Padronização da reação de PCR para detecção de Dirofilaria immitis e determinação da taxa de infecção em mosquitos coletados na Ilha de Santa Catarina

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Programa de Pós-Graduação em Biotecnologia.Dirofilaria immitis is the ethiological agent of canine cardiovascular dirofilariosis or heartworm disease. This onchocercid nematode is widespread among dogs, cats, wild canids and eventually infected the human. The life cycle of parasite alternates between canids vertebrate hosts and mosquito vector. The parasite#s transmission to the dog, its main host, takes place through several mosquitoes species from the gender Culex, Aedes, Mansonia. The parasite#s detection at the vector is usually done by observation through the microscope and in the dog by the modified Knott#s method, where it is possible to observe the presence or absence of microfilarias released by the adult worm within the blood stream. However, these methods set space for errors once other kinds of worms from other filarias species might be found infecting the dog and the vector and they could be taken as D. immitis. For a more precise diagnose to this disease, techniques from molecular biology based on PCR are being developed in order to cope with the usual difficulties. In the present research we have used as a pattern the PCR for detection D. immitis, and we realized a survey of infection rates in mosquitos captured in Santa Catarina Island`s. For such, we have used the primers driven against gene, that transcrible for a surface antigene#s gene segment from the D. immitis. At the patternizing stage we used different DNA amounts withdrawn from D. immits worm, DNA from an infected and uninfected dog blood. We#ve amplified one DNA segment of 378pb of genome`s parasite with DNA extracted from Blood an infected dog. The primers sensibility allows the target segment#s amplification in a range which varies from 100 ng to 1 pg, such result points out the possibility of amplifying a minimum amount of the parasites# DNA at the dog#s blood stream and in the host. A DNA excess of 60 ng/ul extracted of dog`s blood and 80ng/ul DNA extracted the vector#s DNA does not inhibit the specific band#s amplification of the parasite. At the specificity session we did not observe the merge of unspecified bands when DNA of other kinds of nematodes and platelmints that infect dog`s. At the application#s stage in Field, a total of 400 mosquitoes of diferents species, were caugth in the north and central region of Santa Catarina Island`s and tested by PCR. Filarial DNA was found in 2,3% (1/44) of pools tested. These results prove that Oc scapularis is a natural vector of D. immitis in and indicating the existence of the disease at that specific locality Fort#s Beach. We believe that this techniques used as a pattern will be of great value to the diagnosis#s species-specific determination as well as to the canine dirofilariosis actual monitoring