Improved Diagnosis of Bacterial Diseases of Cattle

NON-TECHNICAL SUMMARY: Bacterial diseases have always been important threats to animal production and human health. With the increase in antimicrobial resistance among bacterial pathogens and the limitations on the use of antibiotics, the rapid and accurate detection of bacteria in food-producing animals is essential. Without such ability we risk the failure to detect animal diseases, both existing and emerging, and food safety threats. The current methods used to diagnose bacterial diseases are well established, but being more than a century old, lack both the speed and accuracy required for modern animal production systems. Therefore, we propose here to establish a molecular method as a central means to identify clinically important bacterial pathogens of cattle. As a part of the Animal Health Diagnostic Center, our Clinical Bacteriology Laboratory processes thousands of samples from New York dairy cattle each year. The goal of our proposal is to create a system by which we can identify bacterial pathogens of cattle through the integration of molecular biology with conventional culture techniques. Our plan is to develop the means to identify bacteria cultured from cattle using DNA sequencing. From bacteria obtained from cattle over the next two years, we will isolate DNA and amplify the16S RNA gene. This gene is especially useful for identification, as it exists in all bacteria, but its DNA sequence varies among species. Importantly, it has been used in numerous research applications, and so we can compare our sequences to those of thousands of bacterial species stored in public databases. It is also an extremely useful gene as it varies among bacteria based upon phylogeny; that is, more closely related species have more similar versions of the genes. Thus, it can be used to identify new types of bacteria by their relatedness to existing species. In conjunction with this molecular identification, we will also perform our conventional identification scheme on these isolates, using biochemical and morphological tests to correlate the DNA sequence of the bacterium with its physical characteristics. Although the DNA databases are very large, we may culture bacteria that have been previously uncharacterized. This challenge, however, also presents a great opportunity, as it will allow us to identify new bacterial pathogens of veterinary importance. The long-term goal of this work is thus to make DNA sequencing a practical, routine diagnostic tool in the identification of cattle disease that can be an immediate aid to our clients. This work will have immediate benefits to one of our largest groups of clients, the dairy producers of the state of New York. Once established, this molecular method will become a permanent and integral part of our diagnostic scheme and so will help us to more rapidly and accurately diagnose cattle diseases. The project has obvious implications for dairy producers, but also for the agricultural and safety interests of the state overall, as it will provide a way to better detect food safety threats and new or emerging diseases.

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APPROACH: For this study, we will use 16S rRNA gene sequencing in conjunction with conventional culture methods to identify bacteria from cattle. All samples submitted for aerobic culture to the Clinical Microbiology Laboratory of the AHDC from New York cattle (approximately1500/year) will be tested over two years. Bacteria will be grown on laboratory media, and the 16S gene will be PCR amplified from individual isolates using universal primers. We anticipate an average of two bacterial species per sample, for a total of 6000 isolates. PCR products will be sequenced, and we will analyze the sequences using two databases, Genbank, a large public database, and the Ribosome Database Project, which contains only 16S sequences. The latter also has numerous tools that we will use to assess the quality of the sequence and identify phylogeny with known organisms. Comparisons of sequences will be performed manually to determine the relatedness of the cultured bacteria with those in the databases. We will identify species based upon guidelines established by the Clinical and Laboratory Standards Institute (CLSI), which recommends identification procedures using 16S gene sequencing (Approved Guideline MM18-A). In conjunction, all isolates will be identified using our conventional biochemical scheme. DNA sequence information will then be combined with these phenotypic tests to establish a resident database for cattle pathogens. This work offers the application of a powerful research tool, bacterial identification by DNA sequencing, to the practical service of clinical microbiology. The information we create will be used to establish a comprehensive database of veterinary pathogen sequences that can be used as a resource by the veterinary diagnostic laboratory community. In addition, this effort will provide improved diagnosis of bacterial disease to our clients. We anticipate that this method will become a standard means in our laboratory to diagnosis disease and so will have lasting impacts on our service in years to come.