Production and characterization of monoclonal antibodies against horse immunoglobulins useful for the diagnosis of equine diseases.

Monoclonal antibodies (MAbs) against horse IgG were produced by immunizing Balb/c mice with purified horse IgG and were characterized in indirect ELISA versus purified immunoglobulins from donkey, cow, buffalo, sheep, pig, and chicken. Three MAbs (1B10B6C9, 1B10B6C10, 1B10B6E9) reacted only with horse and donkey IgG and IgM and, in western blotting, were specific for the Fc fragment of equine IgG. MAb 1B10B6E9 was used in chemiluminescent immunoblotting assay for the diagnosis of dourine and in indirect immunofluorescence assay (IFA) for the diagnosis of African horse sickness and dourine.

IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of low molecular weight molecules.

Diagnosis and control of dourine is strongly based on serological evidence, but knowledge of the humoral response of horses during infection is limited. In this study we developed a chemiluminescent immunoblotting (cIB) assay to characterise the Trypanosoma equiperdum antigen pattern recognised by IgGs from naturally or experimentally dourine-infected horses and analyse the kinetics of IgG humoral response following the infection. One compounding factor is that sera from uninfected animals often cross-react with T. equiperdum antigens. Development of the cIB assay was based on the hypothesis that serum IgGs from healthy and infected animals recognise different T. equiperdum antigen patterns. We used sera from 8 naturally infected horses which had recovered from Italian outbreaks and 2 experimentally infected mares. In addition, sera from 10 healthy control animals, eight of which were CFT positive but IFA negative for dourine, were collected from disease free regions. Sera were compared by the complement fixation test (CFT), indirect immune fluorescence (IFA) and the cIB assay. cIB analysis revealed that IgGs from infected horses, in contrast to IgGs from healthy horses, specifically recognise a T. equiperdum antigenic profile with low molecular weight bands ranging between 16 and 35 kDa. A time course experiment indicated that IgGs specific for the 16-35 kDa parasite protein fraction appear 17 days post-infection. The cIB assay confirmed all ten infected animals as positive and all controls as negative. This study demonstrated that analysis of IgGs by cIB can provide clear confirmation of trypanosome infection in horses, suggesting that this technique can be applied as a confirmatory serological test for dourine infection.

Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses.

Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses. Apparent test specificities for the B. equi, B. caballi, T. equiperdum, and B. mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively. Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B. equi, B. caballi, T. equiperdum, and B. mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively. The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use. Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera.

Antigenic variation in Trypanosoma equiperdum.

Trypanosoma equiperdum is an African trypanosome that causes dourine in horses. Like the other African trypanosomes, T. equiperdum escapes elimination by the immune system of its host by using an elaborate system of antigenic variant. The trypanosomes are covered by a coat consisting of a single protein called the variable surface glycoprotein (VSG) that acts as the major trypanosome immunogen. As the host responds to one VSG, trypanosomes covered with another VSG become dominant. There is a loose order of appearance of these VSG during the infection. The factors that affect the timing of VSG expression and the effective size of the VSG repertoire in T. equiperdum are reviewed. The VSG genes are generally activated by a process of duplicative transposition involving the duplication of a silent VSG gene and inserting a copy of the gene into an expression site. The order of VSG expression is related to the amount of homology between the silent gene and the expression site. The genes expressed late in infection lack extensive homology with the expression site and depend on homology with the gene in the expression site. The genes coding for VSG expressed late in infection are hybrid genes because of this mode of transfer. This transfer mechanism allows the trypanosome to create complex VSG genes from parts of several different silent genes that are each pseudogenes. Additionally, data are presented showing that only a limited portion of the VSG is actually seen by the host immune system. These factors indicate that the effective VSG repertoire is greater than the number of VSG genes in the trypanosome genome.