Maristela Vasconcellos Cardoso

Detection of Ureaplasma diversum in cattle using a newly developed PCR-based detection assay

Ureaplasma diversum has been associated with different clinical manifestations including bovine vulvitis, endometritis, salpingitis, spontaneous abortion and infertility. Because the isolation of this ureaplasma from clinical samples is difficult, there is a need for improved detection methods. We developed a PCR assay based on amplification of a region of the gene encoding 16S rRNA. The specificity of the amplification was verified by sequence analysis. Female bovine vaginal swabs (n=168) were collected and the presence of U. diversum evaluated by both culture methods and by the PCR assay. Culture was positive for 60 samples (35.7%), and PCR-specific amplification was obtained for 89 samples (52.9%). These results indicated a high prevalence of U. diversum in the selected animals and the higher sensitivity of this PCR assay as compared to culture.

Ureaplasma diversum and reproductive disorder in Brazilian cows and heifers; first report.

The species Ureaplasma diversum is associated with bovine reproductive illnesses, in particular granular lesions of the vulva and vagina or granular vulvovaginitis (GVV). In Brazil, this pathology is unknown and, until this point in time, the presence of U. diversum in the Brazilian herds has been ignored. With the intention of detecting the microorganism, vulvovaginal mucuses of 152 animals located on seven farms in the São Paulo State, Brazil were analyzed. Those animals had evidence of reproductive disorders at the time of the sample collection. The technique used for microorganism detection was bacterial isolation. Statistical analysis assessed: the exposure of studied farms to U. diversum, relative risks for different symptoms, susceptibility of the animals according to age and breed. The frequency of that microorganism in tested animals was 38.8% and this frequency suggests that U. diversum can be related to GVV in Brazilian herds and possibly with other reproductive illnesses. As a result, the U. diversum differential diagnosis could be very important.

Leptospira spp detection by Polymerase Chain Reaction (PCR) in clinical samples of captive black-capped Capuchin monkey (Cebus apella)

Leptospirosis is a widely distributed zoonosis that affects domestic and wild animals, and that has the man as the end point of its epidemiological chain. Leptospirosis diagnosis in primates is more difficult than in other animal species, as clinical signs and lesions are less evident and antibody response is detected only for short periods. The aim of this article was to describe the detection of Leptospira spp using polymerase chain reaction (PCR), in clinical samples from one captive black-capped Capuchin monkey (Cebus apella), which presented characteristics compatible with leptospirosis (jaundice and haemorrhagic kdney) in the macroscopic post-mortem examination. A friable kidney fragment and urine sample were cultured and submitted to experimental inoculation in guinea pigs and PCR using genus specific primer pair targeting the 16S rRNA region from Leptospira interrogans serovar canicola. Isolation of the agent was negative both in culture and experimental inoculation. The PCR amplification of the clinical samples showed a 330 pb amplified fragment that corresponds to the Leptospira genus. Based on these results PCR was considered an important tool for leptospira detection in nonhumam primates, more sensitive and specific than other techniques, especially considering that the viability of the pathogen was not possible. These advantages enable the detection of the leptospiras in urine and kidney, even when autolysed, frozen or badly conserved, which prevented the isolation and experimental inoculation from positive results.

Evaluation of the counterimmunoelectrophoresis reaction as a serodiagnosis test for swine leptospirosis

The Counterimmunoelectrophoresis reaction (CIE) was tested as a genus specific test for swine leptospirosis diagnosis using three soluble extracts obtained from Leptospira sp, serovars pomona, icterohaemorrhagiae and patoc. The extracts were treated with hot Triton X-100 and applied to serum samples of animals divided in three groups: Group 1, 10 swines experimentally infected with the Pomona strain; Group 2, 50 naturally infected swines and Group 3, 10 swines control. These animals were serologically evaluated by CIE and by the Microscopic Agglutination Test (MAT), the WHO reference technique. Groups 1 and 3 were monitored during a period of 93 days after infection (a.i.). Group 1 serum convertion took place around the 10th day a.i. by MAT but ocurred earlier by CIE using any antigen. When CIE was carried out with the homologous antigen to the experimental infection, the results were consistent with MAT, but not when the heterologous antigens were used. Groups 1 and 3 showed distinct results: in Group 3, diferences between results of CIE accomplished with any three antigen extracts were not significant, indicating lack of dependence on the serovar responsible for the outbreak. Although being safe, fast, easy to perform, inexpensive and ideal for analysis of large number of samples, CIE revealed limited genus specificity, which is not convenient for field screening tests. The advantage of CIE is the capability to detect antibodies earlier than MAT.

Comparison of electrophoretic protein profiles of Campylobacter jejuni subsp. jejuni isolated from different animal species

Electrophoretic protein profiles of Campylobacter jejuni subsp. jejuni strains isolated from feces of seven animal species, including man, were compared. Fourteen strains (two from each species) plus two human strains and the reference one, were ruptured by ultrasound and their total soluble proteins were analyzed by SDS-PAGE technique in a 12% polyacrylamide gel with computerized densitometric reading by the molecular analyst software. All the strains had bands in common that correspond to 45 and 66 Kda molecular weight. The disagreement corresponded to a 97 to 200 Kda molecular weight region. From the 17 strains, 13 (76.5%), were classified as biotype I, three (17.6%) as biotype II and one (5.8%) as biotype III. Since protein extracts were obtained from cells grown under identical conditions, and thus, able to express the same phenotype, this disagreement region could be related to different genotypes or serotypes.