|Clawson, Michael - Mike|
|Briggs, Robert - Bob|
Submitted to: BMC Research Notes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/11/2023
Publication Date: 1/19/2023
Citation: Dassanayake, R.P., Clawson, M.L., Tatum, F.M., Briggs, R.E., Kaplan, B.S., Casas, E. 2023. Differential identification of Mannheimia haemolytica genotypes 1 and 2 using colorimetric loop-mediated isothermal amplification. BMC Research Notes. 16. Article 4. https://doi.org/10.1186/s13104-023-06272-8.
Interpretive Summary: Mannheimia haemolytica is the primary bacterial pathogen involved in bovine respiratory disease complex (BRDC), commonly known as shipping fever, causing extensive economic losses to the beef and dairy cattle industries in the United States. It is an opportunistic bacterial pathogen that primarily resides as a commensal of the upper respiratory tracts of healthy cattle and leads to develop BRDC when the host's immune system is compromised. M. haemolytica isolated from North American cattle were classified into two genotypes, 1 and 2. Genotype 1 is commonly isolated from healthy animals while genotype 2 is largely isolated from lungs of animals affected with BRDC. However, isolation of both genotypes from samples collected from animals suffering from BRDC can occur and complicate BRDC diagnosis. Therefore, we have developed a rapid colorimetric assay (loop-mediated isothermal amplification) to distinguish M. haemolytica genotype 1 from genotype 2 strains.
Technical Abstract: Objective: Mannheimia haemolytica is the primary bacterial pathogen associated with bovine respiratory disease complex (BRDC). While M. haemolytica has been subdivided into 12 serotypes (ST), ST1, ST2 and ST6 are commonly isolated from cattle. More recently, M. haemolytica strains isolated from North American cattle have been classified into genotypes 1 (ST2) and 2 (ST1 and ST6). Of the two genotypes, genotype 2 strains predominantly cause BRDC, however, isolation of both genotypes from pneumonic lung samples can complicate diagnosis. Therefore, the aim of this study was to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay to differentiate M. haemolytica genotypes. Results: The genotype specificity of the LAMP was tested using purified genomic DNA from 22 M. haemolytica strains (10 genotype 1, 12 genotype 2) and strains from four related Pasteurellaceae species; Bibersteinia trehalosi, Mannheimia glucosida, Pasteurella multocida, and Histophilus somni. Genotype 1 (adhesin pseudogene B1) specific-LAMP reactions amplified DNA only from genotype 1 strains while genotype 2 (adhesin G) reactions amplified DNA only from genotype 2 strains. LAMP primers were highly sensitive, and 1-100 target gene copy detection was achieved. LAMP primers designed in this study may help differential identification of M. haemolytica genotypes 1 and 2.