Short communicationDevelopment of ELISA test for determination of the level of antibodies against Rhodococcus equi in equine serum and colostrum
Introduction
Rhodococcus equi infection occurs worldwide and is one of the major causes of losing foals between 1st and 6th month of age (Dawson et al., 2010). Despite many studies, mechanisms of foals’ immunity in rhodococcosis have not been understood so far. Many evidences indicate that virulence of R. equi is related to a large plasmid, which bears genes encoding nine virulence-associated proteins (Vap). Highly immunogenic protein VapA, with molecular weight 15–17 kDa seems to play a crucial role. R. equi produces also catalase, phospholipase C and cholesterol oxidase E, known together as ‘equi factor’. It is believed that severity of the disease is related to the pathogen virulence and the susceptibility of the host. Therefore the scope of available preventive methods is narrow and the efficacy of so far developed vaccines is still insufficient (Takai et al., 1996, Dawson et al., 2010).In the last three decades several serological tests have been developed for the detection of specific antibodies against R. equi (Takai et al., 1985, Takai et al., 1996, Prescott et al., 1996, Martens et al., 2002, Giguere et al., 2003). The tests most commonly used are ELISA-6939, ELISA-33701 and ELISA-VapA (Anzai et al., 1997, Cuteri et al., 2003, Hooper-McGrevy et al., 2005). Other ELISA tests are used seldom (Hietala et al., 1985, Cauchard et al., 2004, Cauchard et al., 2006, Lopez et al., 2002, Taouji et al., 2002, Hooper-McGrevy et al., 2003). Western blotting showed that these tests usually detect antibodies against VapA (Prescott et al., 1996, Takai et al., 1996, Cauchard et al., 2004). Two tests – ELISA-6939 and ELISA-33701 (Takai et al., 1985, Takai et al., 1986) – are based on antigens obtained by extraction from non-virulent strain R. equi ATCC 6939 and virulent strain ATCC 33701 using Tween 20 detergent. Prescott's ELISA-VapA utilizes an antigen extracted with Triton X-114 detergent in which the VapA is a dominant component (ELISA-VapA) (Prescott et al., 1996, Hooper-McGrevy et al., 2001, Hooper-McGrevy et al., 2005). The ELISA-California test (Hietala et al., 1985) is based on lyophilized supernatant of the culture of various R. equi strains. Moreover, other tests based on autoclave-resistant antigen, recombinant VapA, soluble antigen of R. equi, synthetic Vaps: A, D, F and G, whole bacterial cells or synthetic protein PN11-14 are also known (Taouji et al., 2002, Hooper-McGrevy et al., 2003, Lopez et al., 2003, Cauchard et al., 2004, Cauchard et al., 2006, Phumoonna et al., 2006).The application of serological tests to the diagnostics of rhodococcosis is questionable. Nonetheless, serological tests are necessary to evaluate humoral immune response and are most commonly used in the studies on the efficacy of vaccines. The efficacy of a vaccine can be defined as the ability to induce antibodies in the vaccinated mare that are passed via colostrum to a newborn foal to provide passive protection. Serological tests can also be used for quantitative assessment of both anti-R. equi hyperimmune plasma and the results of plasma administration. In both cases, the quantity of antibodies and their kinetics must be investigated (Becu et al., 1997, Varga et al., 1997, Hooper-McGrevy et al., 2001, Hooper-McGrevy et al., 2005, Cauchard et al., 2004).
Hence this study was conducted to develop and standardize ELISA test for the assessment of the level of antibodies against R. equi in equine serum and colostrum.
Section snippets
Bacterial strain and antigen preparation
The virulent R. equi strain (AA 15/03) used in the study was obtained from purulent pulmonary lesions of the 8 week-old Thoroughbred foal. This field isolate was identified on the basis of its biochemical properties using API Coryne and API ZYM tests (bioMériuex, Marcy l’Etoile, France). In order to detect ‘equi factor’ CAMP (Christie, Atkins, and Munch-Peterson) test was performed with Staphylococcus aureus strain (ATCC-25923). Gene vapA encoding VapA protein was detected by the method
Results and discussion
The purpose of the study was to develop the ELISA test for quantitative determination of the level of an antibodies against R. equi in the horse serum and colostrum. Therefore, no attempts to determine the cut-off and specificity and sensitivity were undertaken. The test was standardized on 175 sera obtained from adult horses kept on rhodococcosis-free and endemic farms using Western blot as a reference method (Wright et al., 1993).Due to the substantial similarity in the construction of the
Conflict of interest statement
The authors have not declared any conflict of interest.
Acknowledgements
This study was partially financed by Polish State Committee for Scientific Research 2 P06K 006 27.The authors would like to thank Prof. Shinji Takai from Kitasato University in Japan for his invaluable help in performing the study, Dr. Felix Toka for his support in laboratory analyses and Dr. Michał Czopowicz for the assistance in performing statistical analysis and preparing English version of the manuscript.
References (25)
- et al.
Comparison of tracheal aspiration with other tests for diagnosis of Rhodococcus equi pneumonia in foals
Vet. Microbiol.
(1997) - et al.
Immunoprophylaxis of Rhodococcus equi pneumonia in foals
Vet. Microbiol.
(1997) - et al.
Foal IgG and opsonizing anti-Rhodococcus equi antibodies after immunization of pregnant mares with a protective VapA candidate vaccine
Vet. Microbiol.
(2004) - et al.
Immunogenicity of synthetic Rhodococcus equi virulence-associated protein peptides in neonate foals
Int. J. Med. Microbiol.
(2006) - et al.
A serological survey of Rhodococcus equi infection in foals in central Italy: comparison of two antigens using an ELISA test
Comp. Immunol. Microbiol. Infect. Dis.
(2003) - et al.
Current understanding of the equine immune response to Rhodococcus equi. An immunological review of R. equi pneumonia
Vet. Immunol. Immunopathol.
(2010) - et al.
Virulence-associated protein-specific serum immunoglobulin G-isotype expression in young foals protected against Rhodococcus equi pneumonia by oral immunization with virulent R. equi
Vaccine
(2005) - et al.
Development of an ELISA for the diagnosis of Corynebacterium pseudotuberculosis infections in goats
Vet. Microbiol.
(2001) - et al.
Analysis of anamnestic immune responses in adult horses and priming in neonates induced by a DNA vaccine expressing the vapA gene of Rhodococcus equi
Vaccine
(2003) - et al.
Serum antibody responses of foals to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi
Vet. Microbiol.
(1996)
Serum and mucosal antibodies of infected foals recognized two distinct epitopes of VapA of Rhodococcus equi
FEMS Immunol. Med. Microbiol.
Prevention of Rhodococcus equi pneumonia of foals using two different inactivated vaccines
Vet. Microbiol.
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