Not the Usual Suspects - Understanding the Bacterial Populations of Septic Arthritis Cases in Feedlot Cattle

Background

Septic arthritis (SA), also commonly referred to as joint infection, is typically seen in young calves as a secondary infection to navel infections after not receiving enough colostrum or receiving it too late. This causes joint swelling and acute pain which results in underperforming, lame calves. Unfortunately, there are more and more cases presenting at the feedlot. While a significant cause of lameness, it also packs a higher fatality rate than other causes of lameness accounting for almost 50% of lameness related deaths. These mortalities are also occurring later in the feeding period, meaning producers are not only eating the death loss but also the cost in feeding that animal for most of the feeding period. This not only hurts the pocketbook of producers but is a serious animal welfare concern. With the currently available culture-based diagnostic technology, researchers have routinely identified Mycoplasmopsis bovis and less frequently Histophilus somni as the main culprits in these joint infections. However, new studies using DNA sequencing may have a different story to tell.

Objectives

• Identify mystery organisms found in SA infections from a previous study to understand how they contribute to SA infection
• Accelerate DNA-based diagnostics for infectious disease and antibiotic resistance detection and typing

What they Did

The team used DNA sequencing to characterize bacteria in SA samples and found surprising bacterial diversity, including potentially new organisms represented by novel DNA sequences. A variety of new DNA sequencing strategies were used in this project to attempt to classify the mystery DNA sequences and understand their role in the infection. In doing so, the team discovered that bacterial-targeted DNA sequencing used originally had captured previously unrecognized components of the cattle genome. This discovery allowed them to improve the diagnostic workflow to focus diagnostic efforts and sequencing specifically on bacteria. This led to the application of the pioneering DNA-capture method developed by this team to detect and characterize bacteria types which includes bovine-respiratory disease pathogens Pasteurella multocida and Mannheimia haemolytica as well as species of Mycoplasma not previously detected in SA. The capture method also captures all cattle-associated antibiotic resistance genes at the same time, providing an unprecedented speed and sensitivity for diagnostics.

WHat They Learned

DNA (and RNA) sequencing is rapidly becoming the technique of choice for infectious disease diagnostics. DNA sequencing benefits from incredibly rapid advances in sequencing technologies and computational analysis of DNA data. For example, whereas traditional diagnostics like PCR benefit from speed and sensitivity, they are limited by detecting only the specific pathogen against which the test is designed. Moreover, standard diagnostics cannot effectively type a pathogen, which reduces epidemiological insights into targeted treatment options, infectious disease sources, and transmission routes. DNA sequencing on the other hand has the potential to detect all pathogens in a sample and provide critical diagnostic insights into antibiotic resistance, specific pathogen types, and can characterize co-infections.

This research project was based on the use of multiple DNA sequencing approaches to identify which bacteria cause or exacerbate septic arthritis. Preliminary results revealed an unexpected diversity of co-infecting bacteria in cattle joints. Additionally, using metabarcoding to identify all bacterial species in each sample generated new sequences unknown to science. We proposed to apply multiple strategies to identify these mystery phyla that were computationally assigned as ‘bacteria’. These mystery sequences did not match any known DNA sequences in animals or other forms of life. However, our first discovery in this project was that the mystery sequences are in fact portions of cattle genome that were previously missing from cattle genome sequences. This is an important discovery because it allows researchers to refine the computational workflows to incorporate the newest cattle genome sequences —which are improving every year— to remove spurious sequences that computers might otherwise identify as bacterial in origin.

The more significant breakthrough in this project arose from testing a novel approach to capture bacterial DNA from samples with low abundances of DNA. Infected tissues are dominated by >99% cattle DNA. Thus, otherwise powerful techniques like metagenomics are swamped by resequencing the same cattle DNA over and over, which reduces sensitivity for infectious disease agents. An approach to chemically remove cattle DNA before sequencing was tested; this approach increased bacterial DNA only 2-fold, which was not a significant gain. A probe capture technique specifically for cattle infections currently being developed by the research team was also tested. This capture technique proved useful in advance of this project for detecting and genotyping bacterial BRD pathogens and antibiotic resistance genes. In this project they were able to test a new refinement that captures long pieces of DNA. This new approach generated unprecedented sensitivity and quality of bacterial DNA sequence from infected tissue. Consequently, not only did they confidently detect bacterial pathogens not previously associated with SA, like Pasteurella multocida and Mannheimia haemolytica, they also generated extensive DNA sequence for these pathogens. Improving both sensitivity and the length of DNA sequence provides a new level of disease diagnostics. An improved detection of antibiotic resistance genes was also found with this approach. This technology is now being developed for veterinary diagnostics as it can be applied to all types of infection as well as for surveillance on farms and feedlots. As more infections are sequenced the understanding of which bacteria contribute to lameness and which antibiotic treatments are most appropriate for treatment will be improved.  

What This Means

With BCRC research support, the team made two key advances in infectious disease diagnostics for cattle infections. First, they found that previously un-recognized pieces of the cattle genome can be amplified in standard bacteria-targeting DNA-based diagnostic test. This finding is critical for refining DNA analysis workflow to remove all possible cattle DNA; the workflow will improve further with the pending release of new ‘complete’ cattle genome assemblies that will be released soon.

Second, the team had great success testing a brand-new approach to DNA capture and sequencing and found the technique vastly improves sensitivity and the ability to genotype bacteria, which allowed the team to discover previously undetected bacterial species and antibiotic resistance genes in septic arthritis. Importantly, both technical advances can be tested and applied to all infectious diseases in cattle, including BRD, Johnes, viral infections, and infectious disease surveillance.