Investigação molecular de Leptospira spp em rins de bovinos / Molecular Research of Leptospira spp in Kidneys of Cattle

ABSTRACT

Background: Leptospirosis is a zoonotic bacterial disease caused by Leptospira spp. that affects wild and domestic animals and humans. It has a worldwide distribution and causes public health problems and economic losses in livestock. The genus Leptospira is divided basically into two species the Leptospira interrogans that is pathogenic and Leptospira biflexa that is considered saprobic. Cattle are an important source of infection, mainly to humans who work with these animals such as breeders, cowboys, slaughtermen, veterinarians and agricultural technicians. The temperature and rainfall indices of tropical and subtropical climate regions encourage the continuing disease outbreaks. In humans the symptoms are headache, fever, myalgia, nausea and vomiting. The identification of the disease is possible with some tests such as ELISA, immunofluorescence, bacterial culture, and Polymerase Chain Reaction (PCR). The aim of this work was to perform the molecular research for the presence of Leptospira spp. in kidneys of cattle refuted at a slaughterhouse. Materials, Methods & Results: Two hundred fragments of cattle kidney were collected at slaughterhouse located at Ilhéus, Bahia, Brazil. The macroscopic kidney refutation causes were nefritis, presence of cysts, congestion and presence of whitening and hemorrhagic areas. The kidney samples were placed in plastic containers identified and packed in an isothermal box until arrival at the laboratory, where they were kept at -20°C until the DNA extraction with phenol/chloroform/ isoamyl alcohol (25241). The DNA was quantified by spectrophotometer at 260 nm wavelength. The integrity of DNA was evaluated with 1% agarose gel stained with ethidium bromide and visualized at an ultraviolet light transilluminator. DNA amplification was performed with primers G1- [forward] 5'-CTGAATCGCTGTATAAAAGT-3' and G2- [reverse] 5'-GGAAAACAAATGGTCGGAAG-3' for pathogenic and non-pathogenic Leptospira. Aliquots of 50 ng/µL of extracted DNA were used for PCR reaction. Out of 200 tissue samples, 16 (8%) were positive and the amplified fragments had molecular weight compatible with Leptospira spp. Discussion: The amplicons presented molecular weight compatible with pathogenic and non-pathogenic strains of Leptospira spp. wich may represent a health risk for the ones who may contact animals or consume uncoocked products. Leptospira molecular diagnose may allow the pathogen identification in asymptomatic animals from small DNA amounts. Primers G1 and G2 amplified bacterial 16S rRNA gene area, allowing the identification. The renal tissue alterations identified during inspection, may be associated with Leptospira spp. presence showing the fundamental importance of correct identification and discard of affected organs in order to reduce the bacterial transmission risk. Many articles have being showing the Leptospira cattle prevalence and human risks by contacting these animals. The results presented in this article emphasizes the importance of the cattle as possible transmitters of the pathogen for slaughterhouses workers and meat inspectors, veterinarians as well as rural workers and their families, being critical to conduct orientation and conscientization programs about the problem and ways to combat and control it.