Application of the Cell-Free Turkey Herpesvirus Vaccine by the Aerosol Route for the Prevention of Marek's Disease in Chickens* C. S. EIDSON and S. H. KLEVEN Poultry Disease Research Center, Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602 (Received for publication February 8, 1977)
Poultry Science 56:1609-1615,1977 INTRODUCTION Churchill a n d Biggs ( 1 9 6 7 ) isolated a cellassociated herpesvirus from infectious material, a n d results of s u b s e q u e n t investigations (Eidson et al, 1 9 6 9 ; Witter et al, 1 9 6 9 ) p r o v e d this virus t o b e t h e etiologic agent of Marek's disease (MD). Churchill et al. ( 1 9 6 9 ) f o u n d t h a t t h e pathogenic strain of t h e MD herpesvirus became a t t e n u a t e d after 33 passages in cell culture. When administered t o chicks, it prot e c t e d against t h e d e v e l o p m e n t of MD lesions after challenge with a virulent strain of t h e virus. A l t h o u g h t h e a t t e n u a t e d MD viral vaccine offered considerable p r o t e c t i o n against MD t u m o r d e v e l o p m e n t , Eidson et al. ( 1 9 7 1 ) f o u n d t h a t a herpesvirus, first isolated f r o m t u r k e y s b y K a w a m u r a et al. ( 1 9 6 9 ) , was m o r e effective t h a n t h e a t t e n u a t e d MD vaccine. Calnek et al. ( 1 9 7 0 ) r e p o r t e d o n e x t r a c t i o n of H V T from infected cells b y ultrasonic disruption and preservation in t h e lyophilized s t a t e . A l t h o u g h t h e lyophilized H V T vaccine has been used successfully in m a n y E u r o p e a n countries, questions have arisen as t o w h e t h e r or n o t m a t e r n a l antibodies t o H V T alter t h e response t o vacci-
•Supported by the Georgia Department of Agriculture.
n a t i o n (Patrascu et al., 1972). R e c e n t d a t a indicate t h a t m a t e r n a l a n t i b o d y H V T does n o t significantly affect t h e efficacy of t h e cell-free H V T vaccine in immunizing chickens against MD w h e n t h e r e c o m m e n d e d dose is u s e d (Churchill et al, 1 9 7 3 ; Eidson et al., 1 9 7 5 ; Zygraich and Huygelen, 1 9 7 2 ) . Regardless of w h e t h e r t h e cell-free or t h e cell-associated H V T vaccine is used, t h e p r o d u c t is injected intra-abdominally, intramuscularly or s u b c u t a n e o u s l y . During t h e last 25 years, investigators have observed t h a t animals can b e c o m e i m m u n i z e d as a c o n s e q u e n c e of inhalation of air containing living microbial units. Mass vaccination of p o u l t r y w i t h sprays was first r e p o r t e d b y Hitchner et al. ( 1 9 4 8 ) w h e n chicks were i m m u n i z e d with t h e B1 strain of Newcastle disease vaccine. T h e p u r p o s e of this s t u d y is t o r e p o r t t h e results of field trials in which chicks were vaccinated against MD b y aerosol with cell-free H V T vaccine. MATERIALS AND METHODS Chickens: All broiler chicks used in this s t u d y were derived from breeder flocks t h a t had been vaccinated at 1 day of age with cell-associated H V T vaccine. Selection of Farms: F a r m s were selected t h a t had at least t w o p o u l t r y houses in o r d e r
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ABSTRACT In field trials approximately 300,000 birds were vaccinated against Marek's disease (MD) by aerosol spray with 1,000 to 5,000 PFU of cell-free HVT vaccine per chick. Chicks were sprayed in the trays of the hatchers which still contained dust and down after hatching. Prior to spraying, the incoming and outgoing air ducts were closed. The chicks were sprayed for 12—15 min. with HVT in 250 to 300 ml. of diluent, and after completion of spraying the chicks remained in constact with the aerosol for approximately for 10 minutes. In birds vaccinated by aerosol spray the incidence of MD ranged from 0.0% to 24.4%; while the incidence of MD in birds vaccinatedby subcutaneous inoculation varied from 0.02 t o 23.9%. In trial 1 the incidence of MD in birds vaccinated by aerosol spray was essentially the same as those vaccinated by subcutaneous inoculation; whereas, the incidence of MD (Trial 2 and 3) was higher in birds vaccinated by aerosol as compared to chickens vaccinated by subcutaneous inoculation.
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C. S. EIDSON AND S. H. KLEVEN
Trial 1. Experiment 2. Ninety chickens from the first experiment that were sprayed with
4,000 PFU per bird were brought to the laboratory. Forty of these chickens were challenged at 2 weeks of age by injecting the chicks subcutaneously with 0.2 ml. of MD infectious plasma and the remaining 40 chicks were challenged at 2 weeks by contact exposure. The seeder chicks were 2 week-old hatchmate seeder chicks which had been infected with the MD virus at one day of age. Seeder chicks were brooded in separate facilities until they were distributed at a ratio of 1:4 among the test birds. One hundred-eighty hatchmates were also brought back to the laboratory. Eighty of the birds were injected subcutaneously with approximately 1500 PFU of cell-free HVT vaccine. At 2 weeks of age 40 of the birds were challenged by subcutaneous inoculation of 0.2 ml. of MD-infective plasma while the second group of 40 chickens were challenged by contact exposure. Forty of the controls were challenged by subcutaneous inoculation and 40 chicks were challenged by contact exposure. At the end of the 10 week experimental period, all of the chickens were necropsied and examined for gross MD lesions. * Viremia and subsequent antibody development in chickens vaccinated with the HVT vaccine were studied. A group (10) of chickens obtained from chickens spray vaccinated with 4,000 PFU of cell-free HVT vaccine. Another group (10) of chickens were injected subcutaneously with approximately 1,500 PFU per bird. A final group of 10 chickens served as unvaccinated controls. All of the chickens were bled weekly. For the first 3 weeks an attempt was made to determine the viremia level in chickens from each of these 3 groups. The technique used to detect viremia in chickens vaccinated with the cell-free HVT vaccine was essentially that described by Churchill et al. (1973) with the exception that primary rather than secondary chick embryo fibroblast monolayers were used. To evaluate antibody development blood obtained between 4 and 8 weesk was analyzed by the plaque inhibition test as described by Churchill et al. (1973). Trial I. Experiment 3. In this study there were two poultry houses each holding approximately 6,000 chickens. During the first experiment on this farm the birds in the first house were spray vaccinated with 5,000 PFU per bird; whereas, the chickens in the second house were injected subcutaneously with 1,000 PFU per bird. For the second experiment on the farm
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that the efficacy of graded dose levels of the cell-free HVT vaccine could be compared with the cell-associated HVT vaccine in preventing MD. All of the houses used in the study were of the conventional curtain-type with either dirt or cement floors. No attempt was made to change the usual husbandry procedures. None of the houses were cleaned prior to the placement of the chicks except for the addition of a layer of new shavings to the old litter. In addition to the HVT vaccine all birds were vaccinated at 14—18 days of age against infectious bronchitis and Newcastle disease. Turkey Herpesvirus Vaccine: The cell-associated HVT vaccine was titrated as described by Eidson et al. (1973 a,b). The cell-free HVT vaccine was titrated as previously described by Eidson et al. (1975). Experimental Design. Trial 1. Experiment 1: Approximately 125,000 chickens were exposed by aerosol with 2,000 to 5,000 PFU of cell-free HVT vaccine. The aerosol was generated by Spraying Systems IA sprayer (Spraying Systems, Inc., Wheaton, Illinois) powered by an air compressor which provides 60 pounds per square inch of air pressure. The diluent used in spraying the chickens consisted of 0.15 M sucrose, 0.0038 M monopotassium phosphate, 0.0072 M dipotassium phosphate and 1.5% N Z-Amine. All the chickens were sprayed while they were in the trays of the hatcher. The hatchers measured 4' X 9' X 6' and held approximately 8,000 chicks. Prior to vaccination all air ducts were sealed. The chicks were sprayed for 12 to 15 min. with VT in 2 5 0 - 3 0 0 ml. of diluent, and after completion of spraying the chicks remained in contact with the aerosol for an additional 5 min. The fan inside the hatcher was either turned on intermittently or was turned on for the entire 20 min. experimental period. For comparison, an equal number of chicks were vaccinated subcutaneously with approximately 1000 PFU of cell-free HVT vaccine. Farms selected for the study had at least 2 houses so that one house contained chickens vaccinated with cell-free HVT by spray while the second house was vaccinated by subcutaneous inoculation. Each treatment group was identified separately as the birds were loaded and transported to the processing plants. Personnel of the Consumer and Marketing Service of the U.S.D.A. provided the inspection data.
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AEROSOL VACCINATION AGAINST MAREK'S DISEASE
RESULTS Trial I. Experiment 1. vaccinated by aerosol with protected against challenge MD Virus, (Table 1). Of
All of the birds 5,000 PFU were with the virulent the 30,681 birds
vaccinated with 5,000 PFU only 0.08% were condemned due to MD. The incidence of MD in the group varied from 0.02 to 0.20%. However 2 groups of chickens vaccinated by subcutaneous inoculation had 8.5% and 5.4% condemnation attributed to MD. The average incidence of MD in this group was 3.3% with a range of 0.02 to 8.5%. In contrast one group of birds sprayed with 4,000 PFU had 3.9% condemnation attributed to MD and the average incidence was 0.80% with a range of 0.02 to 3.9%. The incidence of MD in birds vaccinated subcutaneously was 0.20% with a range of 0.01 to 0.48%. In the group of chickens vaccinated with 3,000 PFU there was one group of chickens that had 23.4% condemnation for MD. Although all of the other groups of chickens vaccinated with 3,000 PFU were less than 0.2%, the average incidence of MD for the group was 4.9%. In contrast the average incidence of MD in the group of chickens vaccinated subcutaneously was only 1.6%. In the last group of chickens sprayed with 2,000 PFU of HVT vaccine there was no difference in the incidence of MD when compared to those vaccinated by subcutaneous inoculation. Trial 1. Experiment 2. As shown in Table 2 chickens which were vaccinated by aerosol in the hatcher with 4,000 PFU and brought to the laboratory for challenge, the incidence of gross lesions of MD was 10.0% in those challenged by injection. Whereas, only 5.1% of those challenged by contact exposure developed lesions. In contrast 5.1% of the birds vaccinated subcutaneously and challenged by injection devel-
TABLE 1.—Postmortem condemnations of chickens vaccinated by aerosol spray or by subcutaneous inoculation (Trial 1 — Experiment 1) Condemnations MD
Total
Dose (PFU)
Route of vaccination
No. birds
No.
%
Range %
No.
%
Range %
5000 1000 4000 1000 3000 1000 2000 1000
Aerosol Subcutaneously Aerosol Subcutaneously Aerosol Subcutaneously Aerosol
30,681 48,186 30,025 44,445 30.264 68,722 34,824 32,823
25 1564 254 86 1467 1113 128 126
0.08 3.25 0.80 0.20 4.85 1.62 0.37 0.38
0.02-0.20 0.02-8.50 0.00-3.93 0.01-0.48 0.97-23.40 0.11-2.70 0.09-1.55 0.07-0.66
194 1905 425 379 1630 1618 386 426
0.63 3.95 1.40 0.85 5.39 2.38 1.11 1.30
0.45-0.93 0.54-9.20 0.20-4.42 0.53-1.17 0.53-23.90 0.49-3.70 0.73-2.92 0.79-1.85
Subcutaneously
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the birds in the first house were spray vaccinated with approximately, 1,000 PFU. The birds in the second house were spray vaccinated with 2,000 PFU per bird. In the third successive broiler grow out period, chickens in both houses were spray vaccinated with 2,000 PFU per bird. Trial 2. At company number 2, the houses were paired so that each farm would have 2 houses of the same size. In this trial the birds were sprayed with Spraying Systems 1A sprayer in a hatcher that held 12,000 birds rather than 8,000 birds as in the first trial. Also, all of the birds vaccinated by spray received 5,000 PFU of cell-free HVT vaccine in each of the 3 experiments. For comparative purposes birds vaccinated subcutaneously received 1,000 PFU of cell-associated HVT vaccine. Trial 3. The chickens in study were spray vaccinated within a plastic canopy which measured 9' X 7' X 6'. The birds were sprayed with 2 nebulizers which were provided by an air compressor which provided 40 pounds per square inch of air pressure. Approximately 12,000 birds could be sprayed each time. In 8 experiments 96,000 birds were spray vaccinated with 5,000 PFU per bird and for comparative purposes 96,000 birds were vaccinated subcutaneously 1,000 PFU of the cell-associated HVT vaccine.
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C. S. EIDSON AND S. H. KLEVEN TABLE 2.—MD lesions at 10 weeks of age in chickens vaccinated with cell-free HVT in the hatcher and challenged with MD virus by inoculation or contact (Trial 1 — Experiment 2) 10 weeks Challenge
No. +/No. started
%
Aerosol^ Aerosol Subcutaneous" Subcutaneous None None
Injected^ Contact 0 Injected Contact Injected Contact
4/40E 2/39 2/39 0/37 22/38 12/35
(10%) (5.1%) (5.1%) (0%) (58.9%) (34.3%)
Chickens sprayed with 4,000 PFU/bird dose. B
Chickens injected with approximately 1,000 PFU/bird dose.
"'Chickens injected subcutaneously at 2 weeks of age with 0.2 ml of MD-infective plasma. Chickens exposed to MD virus with 100 seeder chickens/group. E Not including birds dying before 2 weeks of age.
oped gross lesions of MD. T h e groups challenged b y c o n t a c t e x p o s u r e did n o t develop any gross lesions. T h e incidence of MD in t h e g r o u p of non-vaccinated controls t h a t was challenged by injection was 5 8 . 9 % ; whereas, t h e incidence of M D in t h e g r o u p of controls t h a t was challenged b y c o n t a c t e x p o s u r e was 3 4 . 3 % . Birds vaccinated a t o n e d a y of age b y aerosol had developed a viremia ( 2 / 7 ) by o n e week b u t 7/10 chickens vaccinated s u b c u t a n e o u s l y with a p p r o x i m a t e l y 1,500 P F U per bird had devel-
o p e d a viremia. However, b y 2 weeks past vaccination t h e n u m b e r of chickens with viremia t o t h e H V T vaccine was essentially t h e same in b o t h t r e a t m e n t groups. (Table 3). Also, Table 3 shows t h a t significant a n t i b o d y levels were already developing 3 weeks after vaccination in b o t h t r e a t m e n t groups. However, antib o d y titers were slightly lower in t h e g r o u p s vaccinated b y aerosol. Trial 1. Experiment 3. In t h e first p r o d u c tion period 0.20% of t h e birds vaccinated by aerosol from h o u s e # 1 was c o n d e m n e d for M D ;
TABLE 3.—Determination of viremia to HVT and antibody titers following cell-free HVT vaccination of one-day-old chicks by either subcutaneous inoculation or by spray vaccination (Trial 1 — Experiment 2) No. of chicks with viremia
Antibody titersE Age (weeks)
Age (weeks) administration
1
2
3
4
5
6
7
8
Aerosol-^ Subcutaneous^ Control
2/9C 7/10 0/9
8/9 9/10 0/8
6/8D 8/8 0/7
14.7 F 23.5 9.8
19.0 32.0 10.8
32.0 53.8 8.8
69.8 90.5 11.9
234.8 279.2 11.9
Vaccinated with approximately 4,000 PFU of cell-free HVT vaccine. BVaccinated with approximately 1500 PFU. "Numerator = No. of chickens having viremia; denominator = no. chicks tested. No. of birds reduced due to mortality. "Reciprocal of serum dilution to a 50% reduction in virus plaque count. Geometric mean titers.
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Vaccination procedure
AEROSOL VACCINATION AGAINST MAREK'S DISEASE
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TABLE 4.—Post mortem condemnation of chickens vaccinated by aerosol or injection on a single farm during 3 successive 8-week broiler production periods (Trial 1 — Experiment 3) Condemnations No. period
House no. lA
1
2B
2 3
Total
marketed
No.
%
No.
%
Aerosol Sub. Sub. Sub. Sub. Sub.
6,132 6,300 5,993 6,045 5,988 6,012
12 341 1,290 1 48 4
0.20 5.41 21.5 0.01 0.80 0.07
57 409 1,337 46 79 28
0.93 5.41 22.3 0.76 1.32 0.47
Birds sprayed with 5,000 PFU/bird dose. Chickens injected subcutaneously with approximately 1,000 PFU of cell-free HVT vaccine. "Birds sprayed with approximately 1,000 PFU/bird dose. DBirds sprayed with 2,000 PFU/bird dose.
whereas, 5.41% of the birds vaccinated by subcutaneous inoculation were condemned for MD (Table 4). In contrast during the second production period 21.5% of the birds vaccinated with approximately 1,000 PFU was condemned for MD. Birds from house #2 which were sprayed with 2,000 PFU per bird dose had 0.01% condemnation for MD. For the 3rd production period birds from house # 1 were sprayed with 2,000 PFU per bird dose and only 0.80% were condemned for MD. Birds in house # 2 which were also sprayed with 2,000 PFU per bird had 0.07% condemnations for MD. Trial 2. In the first production period, 1.16% of the birds vaccinated by aerosol with 5,000
PFU of cell-free HVT were condemned for MD while only 0.16% of the birds injected subcutaneously were condemned (Table 5). In the second experiment only 0.17% of the birds vaccinated by aerosol were condemned; whereas, 0.83% of the injected birds were condemned. In the last experiment the injected birds (0.07%) had fewer condemnations for MD than the birds vaccinated by aerosol (0.34%). Trial 3. In this study there were 8 experiments in which 96,000 birds were vaccinated by spray and 96,000 birds were injected subcutaneously. Of the birds vaccinated by spray 0.51% were condemned for MD while only 0.15% of those vaccinated subcutaneously were
TABLE 5.—Post mortem condemnations of chickens vaccinated with cell-free HVT by aerosol or by inoculation with cell-associated HVT (company 2) Condemnations MD
Total
period
Vaccine
No birds
No.
%
No.
%
1
CFA CA CF CA CF CA
12,852 12,288 10,656 10,560 12,586 12,432
149 20 54 88 43 0
1.16 0.16 0.17 0.83 0.34 0.07
180 52 104 91 62 87
1.40 0.42 0.97 0.86 0.50 0.70
2 3
CF = Cell-free HVT vaccine; chickens were sprayed with approximately 5,000 PFU/bird dose. CA = Cell-associated HVT vaccine; chickens were injected subcutaneously with approximately 1,000 PFU/bird dose.
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lC 2D ID 2»
MD
vaccination
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C. S. EIDSON AND S. H. KLEVEN TABLE 6.—Post mortem condemnations of chickens vaccinated by aerosol spray inside a plastic canopy or by subcutaneous inoculation Condemnations
Experiment no. 1 2
4 5 6 7 8
Total
Vaccine
House no.
No.
%
No.
%
CFA CA« CF CA CF CA CF CA CF CA CF CA CF CA CF CA
lC 2D 1 2 1 2 1 2 1 2 1 2 1 2 1 2
54 7 20 8 26 11 193 8 83 28 70 67 5 5 34 8
0.45 0.06 0.17 0.07 0.22 0.09 1.61 0.07 0.69 0.23 0.58 0.56 0.04 0.04 0.28 0.07
107 80 59 70 110 96 254 62 122 146 112 107 50 65 100 74
0.89 0.70 0.49 0.58 0.92 0.80 2.12 0.52 1.02 1.22 0.93 0.89 0.42 0.54 0.83 0.62
CF = Cell-free HVT vaccine; chickens were sprayed with approximately 5,000 PFU/bird dose. CA = Cell-associated HVT vaccine; chickens were injected subcutaneously with approximately 1,000 PFU/bird dose. C, "House No. 1 - Contained 12,000 birds. R
DHouse No. 2 - Contained 12,000 birds.
condemned. As shown in Table 6 the MD condemnation in the chickens vaccinated by aerosol ranged from 0.04% to 1.61% while the MD condemnation in birds vaccinated subcutaneously ranged from 0.04% to 0.56%. DISCUSSION In the study reported herein, the objectives were to determine whether aerosol vaccination could be used effectively to protect chickens against MD. Also, attempts were made to determine whether aerosol vaccination was as effective in preventing MD as chickens vaccinated by subcutaneous inoculation. In choosing a vaccine to protect chickens against MD there are many factors such as availability, cost of the vaccine and cost of administering the vaccine must be taken into consideration. However, unlike most poultry vaccines, the vaccines against MD are administered in the hatchery and therefore the number of sites at which vaccination must be performed are considerably less than for conventional poultry vaccines administered to chickens in houses.
One of the most attractive vaccines is one prepared from an apathogenic Marek's disease virus that spreads horizontally. Rispens et al. (1972) were the first to isolate a maternally avirulent virus from a normal flock of chickens. Vaccine viruses that spread horizontally theoretically should have the advantage that only a small proportion of chicks need be inoculated to immunize a flock of chickens. However, in the case of vaccines against MD there is a chance of exposure to the virulent MD virus before the vaccine virus can effectively spread throughtout the flock. Also, a disadvantage to a vaccine virus that spreads is that each time it cycles through chickens there is a chance that it might revert back to a pathogenic form. It has been well documented in the literature that both the cell-free and cell-associated HVT vaccines when administered by subcutaneous inoculation at one day of age are effective in preventing MD (Eidson et al, 1971, 1973 a,b, 1975; Okazaki era/., 1970). Although the cell-free and cell associated HVT vaccines have proven to be effective in preventing MD, the vaccine had to be administered to each chicken. To vaccinate each
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3
MD
AEROSOL VACCINATION AGAINST MAREK'S DISEASE
Other reasons for the erratic results in the aerosol vaccinated chickens were the large amounts of dust, down and egg shells in the hatcher. Also, the density of chicks in the hatchers may have also influenced the results. In Trial 2, there was some extraneous matter in the vaccine that constantly clogged the sprayer. This was the only instance in which we had a problem with the sprayer.
Although the aerosol vaccination was not proven to be as effective in preventing MD as subcutaneous inoculation, it obviously provided a beneficial effect. If we are able to further refine the aerosol technique, perhaps the aerosol vaccination procedure can be made as effective as the subcutaneous inoculation and will prove to be more economical. REFERENCES Calnek, B. W., S. B. Hitchner and H. K. Adldinger, 1970. Lyophilization of cell-free Marek's disease herpesvirus and a herpesvirus from turkeys. Applied Microbiol. 20:723-726. Churchill, A. E., W. Baxendale and G. Carrington, 1973. Viremia and antibody development in chicks following the administration of turkey herpesvirus. Vet. Rec. 92:327-334. Churchill, A. E., and P. M. Biggs, 1967. Agent of Marek's disease in tissue culture. Nature, 215:528-530. Eidson, C. S., D. P. Anderson, S. H. Kleven and J. Brown, 1971. Field trials of vaccines for Marek's disease. Avian Dis. 15:312—322. Eidson, C. S., D. D. King, H. E. Connell, D. P. Anderson and S. H. Kleven, 1973a. Efficacy of turkeys herpesvirus vaccine against Marek's disease in broilers. Poultry Sci. 52:1482-1491. Eidson, C. S., S. H. Kleven and D. P. Anderson, 1973b. Efficacy of cell-free and cell-associated herpesvirus of turkeys vaccines in progeny from vaccinated parental flocks. Amer. J. Vet. Res. 34:869-872. Eidson, C. S., D. J. Ritchey and S. C. Schmittle, 1969. Studies on acute Marek's disease. XI. Propagation of the GA Isolate of Marek's disease in tissue culture. Avian Dis. 13:636—653. Eidson, C. S., P. Villegas, R. K. Page and S. H. Kleven, 1975. Efficacy of lyophilized turkey herpesvirus vaccine against Marek's disease in broilers. Poultry Sci. 54:1868-1874. Kawamura, H., D. J. King and D. P. Anderson, 1969. A Herpesvirus isolated from kidney cell culture of normal turkeys. Avian Dis. 13:853—863. Hitchner, S. B., and E. P. Johnson, 1948. A virus of low virulence for immunizing fowls against Newcastle disease (avian pneumoencephalitis) Vet. Med. 43:525-530. Okazaki, W., H. G. Purchase and B. R. Burmester, 1970. Protection against Marek's disease by vaccination with a herpesvirus of turkeys. Avian Dis. 14:413-429. Patrascu, I. V., B. W. Calnek and M. W. Smith, 1972. Vaccination with lyophilized turkey herpesvirus (HVT): Minimum infective and protective doses. Avian Dis. 16:86-93. Rispens, B. H., H. van Vloten and H. J. L. Maas, 1972. Control of Marek's disease in the Neatherlands. I. Isolation of an avirulent Marek's disease virus (strain CVI 988) and its use in vaccination trials. Avian Dis. 16:108-125. Witter, R. L., G. H. Burgoyne and J. J. Solomon, 1969. Evidence for a herpesvirus as an etiological agent of Marek's disease. Avian Dis. 13:171—184.
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chicken is a time consuming as well as a costly procedure. In an attempt to reduce vaccination costs, the chickens were mass vaccinated at one day of age by aerosol exposure with cell-free HVT vaccine. As shown in Tables 1, 4, 5, and 6 vaccination by aerosol offers a considerable degree of protection against MD. However, the results in each of the 3 tables show that aerosol vaccination was not as effective in preventing MD as in chickens vaccinated by subcutaneous inoculation. In Tables 1, 4, and 5 aerosol vaccination compared favorably with subcutaneous inoculation. As shown in Table 4, spray vaccination was effective in houses that had a high level of exposure to the virulent MD virus. As demonstrated in the first two production periods the virulent MD virus was present as indicated by the MD condemnations at the poultry processing plant. The effectiveness of aerosol vaccination is demonstrated in the 3rd production period by the reduction of MD condemnations. However, the results in Table 6 clearly shows that birds vaccinated by subcutaneous inoculation were more effectively protected against MD than chickens vaccinated by aerosol vaccination. Table 2 gives the results of birds vaccinated by aerosol at the hatchery and hatchmates that were vaccinated by subcutaneous inoculation. Regardless of whether the chickens were challenged by inoculation or by contact exposure to the virulent MD virus, chickens vaccinated by subcutaneous inoculation were more effectively protected against MD than those chickens vaccinated by aerosol. However, there was a significant difference in the incidence of MD in chickens vaccinated by aerosol and the nonvaccinated chickens. Furthermore, Table 3 shows that chickens vaccinated in the hatchery by aerosol developed a viremia at a slower rate than birds vaccinated by subcutaneous inoculation. Also there was a slightly delay in antibody response in chickens vaccinated by aerosol as compared to chickens vaccinated by subcutaneous inoculation.
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Poultry Science 56:1609-1615,1977 INTRODUCTION Churchill a n d Biggs ( 1 9 6 7 ) isolated a cellassociated herpesvirus from infectious material, a n d results of s u b s e q u e n t investigations (Eidson et al, 1 9 6 9 ; Witter et al, 1 9 6 9 ) p r o v e d this virus t o b e t h e etiologic agent of Marek's disease (MD). Churchill et al. ( 1 9 6 9 ) f o u n d t h a t t h e pathogenic strain of t h e MD herpesvirus became a t t e n u a t e d after 33 passages in cell culture. When administered t o chicks, it prot e c t e d against t h e d e v e l o p m e n t of MD lesions after challenge with a virulent strain of t h e virus. A l t h o u g h t h e a t t e n u a t e d MD viral vaccine offered considerable p r o t e c t i o n against MD t u m o r d e v e l o p m e n t , Eidson et al. ( 1 9 7 1 ) f o u n d t h a t a herpesvirus, first isolated f r o m t u r k e y s b y K a w a m u r a et al. ( 1 9 6 9 ) , was m o r e effective t h a n t h e a t t e n u a t e d MD vaccine. Calnek et al. ( 1 9 7 0 ) r e p o r t e d o n e x t r a c t i o n of H V T from infected cells b y ultrasonic disruption and preservation in t h e lyophilized s t a t e . A l t h o u g h t h e lyophilized H V T vaccine has been used successfully in m a n y E u r o p e a n countries, questions have arisen as t o w h e t h e r or n o t m a t e r n a l antibodies t o H V T alter t h e response t o vacci-
•Supported by the Georgia Department of Agriculture.
n a t i o n (Patrascu et al., 1972). R e c e n t d a t a indicate t h a t m a t e r n a l a n t i b o d y H V T does n o t significantly affect t h e efficacy of t h e cell-free H V T vaccine in immunizing chickens against MD w h e n t h e r e c o m m e n d e d dose is u s e d (Churchill et al, 1 9 7 3 ; Eidson et al., 1 9 7 5 ; Zygraich and Huygelen, 1 9 7 2 ) . Regardless of w h e t h e r t h e cell-free or t h e cell-associated H V T vaccine is used, t h e p r o d u c t is injected intra-abdominally, intramuscularly or s u b c u t a n e o u s l y . During t h e last 25 years, investigators have observed t h a t animals can b e c o m e i m m u n i z e d as a c o n s e q u e n c e of inhalation of air containing living microbial units. Mass vaccination of p o u l t r y w i t h sprays was first r e p o r t e d b y Hitchner et al. ( 1 9 4 8 ) w h e n chicks were i m m u n i z e d with t h e B1 strain of Newcastle disease vaccine. T h e p u r p o s e of this s t u d y is t o r e p o r t t h e results of field trials in which chicks were vaccinated against MD b y aerosol with cell-free H V T vaccine. MATERIALS AND METHODS Chickens: All broiler chicks used in this s t u d y were derived from breeder flocks t h a t had been vaccinated at 1 day of age with cell-associated H V T vaccine. Selection of Farms: F a r m s were selected t h a t had at least t w o p o u l t r y houses in o r d e r
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ABSTRACT In field trials approximately 300,000 birds were vaccinated against Marek's disease (MD) by aerosol spray with 1,000 to 5,000 PFU of cell-free HVT vaccine per chick. Chicks were sprayed in the trays of the hatchers which still contained dust and down after hatching. Prior to spraying, the incoming and outgoing air ducts were closed. The chicks were sprayed for 12—15 min. with HVT in 250 to 300 ml. of diluent, and after completion of spraying the chicks remained in constact with the aerosol for approximately for 10 minutes. In birds vaccinated by aerosol spray the incidence of MD ranged from 0.0% to 24.4%; while the incidence of MD in birds vaccinatedby subcutaneous inoculation varied from 0.02 t o 23.9%. In trial 1 the incidence of MD in birds vaccinated by aerosol spray was essentially the same as those vaccinated by subcutaneous inoculation; whereas, the incidence of MD (Trial 2 and 3) was higher in birds vaccinated by aerosol as compared to chickens vaccinated by subcutaneous inoculation.
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C. S. EIDSON AND S. H. KLEVEN
Trial 1. Experiment 2. Ninety chickens from the first experiment that were sprayed with
4,000 PFU per bird were brought to the laboratory. Forty of these chickens were challenged at 2 weeks of age by injecting the chicks subcutaneously with 0.2 ml. of MD infectious plasma and the remaining 40 chicks were challenged at 2 weeks by contact exposure. The seeder chicks were 2 week-old hatchmate seeder chicks which had been infected with the MD virus at one day of age. Seeder chicks were brooded in separate facilities until they were distributed at a ratio of 1:4 among the test birds. One hundred-eighty hatchmates were also brought back to the laboratory. Eighty of the birds were injected subcutaneously with approximately 1500 PFU of cell-free HVT vaccine. At 2 weeks of age 40 of the birds were challenged by subcutaneous inoculation of 0.2 ml. of MD-infective plasma while the second group of 40 chickens were challenged by contact exposure. Forty of the controls were challenged by subcutaneous inoculation and 40 chicks were challenged by contact exposure. At the end of the 10 week experimental period, all of the chickens were necropsied and examined for gross MD lesions. * Viremia and subsequent antibody development in chickens vaccinated with the HVT vaccine were studied. A group (10) of chickens obtained from chickens spray vaccinated with 4,000 PFU of cell-free HVT vaccine. Another group (10) of chickens were injected subcutaneously with approximately 1,500 PFU per bird. A final group of 10 chickens served as unvaccinated controls. All of the chickens were bled weekly. For the first 3 weeks an attempt was made to determine the viremia level in chickens from each of these 3 groups. The technique used to detect viremia in chickens vaccinated with the cell-free HVT vaccine was essentially that described by Churchill et al. (1973) with the exception that primary rather than secondary chick embryo fibroblast monolayers were used. To evaluate antibody development blood obtained between 4 and 8 weesk was analyzed by the plaque inhibition test as described by Churchill et al. (1973). Trial I. Experiment 3. In this study there were two poultry houses each holding approximately 6,000 chickens. During the first experiment on this farm the birds in the first house were spray vaccinated with 5,000 PFU per bird; whereas, the chickens in the second house were injected subcutaneously with 1,000 PFU per bird. For the second experiment on the farm
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that the efficacy of graded dose levels of the cell-free HVT vaccine could be compared with the cell-associated HVT vaccine in preventing MD. All of the houses used in the study were of the conventional curtain-type with either dirt or cement floors. No attempt was made to change the usual husbandry procedures. None of the houses were cleaned prior to the placement of the chicks except for the addition of a layer of new shavings to the old litter. In addition to the HVT vaccine all birds were vaccinated at 14—18 days of age against infectious bronchitis and Newcastle disease. Turkey Herpesvirus Vaccine: The cell-associated HVT vaccine was titrated as described by Eidson et al. (1973 a,b). The cell-free HVT vaccine was titrated as previously described by Eidson et al. (1975). Experimental Design. Trial 1. Experiment 1: Approximately 125,000 chickens were exposed by aerosol with 2,000 to 5,000 PFU of cell-free HVT vaccine. The aerosol was generated by Spraying Systems IA sprayer (Spraying Systems, Inc., Wheaton, Illinois) powered by an air compressor which provides 60 pounds per square inch of air pressure. The diluent used in spraying the chickens consisted of 0.15 M sucrose, 0.0038 M monopotassium phosphate, 0.0072 M dipotassium phosphate and 1.5% N Z-Amine. All the chickens were sprayed while they were in the trays of the hatcher. The hatchers measured 4' X 9' X 6' and held approximately 8,000 chicks. Prior to vaccination all air ducts were sealed. The chicks were sprayed for 12 to 15 min. with VT in 2 5 0 - 3 0 0 ml. of diluent, and after completion of spraying the chicks remained in contact with the aerosol for an additional 5 min. The fan inside the hatcher was either turned on intermittently or was turned on for the entire 20 min. experimental period. For comparison, an equal number of chicks were vaccinated subcutaneously with approximately 1000 PFU of cell-free HVT vaccine. Farms selected for the study had at least 2 houses so that one house contained chickens vaccinated with cell-free HVT by spray while the second house was vaccinated by subcutaneous inoculation. Each treatment group was identified separately as the birds were loaded and transported to the processing plants. Personnel of the Consumer and Marketing Service of the U.S.D.A. provided the inspection data.
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AEROSOL VACCINATION AGAINST MAREK'S DISEASE
RESULTS Trial I. Experiment 1. vaccinated by aerosol with protected against challenge MD Virus, (Table 1). Of
All of the birds 5,000 PFU were with the virulent the 30,681 birds
vaccinated with 5,000 PFU only 0.08% were condemned due to MD. The incidence of MD in the group varied from 0.02 to 0.20%. However 2 groups of chickens vaccinated by subcutaneous inoculation had 8.5% and 5.4% condemnation attributed to MD. The average incidence of MD in this group was 3.3% with a range of 0.02 to 8.5%. In contrast one group of birds sprayed with 4,000 PFU had 3.9% condemnation attributed to MD and the average incidence was 0.80% with a range of 0.02 to 3.9%. The incidence of MD in birds vaccinated subcutaneously was 0.20% with a range of 0.01 to 0.48%. In the group of chickens vaccinated with 3,000 PFU there was one group of chickens that had 23.4% condemnation for MD. Although all of the other groups of chickens vaccinated with 3,000 PFU were less than 0.2%, the average incidence of MD for the group was 4.9%. In contrast the average incidence of MD in the group of chickens vaccinated subcutaneously was only 1.6%. In the last group of chickens sprayed with 2,000 PFU of HVT vaccine there was no difference in the incidence of MD when compared to those vaccinated by subcutaneous inoculation. Trial 1. Experiment 2. As shown in Table 2 chickens which were vaccinated by aerosol in the hatcher with 4,000 PFU and brought to the laboratory for challenge, the incidence of gross lesions of MD was 10.0% in those challenged by injection. Whereas, only 5.1% of those challenged by contact exposure developed lesions. In contrast 5.1% of the birds vaccinated subcutaneously and challenged by injection devel-
TABLE 1.—Postmortem condemnations of chickens vaccinated by aerosol spray or by subcutaneous inoculation (Trial 1 — Experiment 1) Condemnations MD
Total
Dose (PFU)
Route of vaccination
No. birds
No.
%
Range %
No.
%
Range %
5000 1000 4000 1000 3000 1000 2000 1000
Aerosol Subcutaneously Aerosol Subcutaneously Aerosol Subcutaneously Aerosol
30,681 48,186 30,025 44,445 30.264 68,722 34,824 32,823
25 1564 254 86 1467 1113 128 126
0.08 3.25 0.80 0.20 4.85 1.62 0.37 0.38
0.02-0.20 0.02-8.50 0.00-3.93 0.01-0.48 0.97-23.40 0.11-2.70 0.09-1.55 0.07-0.66
194 1905 425 379 1630 1618 386 426
0.63 3.95 1.40 0.85 5.39 2.38 1.11 1.30
0.45-0.93 0.54-9.20 0.20-4.42 0.53-1.17 0.53-23.90 0.49-3.70 0.73-2.92 0.79-1.85
Subcutaneously
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the birds in the first house were spray vaccinated with approximately, 1,000 PFU. The birds in the second house were spray vaccinated with 2,000 PFU per bird. In the third successive broiler grow out period, chickens in both houses were spray vaccinated with 2,000 PFU per bird. Trial 2. At company number 2, the houses were paired so that each farm would have 2 houses of the same size. In this trial the birds were sprayed with Spraying Systems 1A sprayer in a hatcher that held 12,000 birds rather than 8,000 birds as in the first trial. Also, all of the birds vaccinated by spray received 5,000 PFU of cell-free HVT vaccine in each of the 3 experiments. For comparative purposes birds vaccinated subcutaneously received 1,000 PFU of cell-associated HVT vaccine. Trial 3. The chickens in study were spray vaccinated within a plastic canopy which measured 9' X 7' X 6'. The birds were sprayed with 2 nebulizers which were provided by an air compressor which provided 40 pounds per square inch of air pressure. Approximately 12,000 birds could be sprayed each time. In 8 experiments 96,000 birds were spray vaccinated with 5,000 PFU per bird and for comparative purposes 96,000 birds were vaccinated subcutaneously 1,000 PFU of the cell-associated HVT vaccine.
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C. S. EIDSON AND S. H. KLEVEN TABLE 2.—MD lesions at 10 weeks of age in chickens vaccinated with cell-free HVT in the hatcher and challenged with MD virus by inoculation or contact (Trial 1 — Experiment 2) 10 weeks Challenge
No. +/No. started
%
Aerosol^ Aerosol Subcutaneous" Subcutaneous None None
Injected^ Contact 0 Injected Contact Injected Contact
4/40E 2/39 2/39 0/37 22/38 12/35
(10%) (5.1%) (5.1%) (0%) (58.9%) (34.3%)
Chickens sprayed with 4,000 PFU/bird dose. B
Chickens injected with approximately 1,000 PFU/bird dose.
"'Chickens injected subcutaneously at 2 weeks of age with 0.2 ml of MD-infective plasma. Chickens exposed to MD virus with 100 seeder chickens/group. E Not including birds dying before 2 weeks of age.
oped gross lesions of MD. T h e groups challenged b y c o n t a c t e x p o s u r e did n o t develop any gross lesions. T h e incidence of MD in t h e g r o u p of non-vaccinated controls t h a t was challenged by injection was 5 8 . 9 % ; whereas, t h e incidence of M D in t h e g r o u p of controls t h a t was challenged b y c o n t a c t e x p o s u r e was 3 4 . 3 % . Birds vaccinated a t o n e d a y of age b y aerosol had developed a viremia ( 2 / 7 ) by o n e week b u t 7/10 chickens vaccinated s u b c u t a n e o u s l y with a p p r o x i m a t e l y 1,500 P F U per bird had devel-
o p e d a viremia. However, b y 2 weeks past vaccination t h e n u m b e r of chickens with viremia t o t h e H V T vaccine was essentially t h e same in b o t h t r e a t m e n t groups. (Table 3). Also, Table 3 shows t h a t significant a n t i b o d y levels were already developing 3 weeks after vaccination in b o t h t r e a t m e n t groups. However, antib o d y titers were slightly lower in t h e g r o u p s vaccinated b y aerosol. Trial 1. Experiment 3. In t h e first p r o d u c tion period 0.20% of t h e birds vaccinated by aerosol from h o u s e # 1 was c o n d e m n e d for M D ;
TABLE 3.—Determination of viremia to HVT and antibody titers following cell-free HVT vaccination of one-day-old chicks by either subcutaneous inoculation or by spray vaccination (Trial 1 — Experiment 2) No. of chicks with viremia
Antibody titersE Age (weeks)
Age (weeks) administration
1
2
3
4
5
6
7
8
Aerosol-^ Subcutaneous^ Control
2/9C 7/10 0/9
8/9 9/10 0/8
6/8D 8/8 0/7
14.7 F 23.5 9.8
19.0 32.0 10.8
32.0 53.8 8.8
69.8 90.5 11.9
234.8 279.2 11.9
Vaccinated with approximately 4,000 PFU of cell-free HVT vaccine. BVaccinated with approximately 1500 PFU. "Numerator = No. of chickens having viremia; denominator = no. chicks tested. No. of birds reduced due to mortality. "Reciprocal of serum dilution to a 50% reduction in virus plaque count. Geometric mean titers.
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Vaccination procedure
AEROSOL VACCINATION AGAINST MAREK'S DISEASE
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TABLE 4.—Post mortem condemnation of chickens vaccinated by aerosol or injection on a single farm during 3 successive 8-week broiler production periods (Trial 1 — Experiment 3) Condemnations No. period
House no. lA
1
2B
2 3
Total
marketed
No.
%
No.
%
Aerosol Sub. Sub. Sub. Sub. Sub.
6,132 6,300 5,993 6,045 5,988 6,012
12 341 1,290 1 48 4
0.20 5.41 21.5 0.01 0.80 0.07
57 409 1,337 46 79 28
0.93 5.41 22.3 0.76 1.32 0.47
Birds sprayed with 5,000 PFU/bird dose. Chickens injected subcutaneously with approximately 1,000 PFU of cell-free HVT vaccine. "Birds sprayed with approximately 1,000 PFU/bird dose. DBirds sprayed with 2,000 PFU/bird dose.
whereas, 5.41% of the birds vaccinated by subcutaneous inoculation were condemned for MD (Table 4). In contrast during the second production period 21.5% of the birds vaccinated with approximately 1,000 PFU was condemned for MD. Birds from house #2 which were sprayed with 2,000 PFU per bird dose had 0.01% condemnation for MD. For the 3rd production period birds from house # 1 were sprayed with 2,000 PFU per bird dose and only 0.80% were condemned for MD. Birds in house # 2 which were also sprayed with 2,000 PFU per bird had 0.07% condemnations for MD. Trial 2. In the first production period, 1.16% of the birds vaccinated by aerosol with 5,000
PFU of cell-free HVT were condemned for MD while only 0.16% of the birds injected subcutaneously were condemned (Table 5). In the second experiment only 0.17% of the birds vaccinated by aerosol were condemned; whereas, 0.83% of the injected birds were condemned. In the last experiment the injected birds (0.07%) had fewer condemnations for MD than the birds vaccinated by aerosol (0.34%). Trial 3. In this study there were 8 experiments in which 96,000 birds were vaccinated by spray and 96,000 birds were injected subcutaneously. Of the birds vaccinated by spray 0.51% were condemned for MD while only 0.15% of those vaccinated subcutaneously were
TABLE 5.—Post mortem condemnations of chickens vaccinated with cell-free HVT by aerosol or by inoculation with cell-associated HVT (company 2) Condemnations MD
Total
period
Vaccine
No birds
No.
%
No.
%
1
CFA CA CF CA CF CA
12,852 12,288 10,656 10,560 12,586 12,432
149 20 54 88 43 0
1.16 0.16 0.17 0.83 0.34 0.07
180 52 104 91 62 87
1.40 0.42 0.97 0.86 0.50 0.70
2 3
CF = Cell-free HVT vaccine; chickens were sprayed with approximately 5,000 PFU/bird dose. CA = Cell-associated HVT vaccine; chickens were injected subcutaneously with approximately 1,000 PFU/bird dose.
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lC 2D ID 2»
MD
vaccination
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C. S. EIDSON AND S. H. KLEVEN TABLE 6.—Post mortem condemnations of chickens vaccinated by aerosol spray inside a plastic canopy or by subcutaneous inoculation Condemnations
Experiment no. 1 2
4 5 6 7 8
Total
Vaccine
House no.
No.
%
No.
%
CFA CA« CF CA CF CA CF CA CF CA CF CA CF CA CF CA
lC 2D 1 2 1 2 1 2 1 2 1 2 1 2 1 2
54 7 20 8 26 11 193 8 83 28 70 67 5 5 34 8
0.45 0.06 0.17 0.07 0.22 0.09 1.61 0.07 0.69 0.23 0.58 0.56 0.04 0.04 0.28 0.07
107 80 59 70 110 96 254 62 122 146 112 107 50 65 100 74
0.89 0.70 0.49 0.58 0.92 0.80 2.12 0.52 1.02 1.22 0.93 0.89 0.42 0.54 0.83 0.62
CF = Cell-free HVT vaccine; chickens were sprayed with approximately 5,000 PFU/bird dose. CA = Cell-associated HVT vaccine; chickens were injected subcutaneously with approximately 1,000 PFU/bird dose. C, "House No. 1 - Contained 12,000 birds. R
DHouse No. 2 - Contained 12,000 birds.
condemned. As shown in Table 6 the MD condemnation in the chickens vaccinated by aerosol ranged from 0.04% to 1.61% while the MD condemnation in birds vaccinated subcutaneously ranged from 0.04% to 0.56%. DISCUSSION In the study reported herein, the objectives were to determine whether aerosol vaccination could be used effectively to protect chickens against MD. Also, attempts were made to determine whether aerosol vaccination was as effective in preventing MD as chickens vaccinated by subcutaneous inoculation. In choosing a vaccine to protect chickens against MD there are many factors such as availability, cost of the vaccine and cost of administering the vaccine must be taken into consideration. However, unlike most poultry vaccines, the vaccines against MD are administered in the hatchery and therefore the number of sites at which vaccination must be performed are considerably less than for conventional poultry vaccines administered to chickens in houses.
One of the most attractive vaccines is one prepared from an apathogenic Marek's disease virus that spreads horizontally. Rispens et al. (1972) were the first to isolate a maternally avirulent virus from a normal flock of chickens. Vaccine viruses that spread horizontally theoretically should have the advantage that only a small proportion of chicks need be inoculated to immunize a flock of chickens. However, in the case of vaccines against MD there is a chance of exposure to the virulent MD virus before the vaccine virus can effectively spread throughtout the flock. Also, a disadvantage to a vaccine virus that spreads is that each time it cycles through chickens there is a chance that it might revert back to a pathogenic form. It has been well documented in the literature that both the cell-free and cell-associated HVT vaccines when administered by subcutaneous inoculation at one day of age are effective in preventing MD (Eidson et al, 1971, 1973 a,b, 1975; Okazaki era/., 1970). Although the cell-free and cell associated HVT vaccines have proven to be effective in preventing MD, the vaccine had to be administered to each chicken. To vaccinate each
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3
MD
AEROSOL VACCINATION AGAINST MAREK'S DISEASE
Other reasons for the erratic results in the aerosol vaccinated chickens were the large amounts of dust, down and egg shells in the hatcher. Also, the density of chicks in the hatchers may have also influenced the results. In Trial 2, there was some extraneous matter in the vaccine that constantly clogged the sprayer. This was the only instance in which we had a problem with the sprayer.
Although the aerosol vaccination was not proven to be as effective in preventing MD as subcutaneous inoculation, it obviously provided a beneficial effect. If we are able to further refine the aerosol technique, perhaps the aerosol vaccination procedure can be made as effective as the subcutaneous inoculation and will prove to be more economical. REFERENCES Calnek, B. W., S. B. Hitchner and H. K. Adldinger, 1970. Lyophilization of cell-free Marek's disease herpesvirus and a herpesvirus from turkeys. Applied Microbiol. 20:723-726. Churchill, A. E., W. Baxendale and G. Carrington, 1973. Viremia and antibody development in chicks following the administration of turkey herpesvirus. Vet. Rec. 92:327-334. Churchill, A. E., and P. M. Biggs, 1967. Agent of Marek's disease in tissue culture. Nature, 215:528-530. Eidson, C. S., D. P. Anderson, S. H. Kleven and J. Brown, 1971. Field trials of vaccines for Marek's disease. Avian Dis. 15:312—322. Eidson, C. S., D. D. King, H. E. Connell, D. P. Anderson and S. H. Kleven, 1973a. Efficacy of turkeys herpesvirus vaccine against Marek's disease in broilers. Poultry Sci. 52:1482-1491. Eidson, C. S., S. H. Kleven and D. P. Anderson, 1973b. Efficacy of cell-free and cell-associated herpesvirus of turkeys vaccines in progeny from vaccinated parental flocks. Amer. J. Vet. Res. 34:869-872. Eidson, C. S., D. J. Ritchey and S. C. Schmittle, 1969. Studies on acute Marek's disease. XI. Propagation of the GA Isolate of Marek's disease in tissue culture. Avian Dis. 13:636—653. Eidson, C. S., P. Villegas, R. K. Page and S. H. Kleven, 1975. Efficacy of lyophilized turkey herpesvirus vaccine against Marek's disease in broilers. Poultry Sci. 54:1868-1874. Kawamura, H., D. J. King and D. P. Anderson, 1969. A Herpesvirus isolated from kidney cell culture of normal turkeys. Avian Dis. 13:853—863. Hitchner, S. B., and E. P. Johnson, 1948. A virus of low virulence for immunizing fowls against Newcastle disease (avian pneumoencephalitis) Vet. Med. 43:525-530. Okazaki, W., H. G. Purchase and B. R. Burmester, 1970. Protection against Marek's disease by vaccination with a herpesvirus of turkeys. Avian Dis. 14:413-429. Patrascu, I. V., B. W. Calnek and M. W. Smith, 1972. Vaccination with lyophilized turkey herpesvirus (HVT): Minimum infective and protective doses. Avian Dis. 16:86-93. Rispens, B. H., H. van Vloten and H. J. L. Maas, 1972. Control of Marek's disease in the Neatherlands. I. Isolation of an avirulent Marek's disease virus (strain CVI 988) and its use in vaccination trials. Avian Dis. 16:108-125. Witter, R. L., G. H. Burgoyne and J. J. Solomon, 1969. Evidence for a herpesvirus as an etiological agent of Marek's disease. Avian Dis. 13:171—184.
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chicken is a time consuming as well as a costly procedure. In an attempt to reduce vaccination costs, the chickens were mass vaccinated at one day of age by aerosol exposure with cell-free HVT vaccine. As shown in Tables 1, 4, 5, and 6 vaccination by aerosol offers a considerable degree of protection against MD. However, the results in each of the 3 tables show that aerosol vaccination was not as effective in preventing MD as in chickens vaccinated by subcutaneous inoculation. In Tables 1, 4, and 5 aerosol vaccination compared favorably with subcutaneous inoculation. As shown in Table 4, spray vaccination was effective in houses that had a high level of exposure to the virulent MD virus. As demonstrated in the first two production periods the virulent MD virus was present as indicated by the MD condemnations at the poultry processing plant. The effectiveness of aerosol vaccination is demonstrated in the 3rd production period by the reduction of MD condemnations. However, the results in Table 6 clearly shows that birds vaccinated by subcutaneous inoculation were more effectively protected against MD than chickens vaccinated by aerosol vaccination. Table 2 gives the results of birds vaccinated by aerosol at the hatchery and hatchmates that were vaccinated by subcutaneous inoculation. Regardless of whether the chickens were challenged by inoculation or by contact exposure to the virulent MD virus, chickens vaccinated by subcutaneous inoculation were more effectively protected against MD than those chickens vaccinated by aerosol. However, there was a significant difference in the incidence of MD in chickens vaccinated by aerosol and the nonvaccinated chickens. Furthermore, Table 3 shows that chickens vaccinated in the hatchery by aerosol developed a viremia at a slower rate than birds vaccinated by subcutaneous inoculation. Also there was a slightly delay in antibody response in chickens vaccinated by aerosol as compared to chickens vaccinated by subcutaneous inoculation.
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