Rapid detection of Mycobacterium bovis in bovine cytological smears and tissue sections by peptide nucleic acid fluorescence in-situ hybridization

Abstract

Background

Bovine tuberculosis is the leading cause of death in cattle and other species worldwide. Quick and precise identification of mycobacteria is critical to control the occurrence of tuberculosis in cattle.

Methods

We developed a fluorescent peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) approach to detect Mycobacterium bovis and Mycobacterium avium in cytological smears and tissue sections of bovines suspected of having tuberculosis. PNA-FISH was conducted on smears of lung and lymph node tissues. Standard bovine mycobacterial cultures were used to standardize the probes using 50 % formamide for M. bovis and 30 % formamide for M. avium. M. bovis probe (MTBCcy3), which was standardized at hybridization conditions of (55 °C and 40 % formamide) concentrations, was positive in all cytological smears.

Results

Four out of twenty five samples tested positive in tissue sections observed as a bright red fluorescence with a cy3 filter (MTBC probe). No results were observed with (MAV_TAMRA) probe for M. avium which was standardized at hybridization conditions of (55 °C and 30 % formamide). No fluorescence was observed in the control tissue sections. Additionally, the results were juxtaposed with those of other commonly used detection methods such as immunohistochemistry and Polymerase Chain Reaction (PCR) by targeting the esxA gene. None of the samples tested positive for M. avium infection.

Conclusion

PNA-FISH can be used to obtain cytological impression smears and tissue sections. When compared to PCR it consumes less time in the diagnosis of bovine tuberculosis in post mortem cases.