Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos

Abstract

Glanders is an infectious-contagious disease of acute or chronic character which principally affects horses, causing enormous losses in the productive chain of this animal. To control the disease, the Ministry of Agriculture, Husbandry and Supply instituted mandatory sanitation measures in the entire national territory which include an ofϐicial diagnosis through the complement ϐixation (CF) test, maleinization and sacriϐice of the animals that are positive. Nowadays the kits used for the diagnosis of the disease are imported, making their routine application difϐicult and more expensive. The objective of this study was to standardize an indirect ELISA test, using the proteic extract of Burkholderia mallei isolated from a carrier horse in the state of Pernambuco. The samples were cultivated in 10% blood agar and incubated for 48h at 37°C; later, one of the isolated colonies was characterized phenotypically and genotypically and immediately cultivated in brain heart infusion (BHI) for enrichment; then it was peaked (repicada) for the Dor-set Henley medium which was incubated at 37ºC under 60rpm for eight weeks. To standardize the test the Protein G Peroxidase Sigma Conjugate was used in the dilution of 1:90.000, with serums diluted in 1:100 and the antigen in 1:400. Sixty serums were used as negative controls, tested before the CF to determine the cutting point which was 0.042nm. After establishing the standardization, 300 samples were tested, of which 99% (297) were in agreement with the results obtained in the CF. At the end, of assay presented 100% sensibility and 98.2% speciϐicity, with predictive (preditivo) positive and negative values of 97.7% and 100% respectively. The Kappa concordance test was 0.98 and the intra and interplac repeatability were 8.8% and 10.3% respectively. From the results obtained, it is possible to afϐirm that the indirect ELISA test can be used as an efϐicient diagnosis tool. However, more essays must be carried out to consolidate the reliability of this test.

Systematic monitoring of glanders-infected horses by complement fixation test, bacterial isolation, and PCR

“…Usually glanders-positive animals are immediately euthanized after confirmation by CDA, but the seven were included in a larger clinical and epidemiological study in which they could be followed for a longer time. Considering a prevalence of the disease in Brazil of 43% ( Teles et al., 2012 ) and using the lowest values for sensitivity and specificity of the two tests, we calculated a 96.4% predictive positive value for glanders, which is high enough for the analysis of the number of horses available.…”

Non-specific reactions caused by vaccination in agar gel immunodiffusion for diagnosis of equine infectious anemia

“…AGID is used on a large scale, and despite its sensitivity, it does not detect specific antibodies in the early EIA stages. These limitations compromise the strategies for disease control and eradication, as serology has a determining value in a disease with no treatment and ineffective vaccines (VERMA et al, 1990;REIS, 1997;NEUBAUER et al, 2005;NAUREEN et al, 2007;TELES et al, 2012;OIE, 2013aOIE, , 2013b). According to Oliveira et al (2013), the training of veterinarians to ensure the correct execution of the AGID test protocol for diagnosing EIA in Brazil should be intensified.…”