ABSTRACT
The Leptospira serovar Hedjo belongs to the serogroup sejroe and this serovar is the most prevalent in bovine herds worldwide. The sejroe serogroup is the most frequently detected by serology in Brazilian cattle herds suggesting that it is due serovar Hardjo. In the molecular classification, this serovar has two genotypes: Hardjobovis and Hardjoprajitno. This serovar is as considered as fastidious pathogens, and their isolation is one of the bottlenecks in leptospirosis laboratories. In addition, its molecular characterization using genomic approaches is oftentimes not simple and time-consuming. This study describes a method for isolating the two genotypes of serovar Hardjo using culture medium formulations and suggests a get-at-able molecular characterization. Ten cows naturally infected which were seropositive were selected from small dairy farms, and their urine was collected for bacterial isolation. We evaluated three modifications of liquid Leptospira medium culture supplemented with sodium pyruvate, superoxide dismutase enzyme and fetal bovine serum, and the isolates were characterized by molecular techniques. After isolation and adaptation in standard culture medium, the strains were subcultured for 1 week in the three modified culture media for morphologic evaluation using electronic microscopy. Strains were molecularly identified by multilocus variable-number tandem-repeat analysis (MLVA), partial sequencing and phylogenic analyses of gene sec Y. Combining the liquid culture medium formulations allowed growth of the Leptospira serovar Hardjo in three tubes. Two isolates were identified as genotype Hardjobovis, and the other as genotype Hardjoprajitno. Morphologically, compared with control media, cells in the medium supplemented with the superoxide dismutase enzyme were more elongated and showed many cells in division. The cells in the medium supplemented with fetal bovine serum were fewer and lost their spirochete morphology. This indicated that the additional supplementation with fetal bovine serum assisted in the initial growth and maintenance of the viable leptospires and the superoxide dismutase enzyme allowed them to adapt to the medium. These culture strategies allowed for the isolation and convenient molecular characterization of two genotypes of serovar Hardjo, creating new insight into the seroepidemiology of leptospirosis and its specific genotypes. It also provides new information for the immunoprophylaxis of bovine leptospirosis.
ABSTRACT
Identification of Escherichia coli causing porcine postweaning diarrhea requires knowledge regarding the prevalent pathotypes within a given region. A total of 100 Escherichia coli isolates from piglets with diarrhea in Londrina city, Parana State, South Brazil, were screened for the presence of genes for F4, F5, F6, F18, F41 fimbrial antigens by specific probes and for enterotoxins (STa, STb, LT and STx2e) by polymerase chain reaction (PCR). The results showed that 60% of the isolates were positive for one or more of the fimbrial antigens and 92% were positive at least for one of the virulence factors examined. Virulence factor genes detected were F4 (44%), F18 (38%), F5 (30%), F41 (32%), F6 (25%), LTp-I (71%), STa (40%), STb (47%) and STx2e (3%). Twenty four patterns of virulence factor according to the different virulence genes form were found and the most frequent virulence gene pattern was F4, F18, F41, STa, STb and LT. Most of the isolates that carried genes for adhesins also harboured genes for toxins.
