Evaluation of factors influencing replication of serotype 1 Marek's disease vaccines in the chicken lung

Figures & data

Table 1.  Oligonucleotides used for real-time PCR and real-time RT-PCR.

Target	Sequence	Purpose	PCR type used	Reference, Genbank number	Amplicon length
gB	5′-CCAGTGGGTTCAACCGTGA-3′	Forward	PCR	Gimeno et al. (2008), AY129965	67
			RT-PCR
	5′-CGGTGGCTTTTCTAGGTTCG-3′	Reverse
GAPDH	5′-GGAGTCAACGGATTTGGCC-3′	Forward	PCR	Gimeno et al. (2008), GQ184819.1	63
	5′-TTTGCCAGAGAGGACGGC-3′	Reverse
ICP4	5′CGCCACACGAGAACACAATG-3′	Forward	RT-PCR	Levy et al. (2003), M75729.1	74
	5′-GGTTGGAGTAGAGCTGCAACTGT-3′	Reverse
pp38	5′-GTGATGGGAAGGCGATAGAA-3′	Forward	RT-PCR	M74036	147
	5′-AGCTACCCCTTTCGGTTTGT-3′	Reverse
gI	5′-TCTTTCGAGGCATCTGGTCT-3′	Forward	RT-PCR	AY129994	178
	5′-TTCAAGATTTCCGTCGTTCC-3′	Reverse
28S	5′-GACGACCGATTTGCACGTC-3′	Forward	RT-PCR	DQ018756.1	62
	5′-GGCGAAGCCAGAGGAAACT-3′	Reverse

Figure 1.  Replication in the lung of HP vaccines (CVI988-BP5, 648A80, and R2) versus LP vaccines (Clone C, 648A100, and R2-23). DNA load of serotype 1 MDV vaccines was evaluated by real-time PCR. Results expressed as the relative Ct ratio of the amplification of the chicken GAPDH gene and the MDV gB (see Materials and Methods). Results are presented as the mean and the standard error. 1a: Load of DNA of CVI988/BP5 and Clone C in the lung at 3, 5, and 10 days of age (doa). 1b: Load of viral DNA in the lung relative to the load of viral DNA in the spleen (load of viral DNA in the lung – load of viral DNA in the spleen) for serotype 1 MDV vaccines CVI988/BP5 and Clone C. 1c: Load of DNA of 648A80 and 648A100 in the lung at 3, 5, and 10 doa. 1d: Load of viral DNA in the lung relative to the load of viral DNA in the spleen for serotype 1 MDV vaccines 648A80 and 648A100. 1e: Load of DNA of R2 and R2-23 in the lung at 3, 5, and 10 doa. 1f: Load of viral DNA in the lung relative to the load of viral DNA in spleen for serotype 1 MDV vaccines R2 and R2-23. Same letter above the bar indicates no statistically significant differences were detected between the members of each pair(P < 0.05). *Statistically significant differences between the load of vaccine DNA in the lung and in the spleen.

Table 2.  Number of chickens positive for the vaccine DNA and which had transcription of MDV genes in the lung (Experiment 1).

			Detection of vaccine DNA by real-time PCR^a			Detection of MDV genes transcription by real-time RT-PCR^b
Vaccine	Age/route	Dose (PFU)	3 doa	5 doa	10 doa	ICP4	pp38	gB	gI
CVI988/BP5	s.c.	2000	1 (7)	7 (7)	7 (7)	7 (7)	7 (7)	7 (7)	7 (7)
Clone C	s.c.	2000	2 (7)	4 (7)	5 (7)	0 (4)	0 (4)	0 (4)	0 (4)
648A80	s.c.	2000	0 (7)	7 (7)	7 (7)	7 (7)	7 (7)	0 (7)	2 (7)
648A100	s.c.	2000	0 (7)	5 (7)	0 (7)	5 (5)	5 (5)	0 (5)	0 (5)
R2	s.c.	2000	0 (7)	4 (7)	6 (7)	4 (4)	4 (4)	0 (4)	1 (4)
R2/23	s.c.	2000	2 (7)	4 (7)	7 (7)	4 (4)	4 (4)	0 (4)	0 (4)
Control	NA	2000	0 (7)	0 (7)	0 (7)	0 (7)	0 (7)	0 (7)	0 (7)
^aFrequency of chickens positive to the presence of the vaccine DNA in the lung by real-time PCR at 3, 5, and 10 doa. Results presented as number of chickens positive among the total number of chickens sampled in each time point (in parentheses). ^bFrequency of chickens that had transcription of ICP4, pp38, gB, and gI MDV genes in the lung at 5 doa. Results presented as number of chickens positive among the total number of chickens that had been positive to the presence of the vaccine DNA by real time PCR (in parentheses).

Figure 2.  MDV gene transcripts in the lung at 5 doa with HP vaccines (CVI988/BP5, 648A80, and R2) and LP vaccines (Clone C, 648A100, and R2-23). Transcription of MDV genes ICP4, pp38, gB, and gI was measured by real-time RT-PCR (see Materials and Methods). Results presented as the mean and the standard error. Comparisons have been done between the two components of the same pair. 2a: Transcription of MDV genes in the pair formed by CVI988/BP5 and Clone C. 2b: Transcription of MDV genes in the pair formed by 648A80 and 648A100. 2c: Transcription of MDV genes in the pair formed by R2 and R2/23. Same letter above the bar indicates no statistically significant differences were detected (P < 0.05).

Figure 3.  Effect of route of vaccination on the load of vaccine DNA in the lung when vaccines were administered in ovo at 18 days of embryonation (i.o.) and subcutaneously at day of age (s.c.). Load of serotype 1 MDV vaccines DNA was evaluated by real-time PCR. Results expressed as the relative Ct ratio of the amplification of the chicken GAPDH gene and the MDV gB (see Materials and Methods). Results presented as the mean and the standard error. 3a: Load of DNA of CVI988/BP5 in the lung when administered i.o. and s.c. at 3, 5, and 10 days of age (doa). 3b: Load of viral DNA in the lung relative to the load of viral DNA in the spleen (load of viral DNA in the lung – load of viral DNA in the spleen) when CVI988/BP5 was administered i.o. and s.c. 3c: Load of DNA of R2 in the lung when administered i.o. and s.c. at 3, 5, and 10 doa. 3d: Load of viral DNA in the lung relative to the load of viral DNA in the spleen when R2 was administered i.o. and s.c. 3e: Load of DNA of R2-23 in the lung when administered i.o. and s.c. at 3, 5, and 10 doa. 3f: Load of viral DNA in the lung relative to the load of viral DNA in the spleen when R2/23 was administered i.o. and s.c. Same letter above the bar indicates that no statistically significant differences were detected (P < 0.05). *Statistically significant differences between the load of vaccine DNA in the lung and in the spleen.

Table 3.  Number of chickens positive for the vaccine DNA and which had transcription of MDV genes in the lung (Experiment 2).

			Detection of vaccine DNA by real-time PCR^a			Detection of MDV genes transcription by real-time RT-PCR^b
Vaccine	Age/route	Dose (PFU)	3 doa	5 doa	10 doa	ICP4	pp38	gB	gI
CVI988/BP5	i.o.	2000	6 (7)	5 (7)	4 (7)	5 (5)	5 (5)	5 (5)	5 (5)
	s.c.	2000	1 (7)	7 (7)	7 (7)	7 (7)	7 (7)	7 (7)	7 (7)
R2	i.o.	2000	0 (7)	3 (7)	1 (7)	3 (3)	3 (3)	0 (3)	1 (3)
	s.c.	2000	0 (7)	3 (7)	7 (7)	3 (3)	3 (3)	0 (3)	1 (3)
R2/23	i.o.	2000	4 (7)	4 (7)	2 (7)	4 (4)	4 (4)	0 (4)	0 (4)
	s.c.	2000	1 (7)	4 (7)	7 (7)	4 (4)	4 (4)	0 (4)	0 (4)
Control	NA	2000	0 (7)	0 (7)	0 (7)	0 (7)	0 (7)	0 (7)	0 (7)
^aFrequency of chickens positive to the presence of the vaccine DNA in the lung by real-time PCR at 3, 5, and 10 doa. Results presented as number of chickens positive among the total number of chickens sampled in each time point (in parentheses). ^bFrequency of chickens that had transcription of ICP4, pp38, gB, and gI MDV genes in the lung at 5 doa. Results presented as number of chickens positive among the total number of chickens that had been positive to the presence of the vaccine DNA by real-time PCR (in parentheses).

Figure 4.  MDV gene transcripts in the lung of 5-day-old chickens after i.o. vaccination with R2, R2-23, and CVI988/BP5. Transcription of MDV genes ICP4, pp38, gB, and gI was measured by real-time RT-PCR (see Materials and Methods). Results presented as the mean and the standard error. Comparisons are done for each gene among the three vaccine strains. Same letter above the bar indicates that no statistically significant differences were detected (P < 0.05).

Figure 5.  Effect of the dose of vaccine on the load of vaccine DNA in the lung. Load of serotype 1 MDV vaccines DNA was evaluated by real-time PCR. Results expressed as the relative Ct ratio of the amplification of the chicken GAPDH gene and the MDV gB (see Materials and Methods). 5a: Load of DNA of R2 in the lung at 3, 5, and 10 doa after administration of 2000 or 10,000 PFU of the vaccines by the s.c. route. 5b: Load of viral DNA in the lung relative to the load of viral DNA in the spleen (load of viral DNA in the lung – load of viral DNA in the spleen) after administration of 2000 or 10,000 PFU of vaccine R2 via s.c. route. Same letter above the bar indicates that no statistically significant differences were detected (P < 0.05). *Statistically significant differences between the load of vaccine DNA in the lung and in the spleen.