Nova metodologia de diagnóstico para Ehrlichia canis: PCR X LAMP

Abstract

Because the close relationship between men and dogs, possibly some ectoparasites of dogs can be observed parasitizing man. The tick Rhipicecephalus sanguineus is an ectoparasite that has the ability to carry and transmit pathogens to humans. This arthropod, that is blood-feeding, is the main biological vector of the bacteria Ehrlichia canis. Currently, the diagnosis of this disease ehrlichiasis is based on blood, biochemical and serological tests, although they are unreliable for diagnosing the disease, since their clinical and clinicopathological features are largely nonspecific. Tetracyclines are commonly used in the treatment of ehrlichiosis, but studies show that some dogs remain positive for E. canis after treatment or after the loss of spontaneous infection. The chronic ehrlichiosis, having high prognosis fails, can result in high mortality. Therefore, more sensitive and reliable tests may help in the selection of dog carrying the disease. The PCR (polymerase chain reaction) has been used successfully in the diagnosis of E. canis. However, this molecular technology requires expensive equipment and specialized personnel to handle, which limits its use in laboratories routines. A more sensitive, specific, and simple to detect the microorganism method is desirable. In this work we develop the technique for detection of the bacterium Ehrlichia canis using the LAMP (Loop- Mediated Isothermal Amplification), which proved being very effective. Specific sequences of the E. canis dsb gene (disulfide bond) were used as target for the tested techniques. Dsb gene was highly specific in order to detect E. canis, since it is divergent of phylogenetically similar bacteria. Performed molecular tests showed a disease incidence greater than that indicated by authors using traditional diagnostic techniques. In the public kennel, 80% of the samples were infected, while in private clinics and veterinary hospital 40% had the disease. The developed diagnostic technique using LAMP is more sensitive than PCR, highly specific and does not require the prior DNA extraction to amplification. In addition, the product of amplification can be seen with the naked eye. We conclude that the diagnosis by the LAMP method enables the specific and high sensitivity identification of infected animals with minimal laboratory settings. These factors make possible the use of this diagnostic methodology for veterinary clinics, laboratories and educational institutions.