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

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.

Thioglycollate-elicited murine macrophages are cytotoxic to Mycoplasma arginini-infected YAC-1 tumor cells

Macrophages are important components of natural immunity involved in inhibition of tumor growth and destruction of tumor cells. It is known that these cells can be activated for tumoricidal activity by lymphokines and bacterial products. We investigated whether YAC-1 tumor cells infected with Mycoplasma arginini stimulate nitric oxide (NO) release and macrophage cytotoxic activity. Thioglycollate-elicited macrophages from male BALB/c mice were co-cultured for 20 h with YAC-1 tumor cells infected or not with Mycoplasma arginini. The cytotoxic activity was evaluated by MTT assay and nitrite levels were determined with the Griess reagent. Thioglycollate-elicited macrophages co-cultured with noninfected YAC-1 cells showed low cytotoxic activity (34.7 ñ 8.6per cent) and low production of NO (4.7 ñ 3.1 µM NO2-). These macrophages co-cultured with mycoplasma-infected YAC-1 cells showed significantly higher cytotoxic activity (61.4 ñ 9.1 per cent; P=0.05) and higher NO production (48.5 ñ 13 µM NO2-; P=0.05). Addition of L-NAME (10 mM), an inhibitor of NO synthesis, to these co-cultures reduced the cytotoxic activity to 37.4 ñ 2per cent (P=0.05) and NO production to 3 ñ 4 µM NO2- (P=0.05). The present data show that Mycoplasma arginini is able to induce macrophage cytotoxic activity and that this activity is partially mediated by NO.