Development, standardization and validation of a Real Time Polymerase Chain Reaction to the diagnosis of canine brucellosis

Abstract

Brucellosis is a zoonotic infectious disease, affecting various species of hosts. Ten Brucella species are currently described and they are classified into two antigenic groups (smooth or rough). Brucella canis is a rough Brucella species that infects dogs, causing reproductive problems and resulting in economic losses to kennel owners, since animals can be easily infected orally, by aerosols inhalation, and venereal route. Several diagnostic methods have been developed to B. canis in dogs, but not all are effective. The microbiological culture is the gold standard test, however it is a costly method, contamination can occur with other organisms, and it poses risks in view of the laboratory manipulation of Brucella cultures. Serological tests are widely used, but depending on the stage of the infection, they are not able to detect infected dogs, leading to false-negative results. The molecular method using PCR (polymerase chain reaction) is considered a highly sensitive test and an alternative tool for the direct detection of B. canis infected dogs. Real time PCR usually shows higher diagnostic sensitivity and specificity values when compared to conventional PCR, besides it is more simple and rapid to perform. Therefore the aim of this study is the development, standardization and validation of a real-time PCR for the diagnosis of canine brucellosis caused by B. canis using whole blood samples. The samples will be collected from dogs belonging to commercial kennels, which will undergo clinical history and evaluation in order to detect clinical signs suggestive of brucellosis. A total of 50 dogs will be used, 25 infected with B. canis and 25 healthy animals. The blood will be used in blood culture and Real-time PCR, and the results will be compared using the Kappa coefficient and McNemar test. The Chi-square test will determine the association between the presence of clinical signs suggestive of brucellosis and positive results in blood culture tests and Real-time PCR. (AU)