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SELECTED ARTICLES
SOME OBSERVATIONS ON THE BLEEDING OF FOWLS.
BY
A. L. CLAY, B.V.SC., AND W. J. B. MURPHY, B.V.SC.,
Veterinary Officers, Department of Agriculture, New South Wales.
(Reprinted from the Australian Veterinary Journal, Volume XI, No. I, Feb. 1935.)
The authors have now had several years' experience of the blood-testing of
fowls in pullorum disease control work. Poultry farmers generally have been
found on first acquaintance with the operation to be rather anxious as to the effects
of blood-letting on their birds. The observations which are given in this article
reveal that even under field conditions the reaction of the birds to, and any losses
occurring following blood-letting are not such as to give rise to any anxiety.
METHOD OF BLEEDING
An assistant grasps both legs in one hand and suspends the bird with head
down and the ventral surface towards the operator. With his free hand the
assistant extends a wing to one side so as to expose its medial surface and at the
same time in such a way as to form a right angle between the radius and ulna and
the metacarpus. By this means the forearm of the wing is brought into the
most suitable position for operation. If the right wing is intended for operation,
the bird's legs are held with the right hand and wing with the left, and vice versa.
The operator for his part is seated, and, the feathers in the vicinity having
previously been plucked by the assistant, he makes a transverse incision with a
sharp scalpel on the medial surface of the wing a half to one inch proximal to the
distal ends of the radius and ulna.
In the position stated are situated the wing vein and an accompanying artery.
The vessels are made prominent by tensing the skin backwards over the ulna with
the thumb and fore-finger of one hand. The aim is to incise the vein without
actually severing it in twain. Actually the artery is also frequently incised and
sometimes completely severed.
If blood is to be taken from the left wing of the bird, the operator uses his
right hand to make the incision, and vice versa.
Blood collects in a depression which is present at the selected site and about
2 c.c. are run off into small bottles. These are placed in flat wooden boxes provided
with numbered portholes, the boxes being stood on one side in an upright position
until such time as the bloods have coagulated. Under favourable conditions
one hundred birds can be dealt with in an hour.
( 386 )
BLEEDING OF FOWLS
387
AFTER-EFFECTS OF BLOOD-LETTING
The important after-effects of blood-letting are possible mortality and lowered
egg production.
MORTALITY
This occurs either as the result of actual haemorrhage or gangrene of the
wing. With increasing age there appears to be greater risk of fatal haemorrhage,
so that more care is necessary with second year birds than with pullets or first
year stock. Individual birds bleed with varying freedom, and in certain cases
it is necessary to adopt measures to arrest haemorrhage. Cotton wool applied
with the pressure of the fingers is nearly always a satisfactory procedure:. On
rare occasions the use of haemostates has to be resorted to. These are used not
to crush the incised vessel (this being extremely difficult), but to seal the lips of
the skin incision until such time as a clot has formed subcutaneously. This
procedure has two drawbacks, firstly inasmuch as it tends to aid the occurrence of
gangrene, and secondly because of the fact that deviation of the vessels occurs,
making the site unsuitable for future blood-lettings.
The care that should be exercised in securing blood samples under field condi-
tions consists of:—
(a) Restricting oneself to a reasonable speed in carrying out the operation.
An average of 75 birds an hour is regarded as a reasonable thing.
(b) Using only a very sharp scalpel, as when the instrument is dulled greater
pressure must be used and the vessels are more likely to be com-
pletely severed.
(c) The application of pressure over the severed vessels for a minute or two
in the case of individual birds which bleed too freely.
(d) Scrutinizing those birds which have been bled. This will often reveal
an odd bird in which haemorrhage has not been controlled. Such
a bird can then be caught and control measures applied.
One of us (W. J. B. M.) has observed mortality following blood-letting in
2,014 cases to be 0.5 % and one (A. L. C.) in 6,025 cases to be 0.2%. It must
be pointed out that in the latter case 1,775 birds were stud and more than ordi-
nary care was exercised. Of this number five or 0.29% succumbed. Of the
4,250 treated according to usual procedure eight or 0.19% succumbed. These
figures indicate that there is no actua necessity to go to extremes in controlling
haemorrhage at the time of operation.
Speaking generally, it is not considered that a loss of 1% or less justifies the
time taken by the systematic application of specific measures to prevent hae-
morrhage.
388 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY. [V, IV
LOWERED EGG PRODUCTION
No actual figures can be quoted in respect to this aspect of the question, but
inquiries have been made of owners of flocks which had been bled. In only one
instance was it reported that a definite decline had occurred, and the general
impression of the owners was that egg production was not materially affected.
It is probably not the actual taking of blood that is likely to diminish egg
production so much as the handling which is inseparable from the operation.
A great deal depends upon the owner's handling of the birds in preparation for the
operation.
Birds have to be handled on three distinct occasions, viz.:—
(1) For the purpose of putting on leg rings.
(2) For the actual blood letting.
(3) For the removal of reactors.
Leg rings should be placed on the birds the night before the day on which they
are to be bled. In this way the birds are subjected to a minimum of excitement.
Occasionally the leg ringing and bleeding are combined in the one day's activity,
but unless more than ordinary assistance is available, the operation is very consi-
derably slowed.
Every effort should be made to avoid rough handling of the birds at the time
of blood-letting. Expert " catchers " and the provision of coops for holding the
birds will do much in this direction.
Reactors should be searched for at night, as the leg rings can be examined
with the aid of a torch without disturbing the birds to any appreciable extent.
Examination is in wet weather sometimes made difficult owing to mud clinging to
the rings, and if any considerable number of birds are being dealt with, a long and
arduous process results.
The labour involved on the part of the owner in the preparation of the birds,
assistance at the time of the operation, and removal of the positive and suspicious
reactors, are in fact the major items to be considered in blood-testing as at
present constituted. Losses from haemorrhage are of secondary importance,
while it is doubtful when due care is taken whether the question of lowered egg
production need be considered at all.
THE DIAGNOSIS OF JOHNE'S DISEASE BY THE USE OF
JOHNIN
BY
F. C. MINETT,
Research Institute in Animal Pathology, Royal Veterinary College, London.
(Reprinted from the Journal of Comparative Pathology and Therapeutics, Vol. XLVIII.
Part 2, June 1935.)
In the literature on Johne's disease there are many references pointing to the
value of allergic tests for the detection of preclinical cases or as a means of
supporting the diagnosis in cattle showing suggestive symptoms. For this purpose
avian tuberculin or johnin have been used subcutaneously, intravenously or in¬
tradermally. Conclusive evidence as to the value of such tests for detecting
infected animals lies, of course, in the demonstrating of Johne's bacilli in the
intestinal wall or glands, but for several reasons this evidence is not always obtain-
able. One difficulty is the cost of bringing the animal to post mortem. Other
reasons are that there is no convenient animal for test inoculation, that infected
animals do not always show distinct microscopic lesions, and that even on micros-
copic examination of films from the intestine or glands acid-fast bacilli may not
be found. In order to establish the diagnosis, therefore, it may be necessary to
make histological preparations or to attempt cultural isolation of the organism.
In the latter case there is, of course, some technical difficulty and especially
is this so in early cases.
The objects of the work set out as briefly as possible in this paper were to
study the behaviour of animals infected with Johne's disease when subjected at
intervals to a number of intradermal tests with Johnin, and to examine them after
death for Johne's disease and tuberculosis. Nearly all the cases to be described
were derived from seven herds previously reported [Minett, 1933] in which
johnin testing of all the adult stock had been carried on as a routine over a period
of about two years, usually at intervals of about three to four months.* In six
of these herds Johne's disease was known to be causing loss and of a total of 499
adult animals present at the first or second test 16 per cent gave clearly positive
reactions and 72 per cent did not react. In the series of 39 animals mentioned
below where the post mortem examination for Johne's disease was positive, 21 were
tested three to five times, one was tested seven times while 17 could be tested once
or twice only. It was not always possible to apply more than one or two tests,
either because the animals were approaching an advanced clinical stage and, in
consequence, were disposed of, or because the owners were averse to keeping
* The tests were carried out with the assistance of Messrs. R. S. Roberts, W. Orr and
A. J. James.
( 389 )
390 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY. [V, IV
cattle which were known to be reactors. This, however, afforded an opportunity
of purchasing reactors while they were still in good or fair condition and of killing
them for immediate examination. The following classes of animals are
considered:
(1) Thirty-nine, which had shown a definite reaction to johnin on at least
one occasion and which were proved on post mortem examination
to be affected with Johne's disease.
(2) Seven, which had shown a definite reaction to johnin but which were
not proved on post mortem examination to be affected.
(3) Seven, which had shown a definite reaction to johnin but which for
various reasons could not be examined post mortem. The subse-
quent history, however, strongly suggested that they were affected
with Johne's disease.
(4) Six animals, which had not reacted, but which were found post mortem
to be affected.
Of the animals in classes 1 and 2,13 remained throughout in fair bodily condi-
tion and five were fat; 17 others showed symptoms shortly before slaughter. In
seven cases the bodily condition was noted to vary from time to time, there being
periods of unthriftiness with some looseness of the bowels. Four were clinical
cases from the start. Fourteen of the animals were kept under observation at the
Institute for periods varying from three to six months and in two cases as long as
a year, partly in order that their clinical condition might be followed, but parti-
cularly so that further tests might be made with johnin, tuberculin, or with avian
tuberculin.
The johnin used was a ten-fold concentrated product, prepared by Dr. A. L.
Sheather by growing a well-adapted strain of Johne's bacillus on Sauton synthetic
medium containing extract of M. phlei. This product was selected partly as the
result of a preliminary test on infected cattle and partly as being less likely to
cause non-specific reactions than one prepared on a medium containing animal
fluids. It was injected according to the method first suggested by Dunkin [1928]
into the skin of the neck in dose of 0.2 c.c. followed 48 hours later by a similar
injection at the same site. All infections and readings were made by myself.
Caliper readings in millimeters were taken prior to injection and again 48 hours
and 72 hours after the first injection, when notes were also made of the character
of any swellings. In some of the earlier cases a single injection of 0.2 c.c. johnin
was made into one of the anal folds, with or without a simultaneous injection in
the skin of the neck. In the summarised notes below no details are given to
indicate the intensity of the reactions as this would take much space and would
appear to be unnecessary. It may be assumed, however, unless there is a state-
ment to the contrary, that the reactions were unmistakably positive, i.e., a swelling
at the 72nd hour reaching at least twice the normal skin thickness at that point,
DIAGNOSIS OF JOHNE'S DISEASE BY THE USE OF JOHNIN.
391
with diffuse and rather soft oedema and sometimes slight pain. Reactions des-
cribed as " slight " or " indefinite " were those which fell short in some particular,
e.g., where the maximal thickening attained was rather less than twice that of the
normal skin or where the oedema was not diffuse and lacked the easily compressible
texture of a typical reaction. The anal fold test was considered positive if the
injected fold was two to three times the normal thickness or more at the 48th
hour. On some occasions the animals were tested at the same time by the single
intradermal method with ordinary tuberculin or with avian tuberculin, con-
centrated products from glycerine broth cultures being used in doses of 0.1 c.c.
In all cases, save nine, examined after death, the animals were slaughtered at
the neighbouring abattoir and always in the presence of a member of the Institute
staff. The intestine, intestinal and other glands were immediately brought back
to the laboratory and dealt with the same day.
THE EXAMINATION FOR TUBERCULOSIS AND JOHNE'S DISEASE.
The following in outline was the procedure ordinarily adopted in making the
post mortem examination.
Tuberculosis.—When no tuberculous lesions were visible to the naked eye,
guinea-pigs were inoculated subcutaneously with material from the lymphatic
glands after the surfaces of these organs had been cauterised. The glands of the
intestinal tract were often treated separately, two guinea-pigs being injected with
material from several of these glands. Sometimes guinea-pigs were inoculated
with material from thoracic lymph glands only, at other times tissue from thoracic,
pharyngeal, submaxillary and intestinal glands was pooled before injection. In-
oculations were also made from tissues the tuberculous nature of which was in
doubt. The animals were killed for examination two months later. When pre-
mature death took place nine days or more after inoculation, two guinea-pigs
were inoculated from the local lymphatic glands and sometimes also from the
spleen of the first. Premature deaths among the guinea-pigs were not a source
of much trouble. Of 58 guinea-pigs inoculated from non-tuberculous tissues,
eleven died prematurely and of these only two died before the ninth day.
Johne's Disease.—The examination for Johne's disease was made on lines
much like those adopted by previous workers [e.g., M' Fadyean, Sheather and
Edwards, 1912]. The intestine was separated from the mesentery, spread out
on long tables, opened, and the contents removed with water from a hose. The
appearance of the mucous membrance was then noted, smears to be stained by
Ziehl-Neelsen method were taken from various parts of the small and large intes-
tines and small pieces of the wall were fixed in 10 per cent formalin. In this
part of the examination special attention was paid to the ileo-cæcal valve
and ileum and to any areas which had a suspicious appearance. Lengths of intes-
tine measuring some ten inches were then removed from the same parts and,
392 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY. [V, IV
together with some of the intestinal glands, were used for the cultural examination.
For this purpose the bowel was thoroughly washed with tap water, the superficial
layers of the mucosa from several areas scraped with a blunt knife and transferred
to a sterile mortar. In the case of the glands the surfaces were cauterised and
material from the interior removed to a second sterile mortar. From other glands
films were prepared and portions of the tissue transferred to fixative. The material
in each mortar was crushed into a paste, sterile sand being used in some cases, and
was then mixed with about 40 c. c. sterile saline. Each suspension was then divi-
ded between two centrifuge tubes of 80 c. c. capacity and to one tube in each set
was added an equal volume of 20 per cent anti-formin, the other tube in each set
receiving a similar amount of 40 per cent anti-formin. The suspensions were
left at room temperature for 30 minutes, being shaken at intervals, and the tubes
were then filled with sterile saline, centrifuged, the supernatant replaced with fresh
saline, and, after shaking, again centrifuged. The deposit was used for making films
and for sowing tubes of liver extract-phlei-egg medium, prepared in the manner
described by Dunkin [1928] except that gentian violet was omitted. In some
instances, a liquid medium was used, consisting of equal parts of liver extract
and broth and containing glycerine extract of phlei and a small quantity of potato
mash. Two to four tubes of medium were seeded from each deposit; sometimes,
in addition, tubes of medium were seeded direct from the anti-formin mixtures at
intervals according to the method of A. S. Griffith [1916]. The culture tubes were
tightly capped and incubated at 38° C. for six to eight months, being inspected at
monthly intervals. If a growth resembling that of the Johne's bacillus appeared,
this was examined microscopically and if acid-fast organisms were present a subcul-
ture was made to the phlei-egg medium and to plain egg and a portion inoculated
to one or two guinea-pigs subcutaneously. Acid-fast organisms of the proper
morphology, which grew very slowly on the pheli-egg medium and which failed
to grow on plain egg and which produced no lesions in the guinea-pigs killed two
months later, were accepted as Johne's bacilli.
REACTORS PROVED ON post mortem EXAMINATION TO BE AFFECTED WITH JOHNE'S
DISEASE.
(NOTE—In the following account cases where the disease was found to be at an
early stage are placed towards the end of the series. The first johnin test was made on
" day 1." Abbreviations used are :—R = reacted ; NR = did not react (in both
cases, unless otherwise stated, the reference is to johnin used by the double intrad-
ermal method in the neck ); K = killed ; T = tuberculosis J = Johne's disease or
Johne's bacilli ; gp = guinea pigs.)
The indication as to the animals' age has reference to the date of the first test.
Statements as to the presence or absence of lesions of T or J refers to naked-eye exami-
nation only. A test for tuberculosis by inoculating guinea-pigs was made only where
stated.
394
THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY. [V, IV
11. Guernsey heifer, rising two years. NR1, slight R101, R214, NR311, R 401.
R(tuberculin) 21 and 214. In poor condition and scouring after the test of day 311.
K 409. T lesions present in bronchial glands ; gp inoculated from the intestinal glands
did not become infected. Lesions of J in intestine ; histologically there were exten-
sive alternations and very large numbers of J ; cultures were positive.
12. Shorthorn bull. 3½ years old. R 1 (anal). In good condition for 186 days
when active signs of J developed. K 195. No T lesions. Lesions of J in intestine
and J in films, sections and cultures of intestine.
13. Guernsey heifer, rising 2 years. In fair condition at first. R1. Slight R 22
(tuberculin). Reported to be very thin and scouring on day 92 K 102. No T lesions.
Lesions of J in intestine, J in the films of intestine and isolated in culture.
14. Shorthorn. Second calver. In fair condition. R1, NR 92 (also anal).
R 1 (tuberculin), NR 8 (subcutaneous tuberculin). Became very emaciated by day
92. K 99. No T lesions. Lesions of J in intestine and J in films of intestine and
isolated in culture.
15. Shorthorn heifer. About 2 years old. R1, slight R 97, when reported to be
scouring. NR21 (tuberculin). Carcase in moderate condition when K on day 158.
No T lesions. Lesions of J in intestine, J seen in films from intestine and isolated in
culture.
16. Shorthorn. Yearling heifer. NR1, R97 and 204. NR21 (tuberculin),
R 204 (tuberculin). K 220. No T lesions. Lesions of J in intestine, J seen in films
from intestine and isolated in culture.
17. Jersey heifer. From alleged T-free herd. R1 and 97. NR1 (tuberculin).
K 155. No T lesions and 2 gp inoculated from pharyngeal and intestinal glands failed
to become infected. Unfortunately, material from the bronchial glands was not inocu-
lated. Lesions of J in intestine and J seen in antiformin preparations and isolated
in culture.
18. Shorthorn heifer. Nearly 2 years old. Scouring at times, latterly rather
badly, but did not become emaciated. Slight R1, R92 (also anal), and 175, NR 267.
R (tuberculin) 1 and 175. K 273. One mediastinal gland contained calcerous areas,
which could not be demonstrated by gp inoculation to contain tubercle bacilli. 2 gp
inoculated from the bronchial, pharyngeal, submaxillary and intestinal glands did
not become infected. Lesions of J in intestine. J seen in films of intestine and isola-
ted from intestine.
(NOTE.—" Slight R1 " = increase of skin thickness from 6 to maximum of 12
mm., but the swelling was quite firm and well-defined.
19. Shorthorn. 3 years old. R1, 113 and 210. R (tuberculin) 113 and 210.
K246. No T lesions. 2 gp were inoculated from bronchial, pharyngeal and submaxil-
lary glands, 2 from the submaxillary gland and 1 from the mesenteric glands. None
of these animals became infected. Slight congestion of the ileo-cæcal valve, J seen in
anti-formin preparations and in sections of intestine and cultivated from both
intestine and mesenteric glands.