Resumen Los micoplasmas son responsables de varias infecciones en pequeños rumiantes. En Brasil se han detectado micoplasmas potencialmente patógenas en el conducto auditivo externo sano de caprinos y bovinos y en asociación con ácaros, pero nunca se han detectado en ovinos. La presente investigación tuvo como objetivo evaluar la conjuntiva y el oído externo como fuente de infección silenciosa de micoplasmas en ovinos. Se obtuvieron muestras de frotis del conducto auditivo externo y ocular de cuarenta y cinco ovinos (machos y hembras). Fueron realizadas las pruebas de Detección molecular (PCR) y aislamiento de Mycoplasma spp. Se determinó la concentración mínima inhibitoria para el aislamiento, utilizando oxitetraciclina, cloranfenicol, tilosina y ácido nalidíxico. Fue aislado Mycoplasma conjunctivae a partir de la conjuntiva ocular de un ovino sano, además de la detección molecular de M. agalactiae y M. mycoides subsp. capri en ambos sitios anatómicos de ovinos del Estado de São Paulo, Brasil. El análisis filogenético reveló proximidad y similitud con un aislado de ovino y de cabra estadounidenses. La conjuntiva ocular y el oído externo de los ovinos son sitios anatómicos que pueden portar micoplasmas patógenos provocando infecciones a ovinos vulnerables.

Summary: Mycoplasmas are responsible for several infections in small ruminants. Potentially pathogenic mycoplasmas have already been detected in the external ear canal of Brazilian healthy goats and cattle, and in association with mites, but not in sheep. The present research aimed to evaluate the role of the conjunctiva and external ear as a silent source of micoplasmas in sheep. Ocular and external ear canal swab samples were obtained from forty-five male and female sheep. Molecular detection (PCR) and isolation of Mycoplasma spp. were performed. Minimum inhibitory concentration was performed using oxytetracycline, tylosin, chloramphenicol and nalidixic acid. We describe the isolation of Mycoplasma conjunctivae fromthe ocular conjunctivae of a healthy sheep, besides the molecular detection of M. agalactiae and M. mycoides subsp. capri in both anatomical sites in sheep from the State of São Paulo. Phylogenetic analysis revealed proximity and similarity with an American sheep and goat isolates. The ocular conjunctiva and the external ear of sheep are keepers of potentially pathogenic veterinary mycoplasmas which can infect vulnerable sheep.

Resumo: Os micoplasmas são responsáveis por várias infecções em pequenos ruminantes. Micoplasmas potencialmente patogênicos já foram detectados no conduto auditivo externo de caprinos e bovinos brasileiros saudáveis e em associação com ácaros, mas não em ovinos. A presente pesquisa teve como objetivo avaliar o papel da conjuntiva e da orelha externa como fonte silenciosa de micoplasmas em ovinos. Amostras de esfregaço ocular e do canal auditivo externo foram obtidas de 45 ovelhas machos e fêmeas. Detecção molecular (PCR) e isolamento de Mycoplasma spp. foram realizados. A concentração inibitória mínima foi realizada com oxitetraciclina, tilosina, cloranfenicol e ácido nalidíxico. Descrevemos o isolamento de Mycoplasma conjunctivae da conjuntiva ocular de uma ovelha saudável, além da detecção molecular de M. agalactiae e M. mycoides subsp. capri em ambos os sítios anatômicos em ovinos do Estado de São Paulo. A análise filogenética revelou proximidade e similaridade com isolados de ovinos e caprinos americanos. A conjuntiva ocular e a orelha externa das ovelhas são detentoras de micoplasmas veterinários potencialmente patogênicos que podem infectar ovelhas vulneráveis.

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%, respectivamente. A PCR genérica resultou em reações positivas em 63,4% das amostras transportadas em SP-4 e em 69,6% das transportadas em meio de ureaplasma. M. bovis foi detectado, na PCR específica, em 9,8% das amostras e U. diversum, na nested-PCR, em 37,5%. 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%, respectively. With generic PCR, positive reaction was obtained in 63.4% of the samples transported in SP-4 and 69.6% in ureaplasma medium. M. bovis was detected in 9.8% of samples and nested-PCR in a single tube for U. diversum resulted in 35.0% 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.

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.

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 ± 8.6%) and low production of NO (4.7 ± 3.1 µM NO2-). These macrophages co-cultured with mycoplasma-infected YAC-1 cells showed significantly higher cytotoxic activity (61.4 ± 9.1%; P<0.05) and higher NO production (48.5 ± 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 ± 2% (P<0.05) and NO production to 3 ± 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.