The aim of the present study was to consider the wide usage of urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock. A total of 512 adult animals (300 cattle, 138 horses, 59 goats and 15 pigs), from herds/flocks with reproductive problems in Rio de Janeiro, Brazil was studied by serology and urinary PCR. From the 512 serum samples tested, 223 (43.5 %) were seroreactive (cattle: 45.6 %, horses: 41.3 %, goats: 34%and pigs: 60 %). PCR detected leptospiral DNA in 32.4 % (cattle: 21.6 %, horses: 36.2 %, goats: 77.4 % and pigs: 33.3 %. To our knowledge there is no another study including such a large number of samples (512) from different species, providing a comprehensive analysis of the usage of PCR for detecting leptospiral carriers in livestock. Serological and molecular results were discrepant, regardless the titre, what was an expected outcome. Nevertheless, it is impossible to establish agreement between these tests, since the two methodologies are conducted on different samples (MAT - serum; PCR - urine). Additionally, the MAT is an indirect method and PCR is a direct one. In conclusion, we have demonstrated that urinary PCR should be considered and encouraged as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock.
A total of 15 adult ewes from one flock known to be seroreactive for leptospirosis was studied. Urine and vaginal fluid were collected from each animal to test for the presence of leptospires using bacterial culture and conventional PCR methods. One pure culture of Leptospira sp. was obtained from the vaginal fluid sample of a non-pregnant ewe. The isolate was characterized by DNA sequencing of the rrs and secY genes, variable-number of tandem-repeats (VNTR) analysis and serogrouping, and the isolate was typed as Leptospira interrogans serogroup Sejroe serovar Hardjo type Hardjoprajitno. This report indicates the presence of viable Leptospira in the vaginal fluid of a ewe, suggesting the potential for venereal transmission of leptospires in sheep. Case reportA flock of sheep located in Rio de Janeiro, Brazil, was initially studied for leptospirosis by microscopic agglutination test (MAT). The flock was composed of 21 Santa Inês sheep (18 females and 3 males), which were not vaccinated against leptospirosis, semi-extensive breeding and natural mating. No reproductive failures or abortions had been reported in the last breeding season. All animals were kept together throughout the year. This study was submitted to the ethics committee on animal use of the Fluminense Federal University (9 August 2012, protocol number 225). The first screening by MAT revealed 15 seroreactive females (83.4 %) and 1 male (33.3 %) with titres ranging from 100 to 800. Antibodies against serogoroup Sejroe were predominant (81.2 % of seroreactives); however, low titres (100) against serogroup Icterohaemorrhagiae were observed in three animals (18.8 %).Due to the high seroreactivity in this flock, it was selected for further studies. Approximately 1 month after the first sampling, urine, vaginal fluid and blood samples were collected from the 15 seroreactive females in order to confirm the leptospiral infection. Bacterial culture and PCR were performed for urine and vaginal fluid samples, while serum samples were resubmitted for serology testing (MAT); 3 of 15 (20.0 %) ewes were seronegative.Vaginal fluids were collected, after the perineum was first cleaned with water alone, using a tampon (Tampax regular) that was introduced into the vagina of the sheep. After 10 min, the tampon was removed and transferred to a sterile vial containing 20 ml PBS, as described previously (Lilenbaum et al., 2008). Following the collection of vaginal fluids, 0.5-1.0 mg furosemide kg 21 (Teuto Laboratory) was administered intravenously and a second voiding of urine collected into sterile vials.At the Veterinary Bacteriology Laboratory, Fluminense Federal University, the tampons were aseptically squeezed and centrifuged at 800 g for 10 min in sterile vials, and an aliquot (500 ml) of each supernatant was transferred to Fletcher and Ellinghausen-McCullough-Johnson-Harris (EMJH) media tubes (Difco). In addition, 500 ml urine samples were immediately transferred to Fletcher and EMJH media tubes. All samples were processed on the day of sampling. All cultures were...
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