Mycoplasma bovis-pneumonia and polyarthritis in feedlot calves in Argentina: First local isolation / Neumonía y poliartritis por Mycoplasma bovis en terneros de feedlot: primeraislamiento local

Abstract Bovine respiratory disease (BRD) is one of the most frequent clinical concerns inweaned calves after their arrival at the feedlot. This work reports the first local isolation ofMycoplasma bovis from feedlot calves with pneumonia and polyarthritis in Argentina. Twentyfour out of 545 calves showed progressive, subacute to chronic respiratory distress, coughing,and fever. Thirty percent of the affected calves also showed lameness and swelling of elbowor carpal, and knee or tarsal joints. Five necropsies were performed and severe multifocal tocoalescent pulmonary nodules, containing white-yellowish caseous exudate encircled by fibroustissue, and fibrinonecrotic arthritis and tenosynovitis were detected. Mycoplasma was isolatedfrom lung and joint samples. The 16S-23S rRNA ITS consensus sequence obtained from theseisolates showed 100% similarity with the same region of M. bovis strains. Since there are no com-mercially available vaccines in the region for the prevention and control of M. bovis pneumoniaand arthritis, surveillance is a priority to reduce the source of disease to naïve animals.

Resumen La enfermedad respiratoria bovina es uno de los problemas sanitarios más fre-cuentes en terneros recién destetados luego de su arribo a los corrales de encierre (feedlots).Este trabajo describe el primer aislamiento local de Mycoplasma bovis de terneros de feedlotcon neumonía y poliartritis en Argentina. Se vieron afectados 24 de 545 terneros; mostraron enfermedad respiratoria progresiva, subaguda a crónica, tos y fiebre. De los terneros afecta-dos, el 30% presentó, además, problemas en articulaciones carpales o tarsales. Se realizaron5 necropsias de terneros afectados y se observaron nódulos pulmonares multifocales a coa-lescentes, que contenían un exudado caseoso blanco-amarillento, rodeados de tejido fibroso,artritis y tenosinovitis fibrinonecrótica. Se aisló Mycoplasma de muestras de pulmón y artic-ulación. La secuencia consenso del gen codificante del ARNr 16S-23S rRNA obtenido de estosaislamientos mostró un 100% de similitud con la misma región de cepas de M. bovis. Teniendoen cuenta que no hay vacunas disponibles comercialmente en la región para la prevencióny el control de neumonías y poliartritis por M. bovis, es importante realizar una vigilanciaepidemiológica a fin de reducir las fuentes de infección para animales susceptibles.

Risk factors associated with intramammary colonization with Mollicutes in dairy cattle from Southeast Brazil / Fatores de risco associados à colonização intramamária por Mollicutes em bovinos leiteiros do Sudeste brasileiro

ABSTRACT: Bacteria of Mollicutes Class are associated with intramammary infection and decrease in milk production. This study investigated the occurrence of Mollicutes and elucidated their risk factors in dairy herds from Southeast Brazil. For this, milk samples from 387 lactation cows from Minas Gerais, Rio de Janeiro and São Paulo States were subjected to the polymerase chain reaction (PCR) to detect Mollicutes. Species of Mycoplasma were investigated in Mollicutes positive samples by PCR, including Mycoplasma bovis, M. alkalescens, M. bovigenitalium, M. bovirhinis, M. arginini and A. laidlawii. An epidemiological questionnaire was applied to collect data on possible risk factors, which were assessed using Pearson's Chi-square test followed by odds ratio (P≤0.05). Mollicutes were reported in 21% (4/19) of the herds and 4% (16/387) of the animals, while 1% (5/387) were positive for M. bovis and 3% (11/387) for M. arginini. All samples were negative to the other agents. Herds with more than 150 animals [OR=3.51 (95% CI 1.11-11.08)], manual milking [OR=9.97 (95% CI 2.80-35.49)] and not-milking animals with mastitis last [OR=6.54 (95% CI 1.92-22.29)] were risk factors. The presence of these conditions may favor intramammary infection by Mollicutes in dairy herds from Southeast Brazil. This is the first report of M. bovis in Rio de Janeiro and M. arginini in the studied states.

RESUMO: Bactérias da Classe Mollicutes estão associadas à infecção intramamária e diminuição da produção leiteira. O objetivo deste estudo foi investigar a ocorrência de Mollicutes e elucidar seus fatores de risco em rebanhos leiteiros do sudeste brasileiro. Para isso, amostras de leite de 387 vacas em lactação dos estados de Minas Gerais, Rio de Janeiro e São Paulo foram submetidas à reação em cadeia da polimerase (PCR) para detectar Mollicutes. Espécies de Mycoplasma foram investigadas nas amostras positivas por PCR, incluindo Mycoplasma bovis, M. alkalescens, M. bovigenitalium, M. bovirhinis, M. arginini e A. laidlawii. Foi aplicado um questionário epidemiológico para a coleta de dados sobre possíveis fatores de risco, que foram avaliados pelo teste de Qui-Quadrado de Pearson seguido de odds ratio (P≤0.05). Mollicutes foram detectados em 21% (4/19) dos rebanhos e 4% (16/387) dos animais, enquanto 1% (5/387) destes foram positivos para m. bovis, 3% (11/387) para m. arginini, sendo todas as amostras negativas para os demais agentes. Rebanhos com mais de 150 animais [OR=3,51 (95% IC 1,11-11,08)], ordenha manual [OR=9,97 (95% IC 2,80-35,49)] e ausência de linha de ordenha [OR=6,54 (95% IC 1,92-22,29)] foram considerados fatores de risco. A presença dessas condições pode favorecer a infecção intramamária por Mollicutes em rebanhos leiteiros no sudeste do Brasil. Este é o primeiro relato de M. bovis no Rio de Janeiro e M. arginini nos estados estudados.

Microbiological and molecular detection of Mycoplasma bovis in milk samples from bovine clinical mastitis / Detecção microbiológica e molecular de Mycoplasma bovis em amostras de leite oriundas de mastite clínica bovina

The genus Mycoplasma includes more than 200 bacterial species that cause disease in animals. It is responsible for causing mastitis in bovines and may be related to other manifestations, such as arthritis and pneumonia in calves and heifers. The present study aimed to detect Mycoplasma bovis isolated from milk samples of bovine clinical mastitis, and to compare the isolation rates in two culture media: Hayflick and SP4. An initial screening was performed in order to detect the presence of the class Mollicutes in 1166 milk samples from clinical mastitis by the conventional Polymerase Chain Reaction (PCR) technique. According to the 1166 milk samples evaluated, 8.6% (100/1166) were positive to class Mollicutes. Regarding molecular analyses, 1.1% (13/1166) of conventional PCR for positive M. bovis was obtained and 0.9% (11/1166) in real-time PCR. The results of the microbiological culture of the 100 samples previously screened demonstrated that 6% (6/100) of colony growth have been developed when using the Hayflick medium, and 11% (11/100) when using the SP4 medium (including the positive on Hayflick medium). Concerning the 11 isolates obtained in the microbiological culture, conventional PCR confirmed M. bovis in nine of them, and two cultures were negative. In the phylogenetic analysis of the isolates, all of them were grouped in M. bovis and M. agalactiae clusters. The results confirmed the importance of the presence of M. bovis in the etiology of bovine clinical mastitis and reinforced the need for further studies to elucidate other Mycoplasma species that may be involved in bovine clinical mastitis in Brazil.(AU)

O gênero Mycoplasma inclui mais de 200 espécies que causam doenças nos animais. É responsável por quadros de mastite em bovinos, podendo também estar relacionado à outras manifestações como artrite e pneumonia em bezerros e novilhas. O presente estudo objetivou a detecção de Mycoplasma bovis isolados a partir de amostras de leite de mastite clínica bovina, bem como, a comparação da taxa de isolamento em dois meios de cultura: Hayflick e SP4. Para efeito de triagem amostral, foram avaliadas quanto à presença da classe Mollicutes 1166 amostras de leite de casos de mastite clínica pela técnica de PCR convencional. Das 1166 amostras de leite avaliadas, 8,6% (100/1166) foram positivas à classe. Nas análises moleculares, obteve-se 1,1% (13/1166) de positividade para Mycoplasma bovis na PCR convencional e 0,9% (11/1166) na PCR em tempo real. Os resultados do cultivo microbiológico das 100 amostras triadas previamente demonstraram 6% (6/100) de crescimento de colônias ao se utilizar o meio Hayflick e 11% (11/100) ao se utilizar o meio SP4 (incluindo as positivas ao primeiro). A partir dos 11 isolados obtidos no cultivo microbiológico, a PCR convencional confirmou Mycoplasma bovis em nove deles, e dois foram negativos para o agente. Na análise filogenética dos isolados, todos agruparam no cluster Mycoplasma bovis e Mycoplasma agalactiae. Frente aos resultados, ressalta-se a importância da presença de Mycoplasma bovis na etiologia da mastite clínica bovina e reforça a necessidade de estudos mais aprofundados para a elucidação de outras espécies de micoplasmas que possam estar envolvidas na mastite bovina.(AU)

Prevalência de Mycoplasma bovis em rebanhos de vacas leiteiras / Prevalence of Mycoplasma bovis in dairy herds

Mycoplasma é um patógeno altamente contagioso, podendo causar mastite, pneumonia, artrite, entre outras enfermidades. Seu isolamento requer meios e condições específicas devido ao seu crescimento fastidioso. Devido à complexidade do seu diagnóstico, acredita-se que a real prevalência de casos de mastite por micoplasma seja subestimada. O objetivo do presente estudo foi identificar a prevalência de Mycoplasma bovis em diferentes rebanhos de bovinos leiteiros no estado de São Paulo. O estudo foi dividido em fase de triagem, na qual colheram-se amostras de 67 tanques de expansão e a coleta individual, na qual propriedades positivas para M. bovis foram visitadas e colhidas amostras de leite de todos os animais com mastite clínica e subclínica. O diagnóstico laboratorial foi feito por meio da PCR e cultivo microbiológico específico. A prevalência de M. bovis encontrada na fase de triagem foi de 1,4%. Na fase individual, todas as amostras de leite, procedentes de propriedade positiva para M. bovis no tanque de expansão, foram negativas, o que permite concluir pela baixa prevalência do agente nas condições do presente estudo.(AU)

Mycoplasma is a highly contagious pathogen, which can cause mastitis, pneumonia, arthritis, among other diseases. Its isolation requires specific means and conditions due to its fastidious growth. Due to the complexity of its diagnosis, it is believed that the real prevalence of mastitis cases by Mycoplasma is underestimated. The objective of the present study was to identify the prevalence of Mycoplasma bovis in ​​different dairy herds in the state of São Paulo. The study was divided into a screening phase in which samples were collected from 67 expansion tanks and individual collection, in which positive properties for M. bovis were visited and collected milk samples from all animals with clinical and subclinical mastitis. The laboratory diagnosis was made through PCR and specific microbiological culture. The prevalence of M. bovis found in the screening phase was 1.4%. In the individual phase, all milk samples from M. bovis positive property in the expansion tank were negative, which allows to conclude the low prevalence of the agent under the conditions of the present study.(AU)

Innate immune response of bovine mammary epithelial cells to Mycoplasma bovis

Mycoplasma spp. are contagious bacteria, and mycoplasmal mastitis is a serious productivity problem on dairy farms. Bovine mammary epithelial cells (bMECs) have an important role in the elimination of pathogens, but the effect of Mycoplasma bovis on bMECs has not been fully described. To elucidate the immune response against intramammary infection by M. bovis, we undertook microarray analysis to examine and profile mRNA expression in bMECs after stimulation with M. bovis. We also compared the effects of M. bovis, Staphylococcus aureus, and Escherichia coli on immune-related mRNA expression in bMECs. Transcriptome analysis indicated a significant decrease in the level of mRNA-encoding lysine-specific demethylase 4D, suggesting that the immune response is suppressed by a decrease in histone demethylase activity. Interleukin (IL)-1β, IL-6, tumor necrosis factor alpha, toll-like receptor (TLR) 2, and TLR4 mRNA expression levels were significantly increased in bMECs stimulated with heat-killed M. bovis, but the expression levels were lower than those following stimulation by heat-killed S. aureus or E. coli. Our results suggest that M. bovis weakly affects mRNA expression in bMECs compared to the effects of E. coli or S. aureus. Moreover, live M. bovis may induce suppression of the immune response in bMECs.

Demostración inmunohistoquímica de Mycoplasma bovis en lesiones neumónicas crónicas en ganado en corral de engorda / Immunohistochemical demonstration of Mycoplasma bovis in chronic lung lesions in feedlot cattle

Bovine feedlots are intensive production systems of significant importance in México, United States of America and Canadá. Respiratory diseases are the single most important cause of morbidity and mortality in feedlot cattle. Herein, the gross and microscopic findings in the lungs of cattle with unusually persistent pneumonia unresponsive to aggressive antibiotic treatment in feedlots localized in General Escobedo, Nuevo Leon; Morelia, Michoacan; Mexicali, Baja California, and Culiacan, Sinaloa, Mexico are described. Eight chronic suppurative bronchopneumonia cases with multifocal areas of casseous necrosis were selected. Microscopically, these lesions were bronchocentric and characterized by foci of necrosis surrounded by a pyogranulomatous reaction. Immunohistochemical studies revealed positive reaction to Mycoplasma bovis in all cases. Gross and microscopic lesions associated with M. bovis pneumonia have a conspicuous pattern. This is the first report of M. bovis causing pneumonia in feedlot cattle in Mexico.

El corral de engorda es un sistema de producción intensivo de gran importancia en la ganadería de carne en México, Canadá y Estados Unidos de América. Estudios epidemiológicos indican que las enfermedades respiratorias de los bovinos son el principal problema en corrales de engorda. En este trabajo se describen las lesiones macroscópicas y microscópicas en pulmones de ganado de carne con severa neumonía crónica, a pesar de haber recibido tratamiento con antibióticos en más de tres ocasiones. Se realizaron estudios post mortem de ganado en corrales de engorda localizados en General Escobedo, Nuevo León; Morelia, Michoacán; Mexicali, Baja California y Culiacán, Sinaloa, todos en México. Se seleccionaron ocho casos crónicos de bronconeumonía supurativa con múltiples focos nodulares de exudado necrótico-caseoso. Estos focos necróticos estaban microscópicamente centrados en bronquios y bronquiolos y sus bordes estaban claramente limitados por una reacción piogranulomatosa. Los estudios de inmunohistoquímica demostraron positividad a Mycoplasma bovis en todos los casos. Las lesiones neumónicas macro y microscópicas asociadas con M. bovis tienen un patrón conspicuo. Este es el primer informe de M. bovis en neumonías en ganado de corral de engorda en México.

Detección de mastitis bovina subclínica por micoplasmosis mediante ELISA indirecta y aislamiento / Detection of subclinical bovine mastitis caused by mycoplasmosis by indirect ELISA test and isolation

The objective of this study was to determine the effectiveness of ELISA test in serum, for the diagnosis of subclinical bovine mastitis caused by Mycoplasma bovis, having as a reference test, the isolation of the microorganism in milk samples. Two hundred and twenty five blood and milk samples were obtained from herds from the State of Mexico, Coahuila and Hidalgo, all of them in Mexico. Using the Wisconsin test, 139 samples (61.8%) were positives to subclinical mastitis but only in six of them, Mycoplasma bovis was isolated. By ELISA test, 72 samples were positives (32.0%). All animals with positive isolation were also positive to the ELISA test. With a cut point higher or equal to 100, sensitivity and specificity were 83.3% and 83.56%, respectively, although the positive predictive value (PV) (+) was 12.2% and the negative predictive value (PV) (-) was 99.46%. The low PV (+) was associated to a low prevalence. ELISA test could be used as a screen test to detect mastitis associated to Mycoplasma bovis, particularly in herds with high frequencies (> 10%), this with the purpose to improve a positive predictive value (PV), which would allow to establish better diagnoses in cases where the most common tests do not give a specific result, mostly if ELISA test is considered as practical, fast and economic.

El objetivo de este trabajo fue determinar la efectividad de una prueba de ELISA en suero para el diagnóstico de la mastitis bovina subclínica causada por Mycoplasma bovis, teniendo como prueba de referencia el aislamiento del microorganismo a partir de muestras de leche. Se obtuvieron 225 muestras de sangre y de leche provenientes de hatos del Estado de México, Coahuila e Hidalgo, todos en México. Ciento treinta y nueve muestras (61.8%) resultaron positivas a mastitis subclínica mediante la prueba de Wisconsin, y de ellas sólo en seis se aisló M. bovis. A través de la prueba de ELISA, 72 muestras resultaron positivas (32.0%). Todos los animales con aislamiento positivo también fueron positivos a la prueba de ELISA. Con un punto de corte mayor o igual a 100, se obtuvo sensibilidad de 83.3% y especificidad de 83.56%, aunque el valor predictivo (VP) (+) fue de 12.2%, y el VP (-) de 99.46%. El bajo VP (+) se asoció con baja prevalencia. La técnica de ELISA podría ser utilizada como prueba tamiz para la detección de mastitis asociada con M. bovis, particularmente en hatos con frecuencias elevadas (> a 10%), esto con el fin de mejorar un valor predictivo positivo, lo que permitiría establecer mejores diagnósticos en casos donde las pruebas más comunes no dan un resultado concreto, sobre todo si se considera que la prueba de ELISA es práctica, rápida y económica.

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