Short communication: Application of an N-acetyl-l-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subspecies paratuberculosis from milk of naturally infected cows

Abstract

Mycobacterium avium ssp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne's disease and, therefore, is a route of transmission for infection in young stock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of MAP from individual bovine milk samples from known infected herds. Decontamination of milk samples (n = 17) with either 0.75% hexadecylpyridinium chloride for 5h or N-acetyl-L-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15 min before culture in BACTEC 12 B (Becton Dickinson, Franklin, NJ), para-JEM [Thermo Fisher Scientific (TREK Diagnostic Systems, Inc.), Cleveland, OH], and Herrold's egg yolk (HEY; Becton Dickinson) media was compared. Treatment with NALC-NaOH resulted in a lower percentage (6%) of contaminated samples than did treatment with hexadecylpyridinium chloride (47%), regardless of culture medium. The decontamination protocol (NALC-1.5% NaOH) was then applied to milk samples (n = 144) collected from cows at 7 US dairies. Recovery of viable MAP from the milk samples was low, regardless of culture medium, with recovery from 2 samples cultured in BACTEC 12 B medium, 1 sample cultured in para-JEM medium, and no viable MAP recovered on HEY medium. However, 32 cows were fecal culture positive and 13 milk samples were positive by direct PCR, suggesting that several cows were actively shedding MAP at the time of milk collection. Contamination rates were similar across media, with 39.6, 34.7, and 41.7% of samples contaminated after culture in BACTEC 12 B, para-JEM, and HEY media, respectively. Herd-to-herd variation had a major effect on sample contamination, with the percentage of contaminated samples ranging from 4 to 83%. It was concluded that decontamination of milk with NALC-1.5% NaOH before culture in BACTEC 12 B medium was the most efficacious method for the recovery of viable MAP from milk, although the ability to suppress the growth of contaminating microorganisms varied greatly between herds.

Rapid and sensitive diagnosis of live Mycobacterium tuberculosis using clustered regularly interspaced short palindromic repeat‐Cas13a point‐of‐care RNA testing

“…To enable the rapid detection of MTB RNA, we intended to integrate an extraction‐free method of RNA preparation into CRISPR‐Live‐MTB. Thus, the clinical samples were inactivated for 15 min using NALC‐NaOH method 26 followed by a 15 min sonication process for RNA release. The RNA samples were then subjected to CRISPR‐Live‐MTB and the results were resolved on lateral flow strips with densitometry analyses (Figure 5A).…”

“…Specimen processing was performed using the N‐acetyl‐L‐cysteine (NALC)‐NaOH method 26 . Sputum of approximately 5 mL was inactivated by thoroughly mixing with an equal volume of NALC‐NaOH solution and placed at room temperature for 15 min.…”

“…Specimen processing was performed using the N-acetyl-L-cysteine (NALC)-NaOH method. 26 Sputum of approximately 5 mL was inactivated by thoroughly mixing with an equal volume of NALC-NaOH solution and placed at room temperature for 15 min. The sediment was washed once with a sterile 0.9% NaCl solution and resuspended in 1.5 mL sterile 0.9% NaCl solution.…”