Sequence to Discover: The Metagenomic Context Surrounding Mycoplasma bovis Infection in Cattle

Last year Norgen Biotek & Illumina partnered to offer a Sequence to Discover Research Grant. This grant offered researchers $20,000 towards Norgen products as well as Norgen’s sequencing services. A group at the Applied Genomics Centre (AGC) at Kwantlen Polytechnic University (KPU) led by Lyndsey Baillie and Dr. Paul Adams was one of the recipients, and used the funds to help tackle a cattle disease of major concern on dairy farms. The Applied Genomics Centre at KPU uses genomic and metabolomic tools to address the biggest challenges in the Canadian Agricultural Sector. They work together with industry partners to study issues surrounding breeding and pathogens related to livestock, plants, fish.

What is M. bovis?

A significant issue affecting the dairy and beef industry in Western Canada is the management of cattle infected with Mycoplasma Bovis (M. bovis). M. bovis is a bacterium implicated in a variety of diseases. In mature cattle it is associated with mastitis, pneumonia or arthritis, whereas in calves it often presents as pneumonia or otitis media (an ear infection that causes a characteristic ‘head tilt’) .

M. bovis is a unique bacterium. Mycoplasma species are among the smallest free-living species, they have a reduced genome and lack a cell wall. Due to the lack of cell wall, Mycoplasma are inherently resistant to many antibiotics that target the cell wall and can change their shape. They also possess strategies to constantly change their surface antibodies, as well as mechanisms for evading host antibodies.¹ These characteristics make it particularly problematic to treat an infection with many traditional antibiotics.

The disease may be dormant in an animal, causing no symptoms at all. But in times of stress (for example, calving, drying-off, transporting or being exposed to extreme weather), the animal may shed bacteria in milk and nasal secretions. It is thought that other animals may then become infected through close contact, bodily fluids or milking equipment. Moreover, the disease can spread from farm to farm as cattle are transported or through the abattoir.

Is Mycoplasma bovis Related to Bovine Tuberculosis?

Bovine Tuberculosis (bTB) is a disease caused by Mycobacterium bovis, a relative of Mycobacterium tuberculosis. Mycoplasma bovis and Mycobacterium bovis are not the same. Although they have the same species name (bovis) their different first names (Mycoplasma or Mycobacterium) indicate a completely different genera of bacteria. As mentioned, Mycoplasma are small bacteria lacking a cell wall. On the other hand, Mycobacteria are rod shaped bacteria with very thick waxy cell wall, that also makes it difficult to treat with antibiotics. Infection with either bacterium is associated with pneumonia in cattle, however only Mycobacterium bovis can be transmitted to humans through unpasteurized milk, causing Tuberculosis. Due to its ability to infect humans, Mycobacterium bovis has been monitored and controlled in the Canadian cattle industry for many years, while Mycoplasma bovis still remains less well understood.

Mycoplasma bovis Surveillance Programs

Due to its ability to lay dormant, its transmissibility, and its resistance to many antibiotics, combined with the economic importance of the Cattle industry, many governments have begun surveillance and eradication programs for Mycoplasma bovis. In 2017 M. bovis was detected on a farm in New Zealand, the next year they implemented a strict eradication program. Currently, New Zealand is considered M. bovis free with no new cases detected since 2022. However, this came at a huge cost, almost 184,000 cattle culled, including 280 farms depopulated, nearly 3,000 farms subject to movement controls and many more undergoing on-farm testing, the programme touched nearly every farming community across the country.²

Current M. bovis qPCR Detection Methods

In partnership with WestGen, Western Canada’s leading cattle genetics and reproduction company, researchers at Kwantlen Polytechnic University (KPU) have developed a qPCR assay for M. bovis along with a detailed testing strategy. Their M. bovis detection test has been trialed on farms across Western Canada, to help determine the prevalence and possible impact of infection. Going forward, use of this test will assist in decision making around disease management strategies to reduce the overall impact on animal health and economic loss. Initial results have shown that M. bovis is present in at least 17% of Western Canadian dairy farms, and infection with the bacteria appears to be correlated with disease symptoms. This highlights the need for a more complete understanding of the factors that determine disease severity.

Is Mycoplasma bovis the Sole Culprit ?

There is debate as to whether M. bovis is the sole causative agent or is an opportunistic pathogen in the presences of other pathogens or in a dysbiotic community. As such, the group at KPU wants to obtain a fuller picture of the microbial communities present in cattle infected with M. bovis and the corresponding symptoms

 "Our aim is to use Norgen’s DNA extraction kit and perform 16S metagenomics NGS sequencing. Specifically we are interested in looking at Mycoplasma bovis in the context of the microbiome from bovine samples experiencing pathology”
- Lyndsey Baille : Applied Genomics Center, Kwantlen Polytechnic University.

In order to understand how the microbiome correlates with the variety of symptoms, they collected hundreds of samples (ear swabs, nasopharyngeal swabs, milk and joint fluid) After extracting DNA using Norgen’s Milk Bacterial DNA Isolation Kit (Cat. 21550), they performed qPCR for M. bovis as well as 16S metagenomic sequencing. Norgen’s service team prepared sequencing libraries using Norgen’s 16S V4 Library Preparation Kit for Illumina (Cat. 70600), and sequenced the samples using an Illumina MiSeq at Norgen Biotek's laboratory in Thorold, ON.

 "Norgen Biotek provided great customer service and were always happy to spend time answering our questions. We had struggled with troubleshooting library prep on these samples in our own lab due to low DNA concentration and issues with PCR inhibitors, but the technicians at Norgen were able to prepare excellent libraries from all our samples. The NGS results we received were of high quality and allowed us to proceed to data analysis”
- Lyndsey Baille : Applied Genomics Center, Kwantlen Polytechnic University.

Preliminary Results

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Although analysis is still ongoing, the NGS sequencing results provided by Norgen have allowed for a picture of the bovine microbiome in the context of M. bovis infection. Mycoplasma bovis was present in higher abundance in cattle displaying clinical symptoms than in those that were asymptomatic. M. bovis was also still detectable in animals that had previously had clinical symptoms but had recovered at the time of testing. Interestingly, M. bovis was sometimes found at low levels even in completely asymptomatic cows.

Moreover, some of the cattle samples were collected at multiple body sites, for example from both ears as well as the nose. In some of these cases only 1 of these samples tested positive for M. bovis, while the others were negative. This demonstrates the importance of thorough testing on farms, and shows that more research is needed to determine what factors determine how localized versus systemic an infection is.