Entwicklung und Validierung eines kompetitiven Enzym-gekoppelten Immunadsorptionstests für den Nachweis von Antikörpern gegen Theileria annulata im Serum von Rindern

In previous studies, Theileria annulata surface protein (TaSP) was identified as an immunodominant antigen and successfully used to develop a recombinant- protein-based indirect enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antibodies in serum of T. annulata-infected animals. To increase the specificity, a competitive ELISA (cELISA) was developed using recombinant TaSP antigen and a monoclonal antibody (1C7) specifically binding to TaSP. Since the cELISA accurately differentiated T. annulata-infected from uninfected animals, a study was performed to analyse the suitability of the cELISA in the field. For this, 230 sera with unknown status from different governorates in the north of Iraq were analysed using both the indirect and competitive ELISA and were compared. There was a significant (p correlation (r=0.556) between the tests, whereby the cELISA detected more sera as negative (44/230) compared to the indirect ELISA (21/270). Accordingly, less sera were determined to be positive in the competitive (186/230) than in the indirect ELISA (209/230). Sensitivity and specificity of the cELISA taking the indirect ELISA as a reference were 84.2% and 52.4%, respectively. Accordingly, the calculated prevalence of T. annulata infection was 90.9%, and the positive predictive value was determined to be 94.6%. Taken together, the cELISA proved its suitability for field application and was found qualified for use in serological surveys to monitor the prevalence of T. annulata infection and to identify carrier animals. Introduction Theileria annulata, a protozoan parasite transmitted by ticks of the species Hyalomma, is the pathogenic agent of bovine tropical theileriosis, which induces morbidity and loss in productivity in indigenous and more notably in crossbreed cattle, but especially in imported high-grade cattle, the infection of which causes a severe and often lethal disease. Tropical theileriosis is distributed over a wide geographic area ranging from the Mediterranean littoral regions of Europe and Africa through the Middle East to India and China (Dolan 1989). In its early phase, the infection can directly be diagnosed by microscopical examination of biopsy smears and clinical signs, including fever over 41°C, enlargement of lymph nodes, mucus discharge and diarrhoea. In later phases of the disease and particularly in carrier animals, serological tests are more suitable for diagnosis because parasitaemia tends to be at microscopically undetectable levels, and antibody titres are usually significantly higher than in the early phase of infection. Recent molecular biological diagnostic techni- ques like PCR and reverse line blotting have also been successfully used to identify T. annulata DNA in samples of cattle, but these methods have the disadvantages of being expensive and requiring a degree of expertise (d’Oliveira et al. 1995; Gubbels et al. 1999). As the majority of infected animals in the field are indigenous cattle and often carrier animals, there is a great demand for studies monitoring the disease distribution as a part of epidemiological surveys and the implementation of control programmes, including use of attenuated vaccines. An indirect ELISA for the detection of T. annulata infection based on the immunodominant antigen T. annulata surface protein (TaSP) was established, validated and used for epidemiological studies in the field (Bakheit et al. 2004; Stefanie Renneker and Jassim Abdo contributed equally to the paper. S. Renneker : J. Abdo : J. S. Ahmed :U. Seitzer (*) Veterinary Infection Biology and Immunology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany e-mail: useitzer@fz-borstel.de Parasitol Res DOI 10.1007/s00436-009-1625-4 ______________________________________________________________________________ERGEBNISSE