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PRODUCTION, MODELING, AND EDUCATION

Research Note
Field evaluation of the accuracy of vaccine deposition by two different
commercially available in ovo injection systems

C. J. Williams1 and B. A. Hopkins

Pfizer Animal Health, Third Avenue, MS 685-10-1, New York, NY 10017

ABSTRACT The location of injection and vaccine de- in which the vaccine was injected into the amniotic
position in ovo is known to be critical to the efficacy of sac or into s.c. or i.m. regions of the embryo. Incorrect
Mareks disease (MD) vaccine protection against MD vaccine delivery was defined as delivery into the air
viral challenge. Vaccine deposition into the amniotic cell; allantoic sac; any combinations including air cell
sac or a s.c. or i.m. site of the embryo is required for or allantois; the abdominal, cranial, orbital, or thoracic
MD vaccine efficacy. Vaccine deposition into the air cavities of the embryo; or no vaccine delivery at all.
cell or allantoic fluid results in chicks that are not ad- In hatchery 1 (Chick Master, Newton, MS) 1,171 nor-
equately protected against subsequent MD viral chal- mal eggs were processed through the Inovoject system
lenge. A study was conducted in 2 commercial broiler and 1,138 eggs were processed by the Intelliject system.
hatcheries to evaluate the ability of 2 in ovo injection The Inovoject system correctly vaccinated 94.62% of
systems, the Embrex Inovoject system (Pfizer Poultry the normal eggs as compared with 61.16% delivery ac-
Health, Research Triangle Park, NC) and the Intelliject curacy of normal eggs with the Intelliject system. In
system (Avitech, Salisbury, MD; distributed by Merial hatchery 2 (Jamesway Super J, Magee, MS) 926 normal
Ltd., Gainesville, GA) to deliver a vaccine approved eggs were processed by the Inovoject system and 910
for use in ovo accurately and properly. A standard MD normal eggs were processed by the Intelliject system.
vaccine diluent mixed with a protein-staining dye was The Inovoject system correctly vaccinated 91.04% of
delivered through each machine to simulate in ovo vac- the normal eggs, whereas the Intelliject system correct-
cination. The location of the dye within the egg deter- ly vaccinated 71.98% of the normal eggs. The results
mined whether the vaccine was delivered correctly. Each of this study clearly demonstrate that the Inovoject
egg was also evaluated for normal embryo development system accurately delivered in ovo vaccine at a signifi-
(normal eggs). Correct vaccine delivery included eggs cantly higher rate than the Intelliject system.
Key words: site of injection, in ovo, vaccine deposition, embryo
2011 Poultry Science 90:223226
doi:10.3382/ps.2010-00759

INTRODUCTION many commercial broiler hatcheries around the world,


particularly in the Americas, Japan, Australia, and
The first research on the in ovo administration of parts of Europe.
vaccines was completed by Sharma and Witter (1983). The process and technique used to administer in ovo
Subsequent work demonstrated that in ovo adminis- vaccines is critical. Delivering vaccine to an incorrect
tration of Mareks disease (MD) vaccines HVT, SB-1, in ovo site can lead to an ineffective vaccination, thus
and CVI988 to late-stage chicken embryos was safe and reducing the benefits normally seen by vaccinating in
would induce earlier immunity than posthatch adminis- ovo. The complexity of the in ovo route is often under-
tration (Sharma et al., 1984; Zhang and Sharma, 2001). appreciated, and this is attributable in part to the 5
Because of these results, the practice of in ovo vacci- embryonic compartments that can be accessed by the
nation was moved from the laboratory to commercial needle during the in ovo vaccination process: the air
poultry hatcheries (Gildersleeve et al., 1993; Sarma et cell (AC), allantoic sac (AL), amniotic fluid (AM),
al., 1995). Today, in ovo vaccination occurs routinely in embryo body (EM), and yolk sac. Furthermore, EM
injections can be s.c., i.m., intracranial, intraorbital, or
intraabdominal, with the latter 3 causing excessive em-
2011 Poultry Science Association Inc.
Received March 10, 2010.
bryo trauma. The efficacy of a vaccine and the safety to
Accepted September 6, 2010. the embryo can be affected by the embryonic compart-
1 Corresponding author: christopher.williams2@pfizer.com
ment into which it is deposited.

223

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224 Williams and Hopkins

Wakenell et al. (2002) showed that efficacy against Vaccination Procedure


MD virus challenge was dependent on the site of MD
vaccine delivery in ovo. The MD vaccine was not effec- The Inovoject systems used in these studies are de-
tive when delivered onto the AC or into the AL. The signed to deliver 50 L of vaccine per injection cycle to
highest efficacy was found in the AM, with a 94.4% each egg determined as viable, whereas the Intelliject
protective index, and in the EM, with a 93.9% protec- systems are designed to deliver 100 L to all eggs dur-
tive index. Protective efficacy in chicks with vaccine ing each cycle. The test eggs were injected immediately
injected into the AC or AL was less than 50%. This before the beginning of the normal transfer and injec-
work showed that the vaccine must be injected deep tion time. Each vaccine system was purged of alcohol
enough into the egg to be delivered into the amnion, with sterile saline, and then test vaccine was added.
which includes the AM and EM. When vaccines are The sanitation system for both machines functioned as
delivered to the AM, the vaccine is imbibed, breathed normal, using each manufacturers recommended chlo-
in, or both by the embryo before hatching. The more rinated sanitizer solution. The test vaccine bag con-
developmentally mature embryos in the population are tained standard Mareks vaccine diluent and a protein-
injected in the right breast area. Embryo body delivery staining dye without the inclusion of any vaccine.
is normal; however, penetration too deep into the egg
at any stage of development can cause excessive trauma Embryo Euthanasia and Vaccine
to the embryo. Deposition Evaluation
After injection was completed, test eggs, while still in
MATERIALS AND METHODS the original incubation flats, were bagged by treatment,
killed using CO2 gas, and stored for 4 h at low temper-
In Ovo Injection Equipment ature. After euthanasia, eggs were carefully dissected
and vaccine deposition site was determined. Each egg
Technical representatives from both in ovo equipment was also evaluated for normal embryo development.
manufacturers prepared their respective machines and Eggs were segregated by embryo development status
ensured proper function of the equipment during the into abnormal eggs (upside down, early dead, mid dead,
conduct of each trial. The equipment differed by site late dead, cracked shell, malformed or malpositioned
because a specific injector design was required for each embryo, rotten, and infertile) and normal eggs (normal
incubation system. The trial conducted at the Chick embryo development). Only normally developed embry-
Master hatchery (Newton, MS) compared the Embrex os were included in the site of injection analysis.
Inovoject Systems Vaccine Saver CM 108 design (se-
rial no. V 2954, Pfizer Poultry Health, Research Tri-
angle Park, NC) with the Avitech Intelliject CM 162
Experimental Design
design (serial no. 00035, Avitech, Salisbury, MD). The Two separate trials were conducted in 2 commercial
trial conducted at the Jamesway hatchery (Magee, MS) broiler hatcheries. One hatchery used the Chick Master
compared the Embrex Inovoject Systems Vaccine Saver incubation system and the other used the Jamesway
JW 84 design (serial no. V 2252A) with the Intelliject Super-J incubation system. Because of differences in
JW 168 design (serial no. 00063). the design of the incubation tray that nests the eggs
during embryonic development, specific egg injection
Eggs machines from each manufacturer were required for
each incubation system (Chick Master or Jamesway).
Eggs from Cobb breeder flocks were selected by Additionally, other application differences existed be-
incubator tray from the egg incubation racks in the tween the 2 types of egg injection systems (Pfizers Em-
same format and position as they were incubated. In brex Inovoject system or Avitechs Intelliject system),
the Jamesway hatchery, 4 incubator trays (84 eggs/ hence the purpose for this evaluation. Both hatcheries
tray, 336 eggs/age group) were randomly selected from used egg injection equipment specifically designed for
3 separate breeder flocks of differing ages. Eggs were use by either the Chick Master or Jamesway incuba-
injected in ovo at their normal transfer age (Jamesway tion system. Other than the egg flat configuration and
incubation system: d 18 + 7 h of incubation). A total difference in time of injection dictated by either the
of 1,008 eggs were used per injection system. Jamesway or Chick Master incubation system, the pro-
In the Chick Master hatchery, 6 trays (54 eggs/tray, tocol for vaccine application and injection site determi-
324 eggs/age group) were randomly selected from 4 nation were identical between the 2 sites.
separate breeder flocks of differing ages. Eggs were in- Correct injections in ovo for optimal MD efficacy are
jected in ovo at their normal transfer age (Chick Mas- defined as delivery of vaccine into the AM and EM
ter incubation system: d 19 + 1 h of incubation). A or EM-AM injections (Wakenell et al., 2002). For the
total of 1,296 eggs were used for the evaluation of each purposes of this study, incorrect injections were defined
injection system. as AC, AL, combinations that included AC or AL, and

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RESEARCH NOTE 225
Table 1. Number and percentage of eggs with proper and improper vaccine delivery by in ovo injection system and hatchery
Chick Master hatchery Jamesway hatchery

Injection system Correct delivery Incorrect delivery Correct delivery Incorrect delivery

Embrex Inovoject system


Eggs (no.) 1,108 63 843 83
Delivery (%) 94.62a 5.38 91.04a 8.96
Intelliject system
Eggs (no.) 696 442 655 255
Delivery (%) 61.16b 38.84 71.98b 28.02
a,bNumbers within a column lacking a common superscript differ (P 0.05).

all incorrect embryo sites (intracranial, intraorbital, tray to tray associated with eggs from the oldest breed-
intraabdominal). No attempts were made to quantify er flock. Only live normal embryos at injection time
or differentiate volume of vaccine delivered to multiple were included in the evaluation. The Inovoject system
sites in ovo; therefore, suboptimal doses or partial de- correctly injected a higher percentage of normal eggs
livery to correct sites in ovo were considered incorrect (P 0.0001) than the Intelliject system (Table 1). The
applications. Correct injections included only complete same model was then applied to each specific category
delivery of individual vaccine dispensed into the correct (vs. all others). The Intelliject system was significantly
site in ovo. The egg was considered the experimental higher (P 0.0001; Table 2) in all categories of incor-
unit in a generalized randomized complete block design rect vaccine deposition (AC, AL, AC or AL combina-
with flock as the blocking factor. Correct vs. incorrect tions, and EM incorrect), whereas the Inovoject system
injections were analyzed by exact conditional logistic performed significantly better in all categories of cor-
regression, with flock as a stratifying variable and treat- rect vaccine deposition (AM and EM proper) except for
ment as a factor (SAS/STAT Version 9.1.3 Service Pack the combination AM and EM injections, for which the
3, SAS Institute Inc., Cary, NC). In terms of adjust- Intelliject system delivered significantly more than the
ment for multiple comparisons, the overall treatment Inovoject system (P 0.0001).
effect for site of injection was evaluated using a Fishers In the Jamesway hatchery, 1,008 eggs were injected
protected least significant difference type approach for by each injection system. Of those, 926 normal eggs
comparisons for each site without adjustment. Statisti- were injected by the Inovoject system and 910 normal
cal significance was determined with P 0.05. eggs were injected by the Intelliject system. As in tri-
al 1, the Inovoject system (Table 3) had significantly
RESULTS AND DISCUSSION more correct vaccinations (AM) in ovo (P 0.0001)
than the Intelliject system, except for injections of EM
Two separate analyses were completed, one for each proper, for which there was no significant difference (P
type of incubation system involved, because myriad ran- > 0.5984) and injections of AM-EM proper, for which
dom effects were related to each hatchery and respec- the Intelliject system had significantly (P 0.0114)
tive incubation system. In the Chick Master hatchery, more proper injections than the Inovoject system. The
1,296 eggs were injected by each injection system. Of Intelliject system (Table 3) delivered significantly (P
those, 1,171 normal eggs were injected by the Inovoject 0.0001) more incorrect vaccine placements (AC, AC or
system and 1,138 normal eggs were injected by the In- AL combinations, and EM incorrect) when compared
telliject system. Although randomized at injection, the with the Inovoject system, except for AL (P > 0.4308)
difference in the number of normal eggs between the and the combination AM-EM incorrect injections (P >
2 groups was primarily due to fertility variation from 0.9999), for which there was no significant difference.

Table 2. Number and percentage of eggs at each specific site of vaccine deposition1 at the Chick Master hatchery
AC AL AL AM-EM EM Total
Item AC combination AL AM combination AM incorrect AM-EM incorrect EM eggs

Injection system
Embrex Inovoject
system
Eggs (no.) 0 3 1 2 0 673 1 2 56 433 1,171
Delivery (%) 0.00b 0.26b 0.09b 0.17b 0.00b 57.47a 0.09b 0.17b 4.78b 36.98a
Intelliject system
Eggs (no.) 41 26 18 179 78 480 20 48 80 168 1,138
Delivery (%) 3.60a 2.28a 1.58a 15.73a 6.85a 42.18b 1.76a 4.22a 7.03a 14.76b
Total eggs 41 29 19 181 78 1,153 21 50 136 601 2,309
a,bNumbers within a column lacking a common superscript differ (P 0.05).
1AC = air cell; AL = allantoic sac; AM = amniotic fluid; EM = embryo body.

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226 Williams and Hopkins
Table 3. Number and percentage of eggs at each specific site of vaccine deposition1 in the Jamesway hatchery
AC AL AM-EM EM Total
Item AC combination AL AL-AM combination AM incorrect AM-EM incorrect EM eggs

Injection system
Embrex Inovoject
system
Eggs (no.) 2 2 27 48 3 744 0 3 1 96 926
Delivery (%) 0.22b 0.22b 2.92a 5.18b 0.32b 80.35a 0.00a 0.32b 0.11b 10.37a
Intelliject system
Eggs (no.) 17 30 33 134 30 541 1 13 10 101 910
Delivery (%) 1.87a 3.30a 3.63a 14.73a 3.30a 59.45b 0.11a 1.43a 1.10a 11.10a
Total eggs 19 32 60 182 33 1,285 1 16 11 197 1,836
a,bNumbers within a column lacking a common superscript differ (P 0.05).
1AC = air cell; AL = allantoic sac; AM = amniotic fluid; EM = embryo body.

With respect to accuracy of vaccine deposition by the lenge, whereas delivery to the AC shows no embryo
Inovoject system in the Chick Master hatchery vs. the access to the vaccine, thus no vaccinal response. As
JW hatchery, differences could be seen that were caused the embryo matures and begins the hatching process
by embryo age and egg setter tray design. The egg tray (internal and external pipping), more embryos in the
configuration and stability of the egg in the tray varied population are injected either s.c. or i.m. into the right
between the 2 incubation systems, with the Jamesway breast area. Embryo body delivery during in ovo vac-
incubation tray allowing eggs to deviate more than the cination is normal; however, penetration too deep into
Chick Master incubation tray along the upright axis. the egg and embryo during late-stage development can
The design of the Chick Master incubation tray kept cause excessive trauma to the embryo. Therefore, use
eggs in a very tight, upright position and did not al- of an in ovo injection system that does not deliver vac-
low egg movement laterally while the eggs were being cine to the correct embryonic site would compromise
turned in the incubator. In addition, embryos in the embryo safety and vaccine performance. The objective
Chick Master hatchery were 18 h older at transfer com- of commercial in ovo vaccination is to vaccinate every
pared with those in the Jamesway hatchery. The opti- viable embryo safely and uniformly. To accomplish this
mal time to inject eggs, developmentally, is the physi- objective, a commercial in ovo vaccination device must
ological stage between when the stalk of the yolk sac is administer vaccine to sites that result in the greatest
beginning its ascent into the abdomen and the head is safety and efficacy for the vaccine delivered. In this
tucked under the wing up until external pipping is initi- study, the Inovoject system was significantly better at
ated. The optimal target site for uniform automated in meeting that objective.
ovo vaccination is the AM or right breast area of the
developing embryo. Injections into the cranium, ocu- REFERENCES
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