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.

Development and validation of a competitive ELISA kit for the serological diagnosis of ovine, caprine and bovine brucellosis.

A competitive ELISA (Brucella-Ab c-ELISA) was standardized and validated for the detection of Brucella antibodies in cattle, sheep and goat sera using a monoclonal antibody (MAb 4B5A) produced against Brucella melitensis biotype 2. The specificity and sensitivity of the assay were 100% to a 67.5% cut-off point (B/Bo%). When compared with an indirect ELISA, the Brucella-Ab c-ELISA did not demonstrate cross-reactions when testing positive sera for antibodies to some Enterobacteriaceae. A comparison was made between the Brucella-Ab c-ELISA and the complement fixation and Rose Bengal tests. Results demonstrated that the Brucella-Ab c-ELISA is a valuable tool for the serological diagnosis of bovine and ovine/caprine brucellosis.

Production and characterisation of monoclonal antibodies specific for Escherichia coli O157:H7.

Seven monoclonal antibodies (MAbs) specific for Escherichia coli O157:H7, one of the major causes of haemorrhagic colitis in humans, were produced by immunising Balb/c mice with the strain E. coli O157:H7. These monoclonal antibodies do not cross-react with other bacteria such as Salmonella enterica serovar Typhimurium, E. coli O14, E. coli JM109, S. enterica serovar Enteritidis, S. panama, S. saintpaul, S. derby, S. muenchen, S. bredeney, S. hadar, Yersinia enterocolitica, Proteus vulgaris, Shigella flexneri, Listeria ivanovii, L. monocytogenes 13M, L. innocua, Enterobacter cloacae, E. agglomerans, E. amnigenus, Citrobacter freundii, Escherichia fergussoni or Klebsiella pneumoniae. Of the seven MAbs obtained, MAb 8B8C3 was selected to prepare a high-sensitivity sandwich ELISA method specific for O157:H7.