Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates.

Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Taq High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains.

Detection of Mycoplasma pulmonis in laboratory rats and technicians.

Five species of mycoplasma are associated with several rat diseases. Mycoplasma pulmonis is the most important and most studied, possibly causing disease in rats and undermining the validity of laboratory experiments. M. pulmonis was isolated in 144/240 laboratory rats and identified by PCR in 155/240. This species was also detected in 12 human individuals (technicians of a laboratory animal house hold) in contact with these rats. The results were confirmed by sequencing of DNA products. Mycoplasma species are host specific; however, M. pulmonis was identified in humans, suggesting a case of unspecific colonization. Statistical analysis shows a greater risk for M. pulmonis colonizing individuals who are exposed to infected rats in animal facilities than individuals who do not. The detection of M. pulmonis in humans indicates a new status for this mollicute mycoplasmas in animal-holding facilities.

Detecção de Mycoplasma spp. e Ureaplasma diversum em vacas com distúrbios reprodutivos / Detection of Mycoplasma spp. and Ureaplasma diversum in cown with reproductive disorders

Foram utilizadas 112 amostras de muco vulvovaginal, coletados de vacas com distúrbios reprodutivos, para pesquisa de Mycoplasma e Ureaplasma. Para isolamentos, foram usados meios específicos para micoplasmas (SP-4) e para ureaplasmas. PCR genérica, PCR específica para Mycoplasma bovis e nested-PCR em tubo único para Ureaplasma diversum foram realizados com os DNAs extraídos das amostras. Mycoplasma spp. e U. diversum foram detectados em 12,5 e 25,0 por cento, respectivamente. A PCR genérica resultou em reações positivas em 63,4 por cento das amostras transportadas em SP-4 e em 69,6 por cento das transportadas em meio de ureaplasma. M. bovis foi detectado, na PCR específica, em 9,8 por cento das amostras e U. diversum, na nested-PCR, em 37,5 por cento. Houve maior sensibilidade na metodologia da PCR quando comparada à técnica de cultivo para Mycoplasma e Ureaplasma

In the study, 112 samples of vulvovaginal mucus of cows bearing reproductive disturbance were investigated for Mycoplasma and Ureaplasma. Specific media for the culture of mycoplasmas (SP-4) and ureaplasmas were used. PCR with general primers, PCR specific for Mycoplasma bovis, and nested-PCR in a single tube for Ureaplasma diversum were performed to detect DNA of the sample. Mycoplasma spp. and U. diversum were isolated in 12.5 and 25.0 percent, respectively. With generic PCR, positive reaction was obtained in 63.4 percent of the samples transported in SP-4 and 69.6 percent in ureaplasma medium. M. bovis was detected in 9.8 percent of samples and nested-PCR in a single tube for U. diversum resulted in 35.0 percent of positive reaction. Results demonstrated increased sensitivity of PCR methodology compared with culture technique applied to the search of microorganisms of Mycoplasma and Ureasplasma genera

Use of a polymerase chain reaction for detection of Mycoplasma dispar in the nasal mucus of calves.

A polymerase chain reaction (PCR) assay was developed for the detection of Mycoplasma dispar in nasal mucus samples collected from calves. The target DNA sequence was the 16S rRNA gene, and the fragment was selected within a region of high polymorphism. The specificity and detection limit of the method were determined. This method was then used for the detection of M. dispar in nasal swabs collected from 301 calves, including 155 clinical samples from animals showing signs of respiratory disease and 146 samples from healthy animals. PCR with generic primers was applied to the detection of Mollicutes, followed by the detection of M. dispar. Mollicutes were detected in 52.05% of clinical samples from healthy animals and in 90.96% of samples from sick animals. Mycoplasma dispar was detected in 6.16% of healthy animals and in 34.84% of sick animals. The PCR assay was useful in verifying the presence of M. dispar in calves and may be a useful tool in monitoring this mycoplasma in cattle herds.

Molecular characterization of MG isolates using RAPD and PFGE isolated from chickens in Brazil.

In the present study, 27 primers were screened under different cycles by random amplified polymorphic DNA (RAPD) method. Mathematical models were used for analysis of the genetic relationships among strains, including vaccinal, reference strains and nine field isolates previously characterized as Mycoplasma gallisepticum (MG)F by RAPD and pulsed field gel electrophoresis (PFGE). The PFGE was considered as laborious, expensive and time-consuming than RAPD method. These methods improved the typeability for epidemiological studies of MG with regard to differentiation from vaccinal and field strains.

Detection of multiple mycoplasma infection in cell cultures by PCR.

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

Detection of multiple mycoplasma infection in cell cultures by PCR

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9 percent) samples. Although the infection was confirmed by culture for 69 (22.9 percent) samples, PCR with generic primers did not detect the infection in five (5.4 percent). Mycoplasma species were identified with specific primers in 91 (30.2 percent) of the 98 samples (32.6 percent) considered to be infected. Mycoplasma hyorhinis was detected in 63.3 percent of the infected samples, M. arginini in 59.2 percent, Acholeplasma laidlawii in 20.4 percent, M. fermentans in 14.3 percent, M. orale in 11.2 percent, and M. salivarium in 8.2 percent. Sixty (61.2 percent) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6 percent) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.