Introduction

Vaccination of broilers by the in ovo route against Marek’s disease in the hatchery has become routine for most broiler companies in the USA. The in ovo vaccination has advantages related to mass vaccination at a single location, improving the uniformity of vaccination and reducing labor and time expenditures compared to field applications. The Inovoject® system manufactured by Embrex Inc. is the most commonly used in ovo system, while fewer hatcheries use the Intelliject® system manufactured by AviTech LLC. With these in ovo systems in place in most broiler hatcheries in the USA there is a trend to apply other vaccines in ovo as has been done with Infectious bursal disease and Reo virus vaccines. In recent years several broiler rearing regions have experienced infectious laryngotracheitis (ILT) that is suspected to have originated from breeder flocks vaccinated with live CEO lLT vaccine. This has created a need for mass vaccination of broilers against ILT. Live CEO ILT vaccines applied by spray or dinking water at 14 days of age has been the traditional vaccination program used in broilers. In April 2002 a recombinant fowl pox vectored ILT vaccine was licensed by the USDA (Vectormune® FP-LT) for use in chickens by wing web application. A major advantage of this vaccine is the development of protection to ILT without using a live ILT virus, eliminating the risks of rolling ILT reactions within a flock, spreading of ILT virus to other flocks, and of increasing virulence after being passaged through a flock, that are associated with using live CEO ILT vaccines. If Vectormune FP-LT (rVLT) could be effectively and safely applied by the in ovo route the industry would have a new tool against ILT that could be easily and uniformly applied in mass and be neighbor friendly by not introducing live CEO ILT virus in to a flock or region preventing the perpetuation of ILT virus in surrounding flocks by using live ILT vaccines.

Objectives

1. Determine the safety and level of ILT protection in broilers that were in ovo vaccinated with Vectormune FP-LT.
2. Compare the performance of broilers vaccinated in ovo with Vectormune FP-LT to non rVLT vaccinated broilers.

Methods

Commercial broilers were vaccinated using a lyophilized recombinant fowl pox vectored ILT vaccine, Vectormune FP-LT, (rVLT) administered in ovo at a commercial broiler hatchery using the Inovoject® system at 19 days of incubation. The Vectormune FP-LT vaccine was reconstituted with the Marek’s diluent and mixed at half of the labeled wing web dose with the Marek’s and IBDV vaccines and other vaccine components as shown in Table 1. Following hatching 42,600 rVLT vaccinated chicks were placed into two houses on a four house commercial broiler farm. The other two houses on the farm were filled with 44,100 non-rVLT vaccinated broilers that were hatched on the same day in the same hatchery and were from the same breeder flocks as the rVLT vaccinated broilers. The broilers were reared under standard commercial conditions and all four houses were treated the same. At 29 days of age 20 rVLT vaccinated birds and 20 non-rVLT vaccinated birds were removed from the commercial houses and moved to a research facility to perform the ILT challenge. The remaining broilers continued to be reared in the commercial houses until slaughter at 55 days of age. An experimental ILT challenge was not administered to any of the broilers reared in the commercial houses. The hatch, mortalities, feed deliveries and processing lots were kept separate for the rVLT vaccinated houses and the non rVLT vaccinated houses to be able to compare the grow out performance between the two groups.

Table 1.Vaccines and components mixed together and delivered in ovo to broilers using the Inovoject system.
Vaccine / Inoculum ¹	Strain / Contents	Volume Added to Bag	Dose per Embryo	Serial or Lot #	Expiration	Company
Marek’s	HVT 4000 doses	2 vials	1/4 dose	JZ169	9-18-07	Merial
Marek’s	SB-1 4000 doses	2 vials	1/4 dose	JV148	1-28-07	Merial
Diluent Bag	Saline	1600 ml	50ul	DG949		Merial
Sterile Dye	Blue	1.5 ml		ZA215		Merial
Gentapoult	100 mg / ml	32ml/1600ml	0.1mg	4101306	10-06	Am.Tech Labs
Bursal 2+0	S706 4000 doses	2 vials	1/4 dose	EA006	6-22-06	Merial
Vectormune FP- LT	1000 doses	16 vials/1600ml	1/2 dose	420-008A	4-28-06	Biomune

Challenge Model: A direct ILT challenge was administered to two groups of 20 broilers each at 29 days of age using a 5x labeled dose of live CEO ILT vaccine, (Trachivax® Schering-Plough Animal Health Corp.), by placing one eye drop into the left eye & one drop orally. The two broiler groups were either previously vaccinated in ovo with Vectormune FPLT or were non-vaccinated for ILT. Broilers from both groups were reared from hatch until 29 days of age in separate commercial houses. After the ILT challenge both groups of broilers were housed together in a colony house.

Scoring of Lesions Five days post challenge the birds were examined. Normal and abnormal findings regarding the left and right eye, nasal discharge, respiratory noise, trachea, swelling of the periorbital region or head, and physical depression were individually and numerically scored using the scale of 0 = normal, 1 = mild, 2 = moderate, and 3 = severe. Scores 1-3 represented a change from normal function and anatomy as related to lesions consistent with ILT.

Results

Flock of 21 day old broilers vaccinated in ovo against ILT using Vectormune FP-LT	Eye score of 3 : ocular discharge , enlargement of eye lids, bubbles & inflammatory debris inside orbit
Eye score of 1: slight tearing, widening & sagging of the lower eyelid exposing more of the eye	Eye score of 3: ocular discharge, plus a periorbital facial swelling score
Eye score of 2: notice bubbles and thick ocular discharge at the medial canthus	Eye score of 3: plus a periorbital facial swelling and general depression score. Note the dye stained feathers from the ILT eye drop challenge

The hatch for the rVLT inoculated flocks was 88.3% and 86.7% which was above the historic average for the respective breeder flocks. No adverse findings were identified at hatch, in the chicks or at any time during the growing period. Following experimental ILT challenge symptoms consistent with ILT were seen in the left eye of 100% of the control group and 70% of the rVLT vaccinated group. All lesion scores were less in the rVLT vaccinated group and they did not express any signs of depression as seen in the control group. Table 2. contains the lesion scores from the ILT challenge. The performance of the commercially grown flocks vaccinated with rVLT was better than the non-rVLT vaccinated flocks in all production categories as shown in table 3. The photo’s of eye lesions represent severity scores 1-3 recorded in the study.

Table 2. Lesion scores from broilers at 5 days post challenge given a 5x dose of live CEO ILT vaccine
Group ID	Age Days	Lesion Scores	Right Eye	Left Eye	Nasal Fluid	Resp. Noise	Trachea	Swollen Head	Depression
In ovo rVLT	34		Average Score of the rVLT Vaccinated Group
			0.35	0.90	0.05	0	0	0	0
			Percentage of Birds with Each Score
		0	75	30	95	100	100	100	100
		1	25	50	5	0	0	0	0
		2	0	20	0	0	0	0	0
		3	0	0	0	0	0	0	0
Non rVLT Controls	34		Average Score of the non-rVLT Control Group
			0.65	2.35	0.05	0.20	0.20	0	0.95
			Percentage of Birds with Each Score
		0	35	0	95	80	80	100	35
		1	65	10	5	20	20	0	35
		2	0	45	0	0	0	0	30
		3	0	45	0	0	0	0	0

Table 3.Commercial production values of the rVLT vaccinated flocks and non-rVLT vaccinated broiler flocks.
Group ID	Age Days	# Placed	% Live	Dead on Arrival	Condems	Average Daily Gain	Body Weight Lbs.
rVLT 1/2	55	42,600	92.82	73	0.66	0.1278	7.030
Non-rVLT	55	44,100	92.20	104	1.22	0.1203	6.620

Comments

The in ovo administration of Vectormune FP-LT mixed with Marek’s and IBDV vaccines was found to be safe in commercial broilers. Performance advantages in body weight, average daily gain, feed conversion, condemnations, and % livability were obtained from the rVLT vaccinated flocks compared to the non-rVLT vaccinated flocks. Complete or partial protection against ILT was demonstrated in the rVLT vaccinated broiler group by a reduction in lesion scores at 5 days post challenge with live CEO ILT virus. The administration of Vectormune FP-LT in ovo requires no extra equipment or labor and has advantages over live CEO ILT vaccines by not having vaccine reactions that lead to poorer weights, or increased mortality and there is no exposure to live ILT virus eliminating the risks of spreading or shedding of ILT virus after vaccination, making rVLT a user and neighbor friendly vaccine.

Reference

1. Moore K., J.R. Davis, Y. Tsuzaki, D.R. Hout, M. Esaki, T. Okuda, J. D. Leonard. 2002. Efficacy and Safety of a Recombinant Fowl Pox Virus Containing Laryngotracheitis Genes. Western Poultry Disease Conference Puerto Vallarta, Mexico
2. Guy, J. S., and T. J. Bagust. 2003 Laryngotracheitis. Diseases of Poultry. Y. M. Saif, 11 edition, 121-134.