Replication ability of three highly protective Marek's disease vaccines: implications in lymphoid organ atrophy and protection

Figures & data

Table 1.  Viral DNA load by real time PCR and pp38 expression in lymphoid organs¹

dpi	Lot	Spleen		Bursa		Thymus
		DNA Load^2	pp38^3	DNA Load	pp38	DNA Load	pp38
1	CVI988	0.0 ± 0.0^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)
	RM1	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)
	648A80	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)
	Control	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)
3	CVI988	0.5 ± 0.1 ^a	1 (0–1, 0.5)	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)
	RM1	3.7 ± 0.0 ^b	2 (1–2, 0.5)	1.3 ± 0.1 ^b	0 (0–0, NA)	2.2 ± 0.1 ^b	2 (1–2, 0.4)
	648A80	0.7 ± 0.1 ^a	1 (0–1, 0.5)	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)
	Control	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)
6	CVI988	2.7 ± 0.1 ^a	2 (1–2, 0.4)	2.4 ± 0.2 ^a	1 (0–1, 0.5)	2.9 ± 0.3 ^a	1 (0–1, 0.5)
	RM1	5.9 ± 0.1 ^b	4 (3–4, 0.5)	6.0 ± 0.4 ^b	3 (2–4, 0.6)	6.7 ± 0.4 ^b	4 (4–4, NA)
	648A80	3.5 ± 0.1 ^c	2 (1–2, 0.5)	2.4 ± 0.2 ^a	1 (1–1, NA)	2.4 ± 0.2 ^a	2 (1–2, 0.4)
	Control	0.0 ± 0.0 ^d	0 (0–0, NA)	0.0 ± 0.0 ^c	0 (0–0, NA)	0.0 ± 0.0 ^c	0 (0–0, NA)
14	CVI988	0.4 ± 0.5 ^a	0 (0–0, NA)	0.2 ± 0.3 ^a	0 (0–0, NA)	0.4 ± 0.4 ^a	0 (0–0, NA)
	RM1	3.2 ± 0.2 ^b	1 (0–1, 0.4)	3.3 ± 0.3 ^b	0 (0–0, NA)	3.2 ± 0.1 ^b	4 (4–4, NA)
	648A80	0.1 ± 0.3 ^a	0 (0–0, NA)	0.0 ± 0.4 ^a	0 (0–0, NA)	0.7 ± 0.4 ^a	0 (0–0, NA)
	Control	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)	0.0 ± 0.0 ^a	0 (0–0, NA)
26	CVI988	1.2 ± 0.1 ^a	0 (0–0, NA)	1.0 ± 0.3 ^a	0 (0–0, NA)	1.6 ± 0.2 ^ab	0 (0–0, NA)
	RM1	2.8 ± 0.2 ^b	1 (0–1, 0.4)	1.8 ± 0.2 ^b	0 (0–0, NA)	1.9 ± 0.2 ^a	ND
	648A–80	1.7 ± 0.2 ^a	1 (0–1, 0.4)	0.9 ± 0.5 ^a	0 (0–0, NA)	1.2 ± 0.7 ^b	0 (0–0, NA)
	Control	0.0 ± 0.0 ^c	0 (0–0, NA)	0.0 ± 0.0 ^c	0 (0–0, NA)	0.0 ± 0.0 ^c	0 (0–0, NA)
^1 Data presented are the average of six birds per group and time point
^2 Viral DNA load is presented as log 10 (number of viral DNA copies per 50ng of total DNA + 1). Results are presented as average of six birds per group and time point ± Standard Deviation. Same superscript alphabets indicate that differences among groups were not statistically significant in the ANOVA test (p < 0.05).
^3 Expression of pp38 antigen is expressed in a subjective scale from 0–4; being 0 = absence of positive stain and 4 = maximum antigenic expression in a tissue. ND = No data for that time point. Data presented are the median value of six birds per group and time point. In brackets (minimum-maximum value, standard deviation). NA = not applicable.

Figure 1.  Immunohistochemical demonstration of replication of the RM1 strain in the thymus and lung at 6 and 14 d.p.i. 1a: Expression of pp38 antigen in the thymus of a chicken 6 days after subcutaneous (s.c.) vaccination with 10,000 PFU of RM1. Bar = 120 µm. 1b: Expression of pp38 antigen in the thymus of a chicken 14 d.p.i. with 10,000 PFU of RM1 by the s.c. route. Bar = 120 µm. Please note that the thymic tissue has marked depletion of lymphocytes and the pp38 expression is limited to the remnant lymphoid tissues. Arrowheads show the islets of positive cells. 1c: Expression of pp38 antigen in the lung of a chicken 6 d.p.i. with 10,000 PFU of RM1 by the s.c. route. Bar = 40 µm. Please note the distribution of positive cells in the parabronchi (asterisks) and within the interstitium. 1d: Expression of pp38 antigen in the lung of a chicken 6 d.p.i. with 10,000 PFU of RM1 by the s.c. route. Parabronchi are indicated with asterisks. Bar = 60 µm.

Figure 2.  Vaccine DNA load in the lung of chickens vaccinated with 10,000 PFU of CVI988, RM1, and 648A80 strains by the subcutaneous route at 1 day of age. The load of viral DNA is expressed as the log of the number of genome copies per 50 ng total DNA. Data represent the average (and standard error) of six chickens per group and per time point. The same letters above the bars indicate that no statistically significant differences were found in the analysis of variance test within the same time point (P<0.05).

Table 2.  Load of viral DNA in cell suspensions of blood and spleen ¹

dpi	Lot	Blood		Spleen
		Non-adherent	Adherent*	Non-adherent	Adherent*
1	CVI988	0.0 ± 0.0 ^a	ND	0.0 ± 0.0 ^a	0.0 ± 0.0 ^a
	RM1	0.0 ± 0.0 ^a	ND	0.0 ± 0.0 ^a	0.0 ± 0.0 ^a
	648A80	0.0 ± 0.0 ^a	ND	0.0 ± 0.0 ^a	0.0 ± 0.0 ^a
	Control	0.0 ± 0.0 ^a	ND	0.0 ± 0.0 ^a	0.0 ± 0.0 ^a
3	CVI988	0.9 ± 0.5 ^a A	1.6 ± 0.3 ^a B	2.3 ± 0.2 ^a C	3.0 ± 0.2 ^a D
	RM1	2.5 ± 0.2 ^b A	3.0 ± 0.2 ^b B	3.9 ± 0.2 ^b C	4.0 ± 0.1 ^b C
	648A80	1.4 ± 0.2 ^a A	2.1 ± 0.2 ^c B	2.3 ± 0.1 ^a B	0.0 ± 0.0 ^c C
	Control	0.0 ± 0.0 ^c A	0.0 ± 0.0 ^d A	0.0 ± 0.0 ^c A	0.0 ± 0.0 ^c A
6	CVI988	3.6 ± 0.2 ^a A	2.2 ± 0.1 ^a B	5.0 ± 0.1 ^a C	5.0 ± 0.1 ^a C
	RM1	4.7 ± 0.1 ^b A	5.2 ± 0.3 ^b B	6.3 ± 0.3 ^b C	4.2 ± 0.2 ^b D
	648A80	2.9 ± 0.1 ^c A	1.0 ± 0.5 ^c B	5.4 ± 0.2 ^a C	4.5 ± 0.1 ^c D
	Control	0.0 ± 0.0 ^d A	0.0 ± 0.0 ^d A	0.0 ± 0.0 ^c A	0.0 ± 0.0 ^d A
14	CVI988	2.4 ± 0.2 ^a A	1.3 ± 0.2 ^a B	3.6 ± 0.1 ^a C	2.6 ± 0.2 ^a A
	RM1	3.4 ± 0.2 ^b A	2.2 ± 0.3 ^b B	4.0 ± 0.1 ^b C	3.4 ± 0.2 ^b A
	648A80	4.1 ± 0.2 ^c A	2.3 ± 0.2 ^b B	4.9 ± 0.2 ^c C	4.4 ± 0.1 ^c A
	Control	0.0 ± 0.0 ^d A	0.0 ± 0.0 ^c A	0.0 ± 0.0 ^d A	0.0 ± 0.0 ^d A
26	CVI988	2.4 ± 0.2 ^a A	0.0 ± 0.0 ^a B	2.1 ± 0.2 ^a A	1.0 ± 0.7 ^a C
	RM1	3.4 ± 0.2 ^b A	1.6 ± 0.2 ^b B	3.9 ± 0.2 ^b C	2.9 ± 0.1 ^b D
	648A80	2.5 ± 0.2 ^a A	1.7 ± 0.2 ^b B	2.6 ± 0.4 ^c A	1.2 ± 0.3 ^a C
	Control	0.0 ± 0.0 ^c A	0.0 ± 0.0 ^c A	0.0 ± 0.0 ^d A	0.0 ± 0.0 ^c A
^1 Viral DNA load is presented as log 10 (number of viral DNA copies per 50ng of total DNA + 1). Results are presented as an average of six chickens per group and time point ± Standard Deviation. Same superscript lowercase alphabets indicate that differences among groups were not statistically significant in an ANOVA test (p < 0.05). Same superscript uppercase alphabets indicate that differences among adherent and non-adherent subpopulations were not significant in the ANOVA test (p < 0.05). ND = No data for that time point.