Note: Descriptions are shown in the official language in which they were submitted.
- 1 ~329 ~22
5-16215/+/CGC 1241
PREVENTION OF BOVINE RESPIRATORY DISEASE COMPLEX IN CATTLE BY
ADMI~ISTRATION OF INTERFERON
Field of the invention
This invention i9 directed to the field of anlmal health and, more
particularly, to reducing the incidence of bovine respiratory
disease complex.
Back~round of the invention
Several form~ of respiratory diseases in cattle, commonly referred
to as I'Bovine Respiratory Disease Complex (BRD)" are known.
Bovine respiratory disease complex i9 a principal cause of 109~ in
the cattle industry.
One form of BRD, the so-called "Shipping Fever" preferably occurs in
newly weaned calve~, generally within the Eirst week after th~y are
moved to feedlots, where they are exposed to stress as a resuilt of
handling~ transportation or bad weather.
"Shipping Fever" s the most prevaleDt and economically significant
diseases affecting the Noreh American cattle industry with losses
ranging between a quarter to ono billion dollar~ annually.
~nother form of "BRD" is "Enzootic Calf PneumDnia", an outbreak of
pneumonia that particularly-affects calves housed in stables,
generally in the first few months of life.
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Some calves in a group develop acute respiratory disease and die.
Other calves usually develop a milder disease 3 which iæ associated
with irritation of the mucous membranes of the nose and eyes and
with nasal and ocular d~scharge.
These calves may progress to the more acute form, or may recover.
Those that recover, however, risk sufferin~ a relapse within a few
weeks.
While "BRD" is most common in beef cattle, it also occurs in
dairy cattle. All ages are susceptible, but the disaase most
commonly occurs in newly purchased young cattle that have recently
been introduced into a herd. As a result, the introduction of new
dairy stock into a herd will be follow0d by an outbreak of pneumonia
in the herd.
"Bovine Respiratory Disease Complex" is believed to be caused by a
combination of factors, such as damage to the respiratory tract and
stress. Damage to the respiratory tract may be caused by viruses,
such as a bovine herpesvirus, or different mycoplasma and/or
chlamydia strains. Stress may be caused by, for example, weaning9
castration, overcrowding, temperature changes and secondary
infection by bacteria, such as pasteurella, Viruses challenge and
stress may combine to cause immunosuppression of cattle. This
im~unosuppression may in turn lead to secondary colonization of the
lungs with bacteria principally Pasteurella haemolytica (sse Joseph
M. Cummlngs, "Feedlot Diseases and Parasites" in The Feedlot,
G.B. Thompson and Clayton C. O'Mary, ed., Lea and Febiger
Philadelphis, Third Ed.; 1983~.
Predisposing factors, therefore, are of ma~or importance in
susceptibility to "Shipping Fever" or "Enzootic Calf Pneumonia".
Among the most prevalent factors are physical stresses, chemical
agents and psychological factors.
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Physical stresses include weaning, temperature extremes and rapld
changes in tha weathsr, hum~dity, irregular feeding and watering,
changes in ration, dust, overcrowding, mixing and confining of the
calves.
The stre~s felt by~recently arrived calves due to such factors is
considered to be a ma~or factor contributing to pneumonia. In
addition calves are often vaccinated, dehorned, castrated, implanted
and injected with vitamins and antiblotics within a few days after
arrival. These procedures are also performed on cattle that are
exhausted after a long truck ride under crowding conditions.
Chemical agents that cause stress include amonia fumes and toxic
gases generated during agricultural operations.
Concomltant diseases such as para3ite attack have also been
demonstrated to have an adverse effect on the abillty to reslst
infection.
All these factors induce pathological changes such as depression of
the immune system (for example endogenous steroid release with
resulting impairment of neutrophil and lymphocyt function) or
paralysis of or inhibition of mucine secretion in the respiratory
tract.
Among the maJor vlruses implicated in "BRD" are bovine herpes viru~
type I (BHV-l or infectious rhinotracheitis virus), parainfluenza
type 3 (PI-3), bovine viral diarrhea virus (BYD) and bovine
respiratory syncltial virus (BRSY).
Viruses may cause immunosuppression by several mechanis~s: they may,
for exampleS lyse or functionally impalr lymphocyte functions or
lower the activity of the body's own bactericidal mechanism3
especially those involving macrophages and neutrophils, which are
important in clearing gram-negative bacteria from the lungs.
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In addltion, viruses alter the ability of macrophages to ingest
antibody coated bactaria (decreased Fc receptor activity). ~ven if
the bacteria are ingested they are not killed aR efficiently by
virus infected macrophages. Furthermore it has been shown that viral
infection can reduce chemotactic factor production by alveolar
macrophages and therefore dramat~cally influence neutrophll
recruitment into the lung.
Virus infection may also alter neutrophil mobility and induce
neutrophil dysfunction.
Finally, if the alveolar mscrophages sxpress viral antigen on their
surface, either due to replication or phagocytosis of the viruses,
the cytotoxic T cells could be directed against these cells and
further impalr their functions.
As seen earlier, viral infectlon and stress appear to enhance
adherence and colonization of the respiratory tract by both gram
positive and gram negative bacteria.
Although there are a variety of bacteria that can colonize the lung
and cause bacterial pneumonia, the majority of bovine pneumonias are
cau6ed by Pasteurella haemolytica A-I.
In addition to P. haemolytica, P. multocida, and ~aemophilus
somnus are al80 commonly isolated from the respiratory tract of
animals suffering from "BRD", as are various Mycoplasma species
(N. b~vi~ QE and M. bovirh1nis).
After rapid growth of P. hae~olytlca in the nasopharynx the
bacteria canj for example, enter the lung inside droplets.
Furthermore P. haemolytica produces a soluble cytotoxin
(leukotox~n) that can directly impair the function or cause the
death of ruminant leukocytes.
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After phagocytlzing the bacteria, alveolar macrophages and
neutrophils may be damaged or die. Dead neutrophils in the lungs
contribute to the lesions caused by pneumonia.
"BRD" usually occurs in cattle 3 to 15 days after they have beeD
stressed.
Affected calves usually appear depressed and are without appetite. A
~ucopurulent nasal dlscharge and a quiet cough are common. The
respiratory rate is increased from a normal of 30 per minute up
to 80 to 100 per minute. Affected calves have also a fever ranging
from 40 to 41,5C.
Fxaminatlon o~ the lungs with a ststho~cope will usually reveal
evidence of bronchopneumonia or even of pleurisy.
Pasteurella haemolytica produces severe lung leslons. Flbrin
deposition and capillary thro~bosis are the two main pathological
features of pasteurellosis.
For the cattle industry the losses caused by "BRD" are the results
not only of the death of animals, but also of the costs of treat-
ment, weight loss, inefficient feed utilisation, extra labor,
condemnations and poor performers, all of which result from the
disease.
Therefore, while looking for po sibilities for an adequate treatment
of "BRD", suggestion~ have been made to reduce ths incidence and
severity of respiratory diseases in cattle by admini~tering
interferons.
The interferons (IFN) are a family of proteins classified into three
type3, alpha, beta and gamma. The classification is based upon
antigenic ~peciflcl~y, g~ne organisation, structure, p~ sensitivity9
cellular origin and biological activity of the interferon proteins.
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The different types of interferons may be derived from animals such
as cattle, pigs and humans.
The suggestions to treat cattle with interferons in order to reduce
the incidence and severity of respiratory disease have been
supported by model studies. In one model study, cattle were infected
with herpesvirus ~BHV-l)o The administration of human interferon
wa~ shown to reduce the severity of the infection. See Roney et al.,
Am.J.Vet.Res. 46, 1251-1255 (1985).
Model studies are flawed, however, in that they do not subject the
cattle to the combination of stresses and varieties of different
viruses and microorganisms that cattle are subjected to under actual
field condltions as they move through a salebarn, feedlot and
stockyard.
Successful model studies do not, therefore, necessarily mean that
the tre~tment of cattle with bovine lnterferon under actual field
co~ditions will be effective against infectious respiratory
diseases. Nor do such studles provide the optimal conditions ior
interferon inoculation in order to reduce the incidence and
severity of infectious diseases under actual field conditions.
Questions such as the best time and method of inoculation remain.
It is, accordingly, an ob~ect of the present invention to provide a
method for reducing the incidence and severity of bovine respiratory
disease complex under actual field conditions by prophylactic
administration of interPeron to cattle.
This and other ob~ects as will be apparent from the ~ollowing
de~cription of the invention surprisingly have been met by providing
a method for reduci~g the incidence and ~everity of bovine
respiratory disease complex comprising administering systemically a
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propbylactically effective amount of bovine interferon to young
cattle before infectlon by an infectious disease-causing virus or
microorganlsm causes an outbseak of the clinical dlsease.
Description of the invention
The intPrferon useful in the present invention ~ay be alpha, beta or
gamma interferon derived from cattle. The interferon may be isolated
from the cattle and purified, or it may be produced by recombinant
DNA technology.
All three types of IFN can play a direct role in controlling virus
replication within susceptible cells and are involved in the
regulation of the immune function. A11 IFNs can stimulate or enhance
the ~irst line of immune defense-phagocytosi~ and cell killing.
Alpha type interferons are sscreted by lymphocytes and other
leukocytes in response to viral infections.
The cloning and expression of DNA encoding bovine interferons has
showD that the bovine genome contains two distlnct classes of
IFN-genes, namely the BoIFN-~I and BoIFN-aII gene clas~es. These
classes consist of 10-12 and 15-20 subtypes, respectively. The gene
coding for BoI~N-~I1, one subtype of the BoIFN-aI family, has been
cloned and expressed in bacterial cells in nrder to evaluate its
activities in vitro and in vivo.
Recombinant bovine lnterferon alphaI (rBoIFN-~ , trademark
I Interceptor~, is a basic protein composed of 166 amino acids. Its
~ molecular weight i8 19000 Daltons.
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Formulated interferon iB usually stored in its lyophilized (freeze-
dried) fo~. An alternative form comprises already formulated
inJectable solutions.
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For use in the present invention, the lyophilized interferon may be
reconstituted in a diluent, preferably sterile water, for
administration.
The functionally recon~tituted interferon is administered
systemically to young cattle by any convenient method, such as by
means of a hypodermi~ syringe. The systemic administration may be
made lntramuscularly, preferably into the muscles of the rump,
subcutaneously, or intravenously.
Intramusculsr administration is preferred.
For the purposes of this specification young cattlo mean weaned
cattle that are about three days to 18 months old, p~eferably 6 to
l8 months old.
The administration of interferon may be intranasal according to the
present inYentlon, although sy6temic, snd especially intramuscular
administration is preferred since it 19 more convenient and,
surprisingly, at least as effective as intranasal administration.
The amount of interferon administered i9 a prophylactically
effective amount. The lower limit is about 0.5 mg IFN/animal,
preferably about 1 mg IFN/animal, and more preferably
about 3 mg IFN/animal.
The upper limit is the largest amount tolerated by the cattle
without serious side effects. The upper limit is, for example, about
250 mg IFN/animal, preferably about 50 mg IFN/animal, and most
preferably about lO mg IFN/animal.
The optimal amount of interferon is about 3 mg IFN/animal to
10 mg IFN/animal, preferably, about 5 m8 IFN/animal.
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The functionally reconstituted lnterferon may be in any convenient
concentration. For example, in order to inject 5 mg IFN/animal, lt
is convenient to prepare a solution of 2.5 mg IFN/ml diluent (H20),
and tG ln~ert 2 ml/animal of the solution.
As could b~ demonstrated by pharmacokinetic studies, more than 90 %
of the total amount of interferon admlnistered initially is
eliminated within 15 hours after dosing.
It is, therefore, preferable to inoculate the cattle with interferon
before or immediately after infection by an infectlous disease-
causing mlcroorganism.
The preferred time for IFN administration ranges from -72 to
+24 hours preferably from -60 to ~24 hours relative to the exposure
of the animal to a pathogen.
Most preferably the cattle should be inoculated before infection
by an infectious, disease-cau3ing microorganism, particularly
50 to 10 hours before infection.
The exact timing of IFN administration is important, since most
re~piratory disease occurs shortly after animal mixing in ~ale
barns, transportation or entry into feedlot~.
In a preferred embodiment of the present invention, the IFN
inoculation of the young cattle, therefore, occur~ at the fir~t
opportunity the young cattle have to congregate prior to fattening,
i.e. at the salebarn, stockyard or feedlot.
The efficiency and mode of action of intPrferon was demonstrated in
i the course of the present invention by in vitro and in vivo
~ experiments.
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The cllnical efficiency of IFN administration has been supported not
only by a model study using a reproducible di6ease model but also by
field trials.
No treatment-related adverse reactions have been noted during
cllnical field trials9 including more than 3000 calves treated
intrana~ally and of approximately 7000 anlmals treated intra-
muscularyO No adverse effects have been recorded in animals
receiving simultaneously Interceptor~ and treatment reg1men~
associated with routine feedlot processing and fattening, such as
worming, vaccination, treatment with ectopara~iticides, implanting
anabollc preparatlons, branding, implanting in feed growth promotion
and treatment with antibiotics.
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A controlled study demonstrated that concurrent intranasal or
intram~scular treatment with Interceptor~ and vaccination with
modified live IBRIPI-3 virus vaccines resulted in no harmful
effects itl the animals and no impairment of the protective effects
of the vaccines.
The efficiency of IF~ treatment with respect to the reduction of the
incidence and severity of bovine respiratory diseasa complex
results in a significant reduction in mortality rate and the number
of sick days per animal or group 89 well as sn improved weight gain
f and feed efficiency of the treated animals compared to the placebo
Z controls.
In the case of "Enzootic Calf Pneumonia" it proves advantageous to
administer Interferon on days 0, 7 and 14 related to the moment of
infection by an infectious disease-causing virus or microorgani~m.
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In the following reference i8 made to specific example~, which are
i included herein for purpose of illustration only and are not
f intended to be limiting.
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Examples ~ 3291 22
1. Bovine-Interferon
A me~hod ~or producing bovine interferon is disclosed in
Japanese Patent Application 58,224,690 and in European Patent
Application 88,~22 published September 14, 1983 and entitled
"Animal Interferons, Processes Involved in Their Production,
Compositions Containing Them, DNA Sequences Coding Therefore and
Expression Vehicles Containing Such Sequences and Cells
Transformed Thereby". The description of the preparation o~
bovine interferon from Japanese Patent Application 58,224,630 and
European Patent Application 88,622 includes pages 21-37 of the
European application and the corresponding pages of the Japanese
publication and the references, figures and tables cited therein
as well as any other description for the preparation of bovine
inter~eron in these publications.
2. Pharmacokinetic and toxicity
Serum dlsposition and bioavailability of rBoIFN-aI1 was
studied after intranasal, intramuscular and intravenous
administration of a single dose of 10 mg to grouped calves.
The respectlve half-lives were between 130 and 270
minutes. For all methods of application more than 90% of the
total dose was eliminated within 15 hours after dosing Bovlne
lnterferon levels fell below ~he level of detection by 24 hours
after administration.
Approximately ~0% of the intramuscular dose was found to
be avallable to the c1rculatory sy~tom.
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No toxic effects ~rom interferon were seen in animals
treated for 15 consecutive days with doses up to ~5 mg
intranasally ~IN). The serum levels of interferon on the
sixteenth day was found to be only slightly above ~he background
le~el.
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Previous experiments have shown that a single intramuscular (IM~
dose up to 100 mg per head i8 well tolerated.
Translent mild leukopenia and a moderate pyrexic response are
observed following an initial IM dose of interferon. Leukocyte
counts and temperature return to normal within 24 hours to 4 days.
Repeated dosing does not further affect these depressions, which do
not appear to influence the clinical performance of the animals nor
the prophylactic value of interferon.
3. In ~itro Studies of Interferon Efficiencv
a~ The antiviral activity in vitro.
The antiviral potency of rBOIFN-aI1 is assayed on bovine cell
lines challenged with different viruses of bovine origin.
Activity tikres~ commonly expressed in unit U/ml or converted to
units U/mg protein (speciflc activity), represent the reciprocal
of the dilutions that result in 50 % inhibition of virus-induced
cytophathic effects (plaques).
rBoIFN-aIl inhibits replication of the viruse3 most commonly
lsolated from cattle that suffer from "BRD". (BVD~, PI-3, BRSV
and IBR/BHV-1).
b) I~munomodulatory effect in vitro
To assess the immuncmodulatory effect in vitro bovine phagocytic
cells (neutrophils, blood monocytes and alveolar macrophages) are
treated with rBoIFN~
The effects seen are manyfold and comprise enhanced bacterial
uptake by all 3 cell types and incre~sed Fc receptor activity
in alveolar mscrophages, inhibition of both directed and random
migration of monocytes and polymorphs, increased enzyme release
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or inactivation, increased hydrogen peroxyde generatlon and
decrea~ed superoxide amine release by alvenlar macrophages and
PMN leukocytes.
These effects are dose and time dependent.
4. Immunomodulatory effact ex vi~o
A variety of leukocyte functions were measured throughout the cours~
of an experimental infection with BHV-l/P. haemolytica to determine
whether interferon played an immunomodulatory role in vivo and thu3
resulted in rPduced morbidity and mortality.
In the model, peak immunosuppression occurs four days postinfec-
tion, which i8 a time when susceptibility to bacterial infection ia
maximal.
To deter~ine the effect of bovine recombinant interferon on neutro-
phil migration and chemotaxis~ neutrophil3 pre~ent in the peripheral
blood of interferon-treated or placebo animals are measured in a
migration and chemotaxis assay.
In both cases, neutrophil function is suppressed following lnfec-
tion.
In IFN treated groups, however, the neutrophil function returns to
normal much more rapidly than doe~ that of the placebo groups.
~odulation of neutrophil function is further demonstrated in a
chemiluminescence assay by measuring the production of reactive
oxygen apecies. Vsing thi~ a~say, it is demonstrated that IFN
treatment dramatically increases the ability of neutrophil~ to
produce reactive oxygen species in animals challenged with P. haamo
l~tica.
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5. Field trials "Shipping ~ever"
In a flrst study, 770 steers are purchased, shipped by truck to a
salebarn and then to a feedlot. On arrival animsls are randomly
assigned to replicate pens. Each animal receives 5 mg of rBoIFN-~I1
per nostril in 2 ml or an equivalent volume of Placebo.
Clinical signs of depression, loss of appetite or respiratory
symptoms are recorded (see annex). Sick animals ars treated wlth
antibiotics.
The results are summarized in the following table 1.
Table 1
-
aministered disease average utilisation daily
ingredient incidencel amount of the increase
of days fodder of weight
per ani- (kg)
mal when
symptome
could be
recognized
Placebo 38.2 0.956 6.6 O.91
rBoIFN-alphaI 31.9 0.839 6.0 l.O
No. of sick animals/total No. of animals
The number of animals suffering from respiratory disease and the
number of sick days per animal are significantly reduced.
Likewise weight gain and feed efficiency are improved.
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In another study, conducted in a research feedlot, the effect of
rBoIFN~ 10 mgIPN/animal) given as a single intramuscular
injection or by intranasal instillation (5 mgIFN/nostril) with
placebo are compared.
300 single sex calves in the 200 kg weight range are purchased,
passed through a commercial salebarn to ensure adequate exposure to
respiratory infection and trucked to the research site under
conditions resembling commercial shipping practices.
The application of interferon occurs at the salebarn, 62 to 64 hours
before arrival at the feedlot.
In another study IFN is administered only on arrival at the feedlot.
The percent sick animals, the number of sick days, and the percent
mortality for each method of administration are compared to a
control group that received no interferon. The results are shown ln
the table below.
Table 2
Field Study Results of Administration of Bovine Alpha-Interferon-I
to Young Calves
Experi- Time of Method of Number % No. of %
ment of Sick
Admlnistr. Administr. Animals Sick Days Mortality
1 At salebarnIM 107 44 2,02 1,9
(62-64 hoursIN 106 53 2,46 0,9
beEore arrival control 106 63 2,79 5,7
at feedlot)
2 On arrival IM 100 29 1,48 0
at feedlot IN 99 28 1,61 0
control 99 53 1,70 0
Withln the ~roup~: Total No. of sick days/Mo. of animal~ over the
ob~ervation period oF 21 days.
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In ths course of the above described experiments it could be
demonstrated that rBoIF~ I administered either IM or IN clearly
reduced both the severity and the incidence of BRD during the 3
first weeks of ths study.
6. Field trials "Enzootic Calf Pneumonia"
The effect of rBoIFN-~Il against "En200tic Calf Pneumonia" is also
evaluated under commercial conditions.
~0 to 80 Crossbred Simmental and Holstein dairy calves, 2 to 3 week
old, welghing 50 to 65 kg, are bought from various sources, shipped
to a salebarn and then brought to Clba-Geigy research station in
St.Aubin, Switzerland. Only clinically healthy calves will be
admitted. Any animals demonstrating signs of disease prior to the
study will be excluded.
After arrlval they are welghed, individually tagged, and assigned by
stratified randomization (sex, weight~ to 2 replicate pens per
treatment group in a closed stable.
They are fed with unmedicamented milk replacer. Under these
conditions a high proportion of the calves (70 to 90 %~ develops
spontaneous "BRD" within 3 to 15 days of arrival. DAily clinical
examinations are conducted for 3 weeks. The following parameters are
assessed: respiratory rate, coughing, nasal discharge, temperature,
apetite and behaviour (see annsx 1~.
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Table 3
Disease Inctdence
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Experiment ~o. Treatment2 Dose Disease1
Calves (mg) Incidence %
1 58 Control/H2Q - 16/19 ~85
IFN d 0 (lx) 0,S ll/l8 ~62~
IFN d 0 (lx) S,0 13/21 (62)
2 59 Control/H20 - 16118 ~90)
IFN d 0 ~1x) 5,0 16/20 (80)
IFN d 0,7,14 ~3x) 5,0 10/21 (48)
3 78 Control - 26/29 ~89)
IFN d 0,4 (2x) 5,0 2012,2 (90)
IFN d 0 (lx) 5,0 18/27 ~67?
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. 1 Within the groups: No. of sick animals/total No. of animals
2 Intramuscular inJection (2 ml solution) into the neck musculature
d Day(s) of treatment(s) relative to stabling (day 0)
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Table 4
Morbidity/Sick ~ays
Experiment No. Treatment Dose No. oE
Calves (mg? Sick Days
1 19 Control/~2O ~ 1,84
18 IFN d 0 (lx) 0,5 1,2~
21 IFN d 0 (lx) 5,0 1,14
2 18 Control/H20 - 1,2
IFN d 0 (1x) 5,0 1,1
21 I~N d 0~7,14 (3x)5,0 0,90
3 29 Control - 1,34
22 IFN d 0~4 (2x) 5,0 1,36
27 IFN d 0 (1x) 590 1 ~00
~ithin the group3: Total No. of sick days/No. of animals over the
observation period of 21 days.
All treatment groups were clinically improved when compared to the
placebo controls; the cumulative incidence of disease and the number
of sick days per animal were reduced.
The administration of Interceptor~ weekly at days 0, 7 and 14
roduced ~ort~li.y by op to 50 ~.
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Annex I
Description of clinical scoring system
Standardized clinical examination
3 different categories of severity of symptoms are identified with
normal receiving a score of 1 and the most severe receiving a
score of 3.
Respiratory rate 35
35-60 2
~6~ 3
Cough no cough
infrequent~ painless, mild 2
severe, painful, frequent and 3
respiratory distress
Nasal discharge none
serous 2
mucopurulent 3
Auscultatlon normal
increased bronchial tones, 2
a few dry or moist rales
loud rattling noises and 3
a lot of moist rales and
signs of pleuritis
Body temperature 395
395-405 2
~405 3
j The body temperature will be taken twice
daily and the mean will be recorded.
Appetite normal
' decreased 2
anorexia 3
Behaviour normal
moderately depressed 2
severely depressed moribund 3
All other clinical symptoms should also be recorded (diarrhea,
, arthritis.. ~.
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Interpretation of clinical score
A coefficient will be attribuated to every parameter to give an
Overall Mean Value
Respiration rate x 3
Cough x 1
~asal discharge x 2
Auscultation x 2
Body temperature x 3
Appetite x 3
Behaviour x 2
Total
Total/16 = overall mean value
Analysis of disease development will be obtained from evolution of
the overall mean value (OMV~ 7 and lts area under the curve (AUC~ and
the evolution of AUC ant OMV. The comparisons between treatMents
will be made by covariance analysis.
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