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A L O H M A N N A N I M A L H E A LT H N E W S B R I E F August 2008 | Volume 1

Live attenuated vaccines: benefits of water stabilizers

in drinking water and spray administrations
We will techniques for the administra-
tion of live attenuated vaccines.
continue to The stimulation of immunity in a
massively vaccinated popula-
invest in tion (herd immunity) reduces the
A poor administration is still the
most common cause of vaccine
failure in poultry. A successful
administration of live attenuated
vaccines by spray or drinking

research,
development By Iván R. Alvarado, DVM, MS,
Ph.D., ACPV, Technical Services
Veterinarian, Lohmann Animal
and production Health International
Introduction
to assure a
The increasing demand for
economical and rapidly available
steady supply sources of animal protein has
highly influenced the constant
of high quality growth of poultry operations
around the world. The capacity
avian biologics of poultry to adapt to diverse
geographical conditions and the
for the poultry fact that no large areas of land
are required, have also
industry. contributed to the expansion
and efficiency of commercial
Dave Zacek
poultry operations.
President,
Lohmann Animal Health
As a result of their size increase,
it has become necessary to
develop and implement massive
…the ease and low cost have made
spray and drinking water the preferred
methods for massive administration of
live attenuated vaccines in the farms.
water requires an adequate
probability of an individual bird
in a vaccinated flock to become preservation and proper delivery
infected (Marangon and Busani of the vaccine to the host. In
this article, we describe the
2006). Since under field condi-
tions, it is unrealistic to expect adverse effects a live vaccine
might encounter during resus-
a 100% protection of the birds
in a vaccinated flock, the goal pension and administration
in suboptimal water, and the
is to develop immunity in a high
enough proportion of birds to benefits of using water stabilizers
during vaccination.
prevent the transmission or
minimize the adverse effects of
a disease agent. For poultry, the Massive Administration
ease and low cost have made of Live Vaccines
spray and drinking water the During manufacturing, most
preferred methods for massive live attenuated vaccines are
administration of live attenuated subjected to a lyophilization
vaccines in the farms. process. Lyophilization, in

inside Live attenuated

vaccines p.1
President’s Note
p.4
association with adequate refrigeration
temperatures (between 4°C and 7°C
or 39°F and 44°F), guarantees the
preservation of an adequate vaccine
titer able to stimulate a satisfactory
immune response in the bird. However,
once reconstituted, the limited lifespan
of live attenuated vaccines should be
a major concern.
Drinking water is an appropriate method
of administration for live attenuated
vaccines that require an initial infection
within the gut, such as Salmonella,
infectious bursal disease and avian
encephalomyelitis vaccines. Also, due
to the communication of the nasal cavity
with the mouth via the choanal cleft, a
successful application of respiratory virus
vaccines (such as infectious bronchitis
and Newcastle disease virus) should
be expected. Coarse spray is a widely
used and effective method to vaccinate
birds against respiratory viruses, not
only at the hatchery but also in the
farm. By coarse spray, the vaccine is
introduced through the eye, the nostrils
and by ingestion after pecking.
Adverse Conditions
At the farm, a poor condition of the
water used during resuspension and
administration will have a detrimental
effect on vaccine titers. Live vaccines
should be resuspended in fresh water
with a close to neutral pH, moderate
hardness and no chlorine or other
disinfectant residues. The undesirable
effects these factors will have on the
survival of live vaccines are briefly
described in the following paragraph.
Periodic treatments of water wells with
high concentrations of chlorine release
products (CRP), and chlorination of the
drinking water through in-line propor-
tioners, have been effectively used in
poultry operations to control microbial
levels. However, CRP also inactivate
live vaccines. Chlorinated compounds
(such as sodium hypochlorite, chlorine
dioxide and sodium dichloroisocyanu-
rate) are highly active oxidizing agents,
destroying the cellular activity of proteins
(McDonnell and Russell 1999). Sodium
hypochlorite ionizes to produce Na+
and the hypochlorite ion OCL-, which
is transformed in hypochlorous acid
(HOCL), the active moiety responsible
for bacterial inactivation (McDonnell and
Russell 1999). The deleterious effects
of CRP on bacteria include a disruption
of oxidative phosphorylation and other
membrane-associated activity as well as
inhibition of DNA synthesis. Hypochlo-
rous acid levels as low as 2.6 ppm
completely inhibit the growth of E. coli
within 5 minutes, 96% of DNA synthesis
and between 10% and 30% of protein
synthesis (McDonnell and Russell 1999).
The virucidal activity of CRP includes
nucleic acid fragmentation.
pH is a measure of acidity or basicity of
a solution. pH measurements depend
on the concentration of hydrogen (H+)
and hydroxide ions (OH-). Waters with
neutral pH (ph = 7.0), have an equal
concentration of H+ and OH-. The
resuspension of live vaccines in low pH
water will cause structural damage to
cell membranes and macromolecules
such as DNA and proteins. Week acids,
such as some organic acids used in
drinking water for poultry, can pass freely
through the bacterial cell membrane.
Once inside, the week acids dissociate
and increase the acidification of the
cytoplasm, affecting the bacterial
survival in drinking water (Cotter and
Hill 2003). Furthermore, a synergistic
effect between low water pH and
the bacteriocidal/virucidal activity of
chlorine-released agents has been
demonstrated. While low pH values
(below 5) adversely affect water
consumption, high pH values can affect
water palatability and form mineral
deposits which reduce the water flow
rate (Vermeulen, Backer et al. 2002).
Hardness is a measure of the quantity
of salts, mainly of divalent ions such as
calcium, magnesium and/or iron, in the
water. Hardness is expressed as mil-
ligrams of calcium carbonate (CaCO3)
per liter (ppm) and indicates the total
quantity of divalent salts present (Wurts
1993). However, high hardness in the
presence of low calcium but high
magnesium levels can be observed.
A previous research study at Lohmann
Animal Health has shown the detrimental
effect of hard water (20 ppm Fe and
4 ppm NaOCl) on Newcastle disease
virus and Salmonella typhimurium live
vaccines two and four hours after
resuspension. Drastic reductions in viral
and bacterial titers have been observed
2 and 4 hours after resuspension in
hard water. In areas with hard water, the
constant deposit of insoluble minerals
in the nipple drinkers and water lines
can also affect the homogeneous
administration of live vaccines to the
flocks. Spray vaccination using hard
water can create mineral deposits,
clogging the spray nozzles.
Water stabilizers
Live poultry vaccines are produced in
specific pathogen free (SPF) embrion-
ated eggs, tissue cultures or nutri-
tional broths under controlled laboratory
conditions. Vaccine manufacturers have s pH buffering agents: used to stabilize
recognized the adverse conditions live the water pH between 6 and 7, approx-
vaccines face during their administration
in the farm. For such reason, the use
of water stabilizers during vaccination is
recommended worldwide by technical
services veterinarians.
One of the first products used to
stabilize the drinking water during
vaccination was skimmed milk. Dilution
of skimmed milk (approximately 2
g/liter or 10 g/gallon) in the drinking
water at least 20 minutes before the
addition of the vaccine was a gen-
eral recommendation (Cargill 1999).
Skimmed milk has shown to efficiently
overcome the detrimental effects of
chlorine. However, the presence of un-
dissolved residues in cold water, which
can eventually block nipple drinkers,
and the presence of residues inside the
water pipes after vaccination, serving as
a source of nutrients for bacterial growth
(build up of biofilms), has limited its use.
More efficacious dyed water stabilizers
for the protection of live vaccines are
commercially available. These are some
of the characteristics and components
used in water stabilizers:
sReducing agents: used to neutralize
oxidizing sanitizers (i.e. residual
chlorine) or contaminants present
in farm water. One of the most
commonly used reducing agents is
sodium thiosulfate (Simpson 2001),
which is recognized as a safe food
additive. Other reducing agents are
sodium metabisulfite, sodium bisulfite
and ammonium thiosulfate
(Simpson 2001).
imately. Some buffering agents include
sodium phosphate, potassium phos-
phate, sodium citrate, sodium bicarbon-
ate and potassium bicarbonate.
s Water soluble dye: food colored dyes
approved by the Food and Drug
Administration are essential to allow
the detection of the water containing
the live vaccines in the drinker systems
and in vaccinated birds (tongue and
nasal cleft after eye drop administration;
tongue, oral cavity or crop after water
administration, and skin and feathers
after coarse spray vaccination).
s Thermo-stability conferring agents:
bovine serum albumin, sorbitol,
maltose, lactose, sucrose and glycerol
are some of the most common vaccine
stabilizers (Barbour, Abdelnour
et al. 2002).
Conclusions
The administration of live attenuated vac-
cines by massive methods such as drink-
ing water and spray is a very common
practice in poultry operations. Massive
administration methods are not as efficient
Wurts, W. A. (1993). “Understanding water
as individual vaccination. However, they
offer several advantages, such as low cost
and rapid administration of one or several
vaccines. Since live attenuated vaccines
are very fragile organisms, it is important
to neutralize the adverse chemical agents
present in the water used to resuspend
and administer the vaccines. Furthermore,
the inclusion of dyes facilitates the visual-
ization of vaccine in water lines and
vaccinated birds. The use of water
stabilizers during vaccination allows a
proper preservation of live vaccines,
which, in association with a proper
administration technique, will contribute
to a successful vaccination.
References
Barbour, E. K., A. Abdelnour, et al. (2002).
“Evaluation of 12 stabilizers in a developed
attenuated Salmonella Enteritidis vaccine.”
Vaccine 20 (17-18): 2249-2253.
Cargill, P. (1999). “Vaccine administration in
poultry.” In Practice 323-328.
Cotter, P. D. and C. Hill (2003). “Surviving the
acid test: responses of Gram-positive bacteria
to low pH.” Microbiol. Mol. Biol. Rev. 67(3):
429-453.
Marangon, S. and L. Busani (2006).
“The use of vaccination in poultry production.”
Rev. Sci. Tech. Off. Int. Epiz 26(1): 165-274.
McDonnell, G. and D. Russell (1999). “Antisep-
tics and disinfectants: activity, action and resis-
tance.” Clinical Microbiology Reviews: 147-149.
Simpson, G. D. (2001). The reduction of the
Chlorite ion. International symposium on chlorine
dioxide., Las Vegas, Nevada.
Vermeulen, B., P. D. Backer, et al. (2002).
“Drug administration to poultry.” Adv. Drug Del.
Rev. 54: 795-803.
hardness.” World Aquaculture 24(1): 18.
President’s Note
Dave Zacek
President,
Lohmann Animal Health
It has been nearly one year since we
opened our newly expanded pro-
duction/testing/label, pack and ship
site in Maine after having closed our
Vineland site and transferred vaccine
production to Winslow, Maine. Newly
avianinsight
hired additional staff are now fully
integrated and part of the vaccine
manufacturing team in Maine. The
majority of our production consists of
inactivated vaccines.
Increased demand for AviPro® vaccines
both in the USA and internationally,
require we build again. We have begun
a large, state-of-the-art animal testing
complex on property adjacent to our
current manufacturing plant in Winslow.
Included on the site are the Animal
Services Administration Center, a large
pen isolation unit, small test houses and
a large multiple isolation unit. Ground was
broken for phase I of this project June
2008. The multi-million dollar facility will
for more information:
207.873.3989 800.655.1342 www.lahinternational.com
be completed by fall of 2009.
In addition, we are adding a fermentation
suite in the manufacturing plant. This new
production addition will allow increased
volumes of our bacterial products, both live
and killed.
Recognition of AviPro® quality is driving the
demand. We are pleased that customers
value our vaccines and keep demand-
ing more year after year. We will continue
to invest in research, development and
production to assure a steady supply of
high quality avian biologics for the poultry
industry. And we will continue to work hard
to be your Avian Professionals.
375 China Road
Winslow, Maine 04901