RESEARCH PROJECT - ENVIRONMENTAL STRESS AS A CONTRIBUTING FACTOR IN ANIMAL DISEASES

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• Publication Date: April 13, 1964
• Content Type: REPORT

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C-O-N-P-I-D-E-N-T-I-A-L
50X1-H UM
OVNTRY
Poland.
REPORT
UBJECT
Research Project - Environmental Stress
ac a Contributing Factor in Animal
DATE D,c1"
13 Apr 64
?
Diseases
NO. PAGE 1
ATE OF
4FO.
LACE &
1ATE ACQ.
REFER FI''. --7'.
50X1-HUM
THIS is UNFVAIllATF1) imPnftmo-rinm
the 1963 annual report of ffb pages, EngliSig
a Polish research project conducted at the Institute for Veterinary Re-
search at Pulawy. The project, 'which is under the direction of Dr Teodor
Tuskiewicz is entitled "Environmental Stress as a Contributing Factor in
Animal Diseases." The summary of progress reported is as follows:
1. We have found in our previous 'works that after application of
ACTH/Depot-Acethropan, Hoechst/ in doses of 140 I U, the 12-weeks-old
cockerels showed a distinct functional excitation of adrenal cortex.
As the action of ACTH in poultry is a subject of many controversial
reports, 'we made a series of our own investigations. The cockerels
-were given ACTH intramuscularly in large /40 I U/ and small /4 I U/
doses, as veil as the preparation having been introduced into the '
heart blood. A number of biochemical parameters was examined during
the 36 hours from the time of application of the ACTH to cockerels.
No great differences in the action of ACTH have been found after its
application in variousdoses or various ways. It seems, that some
further studies are necessary to explain the action of ACTH in hens.
2. The effects of viral /Newcastle Disease Virus/ and bacterial
/Pasteurella multocida/ influence upon the behaviour of many bio-
chemical parameters in chickens,-'were examined on a large quantity
of cockerels. It has been found that the viral infection, as well
as the bacterial infectionl.produces'in organism several biochemical
changes, which suggest the functional stimulation of adrenal glands.
Due to the action of Prednisolone and also owing to the Influence of
such factors as high temperature of environment or shaking of chickens
on ;he laboratory shaking machine, this process of infection was
acilerated and the death rate of chickens went up.
3. In the reported year, 393 chicks 'were used in the experiments
and the total number of biochemical determinations vas approximately
5800.
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ANNUAL REPORT
OF THE RESEARCH CONDUCTED UNDER GRA 'IS AUTHORIZED BY-PUBLIC.LAW'460.
1. NAME AND ADDRESS OF THE RESEARCH INSTITUTION: The Institute
for Veterinary Research, Pu/awy, Poland.
2. PRINCIPAL INVESTIGATOR IN CHARGE: Docent Dr. Teodor JUSZKIEWICZ,
3., PROJECT TITLE: Environmental Stress as a Contributory Factor
in Animal Diseases.
4. PROJECT NUMBER: E 21-ADP-7
5. GRANT NUMBER: FG-Po-134-62,
6. REPORT PERIOD: From January 1st, 1963 to December 318t, 1963.
?Will?mg1=M1111.11..
. Signature .
of the Principal Investigator
i Charge
Director -
of the Research Institution
Doceft Dr. Teodor JUSZKIEWICZ Professor Dr.StanisZaw KRAUSS
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E 21-ADP-7
-O.
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4
Annual Report 1963
SUMMARY OP PROGRESS.
We have found in our previous works that after application of
ACTH/Depot-Acethropan, Hoechst/ in doses of 40 I.U0, the 12-weeks-
old cockerels showed a distinct functional excitation of adrenal
cortex. As the action of ACTH in poultry is a subject of many con-
troversial reports, we made a series of our own investigations.
The cockerels were given ACTH intramuscularly in large /40 'O./
and small /4 I.U./ doses, as well as the praparation having been
introduced into the heart blood. A number of biochemical parameters
was examined during the 36 hours from the time of application of
the ACTH to cockerels. No great differences in the action of ACTH
have been found after its application in various doses or various
ways. It seems, that some further studies are necessary to explain
the action of ACTH in =hens.
The effects of viral /Newcastle Disease Virus/ and bacterial
/Pasteurella multocida/ influence upon the behaviour of many bio-
chemical parameters in chickens, were examined on a large quanti-
ty of cockerels. It has been found thatt the viral infectionos well
as the bacterial infection, produces in organism several biochemi-
cal changes, which suggest the functional stimulation of adrenal
glands. Due to the action of Prednisolone and also owing to the in-
fluence of such factors as high temperattre of environment or sha-
king of chickens on the laboratory shaking machine, this process
of infection was accelerated and the death rate of chickens went
Up*
In the reported year, 393 chicks were used in the experiments
and theAotal number of biochemical determinations was approximate-
ly 5800.
?R 0:' '_
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Annual Report 1963
DETAILED REPORT
EFFECTS OP ACTB-ACTION IN CHICKENS.
It has already been mentioned in the previous report that the action
of ACTH in chickens was a subject of controversy in the available scien-
tifical literature. A short review of literpture in this respect was in-
eluded into our'"Annual Report 1962" and presented in the paper "Patho-
physiology of the Pituitary-Adrenal Axis in Birds" /3, 4, 5/.
In our previous investigations we have found that after intramuscular
application of ACTH /DepotTAcethropan, Hoechst/ in doses of 40 IX" the
12-weeks-old cockerels used to dhow, among others, a reaction similar to
that which could be observed in such cases in mammals /3, 4, 5/. In some
later experiments, however, done with the preparations of other firms,
we have not been able to find the confirmation of this observation.
Therefore, it was of interest to re-investigate the action of ACTH in
chickens within a specially designed.experiment.
Material and Methods
Experiment I. Seventy two 11-weeks-old cockerels had been
subdivided into three groups. Those of group No.1 were given single
intramuscular injection of 40 ACTH /Polfa/ per bird; group No.2
- 4 I.U. ACTH per cockerel, and the group No.3 /control/ - saline
solution.
After 10 6, 12 and 24 hours respectively from the time of injection
of the preparation, 6 cockerels from every group were killed and the ma-
terial for analyses was collected. The following determinations have been
made: weighing of adrenal glands, spleen and thymus; concentration of
adrenal and blood .ascorbic acid; concentration of adrenal and blood
cholesterol; concentration of liver glycogen and blood glucose; and
concentration in blood serum of sodium, potassium, calcium, inorganic
phosphorus and magnesium. The methods of determinations were given in
the "Annual Report 1962"*
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E 21**ADP-7
Annual Report 1963
Experiment II. One hundred and ten t2-week-old cockerels
were divided into three groups. The cockerels of group Nool were gi-
ven 4 I.U. of ACTH per bird, injected into the heart blood; Those of
group NO.2 - 4 I.U. of ACTH per bird, intramuscularly; and those of
group No.3 /control/ - 4 ml of sterilized water, the same as used for
dissolving the ACTH, into the heart blood.
Then after 1, 6, 12, 24 and 36 hours from the time of application
the preparations, an appropriate number of birds from every group was
killed and the material collected for analyses. All the same determina-
tions as in the Experiment I were made, with an addition that also the
concentration of liver cholesterol was determined.
'Results and Discussion
It was found in the Experiment I, that the applied preparation of
ACTH /P6Ifa/ induced rather faint changes in the examined parameters*
Most of the found changes were more distinct at the beginning of the
experiment. Those chickens which were given 4 I.U. of ACTH, showed af-
ter 1 hour, a decrease in weight of the spleen and thymus and also a de-a
crease in contents of adrenal cholesterol. Instead, and what was ra-
ther tnteresting, some increase in ascorbic acid was noted in the adi-
renals and a decrease in weight of the adrenals. However, after the
tenfold doses of ACTH, ice* after 40 I.U., certain decrease of ascor-
bic acid concentration in the adrenals was noted together with the in-
crease in weight of adrenals. These changes were statistically signifi-
cant only after 1 our of ACTH action. The other examined parameters
did not present any significant differences.
It is, however, to be emphasized that in spite of such differing
doses being compared as 4 I.U. and 40 1.1. per speciten, the differen-
ce in response were relatively minor. When analyzing these results we
were rather surprised with the limited activity of the applied prepara-
.
tions. Especially, that when in our previous experiments the long-ac-
ting ACTH /Depot-Acethropan, Hoechst/ was used, a significant reaction
to 40 I.U. of ACTH was found. It seemed therefore, that these results
would confirm the assumption of Garren et al./2/, Zarrow et al./1/ and
of some other investigators, that the avian tissues Cause a rapid des-
truction of the ACTH molecule, ./
With that point df view in mind it has been decided to administer
the ACTH in the next experiment intravenuously in/doses of 4 I.U.
For that purpose the technique has been adopted to introduce the drug
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d E 21 -ADP-7.
Annual Report 1563
directly into the heart. At thesame, in another group of cockerels, the
same preparation of ACTH was administered intramuscularly, and the conr.
trol group was given only the sterilized water into the heart blood*
The obtained results show that in the chickens whichrmere given in-
jections of ACTH intramiscularly, the eXamined parameters behaved simi-
larly to those in Experiment I. And to our surprise, the effects of ACM,
when aftinistered directly into the heart blood, did not much differ
from its action after the intramuscular application. Although our inves-
tigations have been conducted on a comparatively large number of cocke-
rels, we think it is still too early to draw any definite conclusions*
At present, we are considering the possibility to make a similar expe-
riment on chickens with the ACTH made by Hoechst. We shall also endeavour
to test the activity of extracts prepared from the anterior pituitary
of chickens. For, it seems, that the ACTH action in poultry may chiefly
depend upon the composition of ACTH and upon the initial structure of
the amino acids found in corticotrophins as well. It is a known fact
that the preparations of various firms differ widely in this respect.
Perhaps this is the reason of such a discrepancy in the reported effects
of ACTH action in poultry.
Our point of view is that no sooner than after the completion of just
mentioned additional experiments, the up to now results ofoour investi-
gations will be ready for closer analysis and their publication.
II. IbtFLOTS OF VIRAL INFECTION STRESS ON PITUITARY-ADRENAL AXIS
IN CHICKENS* 1
Asdel and 'Hanson found that,the increase in,body\virus titer of'
Newcastle disease virus was usually interrupted at .about the 36th'hoUr
and this interruption lasted 12 to 24 hours. injecn'ef hydrocerti**
sone usually eliminated the period of interruption and shortened the
period between inoculation and appearence of clinical Signs and'even-
tual death /6/0 /.
In our experiments we wanted to determine .to jhat extent the changes
i
caused by the viral infection in an organism are! similar to those Which
are noted under the influence of corticosteroid hormones action. At the
same time' we want to inquire into the question ok?.Whether'.the, large '
doses of. Prednisolone would influence the development of.viral'infec7
tion in chickens* 0
v
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7 E 214a77: ? Annual Report 1963
Material and Methods
The experiment was conducted on 145 ieleven-week-old cockerels of
bar breed. From that number 17 untreated cockerels were allocated at ran-
dom for the starting point determinations, or normal values of the exa-
mined parameters. The remaining cockerels were divided into three groups.
Group No01 amounted to 48 chickens. Every chicken of this group was,
inoculated intramuscularly with 0.2 ml of 10-14 dilution of the highly,
virulent field strain of Newcastle disease virus /NDV/. After 12, 24, 48.
and 72 hours from the time of infection, 8 cockerels were selected at
random, killed and the representative body tissues or,fluids were taken
for examinations. After the remaining 18 chickens had been kept for 100 ,
hours, 50 per cent of them died from the virus infection and then 9 sur
viving chickens were killed for examination.
Group No.2 of 40 chickens was given intramuscularly 10 mg of Predni-
solone acetate /Hostacortin H, Hoechst/ per cockerel twice, ice4 the
first time 24 hours before the time of infection of the first group and
then, the second time at the same time when the first group was being
infected. Then, after 6, 12, 24 48 and 72 hours from the second inject-
tion of Prednisolone, 8 chickens were selected at random every time for
the chemical and virological determinations.
Group No.1,1 of 42 cockerelsw was given in the same manner and in the
same doses the Prednisolone, and one hour later, after the second appli-
cation of them preparation, the chickens were infected with the same
dose of NDV as the chickens of the group No.1 The times of killing the
chickens and collecting the material were 12, 24 and 48 hours from the
time of infection. The remaining chickens of this group were killed,
when half of their number died, what for this group happened 74 hours
after the infection.
The following parameters have been determined with this experiment:
body weight; weight of spleen, adrenals and thymus; concentration of
adrenal- and blood cholesterol; concentration of liver glycogen and of
blood tlucose; and the concentration in blood serum of sodium, potassium,
calcium, inorganic phosphorus and magnesium. The methods of determina-
tions were quouted in the "Annual Report 1962".
Apart from that, the virus content in blood, brain, spleen, lungs
and muscles was determined by chicken embryo inoculation.
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1???1?111.
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9?..7 ?
100.
? ??
80_
81000
?t OR 051(Ni Al 119,17:
. _
0
12
0 VIRUS
?
INFECTION, %
4
48 72 MRS
VIRUS ? PREDNISOLONE
.100: . LIVER
80.
60.
40.
o A
Fr G. 1,
4.8 MRS
0 VIRUS VIRUS. PREOMISOLOME
INFECTION. X
100.
80.
60.
40.
Z0.
BRAIN
12
0 VIRUS
100_
80_ MUSCLES
60.
40.
20 _
24
72 .100 MRS
VIRUS + PREDMIS0L0M6
100 MRS
VIRUS ? PRE0MISOIONE
INFECTION, X
100.
80 _
60.
+0.
so.
SPLEEN
12
0 VIRUS
LUNGS
We.
80.
60.
40-
20-
o.
f2
0 VIRUS
TOR OFFICIAL USE ORLY,
FIG.II
2.
r
1,
48 MRS
VIRUS * PREDNISOLONE
MRS
riltat? IWEDNISOIONS
?
:FOR OFFIVI
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Determinations
4
Res:Pons? of 11-week old cockerels to Vewcastle diseases virus' inoculation /VIR/1
prednisolone injection /PR/, and a combined virus and prednisolone injeCtion /Via+FR/".
All values are means + standard errors. P compered with normal values '
/untreated subject. Numbers in parenthesis represent number a animals Studied,
.0041.1.10 -POO .
,Normal
values- Group
OPOialaaadaira4a0agala**004.441a4.4.0.0a0WOWOOliaa4Malaattainlait
' 4 ?
M 0
in
o u r
6
afaeampoaalaalaula
, Spleen weight,
vtu
2.13 09/
g/kg bedy ,weig4 +0.13'
/17/
12 24
ilaamplabmpaaa diaaaapalagi
72 100
wipaaalpippriaaliamapaspa.. aapaowo? aroasaolaas.
1.81+020 2.48+0,20 3.33+0.29 2.34+0,25 1.99+0.28
? P 0.2 P>.0.2 - 10.5 0.5
PR 0,90+0'06 0.860.O% 0.86+0.09 MOO, 05 0..75+0.07
/40/ P40.001 1Z0.001 P0.001 140,001 - 14.0.001
Ana
VIR+PIr
/33/
0.80+0.09 0.97+0.07 0.97+0.10 0.19+0.11
40.001 P.4-0.001 ,PO.O01 P40.001
T.471mY3 weighti 4.31
i .
i. ..,..s. .
i g/kg body .weight :
i
410
139/
- 2.18+0.34
pa.00i
2.03?0. 19 1 ? 784**12
P4 0.001 P40.001
.64+0.39 3.30+0.34
P7.0.1 P0.1
2,88+0.41 1.88+0.1
P 40.05 P4.0 001
0,96+0.1
1z-0.001
1.61+0. 29 0.70+0.10
P40.001 .P0,001
OP
ITIR+PR
/33/
tap
Adrenal vrei ght
mg/kg body weight
99..1
VTR
/39/
PR
/40/
90+5.
P 0.2
, VIR+PR
T171 /33/
Ainorbic. acid,
adrenals',
mg/loo
170.7
1%89
/11/
VIR
/33/
art
1.45+0,31
PLO
114+7.3
86+6.1
061
97+4.4
P>0.5
184t5+3 6
P 0.4
197.816J
P
+14.8
>0.2
91+4:4
0.2.
117+63
148+21.7
- P4(0.05
160.7+15.5 149.8110.8 147,0+3.8
P!0.2. P>0.05 ....0. P>0.2 ,P4,0.001
15846+11.8 139.2+16.4 171.6+23.8 ?
P >Oa P> 0.05 ,P>-0.5
141.2+5.9
,P,(0.005
Choletierol,
adrenala-
mg/gm
55445
VIR
n9/
55.39+2.43 50.56?1.58 49.
>0.4 P0.2
P0.00
4.09+3. 24 ,
? PC..0.001
PR
/40/
59.3313.09 58.41+5.86 57.809.55 61.)6+3.53 50.0513.90
-P70.5? 12,> 0.1
P>0.5 P>0.5 P>0.2
Ola
V1R+1711
/171/ /33/
aft
54.41+1
1> 0.1
44 50.95+3.01 55,11+8
1. :12> 0,05 P>_ 0
69 . 41.72+2.86
2 - --1.40.001
Glycogon,
livers
raBlen
13.05
/17/
VIE
/39/
-PR
/39/
VIE+ R
/33/
15.01+1.54 13.00+0.71 10.01+0.68 7.98+1.16
?P > 0.05 P>0.2 P0.005
25.12+0.95 22.20+1.32 25:85+3.44, 26.20+1.24 21.53+3.9 -
P0.001 140.001 14.0.005 /40.001 ? p 4o.o5
21.61?1.36 23.70+4.72 19.07+1.35 9.47+1.08
P >0.1 P40.05 140.001 140.05
5.11+0.25
PL0.001
Ascorbic acid,
blood,
mg %
VIR
/39/
1.68
PR
+0.06 ' /40/
117/
1.50+0.07
P> O.5
2.0340.11 1.50+0,08 '
140.025 4 0.05
105+0.08
P4 0* 005
218.7+9,4
P 0.05'
VIR+PR
/33/
Glucose, blood/
VIR
/39/
235.03 ?
3.6p
/17/
PR
/40/
Ora
1.8210.06
? 0.
1.7W,08
1.8610.11
P 0.1
1.52+0.05
P>0.5
1,63+0.15
? P>0.5
1.58+0.05
P)'0.5
2.10+0.12
P4.0.0
1.74+0.08
P >0.5
221.717.8
1.47+0.10
P>-0.05
1.24+0.09
P 40.001
181.0?34.6 207.6111.5
1_0425 1)0.05 P(0.001
507.5?34.2 658.3+124 700.6+12.7 656.0+18.1
P40.001`, /40,001 00.5
340.+13.5 330+14.3 315+14.7 310?12.6 344+10.2
P)o.1 P 0.05 140.025 P0.005 >0
340+8.7 343+7.2
---P->0.05-
3230 0.7 302?9.9-
389?15.8 339+,11.5
5 _ 0.1 P>0 1
21.0+0.8
P40.001
YEt 20.6+$.2 21.1+0,9.
+0,85 (391 P0.005 14 0,901
VIR+PR
/16( /31/
'/37/'
CsiCium?
serum
f
flag ?m,
Phosphorous,
serum,
mg %
? '2.46
0.12
?
)11
21.8+1..1
240.001
. 11.7+0.5 ?
P>0.
09/ P.3 P 0.3 >0.
18.5+1,5
P>0.05
19.2+1,6
P