Replication of recombinant herpesvirus of turkey expressing genes of infectious laryngotracheitis virus in specific pathogen free and broiler chickens following in ovo and subcutaneous vaccination

Figures & data

Table 1.  Oligonucleotide primers used for real-time PCR.

Target gene	Sequence	Orientation/type
GAPDH	5'-GGAGTCAACGGATTTGGCC-3 '	Forward
	5'-TTTGCCAGAGAGGACGGC-3'	Reverse
Non-coding region HVT	5'-CGGGCCTTACGTTTCACCT-3'	Forward
	5'-GCGCCGAAAAGCTAGAAAAG-3'	Reverse
	5'-FAM-CCCGGGTCGCCTCATCTGGA-3' 5'-TAMRA-7'	TaqMan Probe
LTV gI	5'-TGTGGGCAGTGGACTATGTT-7'	Forward
	5'-TCAGGGTCAGCAAGTATTGG-7'	Reverse
HVT gB	5'-CTTCGCGCCACAACACTAATTCC-7'	Forward
	5'-GGCCATCTTGAACGACTCCGTAA-7'	Reverse
HVT ICP4	5'-GGGTTCTACAGTGGCGTGAT-7'	Forward
	5'-CGCGGGCTACTAGTTTCTTG-7'	Reverse

Figure 1.  Level of maternal antibodies against LTV measured by ELISA. 1a: Level of maternal antibodies against LTV in 50 unvaccinated 1-day-old chicks derived from a 30-week-old parent flock and 50 unvaccinated 1-day-old chicks derived from a 50-week-old parent flock. 1b: Level of maternal antibodies against LTV in broiler chickens used in Experiment 3 at 2 weeks of age. There were 30 rHVT-LT-vaccinated 2-week-old chickens derived from the same 30-week-old parent flock and 30 rHVT-LT-vaccinated 2-week-old chickens derived from the same 50-week-old parent flock. Results are presented as the mean±standard error (SE)±standard deviation (SD) in a box and whisker plot graphic.

Table 2.  Comparisons of vaccine DNA load and transcription o f LTV gI, HVT gB, and HVT ICP4 genes in various tissues at 3 weeks of age in broilers coming from 30-week-old and 50-week-old flocks.^a

		Features compared
		DNA load^b			gI LTV transcripts^c		gB HVT trancripts^c		ICP4 HVT transcripts^c
Treatment group	Dam age (weeks)	Spleen	Lung	FP	Spleen	Lung	Spleen	Lung	Spleen	Lung
ED 18	30	0.6±0.5^A	0.2±0.4^A	0.8±0.5^A	2.8±4.1^A	2.1±3.1^A	0.0±0.0^A	0.0±0.0^A	3.6±3.6^A	1.9±4.2^A
	50	0.8±0.4^A	0.2±0.4^A	0.9 ±0.1^A	4.3±4.0^A	10.8±19.7^A	4.1±9.3^A	0.9±2.1^A	6.0±7.6^A	1.7±3.8^A
ED 19	30	0.6±0.5^A	0.3±0.5^A	0.7±0.5^A	3.1±4.3^A	1.7±3.7^A	0.0±0.0^A	0.2±0.4^A	3.8±3.9^A	0.0±0.0^A
	50	0.8±0.4^A	0.4±0.5^A	0.6±0.6^A	2.8±3.9^A	0.0±0.0^A	0.0±0.0^A	0.0±0.0^A	2.1±2.8^A	7.0±11.0^A
s.c.	30	0.9±0.0^A	0.5±0.5^A	0.9±0.0^A	7.2±4.2^A	5.7±12.8^A	0.0±0.0^A	2.4±5.4^A	2.8±2.6^A	7.2±4.6^A
	50	0.6±0.5^A	0.4±0.5^A	0.7±0.4^A	3.4±4.7^A	4.0±5.3^A	0.0±0.0^A	0.0±0.0^A	1.6±3.5^A	4.0±3.7^A
^aChickens were vaccinated in ovo, either at 18 days of embryonation (ED 18) or 19 days of embryonation (ED 19) or at 1 day of age by the subcutaneous (s.c.) route with a full dose of rHVT-LT following manufacturer recommendations. Samples of the spleen, FP, and lung were taken at 3 weeks of age. ^bViral DNA load is expressed as a Ct ratio (Ct GAPDH/Ct HVT). Results are presented as the average of five chickens per time point±standard deviation. Comparisons between broilers derived from a 30-week-old flock and a 50-week-old flock were conducted by Student's t test (P<0.05). Comparisons between age groups within each treatment group and within each tissue are indicated by uppercase superscript letters; the same letter indicates that no statistically significant differences were found. ^cTranscription of LTV gI, HVT gB, and HVT ICP4 genes is expressed as total number of cycles (50) minus the corrected gI mean (see Materials and Methods for more detail). Results are presented as the average of five chickens per time point±standard deviation. Comparisons between broilers derived from a 30-week-old flock and a 50-week-old flock were conducted by Student's t test (P<0.05). Comparisons between age groups within each treatment group and within each tissue are indicated by uppercase superscript letters; the same letter indicates that no statistically significant differences were found.

Table 3.  Chronological evaluation of rHVT-LT DNA load in the spleen, FP, and lung of SPF chickens (Experiment 1).^a

			Treatment group^b
Tissue	Week	Number of samples	rHVT-LT ED 18	rHVT-LT ED 19	HVT ED 18	HVT ED 19
Spleen	1	5	1.0±0.6 (80%)^A A	1.2±0.1 (100%)^A A	1.3±0.1 (100%)^A A	1.2±0.0 (100%)^A A
	2	5	0.9±0.1 (100%)^A A	0.8±0.4 (80%)^A B	1.0±0.0 (100%)^A A	1.0±0.0 (100%)^A A
	3	5	1.1±0.0 (100%)^A A	1.1±0.0 (100%)^A A	1.2±0.0 (100%)^A A	1.2±0.0 (100%)^A A
	4	5	0.8±0.4 (80%)^A A	1.1±0.0 (100%)^A A	0.9±0.5 (80%)^A A	0.9±0.5 (80%)^A A
	5	5	0.4±0.5 (40%)^A B	0.9±0.5 (80%)^B A	0.9±0.5 (80%)^B A	1.0±0.5 (80%)^B A
FP	1	5	1.0 ±0.6 (80%)^A A	1.2±0.1 (100%)^A A	1.3±0.0 (100%)^A A	1.4±0.1 (100%)^A A
	2	5	0.9±0.0 (100%)^A A	0.9±0.1 (100%)^A A	1.1±0.1 (100%)^A A	1.0±0.1 (100%)^A A
	3	5	0.7±0.5 (60%)^A A	1.0±0.1 (100%)^AB A	1.3±0.3 (100%)^B A	1.2±0.1 (100%)^AB A
	4	5	1.0±0.1 (100%)^A A	0.8±0.4 (80%)^A A	1.1±0.1 (100%)^A A	1.1±0.1 (100%)^A A
	5	5	0.6±0.6 (60%)^A B	0.8±0.4 (80%)^AB A	1.1±0.1 (100%)^B A	0.9±0.5 (80%)^AB A
Lung	1	5	0.9±0.5 (80%)^A A	1.2±0.0 (100%)^A A	1.0±0.6 (80%)^A A	1.3±0.1 (100%)^A A
	2	5	0.6±0.5 (80%)^A A	0.9±0.0 (100%)^A A	0.9±0.0 (100%)^A A	0.9±0.1 (100%)^A A
	3	5	1.0±0.0 (100%)^A A	1.0±0.1 (100%)^A A	1.1±0.1 (100%)^A A	1.0±0.1 (100%)^A A
	4	5	1.0±0.1 (100%)^A A	1.0±0.1 (100%)^A A	1.1±0.1 (100%)^A A	1.0±0.1 (100%)^A A
	5	5	0.4±0.6 (40%)^AC B	0.2±0.6 (20%)^A B	0.8±0.6 (60%)^BC A	0.9±0.5 (80%)^B A
^aChickens were vaccinated in ovo, either at 18 days of embryonation (ED 18) or 19 days of embryonation (ED 19) with a full dose of rHVT-LT or HVT following manufacturer recommendations. Spleen, FP, and lung samples were taken at 1, 2, 3, 4, and 5 weeks of age. ^bViral DNA load is expressed as a Ct ratio (Ct GAPDH/Ct HVT). Results are presented as the average of five chickens per time point±standard deviation. The percentage of positive samples is indicated in parentheses. Comparisons among groups and among time points were conducted by an ANOVA test (P<0.05). Comparisons among treatment groups within each time point are indicated by uppercase superscript letters. Comparisons among time points within treatment groups are indicated by bold italic uppercase superscript letters. The same letter indicates that no statistically significant differences were found.

Table 4.  Effect of age and route of vaccination on the DNA load of rHVT-LT and on the transcription of LTV gI gene in the spleen, FP, and lung of SPF chickens (Experiment 2) and broiler chickens (Experiment 3) at 3 weeks of age.^a

				Treatment group
Chicken type	Assay detection	Tissue	Number of samples	rHVT-LT ED 18	rHVT-LT ED 19	rHVT-LT s.c.-1d
SPF	HVT DNA^b	Spleen	5	1.1±0.0 (100%)^A A	1.1±0.0 (100%)^A A	1.1±0.0 (100%)^A A
		FP	5	0.9±0.5 (80%)^A A	1.0±0.1 (100%)^A A	0.8±0.5 (80%)^A A
		Lung	5	0.8±0.5 (80%)^A A	0.6±0.6 (60%)^A A	1.1±0.0 (100%)^A A
	LTV gI transcripts^c	Spleen	5	5.0±2.8 (80%)^A A	2.5±3.2 (60%)^AB A	0.0±0.0 (0%)^B A
		Lung	5	0.0±0.0 (0%)^A B	1.6±3.6 (20%)^A A	1.6±3.5 (20%)^A A
	HVT gB transcripts	Spleen	5	3.5±5.0 (40%)^A A	2.3±5.2 (20%)^A A	0.0±0.0 (0%)^A A
		Lung	5	0.0±0.0 (0%)^A A	0.0±0.0 (0%)^A A	0.0±0.0 (0%)^A A
	HVT ICP4 transcripts	Spleen	5	3.8±4.1 (40%)^AB A	5.0±2.9 (80%)^A A	0.0±0.0 (0%)^B A
		Lung	5	2.0±4.6 (20%)^A A	2.3±5.1 (20%)^A A	1.1±2.5 (20%)^A A
Broilers	HVT DNA	Spleen	10	0.7±0.5 (80%)^A A	0.7±0.5 (70%)^A A	0.8±0.40 (80%)^A A
		FP	10	0.9±0.3 (90%)^A A	0.7±0.5 (70%)^A A	0.8±0.3 (90%)^A A
		Lung	10	0.2±0.4 (20%)^A B	0.4±0.5 (40%)^A A	0.5±0.5 (50%)^A A
	LTV gI transcripts	Spleen	10	3.6±3.9 (50%)^A A	3.0±3.9 (40%)^A A	5.3±4.6 (60%)^A A
		Lung	10	6.5±14.1 (20%)^A A	0.9±2.6 (20%)^A A	4.9±9.3 (30%)^A A
	HVT gB transcripts	Spleen	10	2.1±6.6 (10%)^A A	0.0±0.0 (0%)^A A	0.0±0.0 (0%)^A A
		Lung	10	0.5±1.5 (20%)^A A	0.1±0.3 (10%)^A A	1.2±3.8 (10%)^A A
	HVT ICP4 transcripts	Spleen	10	4.8±5.8 (60%)^A A	3.0±3.4 (60%)^A A	2.2±3.0 (40%)^A A
		Lung	10	1.8±3.8 (20%)^A A	3.6±8.2 (20%)^A A	5.6±4.3 (30%)^A A
^aChickens were vaccinated in ovo, either at 18 days of embryonation (ED 18) or 19 days of embryonation (ED 19) or at 1 day of age by the subcutaneous (s.c.) route with a full dose of rHVT-LT following manufacturer recommendation. Samples of spleen, FP, and lung were taken at 3 weeks of age. ^bViral DNA load is expressed as a Ct ratio (Ct GAPDH/Ct HVT). Results are presented as the average of five chickens per time point±standard deviation. The percentage of positive samples is indicated in parentheses. Comparisons among groups and among time points were conducted by an ANOVA test (P<0.05). Comparisons among treatment groups within each tissue are indicated by uppercase superscript letters. Comparisons among tissues within a treatment group are indicated by bold italic uppercase superscript letters. The same letter indicates that no statistically significant differences were found. ^cTranscription of the LTV gI gene is expressed as the total number of cycles (50) minus the corrected gI mean (see Materials and Methods for more detail). Results are presented as the average of five to ten chickens per time point±standard deviation. The percentage of positive samples is indicated in parentheses. Comparisons among groups and between tissues were conducted by an ANOVA test (P<0.05). Comparisons among treatment groups within each tissue are indicated by uppercase superscript letters. Comparisons between tissues within a treatment group are indicated with bold italic uppercase superscript letters. The same letter indicates that no statistically significant differences were found.