Cultural Requirements of Different Strains of MAP

A variety of types of MAP occur in cattle, sheep, goats, deer and other hosts (Stevenson, 2015). Molecular genetic differences between MAP isolates led to the naming of so-called C (cattle) and S (sheep) strains based on the hosts of origin of the isolates (Collins et al., 1990a).

Other types of MAP have been reported: yellow-orange pigmented strains in the UK and so-called bison strains in the USA and India (see Chapter 6, this volume). Compared with MAP-C, the MAP-S, pigmented and bison strains grow less well, poorly or not at all on HEYM (Collins et al., 1990a; Juste et al., 1991; Whittington et al., 1999; Whitlock et al., 1999; Stevenson et al., 2002). Furthermore, MAP-S strains do not grow readily in MGIT ParaTB medium due to the inclusion of vancomycin (Gumber and Whittington, 2007). Kawaji et al. (2014) excluded vancomycin from MGIT medium and improved the detection time for growth of a laboratory adapted MAP-S strain, but the MAP-S strain still required a prolonged incubation in this medium compared with the MAP-C strain.

In an attempt to understand apparent disparities in culture success between strains and countries, 35 field isolates comprising 14 different BstEII IS900∕IS1311 genotypes and representing MAP-C, MAP-S and bison strains that originated from the USA, Spain, Northern Ireland and Australia were propagated in BACTEC 12B medium, Middlebrook 7H10 agar, LJ agar, HEYM, modified Middlebrook 7H10 agar, Middlebrook 7H11 agar and WatsonReid (WR) agars (Whittington et al., 2011). Most MAP-S strains grew poorly on HEYM and pyruvate was inhibitory to some isolates, but

bgcolor=white>3.33 mg^h

Table 18.3. Comparison of the composition of the Middlebrook-based liquid culture media M7H9C, BACTEC 12B and MGIT ParaTB.
Ingredient	Amount/litre			Amount/vial
Ingredient	M7H9C	BACTEC	MGIT	M7H9C	BACTEC	MGIT
Base-Middlebrook 7H9	3.1 g			18.6 mg	4.0 ml	7.0 ml
C-14 palmitic acid	-	667 uCi	-	-	4 uCi	-
Fluorescent indicator	-	-	13.25 ml	-	-	110 μl
Casein	667 mg^a	667 mg^b	48.2 mg ^c	4.0 mg	4.0 mg	0.4 mg
Bovine albumin	1340 mg^d	3300 mg^b	4820 mg^c	8.0 mg	19.8 mg	40 mg
Dextrose	534 mg^d	-	-	3.2 mg	-	-
Catalase	0.8 mg^d	32,000 U^b	4627 U^c	4.8 μg	192 U	38.4 U
Oleic acid	-	-	9.64 mg^c	-	-	0.08 mg
Egg yolk	167 ml^e	167 ml^e	30.1 ml^f	1.0 ml	1.0 ml	0.25 ml
Mycobactin J	0.83 mg^e	0.83 mg^e	Included^g	5 μg	5 μg	Included
Polyoxyethylene stearate	optional	133 mg^h	-	Optional	0.80 mg ^h	-
Amphotericin B	3.33 mg^h	3.33 mg^h	7 mgⁱ	20 μg	20 μg	60 μg
Nalidixic acid	13.3 mg^h	13.3 mg^h	18 mgⁱ	80 μg	80 μg	150 μg
Vancomycin hydrochloride	-	-	18 mgⁱ	-	-	150 μg
Polymyxin B	33,300 U^h	33,300 U^h	-	200 U	200 U	-
Trimethoprim	3.33 mg^h	3.33 mg^h	-	20 μg	20 μg	-
Azlocillin	3.33 mg^h	-	20 μg	20 μg	-
Ampicillin (optional)	100 mg	100 mg	100 mg	0.6 mg	0.6 mg	0.83 mg
Final volume	1000 ml	1000 ml	1000 ml	6.0 ml	6.0 ml	8.3 ml

^aAdded by user as casitone.

^bAlready added by manufacturer to Middlebrook 7H9 broth to form BACTEC 12B medium.

^cAdded by user to each MGIT ParaTB medium vial as MGIT ParaTB supplement.

^dAdded by user as ADC, 26.7 ml per litre or 0.16 ml per vial.

^eAdded by user after dispensing medium to each M7H9C or BACTEC 12B medium vial (100 μl of 50 μg∕ml mycobactin J solution, 600 μl water, 1 ml egg yolk) resulting in a final volume of 6 ml medium.

^fEgg yolk (0.25 ml plus 0.25 ml water) added by user to each MGIT ParaTB medium vial resulting in a final volume of 8.3 ml; BD recommends egg yolk enrichment, a 50% egg yolk solution, at a rate of 0.5 ml per MGIT ParaTB medium vial.

⁹Amount not specified by the manufacturer.

^hAdded by user to each M7H9C or BACTEC 12B medium vial as 200 μl PANTA PLUS; an alternative form of antibiotics such as BD BBL MGIT PANTA, which does not contain polyoxyethylene stearate, may be used.

Added by user to each MGIT ParaTB medium vial from stock antibiotic solutions.

all 35 grew on modified Middlebrook 7H10 agar. Growth was slower and less prolific on LJ agar and mycobactin J was required for growth on all media except 7H11 agar. These results were generally supported by those of Dimareli- Malli et al. (2013), who compared the suitability of HEYM, 7H11 and LJ agars to support growth of MAP from small ruminants where both MAP-C and MAP-S strains were present in the livestock population of northern Greece.

The suboptimal growth of MAP that has been reported historically in diverse settings, including for example reports of extremely long incubation periods and minute colony sizes, likely has been strongly influenced by the interaction between strain of MAP and type of culture medium. When an optimal culture medium is used (such as a suitably modified Middlebrook medium), less difference between the growth phenotypes of diverse strains of MAP is observed. Caution should be exercised in the use of a commercial liquid culture system marketed for detection of MAP unless it has been proven to support the growth of all common strains of MAP. Otherwise it is not possible to interpret negative culture outcomes with any confidence.

growth of MAP from the faeces of cows with low- compared with high-shedding rates (Kim et al., 2002). Overall, these data suggest that the environment in which MAP lies immediately prior to incubation in culture medium has a large impact on its subsequent in vitro growth.

18.10

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Source: Behr Marcel A., Stevenson K., Kapur V. (eds.). Paratuberculosis: Organism, Disease, Control. 2nd edition. — CAB International,2020. — 439 p.. 2020

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