Microplate Agglutination Test for Canine Brucellosis Using Recombinant Antigen-Coated Beads

Yussaira Castillo,Yasuyuki Endo,Suk Kim,Yui Kimura,Masato Tachibana,Takashi Shimizu,Masahisa Watarai,Yasuaki Ichikawa,Kenta Watanabe

doi:10.1155/2014/348529

Abstract

Brucella canis, a facultative intracellular pathogen, is the causative agent of canine brucellosis. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods, including agglutination and gel diffusion tests. In this study, four recombinant antigens, heat shock protein 60, rhizopine-binding protein, Cu-Zn superoxide dismutase, and hypothetical protein (Ag 4), were constructed. These antigens were coated on latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. All recombinant antigens showed specific reaction with sera from B. canis-infected dogs in Western blotting. In a microplate agglutination test, mixing sera from B. canis-infected dogs, but not sera from B. canis-free dogs, with single or multiple antigens-coated latex beads produced clear agglutination. Moreover, the antigen-coated latex beads did not show nonspecific agglutination in hemolyzed serum samples. A survey of canine serum samples conducted by the microplate agglutination test using single antigen-coated latex beads showed that this method would be useful in the serological diagnosis of canine brucellosis. Further investigations using more serum samples are required to confirm the usefulness of our method.

Highlights

Brucellosis is one of the world’s major bacterial zoonosis and an important cause of a serious debilitating disease in humans; further, it is the cause of abortion and sterility in animals
This paper describes the development of latex beads coated with purified recombinant versions of these antigens and demonstrates the usefulness of these antigen-coated latex beads in the serological diagnosis of canine brucellosis
To identify B. canis-infected sera, we performed tube agglutination test (TAT) on canine serum samples (n = 743) collected from dogs consecutively admitted to animal hospitals in Japan by the hospital staff

Summary

Introduction

Brucellosis is one of the world’s major bacterial zoonosis and an important cause of a serious debilitating disease in humans; further, it is the cause of abortion and sterility in animals. Canine brucellosis is widely distributed around the world and this is an important disease due to the economic losses in animal production and its risks for human health [1]. Diagnosis of brucellosis is primarily based on bacteriological and serological tests [2]. Agglutination tests often give false positive results because of cross-reactions with other pathogens [2]. A general strategy for eliminating cross-reactions is to use purified antigen with unique epitopes in the serological tests. Bacterial cell wall antigens of B. canis can be prepared by hot saline extraction and are reportedly useful in serological diagnosis [5]. We reported the characterization of crude hot saline extracts and identified three antigens, namely, rhizopine-binding protein (RBP, former assigned ribose ABC transporter), Cu-Zn superoxide dismutase (SOD), and a hypothetical protein (Ag 4) [6]

Methods

Results

Conclusion