anti-mouse antibodies conjugated with HRP (Santa Cruz Biotechnology) for 1hr and then washed with TBST buffer. For detection of antigen-antibody immune complexs, the membrane was incubated in enhanced chemiluminescence reagent for 5min and then scanned on C-digit blot scanner (LI-COR Biosciences) to obtain images which were analyzed by ImageStudioLite programme. III. RESULTS
A. Analysis of E/S-Ags of T. canis larvae by SDS-PAGE and 2D-gel electrophoresis After 4 weeks of culture of T. canis larvae, E/S-Ags were harvested and concentrated as showed in Materials and methods. The E/S-Ags were then subjected to SDS-PAGE (Fig. 1A) and then 2D-gel (Fig. 1) analysis. SDS-PAGE presented in Fig. 1A showed that the majority of E/S constituents was made off 33.1kDa, 97.2kDa and 105.5kDa proteins. 2D-gel analysis showed that these bands mostly have pI at pH 9-10 (Fig. 2) and likely contain one spot in each. There are four spots separated from 67.8kDa band at pH 6, six spots from 49.4kDa band at pH 7-10. We also found some spots derived from bands which have MW
