Toxoplasma gondii: Prolonged in-vitro maintenance of virulent tachyzoites in fluid media at low temperatures

Peer review under responsibility of Alexandria University Faculty of Medicine.

Available online 27 November 2018

Figures & data

Table 1 Vitality % (mean ± SD) and infectivity of tachyzoites maintained in fluid media at 37 °C in the darkness.

Days post incubation (dpi)	PBS		PBS + 3% FBS		RPMI		RPMI + 3% FBS		P value^2
	% of Viability	% mortality of mice	% of Viability	% mortality of mice	% of Viability	% mortality of mice	% of Viability	% mortality of mice
2 dpi	100 ± 0	NP	100 ± 0	NP	100 ± 0	NP	100 ± 0	NP	≤ 0.001^**
3 dpi	89.5 ± 2.12	NP	83.4 ± 1.1	NP	100 ± 0	NP	93.5 ± 2.12	NP
4 dpi	73 ± 1.4	NP	55.5 ± 0.5	100	77.8 ± 1.8	NP	66 ± 1.3	100
5 dpi1	55 ± 0.7	100	32.3 ± 1.0	0	68.2 ± 1.7	100	45.6 ± 0.1	100
6 dpi	35.5 ± 0.7	0	21.7 ± 0.4	0	42.7 ± 1.14	100	31.6 ± 2.0	0
7 dpi	20 ± 1.4	0	17.4 ± 0.84	0	32.9 ± 1.5	0	23.6 ± 1.95	0
8 dpi	14.75 ± 0. 35	0	8.8 ± 1.3	0	22 ± 3.6	0	18.1 ± 1.3	0
9 dpi	8.35 ± 0.5	0	0 ± 0	0	14 ± 0.4	0	11.5 ± 0.7	0
P value^1	0.005^*		0.017^*		0.002^*		0.005^*

NP: the test was not performed at this level of infection.

P value^1*: comparison of viability rate at different dpi within the same group (student t test).

P value^2**: comparison of viability rate at the same dpi between different groups (oneway ANOVA).

Table 3 Vitality % (mean ± SD) and infectivity of tachyzoites maintained in fluid media at refrigerator temperature ( + 4–6 °C) in the darkness.

dpi	PBS		PBS + 3% FBS		RPMI		RPMI + 3%FBS		P value^2
	% of Viability	% mortality of mice	% of Viability	% mortality of mice	% of Viability	% mortality of mice	% of Viability	% mortality of mice
5 dpi	100 ± 0	NP	100 ± 0	NP	100 ± 0	NP	100 ± 0	NP	≤ 0.001^**
7 dpi	92 ± 0.7	NP	88.9 ± 0.7	NP	94.3 ± 1.4	NP	90.5 ± 2.8	NP
9 dpi	89.6 ± 2.3	NP	82.0 ± 0.04	NP	90 ± 1.4	NP	85.6 ± 2.1	NP
10 dpi	82.5 ± 1.6	NP	73.1 ± 3.4	NP	84.2 ± 0.27	NP	76 ± 2.1	NP
11 dpi	72.2 ± 1.8	NP	60.5 ± 2.1	100	76.5 ± 0.7	NP	66 ± 2.8	100
12 dpi	64.4 ± 0.8	100	53.0 ± 2.9	100	68.13 ± 1.2	100	57.13 ± 2	100
13 dpi	54.7 ± 0.5	100	48.6 ± 0.4	100	59.8 ± 1.6	100	51.0 ± 1.4	100
14 dpi	48.7 ± 0.7	100	42.2 ± 1.1	0	50.5 ± 0.7	100	45 ± 0.0	100
15 dpi	42 ± 0.8	0	35.5 ± 3.23	0	45.3 ± 0.4	100	38 ± 2.1	0
16 dpi	33.6 ± 0.64	0	28.2 ± 1.70	0	38.13 ± 0.18	100	28.1 ± 2.7	0
17 dpi	22.2 ± 1.3	0	15 ± 1.4	0	26.4 ± 0.9	0	19 ± 7.0	0
18 dpi	10.4 ± 0.5	0	0 ± 0	0	12.2 ± 1.14	0	8 ± 4.2	0
P value^1	≤ 0.001		≤ 0.001		≤ 0.001		≤ 0.001

NP: the test was not performed at this level of infection.

P value^1*: comparison of viability rate at different dpi within the same group (student t test).

P value^2**: comparison of viability rate at the same dpi between different groups (oneway ANOVA).

Table 2 Vitality % (mean ± SD) and infectivity of tachyzoites maintained in fluid media at room temperature (18–22 °C) in the darkness.

Dpi	PBS		PBS + 3% FBS		RPMI		RPMI + 3%FBS		P value^2
	% of Viability	% mortality of mice	% of Viability	% mortality of mice	% of Viability	% mortality of mice	% of Viability	% mortality of mice
4 dpi	100 ± 0	NP	100 ± 0	NP	100 ± 0	NP	100 ± 0	NP	≤ 0.001^**
6 dpi	85.1 ± 1.0	NP	73 ± 1.6	NP	87.5 ± 0.9	NP	76.5 ± 0.7	NP
8 dpi	67.4 ± 1.8	100	53.5 ± 1.8	100	72.8 ± 1.0	NP	64.4 ± 2.2	100
9 dpi	49.3 ± 1.5	100	42.8 ± 1.0	100	60.9 ± 1.2	100	51 ± 1.3	100
10 dpi	34.5 ± 0.9	0	31.9 ± 2.4	0	42.3 ± 1.9	100	35.9 ± 0.18	0
11 dpi	25.6 ± 0.5	0	15.3 ± 0.4	0	35.8 ± 1.1	0	18.5 ± 0.7	0
12 dpi	15.0 ± 0.6	0	10.5 ± 0.7	0	22.4 ± 0.9	0	12.6 ± 0.6	0
P value^1	0.004		0.008		0.001		0.005

NP: the test was not performed at this level of infection.

P value^1*: comparison of viability rate at different dpi within the same group (student t-test).

P value^2**: comparison of viability rate at the same dpi between different groups (oneway ANOVA).

Table 4 The degree of significant difference between the results obtained at two different temperatures circumstances for the same fluid medium.

Groups	(P value for significance)
	37–18 °C	18–4 °C	37–4 °C
PBS	0.001	≤ 0.001	≤ 0.001
PBS + 3% FBS	0.001	≤ 0.001	≤ 0.001
RPMI	0.001	≤ 0.001	≤ 0.001
RPMI + 3%FBS	0.001	≤ 0.001	≤ 0.001

Paired sample test.