PCR compared to bacterial culture for detection of sheep respiratory pathogens

For years culture techniques were considered the gold standard for detection of bacterial pathogens involved in infectious disease in animals. The advent of real-time hydrolysis probe PCR is making us re-think the gold standard for diagnosis of a range of animal diseases.

Diagnostic samples from cattle or sheep are often contaminated from field collection, have undergone autolysis or bacterial overgrowth during transport to the laboratory, or are from animals that have chronic disease. This presents challenges that PCR can help overcome.

Bacterial pneumonia and pleurisy in sheep and lambs  involves multiple, opportunistic pathogens. When stress from adverse weather conditions, mixing groups of lambs and sheep, overcrowding and transport occur on top of an existing Mycoplasma ovipneumoniae or viral infection the sheep’s immune system is suppressed. When this happens, bacteria, such as Mannheimia haemolytica, that normally live inside the nose and throat of ruminants without causing harm, colonize the trachea and bronchi, where they grow and secrete toxins. These toxins cause inflammation and tissue destruction, and  acute bronchopneumonia develops.

For highly fastidious and slow growing bacteria like Mycoplasma the benefits of PCR are obvious – more rapid and reliable detection of outbreaks and shedding [1, 2]

But what about less fastidious and faster growing bacteria like M. haemolytica?

Recently published research has demonstrated how PCR can enhance detection of M. haemolytica in bacterial pneumonia and pleurisy in sheep and lambs .

Working with North American Bighorn sheep, researchers in the western USA found that PCR for  M. haemolytica detected the pathogen in 77% of lung samples that were negative using traditional culture techniques [3]. The researchers thought this could be because two other bacterial pathogens involved in bacterial pneumonia and pleurisy in sheep and lambs, Biberstenia trehalosi  and Pasteurella multocida, can outgrow and inhibit the growth of M. haemolytica during culture [4, 5].

Researchers at the University of Nebraska have developed a multiplex PCR test for M. haemolytica  and P. multocida and used this to demonstrate that culture detects only  46% of M. haemolytica positive samples and 77% of P. multocida  positive samples [6]. Co-infections with M. haemolytica  and P. multocida were also more commonly found using PCR.

The PCR test for M. haemolytica  and P. multocida is now up and running in our laboratory. We are using the assay in our current abattoir survey of pneumonia and pleurisy pathogens in Australian sheep and lambs with good results. The assay is helping us detect co-infections with M. haemolytica and P. multocida.

References

[1] K. Sachse, H. S. Salam, R. Diller, E. Schubert, B. Hoffmann and H. Hotzel, “Use of a novel real-time PCR technique to monitor and quantitate Mycoplasma bois infection in cattle herds with mastitis and respiratory disease,” The Veterinary Journal, vol. 186, pp. 299-303, 2010.

[2] F. Yang, X. Dao, A. Rodriguez-Palacios, X. Feng, C. Tang and X. Yang, “A Real-Time PCR for Detection and Quantification of Mycoplasma ovipneumoniae,” Journal of Veterinary Medical Science, vol. 76, no. 12, pp. 1631-1634, 2014.

[3] S. Shanthalingnam, A. Goldy, J. Bavanathasivan, R. Subramaniam, S. A. Batra, A. Kugadas, B. Raghavan, R. Dassanayake, J. Jennings-Gaines, H. Killion, W. H. Edwards, J. M. Ramsey, N. J. Anderson, P. L. Wolff, K. Mansfield, D. Bruning and S. Srikumaran, “PCR assay detects Mannheima haemolytica in culture-negative pneumonic lung tissue of bighorn sheep (Ovis canadensis) from outbreaks in the western USA, 2009-2010,” Journal of Wildlife Diseases, vol. 50, no. 1, pp. 1-10, 2014.

[4] R. P. Dassanayake, D. R. Call, A. A. Sawant, N. C. Casavant, G. C. Weiser, D. P. Knowles and S. Srikumaran, “Biberstenia trehalosi inhibits growth of Mannheima haemolytica by a proximity-dependent mechanism,” Applied and Environmental Microbiology, vol. 76, no. 4, pp. 1008-1013, 2010.

[5] J. Bavananthasivam, R. Dassanayake, A. Kugadas, S. Shanthalingam, D. R. Call, D. P. Knowles and S. Srikumaran, “Proximity-dependent inhibition of growth of Mannheimia haemolytica by Pasteurella multocida,” Applied and Environmental Microbiology, vol. 78, no. 18, pp. 6683-6688, 2012.

[6] J. D. Loy, L. Leger, A. M. Workman, M. L. Clawson, E. Bulut and B. Wang, “Development of a multiplex real-time PCR assay using two thermocycling platforms for detection of major bacterial pathogens associated with bovine respiratory disease complex from clinical samples,” Journal of Veterinary Diagnostic Investigation, vol. 30, no. 6, pp. 837-847, 2018.