Prevention of Virus Inactivation During Drinking

Water Vaccination of Poultry

R. F. GENTRY AND M. 0 . BEAUNE
Wiley Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802
(Received for publication December 17, 1971)

ABSTRACT Newcastle disease (N.D.) and infectious bronchitis (I.B.) drinking

water type vaccines secured at the time of actual field application all had adequate
viable virus to produce an immune response. I.B. vaccine having only 0.7 logs (E.I.D.a,) titer
at the final drinking water concentration stimulated an immune response in 4-week old
chickens. Titer of both N.D. and I.B. vaccines from manufacturing source A average approxi-
mately 1.0 logs higher than vaccines from source B.
N.D. and I.B. test virus suspensions were prepared in phosphate buffered saline
(P.B.S.), distilled water and filtered tap water. I.B. virus titers were markedly reduced
when held at room temperature. The greatest reduction was from filtered tap water, both at
room temperature and when held in an ice water bath. N.D. virus filtered tap water at
room temperature was also markedly reduced. The addition of powdered skim milk
(P.S.M.) at 1:400 concentration overcame the adverse effects of both temperature and
diluent resulting in titers which were higher than the control tests using P.B.S. and an ice
water bath.
As little as 1 p.p.m. of chlorine or quaternary ammonium sanitizers greatly reduced
the titer of N.D. test virus. The addition of 1:400 concentration of P.S.M. neutralized the
adverse effects of up to 5 p.p.m. chlorine or IS p.p.m. quaternary ammonium. When
N.D., I.B. and A.E. viruses were prepared in concentrations to approximate those used
in drinking water vaccination, they were inactivated by 1 p.p.m. of either chlorine or
quaternary ammonium sanitizers. The addition of P.S.M. neutralized the effect of the
sanitizers.
Virus neutralization tests on serums from 34 field flocks vaccinated against N.D.
and I.B. without P.S.M. in the drinking water showed that 32% had not responded to the
N.D. vaccine and 44% were still susceptible to I.B. When P.S.M. was used, tests on 42
flocks showed that all had given an immune response to the N.D. vaccine and only 2
(4.8%) had failed to respond to the I.B. vaccine. Results indicated the value of P.S.M.
in drinking water vaccination of chickens.
POULTRY SCIENCE 5 1 : 1450-1456, 1972

T HE administration of Newcastle dis- gal contamination in water troughs or cups

ease (N.D.) and infectious bronchitis as described by Taylor (1969). Continuous
(I.B.) vaccines through the drinking water or intermittent addition of chlorine or qua-
as developed by Luginbuhl et al. (1955) has ternary ammonium type water sanitizers
become a routine procedure for most poul- has been recommended for good manage-
try flocks. Calnek et al. (1961) reported ment programs. Under such a program, the
drinking water vaccination against avian drinking water would contain relatively
encephalomyelitis (A.E.). When potent high levels of sanitizer. They are advertised
vaccine is added to fresh untreated water as potent bactericidal, fungicidal and viru-
and properly administered to chickens, an cidal agents and are used to control con-
immune response can be expected. How- tamination. Therefore, none of these mate-
ever, no immunity would develop if the wa- rials should be present in drinking water
ter contained a substance which destroyed used to administer N.D., I.B. or A.E. vac-
some or all of the virus. Such a condition cines. If small amounts of these materials
could exist when sanitizers are added to the remained in the watering system, even after
water for control of the bacterial and fun- rinsing with untreated water, partial or
' ; '. ~7~, ~7 ; ~ "~~ complete inactivation of vaccine might oc-
Authonzed for publication as paper No. 3732 * , .
in the journal series of The Pennsylvania Agri- c u r - Without adequate amounts of virus, a
cultural Experiment Station. protective immunity could not develop.
1450
 WATER VACCINATION
Reports of N.D. and I.B. outbreaks in
field flocks that had been vaccinated
through the drinking water with N.D.-I.B.
combination vaccine was considered due to
one of two factors. Either the original vac-
cine was not of sufficient potency to stimu-
late an immune response, or during its ap-
plication the vaccine was exposed to ad-
verse conditions and inactivated. The latter
would occur if sanitizers remained in the
watering system. Burrows (1963) reported
the activity of sanitizers to be markedly re-
duced in the presence of protein material.
Therefore, the addition of a protein mate-
rial might be beneficial. Previous work by
Gentry (1969) on the evaluation of disin-
fectants had shown that a solution contain-
ing as little as 500 p.p.m. of egg yolk or
powdered skim milk (P.S.M.) greatly re-
duced the activity of both chlorine and
quaternary ammonium based disinfectants.
P.S.M. solution was much easier to stan-
dardize and was selected for use in this
study.
The work reported here was a combina-
tion of field and laboratory studies. Sam-
ples of vaccine used for field vaccination
were tested in the laboratory to evaluate
their potency after exposure to field condi-
tions. Several vaccines having relatively
low titers were used for laboratory expos-
ure of chickens to determine if they were of
adequate potency to stimulate an immune
response. The effect of sanitizers on labora-
tory strains of N.D. and I.B. viruses and
the ability of P.S.M. to neutralize this
effect was then measured. The immune re-
sponse in field flocks that had received
drinking water vaccination with P.S.M.
added was then compared to flocks vacci-
nated prior to the use of P.S.M. The objec-
tives of this work were: (1) to measure the
potency of commercial vaccines (2) deter-
mine the minimal amount of sanitizers that
would inactivate N.D. and I.B. viruses and
(3) evaluate the use of P.S.M. for protec-
1451
tion of virus against residual sanitizer.
MATERIALS AND METHODS
The techniques used in these studies
followed the procedures outlined in the
manual Methods for the Examination of
Poultry Biologies (1959). Titrations and
virus neutralization (V.N.) tests for N.D.
and I.B. viruses were done in specific
pathogen free (S.P.F.) eggs* by inocula-
tion of the chorioallantoic sac (CAS) at 9
or 10 days incubation. The GB-Texas and
Beaudette egg adapted strains of N.D. and
I.B. viruses respectively were employed for
V.N. tests. The embryo infective dose—
50% (E.I.D.50) for N.D. virus was based
on embryo mortality and hemagglutination
(HA) activity of surviving embryos at 5
days post inoculation (P.I.). For I.B. vi-
rus, the E.I.D.50 was calculated on the ba-
sis of mortality and I.B. type lesions in em-
bryos at 6 days P.I. The yolk sac route at 8
days incubation was used for AE inocula-
tions. An egg adapted strain of A.E. virus
originally secured from Dr. R. Luginbuhl,
University of Connecticut was used for
A.E.-V.N. tests and embryos were exam-
ined for typical changes at 10 days P.I. as
described by Jungherr et al. (1956).
Vaccine evaluation: In order to test indi-
vidual N.D. and I.B. vaccines, it was re-
quested that flock owners purchase the vac-
cines separately and then administer them
through the drinking water as combination
vaccine. The vaccines tested had been pur-
chased by the flock owners from either of
two commercial sources designated as
sources A and B. An extra bottle of each
vaccine (N.D., LB., and A.E.) was taken
to the poultry farm where drinking water
vaccination was being administered. When
vaccination was completed, the extra bot-
tles of vaccine were packaged with coolant
and sent to the laboratory (Penn State
* SPAFAS, Inc., Norwich, Conn. 06360
1452 R. F. GENTRY AND M. 0 .
University) for testing. For titration, each
bottle of vaccine was reconstituted to its
original volume (liquid vaccine prior to
freeze drying) with sterile distilled water
and considered the undiluted vaccine sam-
ple. Serial 10-fold dilutions were prepared
and inoculated into embryonated S.P.F.
eggs. A total of 36 N.D. vaccine samples
(12 from source A and 24 from source B)
and 24 I.B. vaccine samples (9 from source
A and 15 from source B) were tested. In
most cases a vaccine sample also repre-
sented a different manufacturer's serial lot
of vaccine.
Tests to determine the response of isola-
tion reared chickens to drinking water vac-
cines were conducted using S.P.F. chicks
which had been hatched and maintained in
an isolation pen to 4 weeks of age. Two 1000
dose samples of I.B. vaccine having E.I.D.50
titers of 4.5 logs (source A) and 5.0 logs
(source B) were each suspended in 5 gallons
of fresh distilled water. This was equivalent
to a dilution of 1:6308 or 3.8 logs and ap-
proximated the concentration employed in
the field for birds of this age. The final water
preparation, therefore, contained only 0.7
logs or 5 E.I.D.50 of virus per ml. for source
A vaccine and 1.2 logs or 158 E.I.D.50 of
virus per ml. for source (3 vaccine. Groups
of 25 chicks were placed in modified Hors-
fall units without feed or water. After 2
hours, feed was added and 30 minutes later
the water-vaccine mixture was prepared and
given for a period of 30 minutes. All birds
ate and drank during this period. Waterers
were then emptied, flushed and regular
water supplied. Birds were observed daily
but no evidence of clinical reactions were
detected. Five weeks after exposure to the
drinking water vaccine, 10 birds were bled,
the serum collected and stored at 4°C. V.N.
tests were conducted on the serum of in-
dividual birds using the egg adapted Beau-
dette strain of I.B. test virus.
BRAUNE
Drinking water vaccination with N.D.
vaccine was conducted similar to I.B. vac-
cination except that each sample was di-
luted equivalent to 1000 doses per 15 gal-
lons of water. This resulted in a dilution
factor of 1:18,925 or 4.3 logs. The original
E.I.D.50 titers of the N.D. samples was 7.2
logs for source A and 8.3 logs for source B
vaccine. Therefore, the concentration in the
drinking water of source A vaccine was 2.9
logs and for source B vaccine 4.0 logs. The
N.I. of serums collected 5 weeks post expo-
sure from 10 chickens was determined us-
ing GB-Texas strain N.D. as the test virus.
Effect of diluent on virus titers: Allantoic
fluid harvests from embryo passage of GB-
Texas strain N.D. and Beaudette egg
adapted strain I.B. were used as sources of
test virus. Diluents employed were sterile
phosphate buffered saline (P.B.S.) pH 7.2,
sterile distilled water pH 6.8, sterile dis-
tilled water with PSM pH 7.0, filtered tap
water pH 5.8, and filtered tap water with
PSM pH 7.0. The filtered tap water was
prepared by passing fresh tap water
through a 0.22 mpi. Millipore membrane
filter, then allowing it to stand 24 hrs. at
room temperature. Tests for chlorine (sen-
sitive to < 0.1 p.p.m.) were negative when
filtered tap water was checked at time of
use. The PSM was added in the amount of
1.0 gram per 400 ml. of water. Duplicate
tubes of 9.0 ml. were prepared with each
diluent. One tube was placed in an ice-wa-
ter bath (approximately 0°C.) and the
other at room temperature (22°C.) and
held for 15 minutes. Each tube then re-
ceived 1.0 ml. of test virus in PBS which
had been kept in an ice-water bath. In or-
der to permit consistent time factors, test
virus was added to the different diluent
tubes at 2 minute intervals. After 30 min-
utes, samples were removed in the same se-
quence, dilutions made in P.B.S. (in ice-
water bath) and 0.2 ml. inoculated immedi-
 WATER VACCINATION
ately using 5 eggs per dilution. Virus titers
were then determined as previously de-
scribed.
Effect of sanitizers on N.D. virus titer:
Sodium hypochlorite solution and a quater-
nary ammonium compound were used to
prepare solutions containing 50, 30,10, 5, 2
and 0 parts per million (p.p.m.) in sterile
distilled water. Four and one-half ml. of
each solution were then transferred to 2 vi-
als. An equal volume of sterile distilled wa-
ter was added to one vial. The second vial
received the same volume of P.S.M. pre-
pared by mixing 1 part P.S.M. with 200
parts sterile distilled water. The final con-
centrations of chlorine and quaternary am-
monium were thus 25, 15, 5, 2.5, 1 and 0
p.p.m. with and without PSM at 1:400
concentration.
After thorough mixing, 0.5 ml. of undi-
luted N.D. test virus (held in ice-water
bath) was added to the vials containing the
highest concentrations of sanitizers. Virus
was then added to the lesser concentrations
at 3 minute intervals. Samples were re-
moved after 30 minutes at room tempera-
ture, 10-fold dilutions prepared in P.B.S.
(in an ice-bath) and inoculated into em-
bryonated eggs. The E.I.D.50 titers were
determined and the amount of virus that
had been inactivated by the sanitizer was
calculated by subtracting these values from
the titers of control tests containing 0
p.p.m. of sanitizer.
Effect of sanitizers and P.S.M. on virus
at approximately drinking water concen-
tration: Three groups of 6 water dilution
bottles were filled to the 99 ml. mark with
sterile distilled water. Sodium hypochlorite
solution was added to the first group and
quaternary ammonium solution added to
the second group in sufficient amounts to
make final concentrations of 1 p.p.m. The
third group was plain distilled water. Three
bottles of each group (distilled water, chlo-
1453
rine solution and quaternary ammonium
solution) then received 0.25 grams (1:400)
of P.S.M. They were then redivided into
three groups of 6 consisting of one bottle
each of distilled water, 1 p.p.m. chlorine
and 1 p.p.m. quaternary ammonium with
and without P.S.M. One group (2 bottles
with sanitizers and one of distilled water
with and without P.S.M.) was used for
tests on N.D., the second group for I.B.
and the third group for A.E. virus. N.D.
and I.B. test viruses were first diluted 1:
100 in P.B.S. and 1.0 ml. then added to
each of the 6 bottles containing 99 ml. of
the different preparations. This constituted
a virus dilution of 1:10,000 and the re-
maining virus approximated the concentra-
tion used for field application of drinking
water vaccine. A.E. egg adapted test virus
was used undiluted and 1.0 ml. of stock
material added to each of the 6 bottles. Af-
ter 30 minutes at room temperature, a sam-
ple was removed and 10-fold log dilutions
prepared with P.B.S. in an ice-water bath.
N.D. and I.B. viruses were titered in 9 to
10 day embryonated eggs as described.
A.E. was inoculated in 8 day embryonated
eggs by the yolk sac route and titers deter-
mined by examination of embryos for gross
lesions at 18 days incubation. The effect of
sanitizers with and without P.S.M. on virus
titers was determined.
Field application of P.S.M. for drinking
water vaccination against N.D. and I.B.:
The addition of 1 pound P.S.M. to 50 gal-
lons of drinking water (approximately 1:
400 w./v.) before addition of N.D. and
I.B. vaccine was used in field flocks. The
effect of P.S.M. on the ability of vaccines
to produce the desired immune response
was measured by conducting V.N. tests on
serum samples collected 5 weeks post vacci-
nation. Vaccination was done when chicks
were 10 to 21 days of age. Blood samples
were secured from 10 birds of each flock
1454 R. F. GENTRY AND M. 0 . BRAUNE
and 1.0 ml. of serum from each bird com-
bined to constitute a pooled sample for
testing. The N.I. titer was calculated and
flocks having values over 2.0 logs were con-
sidered immune. Samples were first secured
from 34 flocks that had been vaccinated by
the drinking water route without the addi-
tion of P.S.M. These served as control
flocks. Samples were then secured from 42
flocks where P.S.M. had been added prior
to mixing the vaccine in the drinking water.
The immune response for the two groups of
flocks was evaluated to determine if a bene-
ficial effect had resulted from the use of
P.S.M.
RESULTS AND DISCUSSION
Vaccine evaluation: The vaccines, tested
for potency by inoculation of embryonated
eggs had E.I.D.50 titers as shown in Table
1. Vaccines from source B averaged 1.1
logs higher for N.D. and 0.8 logs higher for
I.B. than those from source A. With N.D.
vaccine, titers for 3 of 12 samples from
source A were below 7.0 logs whereas from
source B, none were below 7.0 logs and
only 2 of 24 were below 8.0 logs. Similarly
with I.B. vaccine, titers for 4 of 9 from
source A compared to only 1 of IS from
source B were below 5.0 logs. If titer alone
were used as the measure of quality, vac-
cine from source B would be considered su-
perior.
The immune response was measured 5
weeks after drinking water vaccination by
V.N. tests. The neutralization index (N.I.)
of serums from 10 birds exposed to I.B.
vaccine from source A (0.7 logs concentra-
tion in the drinking water) ranged from 2.7
to 3.6 logs. Considering a N.I. titer of
greater than 2.0 as significant, it was evi-
dent that although the drinking water con-
tained only 0.7 logs (E.I.D.50) concentra-
tion of virus per ml., it was sufficient to
stimulate an immune response. Serums
from birds exposed to I.B. vaccine from
source B (1.2 logs of virus per ml.) gave
N.I. titers of 2.6 to 4.0 logs.
The N.I. titers against N.D. test virus
showed that an immune reaction had
occurred with both vaccines. Serums from 10
birds that received source A vaccine had
N.I. titers ranging from 2.8 to 3.9 logs
while N.I. titers for those receiving source
B vaccine ranged from 2.7 to 4.1 logs.
Although the concentration of virus in
the drinking water was much higher with
N.D. vaccines (2.9 and 4.0 logs) than for
I.B. vaccine (0.7 and 1.2 logs), there was
little difference in the immune response as
measured by the N.I. titers. The I.B. vac-
cines selected for use in these trials had rel-
atively low titers (4.5 logs source A and 5.0
logs source B) when compared to other
vaccine samples (Table 1). Since an im-
mune response was obtained with these low
titered vaccines, it must be assumed that
higher titered samples would produce equal
or better responses. Results thus indicated
that commercial vaccines were sufficiently
potent to stimulate the desired immune re-
sponse when properly administered.
Effect of diluent on virus titer: Various
diluents were used with N.D. and I.B. vi-
ruses to determine their effect on the
E.I.D.5o titers. Identical preparations were
held in an ice-water bath or at room tem-
perature for 30 minutes to measure the
effect of temperature. Duplicate sets of
tests were conducted and the maximum dif-
ference between comparable samples was
0.3 logs. Therefore, the results of the two
TABLE 1.—Potency of commercial Newcastle disease
and infectious bronchitis drinking water vaccines
Maim- , r XT , E.I.D.50 titer (logs)
facturer ^ ^ *%£? — —
source JH Range
Ranee Ave.
A N.D.* 12 6.3 to 7.8 7.3
B N.D. 24 7.2 to 9.4 8.4
A I.B.** 9 4.3 to 6.2 5.1
B I.B. 15 4.4 to 6.8 5.9
* N.D.—Newcastle disease.
** I.B.—Infectious bronchitis.
 WATER VACCINATION 1455

tests were averaged and are shown in Table TABLE 3.—Effect of sanitizers on Newcastle disease
virus titer with and without the addi-
2. The incubation temperature had no tion of powdered skim milk
marked effect on N.D. titer except when fil-
tered tap water was used. This was possi- Reduction of E.I.D.50 titer
Sanitizer Cone.
bly due to the combined effects of higher (p.p.m.) Without With
temperature and the lower pH (5.8) of tap P.S.M.* P.S.M.
water. However, the presence of P.S.M. not Chlorine 25 7.0 1.3
only overcame these effects but resulted in 15 6.9 1.2
5 6.0 0.3
a higher titer whenever it was used. 2.5 5.9 0.0
I.B. virus was more susceptible to the 1 5.0 0.0
Quaternary
effects of temperature. Filtered tap water Ammonium 25 6.0 1.0
at room temperature showed the greatest 15 5.3 0.5
5 4.5 0.2
reduction in titer. Again, addition of 2.5 3.0 0.0
P.S.M. not only protected the virus but re- 1 2.4 0.0
sulted in even higher titers than were ob- * P.S.M.—Powdered skim milk present at 1:400
tained with P.B.S. at ice-bath temperature concentration.
which had been considered as optimal. Al-
though the exact mechanism for protection trations. In the presence of P.S.M., 5
and/or enhancement of virus is not under- p.p.m. quaternary ammonium reduced the
stood, the presence of skim milk consis- titer only 0.2 logs compared to 4.5 logs
tently resulted in higher titers. when no P.S.M. was used. It was evident
Effect of sanitizers on N.D. virus titer: that P.S.M. provided protection of N.D. vi-
The E.I.D.50 titer of N.D. test virus in dis- rus from the effects of low concentrations
tilled water alone was 7.0 logs and distilled (up to at least 5 p.p.m.) of chlorine or qua-
water with P.S.M. was 8.2 logs. Titers of ternary ammonium sanitizers. The nature
samples containing sanitizer were sub- of this protection is believed to be due to
tracted from these control values to deter- the nonspecific adsorption of the sanitizer
mine the amount of virus inactivated. As by the protein fraction of P.S.M.
shown in Table 3, as little as 1 p.p.m. of Effect of sanitizers and P.S.M. on virus
chlorine reduced the titer 5 logs. When 5 at approximately drinking water concen-
p.p.m. were present in distilled water, there tration: Preparation of vaccine for drink-
was a reduction of 6 logs compared to only ing water application results in a relatively
0.3 logs when P.S.M. had been added. Qua- high dilution of the virus. Therefore, N.D.
ternary ammonium caused less reduction of and LB. test viruses were diluted 1:10,000
titer than chlorine at comparable concen- for these tests. A.E. test virus stock had a
titer of 5.5 logs and was diluted only 1:100
TABLE 2.—Effect of diluent on virus titer after so that adequate virus would remain for ti-
30 minutes incubation in an ice-bath
or at room temperature tration. In all cases, virus titers after 30
minutes at room temperature, were much
E.I.D.so Virus Titers (logs)
lower in plain distilled water than wheK
Newcastle Infectious
Diluent Disease Bronchitis P.S.M. was present (Table 4). The pres-
Ice Room Ice Room ence of 1 p.p.m. chlorine or quaternary am-
bath Temp. bath Temp.
monium completely inactivated all three vi-
Phosphate buffered saline 7.5 7.5 6.6 5.1
Distilled water 7.0 7.0 6.5 4.6 ruses (N.D., LB. and A.E.) at these re-
Distilled water plus
skim milk 8.2 8.1 7.1 6.9 duced concentrations. However, when
Filtered tap water 7.6 5.5 5.4 3.3
Filtered tap water plus P.S.M. was present, the sanitizers were ap-
skim milk 8.3 8.1 6.9 6.8
parently neutralized and there was little or
1456 R. F. GENTRY AND M. 0 . BRATJNE

TABLE 4.—The E.I.D.w titers of viruses at approx- water vaccination: Many of the flocks vac-
imately drinking water vaccine concentration
in the presence of sanitizers with and cinated by the drinking water route with-
without powdered skim milk out the addition of P.S.M. failed to develop
the desired immune response. A N.I. titer
E.I.D.50 titer after 30 min.
at room temp. of 2.0 logs or greater was considered neces-
Diluent sary to indicate immunity. Ten (32%) of
Newcastle Infectious Avian
disease bronchitis Encephalo- the 34 flocks tested had not developed im-
myelitis munity (N.I. titers of less than 2.0 logs)
Distilled water 3.8 1.8 2.8 against N.D. and IS (44%) flocks had not
developed LB. immunity. The addition of
Distilled water
plusP.S.M.* 4.8 3.4 3.2 P.S.M. to the drinking water prior to mix-
Distilled water+
ing the vaccine greatly increased the num-
1 p.p.m. chlorine <0.5 <0.6 <0.5 ber of flocks showing good immune re-
sponse. All 42 flocks tested showed immu-
Distilled water+
1 p.p.m. chlorine nity to N.D. with N.I. titers ranging from
and P.S.M. 4.8 3.5 3.0 2.7 to > 4.5 logs. Only 2 (4.8%) of the 42
Distilled water+ flocks failed to show LB. immunity. The
1 p.p.m. quater- addition of P.S.M. had eliminated N.D.
nary amm. <0.5 <0.5 <0.6
vaccination failures and reduced LB. fail-
Distilled water+ ures from 44 to 4.8%. These results indi-
1 p.p.m. quater-
nary amm. and cated that the application of P.S.M. for
P.S.M. 4.6 3.4 3.2
drinking water vaccination resulted in a
* P.S.M.—Powdered skim milk at 1:400 concen- much higher incidence of immunity and
tration. should be used for drinking water vaccina-
tion of field flocks.
no loss of virus titer. It was therefore evi-
REFERENCES
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Burrows, W., J. W. Moulder and R. M. Lewert,
as low as 1 p.p.m. could inactivate the vi-
1963. Textbook of Microbiology, 214-228, 18th
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water route. London.
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ment and application of an oral vaccine. Avian
concentrated virus had neutralized the Diseases, 5: 297-312.
effect of at least 5 p.p.m. of chlorine or Gentry, R. F., 1969. Unpublished data.
quaternary ammonium sanitizers (Table Jungherr, E., F. Sumner and R. E. Luginbuhl,
3). When virus was present at higher dilu- 19S7. Studies on avian encephalomyelitis I. Egg
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717-719.
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Luginbuhl, R. E., E. L. Jungherr and T. W.
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pletely inactivated by as little as 1 p.p.m. disease and infectious bronchitis vaccine
sanitizer. However, N.D. (4.8 logs) LB. through the drinking water. Poultry Sci. 34:
(3.4 logs) and A.E. (3.2 logs) viruses were 1399-1403.
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Field application of P.S.M, for drinking it! Poultry Tribune, May: 12-14.