Immunomagnetic recovery of Mycobacterium bovis from naturally infected environmental samples

Abstract

Aims: To adapt an immunomagnetic capture (IMC) technique to concentrate and cultivate Mycobacterium bovis from environmental samples including soil, faeces and urine. Methods and Results: Cells of Myco. bovis BCG and wild‐type Myco. bovis were successfully isolated and cultured from seeded and naturally infected materials respectively. The IMC cell recovery estimated by colony forming units (CFUs) counts ranged from 0·10% to 0·16% for spiked media, and 0·15–0·36% for naturally infected soil and faeces. Recovery estimated by cell counts calculated using semi‐quantitative PCR ranged from 80·3% to 88·6% for spiked and 84·1–88·2% for naturally infected material. The differences in the recovery rates estimated by CFUs compared with pixel intensity is likely to be due to clustering of cells on culture plates, thereby underestimating the true cell count. Conclusions: The IMC techniques can be applied to isolate viable wild type Myco. bovis from naturally contaminated environmental samples. Significance and Impact of Study: Cultivation of Myco. bovis from environmental samples using traditional methods is extremely problematic. Here, we demonstrate a novel development of IMC techniques that will greatly facilitate the study of the organism in situ in order to assess its epidemiological importance in bovine tuberculosis persistence.

Detection of Mycobacterium tuberculosis based on H37Rv binding peptides using surface functionalized magnetic microspheres coupled with quantum dots – a nano detection method for Mycobacterium tuberculosis

“…IMS has been applied to the enrichment of mycobacteria, including MTB, Mycobacterium bovis bacillus Calmette-Guérin (BCG), M. bovis, etc, in sputum, [9][10][11][12] cerebrospinal fluid, 13 and environmental samples. 14 Coupled with microscopy, polymerase chain reaction or culture has been used as the detection end-points. 15,16 The latest research reported the ligand-coated magnetic beads, TB-beads, that show major advantages over the conventional direct smear detection of MTB on sputum samples.…”

Production and Evaluation of Antibodies and Phage Display-Derived Peptide Ligands for Immunomagnetic Separation of Mycobacterium bovis

“…Previous studies describing IMS methods for detection of M. bovis (10,27,28) have employed polyclonal or monoclonal antibodies (PAbs or MAbs) to M. bovis, and little or no optimization of either magnetic beads or the antibody coating was undertaken. Sweeney et al (27) reported capture efficiencies for M. bovis from spiked environmental samples (soil, badger feces, and urine) of 80.3 to 88.6%.…”

“…Previous studies describing IMS methods for detection of M. bovis (10,27,28) have employed polyclonal or monoclonal antibodies (PAbs or MAbs) to M. bovis, and little or no optimization of either magnetic beads or the antibody coating was undertaken. Sweeney et al (27) reported capture efficiencies for M. bovis from spiked environmental samples (soil, badger feces, and urine) of 80.3 to 88.6%. The capture efficiency was assessed by quantitative PCR using secondary coated Dynabeads and a commercially available polyclonal antibody to M. bovis BCG (DakoCytomation, Glostrup, Denmark) in an indirect IMS approach (i.e., primary coated beads and antibody added separately to sample).…”

Utility of a Fecal Real-time PCR Protocol for Detection of Mycobacterium bovis Infection in African Buffalo (Syncerus caffer)

“…In our limited sample size, infected buffalo might not have shed M. bovis in feces because, in contrast to badgers, M. bovis discharged from the lungs was destroyed in the complex gastrointestinal tract, or the real-time PCR assay was not sensitive enough to detect M. bovis diluted in the large fecal volume. Combining real-time PCR with immunomagnetic capture (Sweeney et al 2006) …”