Contamination Rate

An important characteristic of any MAP cul­ture protocol is its capacity to prevent growth of irrelevant microbes that may be present in a clinical sample. On solid media, colonies of MAP may not be visible among colonies of other or­ganisms, but sometimes MAP can still be detect­ed by PCR from the surface of the slope (Secott et al., 1999; Whittington, 2009).

The reported contamination rates in MAP faecal culture are extremely variable and there is still a need for a better culture protocol (Table 18.5). Contamination rates for culture on solid media in human clinical mycobacteri- ology are also highly variable, and in one study ranged from 0.4 to 41% (van Griethuysen et al., 1996).

Contamination of faecal cultures is due to the presence of microbes that resist decon­tamination; not unexpectedly their prevalence can vary with the diet of livestock and their geographical location and typically is clustered within groups of samples from certain farms or localities (Whitlock et al., 1989; Whittington, 2009; Rangel etal., 2017). In one study of 2599 faeces from 13 7 farms, between 1.7 and 11% of samples among farms yielded growth of irrel­evant organisms (Whittington, 2009). Culture protocols may need to be modified, for example by inclusion of additional antibiotics, in order to obtain satisfactory results for samples from some farms.

Contamination of intestinal tissue and mesenteric lymph node cultures is less prob­lematic.

HPC HEYM bgcolor=white>131 (3) 26 and 60% Presence of contaminants Kimef al. (1989) Cattle 1. Sedimentation, 3μm filtered

HPC BACTEC 12B 603 3.9% Uncertain Collins etal. (1990b) Cattle 1. Sedimentation

HPC BACTEC 12B 453 7.7% Presence of contaminants Cousinsef al. (1995) Alpaca 1. Sedimentation

HPC BACTEC 12B 137 7.3% Presence of contaminants Cousins etal. (1995) Cattle 5. Centrifugation NaOH- OA LJ 2989 samples (4) 0.13% of samples

7.0% of slopes Overgrowth Kalis etal. (1999) Cattle 3. Sedimentation, centrifugation HPC HEYM 463 samples (4) 30% Overgrowth of contaminants on >1 slope Secott ef al. (1999) Cattle 5. Centrifugation NaOH-OA LJ and HEYM 2513 (4 HEYM;

2 LJ) 13 to 14% of samples Overgrowth Nielsen ef al. (2004) Sheep 3.

^aThe number corresponds to a method in Table 18.4. HPG₁ hexadecylpyridinium chloride;

284 R. Whittington

practice is to keep clinical samples out of direct sunlight and chilled during transport to the laboratory. The viability of MAP in samples de­clines during storage at -20°C (e.g. see Hahn et al., 2017; Schwalm et al., 2018). If there is likely to be a delay in set up of cultures, samples should be stored at -80°C where viability in fae­ces is many years (unpublished data). Recovery of MAP from faecal samples that had been ther­mally stressed by eight freeze thaw cycles from -20°C was greater on M7H9C liquid medium (75% of 20 samples) than on solid HEYM (25%) (Schwalm et al., 2018).

There can be a dramatic loss of viable MAP when samples are cultured. The factors are both physical and chemical. A progressive loss of vi­able organisms occurs as only part of the sam­ple material from one step is taken forward to the next step in the protocol. Decontamination solutions kill some MAP and some of the anti­microbials in the culture media may also have a deleterious effect on the viability and growth of MAP. In fact, about 99% (two logs) of the viable MAP organisms in a sample can be killed or lost during routine processing.

Protocols with NaOH or OA reduced the concentration of MAP from cattle by one to two logs in 4 h and BAC reduced it by one log; HPC at concentrations of up to 1% did not af­fect the viability of MAP over a 5-day incuba­tion (Merkal et al., 1982; Whipple and Merkal, 1983). The NaOH-OA protocol was confirmed independently to cause a two-log loss of viable MAP (Jorgensen, 1982). The double incuba­tion protocol (method 3 in Table 18.4), which involves both HPC and the antibiotic mixture vancomycin, amphotericin B and nalidixic acid (VAN) caused a 2.7-log loss of the MAP-S strain, of which 1.3-1.7 logs loss was due to the VAN step and little due to HPC (Reddacliff et al., 2003b).

The situation with tissue samples is simi­lar or worse; there was a 3.1-log loss with the HPC protocol (Reddacliff et al., 2003b). There is a striking deleterious impact of HPC on MAP in milk: the temperature (22°C optimal) and duration of incubation (2-5 h optimal) in this decontaminant can be critical to minimize inac­tivation of MAP. After 5 h of incubation in HPC, losses are such that analytical sensitivity in liq­uid media is no better than 10² to 10³ MAP cells per millilitre of milk (Grant et al., 2003). These results have been confirmed, leading to recom­mendations that NALC-NaOH be used instead of HPC (Bradner et al., 2013b, 2014) (see Section 18.11.4).

Conflicting data for the impact of HPC on the recovery of MAP from faeces, tissues and milk suggest that there might be complex inter­actions between this chemical, the type of sub­strate, the strain of MAP and the culture media.

18.15