Epidemiology of mastitis and interactions of environmental factors on udder health in the compost barn system / Epidemiologia da mastite e interações de fatores ambientais na saúde do úbere no sistema de Compost Barn

This study aimed to describe the epidemiological indexes of mastitis, milk quality and udder hygiene in the Compost Barn system, as well as to search for associations between isolated pathogens from milk with compost characteristics. Three dairy herds participated in the study, and the samples were collected during different periods on each farm. Individual milk samples were collected in duplicate for SCC analysis and microbiological culture. Environmental pathogens caused most cases of clinical mastitis on farm 2, and contagious pathogens caused the most cases on farm 1. Bed moisture was not associated with the incidence of environmental pathogens. Most of the animals remained in good udder hygiene during the study. Poor udder hygiene contributed to the increased incidence of environmental pathogens in one of the farms. A higher number of animals with a hygiene score of ≥ 2 were observed during the warmer and rainfall periods. There was no association between hygiene scores and somatic cell counts. The results suggest that pathogens isolated from milk in animals confined in Compost Barn under tropical climate are like other confinement systems adopted elsewhere. The year period influenced the udder hygiene score, reinforcing the importance of bed management throughout the year.

O objetivo deste estudo foi descrever os índices epidemiológicos da mastite, da qualidade do leite e da higiene do úbere em animais confinados no sistema Compost Barn, bem como buscar associações entre patógenos isolados do leite e características do composto. Três rebanhos leiteiros participaram do estudo e as amostras foram coletadas em diferentes períodos em cada fazenda. Amostras individuais de leite foram coletadas em duplicata para análise de CCS e cultura microbiológica. Patógenos ambientais causaram a maioria dos casos de mastite clínica na fazenda 2, e na fazenda 1 a maioria dos casos foi em decorrência de patógenos contagiosos. Amostras de material da cama foram coletadas em duas fazendas para análise da densidade bacteriana e da umidade. A umidade da cama permaneceu dentro da faixa de controle durante o estudo e não foi associada à incidência de patógenos ambientais. A maioria dos animais permaneceu com boa higiene de úbere durante o estudo. A falta de higiene do úbere contribuiu para o aumento da incidência de patógenos ambientais em uma das fazendas. Durante o período mais quente e mais chuvoso, um número maior de animais com escore de sujidade ≥2 foi observado em todos os rebanhos. Não houve associação entre escore de sujidade e contagem de células somáticas em qualquer uma das três fazendas. Os resultados indicam que o perfil de patógenos isolados do leite em animais confinados em Compost Barn sob clima tropical é semelhante a outros sistemas de confinamento adotados em outros lugares. O período do ano influenciou o escore de higiene do úbere, o que reforça a importância do manejo da cama ao longo do ano.

Integrity of prokaryotic mRNA isolated from complex samples for in vivo bacterial transcriptome analysis.

Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs.

Characterization of the microbial community in a lotic environment to assess the effect of pollution on nitrifying and potentially pathogenic bacteria.

This study aimed to investigate microbes involved in the nitrogen cycle and potentially pathogenic bacteria from urban and rural sites of the São Pedro stream. Water samples were collected from two sites. A seasonal survey of bacterial abundance was conducted. The dissolved nutrient content was analysed. PCR and FISH analysis were performed to identify and quantify microbes involved in the nitrogen cycle and potentially pathogenic bacteria. The seasonal survey revealed that the bacterial abundance was similar along the year on the rural area but varied on the urban site. Higher concentration of dissolved nutrients in the urban area indicated a eutrophic system. Considering the nitrifying microbes, the genus Nitrobacter was found, especially in the urban area, and may act as the principal bacteria in converting nitrite into nitrate at this site. The molecular markers napA, amoA, and nfrA were more accumulated at the urban site, justifying the higher content of nutrients metabolised by these enzymes. Finally, high intensity of amplicons from Enterococcus, Streptococcus, Bacteroides/Prevotella/Porphyromonas, Salmonella, S. aureus, P. aeruginosa and the diarrheagenic lineages of E. coli were observed at the urban site. These results indicate a change in the structure of the microbial community imposed by anthrophic actions. The incidence of pathogenic bacteria in aquatic environments is of particular importance to public health, emphasising the need for sewage treatment to minimise the environmental impacts associated with urbanisation.