Detección de Mycoplasma canadense y Mycoplasma californicum en ganado lechero de Argentina / Detection of Mycoplasma canadense and Mycoplasma californicum in dairy cattle from Argentina

Diferentes especies del género Mycoplasma pueden afectar al ganado bovino y causar varias enfermedades. La técnica de PCR, secuenciación y posterior análisis de la región ITS 16S-23S ARNr ha mostrado que existe una importante variabilidad interespecies entre Mollicutes. Se realizó la amplificación (región ITS 16S-23S ARNr) de 16 aislamientos sospechosos de corresponder a alguna especie de Mycoplasma, que habían sido obtenidos de muestras de leche provenientes de rodeos lecheros. Catorce de esos aislamientos fueron PCR positivos. Para confirmar la identidad de Mycoplasma bovis, dichos aislamientos fueron evaluados por otra PCR especie-específica. Siete aislamientos dieron un resultado positivo. Los productos de la PCR de la ITS 16S-23S ARNr de un aislamiento identificado como M. bovis y de otros dos aislamientos identificados como no-M. bovis fueron seleccionados al azar, secuenciados y analizados. Las tres secuencias (A, B y C) mostraron 100 % de similitud con cepas de M. bovis, Mycoplasma canadense y Mycoplasma californicum, respectivamente

Different species of Mycoplasma can affect bovine cattle, causing several diseases. PCR sequencing and further analysis of the 16S-23S rRNA ITS region have shown a significant interspecies variability among Mollicutes. Sixteen suspected isolates of Mycoplasma spp. obtained from milk samples from dairy herds were amplified (16S-23S rRNA ITS region). Fourteen out of those 16 suspected Mycoplasma spp. isolates were PCR-positive. To confirm the identity of Mycoplasma bovis, these 14 isolates were tested by another species-specific PCR. Seven of the isolates rendered a positive result. The products of 16S-23S rRNA ITS PCR from one isolate that was identified as M. bovis and from two other isolates, identified as non- M. bovis were randomly selected, sequenced and analyzed. The three sequences (A, B and C) showed 100% similarity with M. bovis, Mycoplasma canadense and Mycoplasma californicum respectively

Ocorrência de Mollicutes e Ureaplasma spp. em surto de doença reprodutiva em rebanho bovino no Estado da Paraíba / Occurrence of Mollicutes and Ureaplasma spp. in outbreak of reproductive disease in cattle herds, State of Paraíba, Brazil

Em março de 2012 foi diagnosticado um surto de doença reprodutiva em rebanho bovino no Estado da Paraíba, Brasil. Foram examinadas 32 vacas e dois touros da raça Girolando. As vacas apresentaram sinais de doença reprodutiva como repetição de cio, vulvovaginite granular, infertilidade e abortos. As amostras de suabes vaginais e prepuciais foram colhidas e submetidas a isolamento bacteriano e PCR. As reações da PCR para Mollicutes e Ureaplasma spp. foram realizadas com os iniciadores MGSO-GPO3 e UGP'F-UGP'R, respectivamente. Na Nested PCR para Ureaplasma diversum, os iniciadores usados foram UD1, UD2, UD3 e UD4. Para isolamento bacteriano, as amostras foram diluídas de 10-1 até 10-5, semeadas em meio "UB", líquido e placa, sendo incubadas por até 21 dias a 37ºC em jarra de microaerofilia. A frequência de Mollicutes detectada na PCR foi de 65,6% e para Ureaplasma spp. foi de 50,0%, enquanto que para U. diversum foi de 15,6%. No isolamento a frequência de Mollicutes foi de 57,1% e para Ureaplasma spp. foi de 28,6%. No ágar "UB" foi visualizado o crescimento misto de Mycoplasma spp. e Ureaplasma spp. em seis amostras. Foi confirmado o envolvimento de micro-organismos da Classe Mollicutes em surto de doença reprodutiva em vacas no sertão paraibano.

In March of 2012 was investigated a reproductive disease outbreak in cattle herds from Paraíba State, Brazil. Were examined 32 cows and two bulls Giroland breed. The cows showed signs and symptoms of reproductive failure such as repeat breeding, granular vulvovaginitis, infertility and abortions. Vaginal and preputial mucous samples were collected for analysis by PCR and isolation. The PCR reactions for Mollicutes and Ureaplasma spp. were realized with primers MGSO and GPO3, and UGP'F and UGP'R respectively. The nested PCR assay for Ureaplasma diversum was realized with primers UD1, UD2, UD3 and UD4. For bacteriologic isolation, obtained samples were diluted up to 10-1 at 10-5, inoculated into liquid and solid "UB" medium, and incubated for up to 21 days, at 37ºC in microaerophilie jar. In the PCR reactions the frequency of Mollicutes detected in the analyzed vaginal mucous samples was 65.6, for Ureaplasma spp. was 50.0, while for U. diversum was 15.6. The frequency for isolation of Mollicutes was of 57.1 and for Ureaplasma spp. was of 28.6. In the UB agar was visualized growth of Mycoplasma spp. and Ureaplasma spp., associated in six of the samples. In the cows the presence of Mollicutes and Ureaplasma spp. was confirmed for the reproductive disease outbreak in the semiarid region of Paraiba.

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.

Detección por técnicas de biología molecular de Mycoplasma hominis y Ureaplasma urealyticum en muestras urogenitales / Detection by molecular biology techniques of Mycoplasma hominis and Ureaplasma urealyticum in urogenital samples

Mycoplasma homonis y Ureaplasma urealyticum están estrechamente relacionadas con enfermedades del tracto urogenital como pielonefritis, uretritis no-gonocóccica, cálculos urinarios, epididimitis, inflamaciones pélvicas, infertilidad, abortos y fiebre post-parto; en recién nacidos también pueden causar neumonías y meningitis. Estas bacterias pueden ser diagnosticadas por diferentes métodos. En este trabajo utilizamos la hibridación de ácidos nucléicos y la reacción en cadena de la polimerasa para analizar 22 muestras de pacientes con diferentes síntomas urogenitales, en busca de micoplasmas y ureaplasmas. Como resultado obtuvimos 10 muestras positivas y 12 negativas. Entre las muestras positivas se identificaron 2 como Mycoplasma hominis, 2 como Ureaplasma urealyticum y 6 con ambas especies. los resultados obtenidos por las técnicas moleculares fueron comparados con los métodos de referencia, encontrándose en 18 muestras resultados coincidentes, mientras que en 4 los resultados fueron discordantes, siendo esta diferencia estadísticamente no significativa