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Studies in Lamb Dysentery
THE
JOURNAL OF
COMPARATIVE PATHOLOGY AND
THERAPEUTICS. Vol. XLI.-No. 3.
SEPTEMBER 29, 1928.
STUDIES IN LAMB DYSENTERY.
PRICE 3s. 6d.
*
By
W. A. POOL, H. PRESTON,
A. BROWNLEE,
Animal Diseases Research Association of Scotland.
THE opmlOns held by different investigators concerning the nature of the dysentery of new-born lambs prevalent in the counties bordering Scotland and England have been very varied. The earliest view was that infection is acquired after birth by the ingestion of the causal agent, but whether infection occurs before or after birth, and in the latter event whether the path of entry is via the mouth or otherwise, are questions which have been disputed. Pathologists have even doubted if the disease is a specific infectious condition and capable of being transmitted directly from lamb to lamb. This uncertainty concerning the nature of the disease has led to revision of available data and has shown the need for further investigation. Different authors have obtained curiously varying results from transmission experiments.
* Received for publication,
14th March, 1928. A2
174
GENERAL ARTICLES.
When using material which might be expected to be most virulent, some have failed to set up the disease in experimental conditions even when the most likely paths of infection have been followed. The results now described are part of an investigation planned to review the whole question from the beginning. Much of the work at present in progress is still incomplete. It is deemed advisable, however, to publish certain parts which are complete as far as the material available in the 1927 lambing season is concerned. A subsequent paper will record the continuation and amplification of the inquiry in the next season's field work.
I. A
SEARCH FOR PATHOGENIC MICRO-ORGANISMS IN THE GENITAL ORGANS OF EWES WHOSE LAMBS HAD DIED FROM DYSENTERY.
For this work ewes were obtained from farms on which lamb dysentery had caused considerable losses. Ewes were obtained in the winter and spring of 1926-27, while in lamb or barren, which had lost their lambs from the disease in the 1926 lambing season. The barren ones were received at the laboratory before the rams were put out on the farms in the winter of 1920. Recently lambed ewes were dealt with as soon as they could be obtained after their lambs died from the disease in the 1927 lambing season.
Abbreviations used in the text.
Technique.
Culture Media Used. Robertson's bullock's heart medium with a BHM. liquid paraffin seal. pH 7·4 Ditto, plus 10 per cent. sterile unheated sheep serum, plus a piece of sterile unheated rabbit kidney, the depth of the column of liquid being about 7·5 ems. and a vaseline seal being provided. pH 7·4 Hall's glucose (0·2 per cent.) agar (1 per cent.) medium used in deep tubes for shake cultures. HSM. pH 7·4 Plain agar slopes. Blood agar slopes. About 30 per cent. of sterile sheep blood added to 3 per cent. agar when at 45 C., mixed and set • Incubated aerobically BAA. Incubated anrerobically BAN. Anrerobic conditions for the surface cultures were provided by M'Intosh and Fildes jars. The tubes of BHM and HSM were always boiled for 15 minutes and cooled immediately before they were inoculated. In each case at least three tubes of each type of culture medium mentioned above were inoculated from each tissue studied. 0
GENERAL ARTICLES.
175
The fleece over the whole of the ventral surface of the body was clipped. The ewe was fixed to a table with the ventral surface upwards and bled to death under chloroform anresthesia. The fleece was wetted with 2 per cent. lysol and a doth which had been soaked for 24 hours in 1 : 1,000 perchloride of mercury was spread over the body. A hole of such a size as to expose the operation area had been cut out of the centre of the cloth before it was disinfected. Wide lines were seared with red hot firing irons around the line of proposed incision. The abdominal wall was removed in one piece, the incisions being made with sterile instruments along the seared lines. In the case of barren ewes the vagina was clamped close to the os uteri and cut through distally with sterile scissors. The attachments of the uterus were similarly freed, and it was transferred immediately to a sterile glass dish with a cover. . The subsequent manipulations were carried out in an inoculating hood. The examination of ewes which had lambed recently and of ewes due to lamb was carried out at a field laboratory established on a farm in Roxburghshire. In the former case, after exposing the uterus as described above, it was seared with a hot firing iron and an incision was made with a sterile scalpel over the seared area. In the latter case the amniotic fluid was first aspirated with precautions to avoid contamination, so that after exposure of the lumen of the uterus there would be no fluctuation of the liquid and consequent possible infection from back wash. In all cases when uterine cotyledons were examined culturally pieces were removed and crushed with sterile sand in the improvised pestle and mortar composed of two modified test tubes described by Hagan:l A separate sterile improvised pestle and mortar was used for each manipulation. The Direct Microscopical Examination of Films Prepared from Uterine Exudate. In all cases the films were prepared within at least an hour of the completion of the autopsy. They were always prepared immedi~tely after the cultural work had been completed. The moist films were prepared by placing a drop of a suitable suspension on a thin slide, placing a cover glass in position, and sealing it with hard paraffin. They were examined under an oil immersion lens by dark ground illumination. Dry films were stained with warm 10 per cent. carbol-fuchsin and by the methods of Gram-Jensen & Leishman The results of the microscopical and cultural examinations carried out are given in Table 1. 1
J I. Exp. Med., Vol. xxxvi,
1922, p. 711.
176
GENERAL ARTICLES. CONCLUSIONS.
Direct observation of films from 11 ewes has given negative results. Observations by cultural methods were also negative except in two instances. As the growths in each of these were mixed and they were from ewes which had lambed a few days previously, it is considered that they were due to contamination or were of accidental occurrence in the tissues from which they were obtained.
II. TRANSMISSION EXPERIMENTS TO DETERMINE THE PATH BY WHICH LAMBS BECOME INFECTED WITH LAMB DYSENTERY.
The delay in acquiring accurate knowledge concerning Iamb dysentery has been caused by the short interval in each year in which natural infective material is available, and by the fact that in normal circumstances the only satisfactory test animal, the new-born lamb, is unobtainable throughout most of the year. Thus; by the end of the lambing season, when investigators have studied and selected the material available, it is not possible to buy lambs for experimental purposes. The alternative of maintaining a special flock of ewes of suitable breed at headquarters and arranging for them to lamb later than the normal season and over a more prolonged period, is only possible in special circumstances. To meet the requirements of the present investigation, Scotch Blackface ewes and a ram were obtained from an area remote from any history of Iamb dysentery infection and kept on clean ground until the lambs were born. The writers are indebted to Mr. T. M. Macdonald, of Barguillean, TaynuiIt, Argyllshire, for purchasing thirty (30) ewes from the neighbourhood of Oban and for presenting them with a reliable ram of the same breed. The ewes and ram arrived in Edinburgh in the autumn of 1926, and the ram was put with the ewes at intervals to provide a sequence of about two lambings a week from the middle of March to the end of June. The arrangement worked fairly well. Several of the ewes, as was expected, took the ram more than once. It was not always possible to forecast accurately when Iambs would be available to test particular materials, and in one or two instances when they were available the material required was unobtainable. When the transmission experiments were about half finished it was decided that every effort must be made to get more pregnant Scotch Blackface ewes, as there were insufficient lambs for the work which had been planned. Blackface ewes due to lamb early in May were accordingly purchased. They were said to have been put to a Blackface ram, but all the Iambs were Greyfaces (progeny of Border Leicester ram). The writers consider that the more robust Greyface Iambs were much more difficult to infect. The healing ulcers found in two of them, which would almost undoubtedly have recovered if they had been
GENERAL ARTICL ES
177
allowed to live, supports this view. The experiments with these lambs are included for the sake of completion.
Preparation of Material. Uterus.-C'otyledons were collected with pre<:autions to avoid
contaIIfination, ground with sterile sand, and suspended in sterile normal saline solution. Scrapings from the lumen of the uterus were mixed with this suspension. Intestinal ulcers.-Ulcers were separated from the healthy surrounding tissue with sterile instruments, washed in five changes of sterile normal salt solution, ground up with sterile sand, and suspended in sterile normal saline solution. Fa'ces.-F
(a)
Through the agency of the ewe. 1. With material from the, uterus of ewes whose lambs had died from dysentery. i. By Introduction into the vagina. Test Animals. Ewes 11, 14 and 17. Dosage. 20 c.c. of a turbid suspension. Age of material tested. Used immediately. Source of material. Ewe 100. Result. All three ewes lambed normally and the lambs remained healthy. 11. By Ingestion. Test animal. Ewe 27. Dosage. 250 c.c. of a turbid suspension. Source of material. Ewe B1. Age of material. 6 hours. It was sent by passenger train to Edinburgh from the field laboratory. Result. 1 lamb born. Remained healthy. 111. By Subcutaneous Inoculation. Test animal. Ewe 19. Source of material. Ewe B1. Age of Material. 6 hours. Dosage. 20 c.c. of a turbid suspension. Result. 1 lamb born. Remained healthy. IV. By Intravenous Inoculation.· Test animals. Ewes 20 and 26. Source of material. Ewe 101. Age of material. 18 hours (kept in cold store). Dosage. 20 c.c. of supernatant fluid after standing for 1 hour. Result. 1 lamb born to each ewe. Remained healthy. 2. With intestinal ulcers and with freces from lambs affected with the disease. By Ingestion. Using intestinal ulcers as test material. Test animals. Ewes 12 and 15. Age of material. Half an hour.
178
(b)
GENERAL ARTJCLFS.
Dosage. 100 C.c. of a turbid suspension. Result. One lamb born to each ewe. Both lambs remained healthy. Using freces as test material. Test animal. Ewe 28. Age of material. Under 18 hours. Dosage. 100 c.c. Result. One lamb born. Remained healthy. By direct infection of the lamb. 1. With material from the uterus of ewes whose lambs had died from dysentery. By Ingestion. Test animal. Lamb 49. Source of Material. Ewe Bl. Age of material. Two days (in cold storage at about 5° C.). Dosage. 5 c.c. of a turbid suspension. Result. Remained healthy. 2. With intestinal ulcers and with freces from lambs affected with the disease. 1. Through the umbilicus. Blackface Lamb 53. Dosage. Freces smeared over the umbilical cord. Age of material. Under 18 hours. Result. Remained healthy and was sold fat at 4 months old. Blackface Lamb 56. Dosage. 2 c.c. of frecal matter was inserted into umbilicus and a ligature was applied to prevent escape. The ligature was removed 6 hours later. Age of material. About 40 hours (last 24 hours in cold store). Result. The umbilicus became swollen but healed and the lamb was sold fat at 4 months old. 11. By Ingestion. (a) With Freces. Blackface Lamb 54. Dosage. 3 c.c. of undiluted freces. Age of material. Under 18 hours. Result. Well up to 4th day, when diarrhrea started and lamb was visibly ill. At times it lay on its side and appeared to be almost in a state of collapse. Its condition gradually improved and it finally recovered. Blackface Lamb 57. Dosage. 3 c.c. of freces. Material from the same sample was also smeared over the udder and teats of the ewe. Age of material. About 40 hours. Result. Well up to second day, when a yellowish-brown diarrhrea began. It died on the third day. Lesions. Typical L.D. ulcers were found in the small intestine. Greyface Lamb 63. Dosage. 3 c.c. per os, and udder and teats of ewe smeared. The dosage was repeated at the 24th and 48th hour. Age of material. Under 10 hours for first dose. Result. Well up to 3rd day, when it was dull, lying in a corner, unwilling to move and showing diarrhrea.
GENERAL ARTICLES.
179
There was steady improvement up to the 11th day, when diarrhrea and straining were still present. It was considered that the lamb might recover if allowed to live. As in that event there would be no knowledge about the result of the experiment, it was decided that the lamb should. be killed. Lesions. A few dozen healing ulcers were present along the whole length of the small intestines. Four small discrete healing ulcers were present in the large colon. Blackface Lamb 72. Dosage. 5 C.c. of freces per os, and teats and udder of ewe smeared. Repeated at the 24th and 48th hours. Age of material. About 6 hours for first dose. Result. Well up to the second day, when slight diarrhrea started. This continued till death occurred on the 4th day. Lesions. About the junction of the middle and lower thirds of the small intestine there was an ulcer 4 m.m. in diam. In the succeeding 5-ft. of intestine there was continuous ulceration. The remainder of the small intestine to the ileo-caecal valve showed diffuse inflammation. There were patches of inflammation in the large intestine. The kidneys were very congested. Greyface Lamb 68. Dosage. 5 c.c. per os, repeated at the 24th and 48th hours. The Ildder and teats of the ewe were not smeared. Age of material. From a lamb found dead. Date of death unknown . . Result. There was diarrhrea on the 2nd, 3rd, and 4th days. On the 6th day appeared to be healthy. Was killed and examined. Lesions. All organs healthy. Greyface Lamb 82. Dosage. 5 c.c. per os, and udder and teats of ewe smeared. Repeated at the 24th and 48th hours. Age of Material. Under 6 hours for first dose. Result. Diarrhrea from 2nd to 5th days. Killed on 7th day as appeared to be healthy. Lesions. There were 3 healing ulcers in the small intestine. Greyface Lamb 85. Dosage. 3 C.c. of freces per os, and udder and teats of ewe smeared. Repeated at 24th and 48th hours. Age of Material. From a lamb found dead. Date of death unknown. Result. There was slight diarrhrea on the second and third days. Remained healthy.
(b)
With washed ulcers. Greyface Lamb 71. Dosage. 5 C.c. of a turbid suspension per os, and udder and teats of ewe smeared also. This was not repeated. Age of material. 6 hours. Result. Lamb remained healthy.
180
GENERAL ARTICLES. TABLE
I
I
'I,,
No. of Ewe.
Dale 0/ Autopsy.
Breed.
!
·1
I.
1
History .
i
i
i
-100-1- Scotch
1- - ;;;1/2"7.- ,
_ _ _ ' _ _ Blackface ___ _ _
I
I
101
I, I
Scotch Blackface
I '
I
i
Lamb said to have died I Ba-r-ren-.-S-m-o-n-th-s i -N-o-r-m-al-.-N-o-c-hoc-,-o-Ia-te-,from L.D. in 1926. I since lamb died coloured exudate. !
id. t,'on lh9a2v6e. died , Due to lamb. Lfarmomb Lsa.D
- -B-2-.-II-SB-C01-atcc-hk-fa-cel
12/4/27.
1
- Lamb said to have died : Due to lamb. from L.D. in 1926.
1
1,'- - I 0 3I' s ,
-1'--7/4/27·
cotch Blackface
,i
I "
Lamb said to have died from L.D. in 1926.
- --1--- - ' - - - - ' - - - -- B1.
:
Cheviot
'
!
I, I \
B3.
I
-23/4/27.
Cheviot.
i
1I--1 i- --- ----iI '
!
B4.
Cheviot.
B5.
Cheviot.
23/4/27.
I
I
2/5/27.
I
1
!
!
Her lamb was found to be affected with L.D. on the 22/4/27 when said to be 5 days old.
: One lamb in uterus. No ' abnormality. There was no chocolate. coloured exudate.
i Now !:>3rren. Her \'
Normal. No chocolatecoloured exudate.
lamb had died
II
' 12 months pre· viously.
i
--~--,-------
Lamb said!o have di~d 1 from L.U. III 1925. Th,s , year her lamb died from ! L.D. when said to be 4 : days old. She was killed I the day after i.t died.
12/4/27. :
:1
I
One lamb in uterus. There was a chocolate.coloured exudate around some of i the cotyledons . The lamb was healthy.
, __________ i______ :
I
, I
'
7/4/27.
Appearance Of lumen 0/ ulerlls.
Present state.
I Some of the cotyledons of °bnroewnho,'nncowloeurre anddarka
1
I
yellowish.grey mucus covered some others. The cotyledons of the , other horn were dar k red , in colour and free from any coating of mucus. There was a chocolate. coloured exudate around the cotyledons.
I'
1_
_
_ _ _ _ _ _ _ _ _ __ __
Her lamb died on the 30/4/27 from L.D. when said to be 4 days old.
I
I !
I The cotyledons o f the right horn were dark
'
~~~b~~t~e~~o~~ ~~vecr~
or surrounded by any exudate. They were rather easily detached from the uterus. The cotyledons of the left horn were dark red in colour and there was no exudate.
II
1 1
_
Said to have lost twin I Normal. lambs in 1926 from L.D. i She was killed as soon as I her lamb was born. ' i _____.__________
I
[ 1- -B-7!" ,'
•
' Ii
Cheviot.
I 1
!
- II- La-m-b-w-as-sa-id-t-O-h-a-ve-:i I All the cotyledons showed died from L.D. when a brownish colouration. about 7 days old.. This , was four days before the I ewe was killed. I- - - - - -I- - - - - - - - -! 7/5/27. I' Lamb, died from L.D. [ The cotyledons were eov- ! when about 4 days 0 d. ~~"i:o~~~e~~u~~n:;~~ ! I or I date. This involved the b~~eh~:n:~e IUmen of
, I,'
I' ,
- -I 88.
-1·--7-/5~/2-7~.
-Ch-e-v-io-t
86.
~fe~ ili~s d~~:~ I:b~
I
I----~·II -----------------1 I,
Cheviot.
14/5127.
I I
Lamb died from L.D .• said to be about 4 days I old. Ewe was killed the day arter the lamb died. !
[I
There was a chocolate. coloured exudate arounj the cotyledons.
181
GENERAL ARTICLES. TABLE
L
Micr'!!..~~m... Exami':!!!ion. _______ '
Cultural Examinatio'!:. ____ Foet us. Ewe. I Foet us. , S~;;g;j;:;;;;;-i;;n-;;;-;;-;i;;;·-1 Scrapings of sto-;;'ach , - - - - - - - - - contents by dark, Crushed cotyledon Stomach: Dark RTDund ground illumination. , from uterus. :, contents. Stained. illumination. I I Neg. I Neg-.- - - - j - - - - - - - - - I "N-;g-. '1 _ _ _ _ _ _
1
Ewe.
I
--I
I
I
---------,\'-----1
,----~--- ·--~---i--~------II------------':-----Neg. i Neg. Neg. , Neg.
1
:
'
I Neg.
I,-------,----,----------,-----_·_---,---1 I· I
Neg.
!
I
INeg. ;
'
' Neg.
I
I
'
Neg.
'
----I Neg~--.--1-------1
---I
I
i
iN-;g-.
I
Neg.
I
1
I
Neg- : - - - - - - - - - -
:
I
rN~--I- ---
I
N~
;
I
I
----!
I I
I i i
I
'
1
I I ' I I
Neg.
I
Neg~-----i-------
Neg.
i
---I ""~---- --I--
I
[!
I
.
' - _ · __ ·_------1-----', : \ - - - - - - - -" - - - - - - - , !- - - - - - - - _ · _
i
I Neg.
I
'I,
I Neg.
Neg.!
!N-;g-:-----rN-;g~---I,--------1 Two
,
I
' I ''
'
,
I
I
,
I
!
, I
i
I
'
I
'-·----I------__________ ,
Neg
I
,
I
---I N~
Neg.
i,
'
other media rem~n.:d~~rile·l----, Neg. :
1
I
• _ _ _ _ _ .1
'-N-eg-.
out -.;J;;;;-SR.M.';-j'----: One con" tained a bacillus like B. wel- i chii, a coliform bacillus, and I a streptococus. The other ' tube showed a slender,Gram- I positive, non-motile bacillus, I' a short stout, Gram-positive non-motile bacillus, and 1 cocci. One tube out of 6 H.S.M.' s showed growth. There was a bacillus like B. welchii, a smaller and more I slender Gram-positive bacillus, and a coliform bacilluo. I No growth occurred on the B.A.N.tubes till the third day, j when a greyish thick, white film appeared in one out of 6 , tubes. These cultures were I not studied further. All the
, showed growth.
I
Neg~------ ----I ---
I ' -N-e-g-.----! Neg. I
----I
•
'
I I,
I
----------
Lefthorn.-l out of 6 B.H.M.s
I·
1------1
showed a bacillus like B. welchii. Right horn.-2 out of 6 B.H.M.'s showed growth I consisting of a long, slender, Gram-positive,gas-producing bacillus in one and a mediumsized motile bacillus in the , other. These cultures were not studied further. All the other media remained sterile.
I
._-
_.
'I'
' I , '[
!
I
I
II
20/4/27.
19/4/27.
26/4/27.
13/6/27.
7/5/27.
16/6/27.
6/5127.
21/4/27.
23/4/27.
1516127.
6
4
10--12
17
II
16
20
20
10--11
6/1/27.
6/1/27.
14/4/27.
12/4/27.
8/4/27.
12/4/27.
18/4/27.
18/4/27.
11/4/27.
14
17
27
19
26
20
12
15
28
~--.
Dateo of Lambing.
6-8
Week of Gestation at the time of dosing.
6/1/27.
Date of Dosing.
11
Number of Ewe.
TABLE
II.
+ + +
By introduction into the Vagina.
+
By Ingestion.
+
Subcut.
+ +
Intraven.
By Inoculation.
Through the Agency of the Ewe.
I
I
I
I
Ewe by Ingestion of
+ +
Ulcers.
I
-'-
Faeces.
I Through the Agency of the
A Mixture of ground cotyledons and scrapings from the lumen I of the uterus. I
Table Showing Results of attempts to Infect Lambs with Dysentery through the Agency of the Ewe.
Result.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Scotch Blackface.
Breed.
r.j
rn
rl r
~
~
~
...... ~
Date of Dosing.
S.B.F.
S.B.F.
G.F.
S.B.F.
G.F.
G.F.
G.F.
G.F.
G.F.
S.B.F.
Under 8
Under 8
Under 8
4
Under 8
Under 8
Under 8
Under 8
4
20/4/27.
21/4/27.
30/4/27.
6/5/27.
2/5/27.
12'5/27.
21/5/27.
5/5/27.
11/5/27.
6/5/27.
15/5/27.
54
57
63
72
68
82
85
71
80
75
83
G.F.
S.B.F.
I
+ +
'I
-I-
+
+ +
' . 1 LD UnfIltered Faeces OJ • • Lamb. B Through Y. I Umbilicus. IngestIOn.
+ +
of L.D. Lambs I by Ingestion.
I Filtered Faeces
+ +
Tri/ urated I1lcers.
I
I
.
Results.
+
+
I I
I
+
+
+ +
+
+
+ +
+ + +
+
Died. Killed. ljtm'f/;ed ea Y, _ _ _ _ _ _.
GF=Greyface (Border Leicester-Scotch Blackface).
Suspension of cotyledon of Ewe .by IngestIOn.
SBF=Scotch Blackface.
2
21/4/27.
56
S.B.F.
20/4/27.
53
, S.B.F.
I
I
14/4127.
Under 8
I
Breed.
49
----~-
I .
i
l
I
4 e of Lamb n
I' g HOUTS.
'I
----- -------
1
ljuLmbebr OJ am.
I-~--~-------
III.
Table Showing Results of attempts to Infect Lambs with Dysentery.
TABLE
+
+ + +
Lesions of L.D.
______
I
00 <:0
......
~
t"'
(")
i§ C)
184
GENERAL ARTICLES.
Greyface Lamb 80. Dosage. 5 C.c. of a turbid suspension per oSi,lld udder and teats of ewe smeared. Repeated at the 24th, 48th, and 72nd hour. Age of material. 1 hour for first dose. Result. Killed at tenth day, as was healthy. Organs all normal. (c) With sterile filtrates of fresh freces. The freces were passed through sand and pulp and then through a Seitz filter. About 1 C.c. of the filtrate was inoculated into B.H.M. and failed to grow. Greyface Lamb 75. Dosage. 5 c.c. per as, and smeared over udder and teats of ewe. This was not repeated. Result. Remained healthy. Blackface Lamb 83. Dosage. 5 c.c. per as, and smeared over udder and teats of ewe. This was not repeated. Result. Remained healthy. These results are shown in Tables 2 and 3. In the course of the work the evidence obtained always appeared to negative the possibility that infection occurs in utero. The only factor suggestive of any abnormality within the lumen of the uterus was the presence of the chocolate-coloured exudate around some of the cotyledons in a few of the cases. The positive results obtained from introducing infected freces into the alimentary tract of new born lambs are very significant. They prove beyond all possible doubt that infection can occur from ingestion and suggest that in lamb dysentery infection occurs after birth and through the alimentary tract.
1.
CONCLUSIONS.
Attempts to demonstrate by means of transmISSIOn experiments an infective agent in the uterus of ewes whose lambs had died from dysentery gave negative results. In the course of these experiments materials under test were introduced by the following paths. (a) Through the agency of the pregnant ewe i. By introduction into the vagina, in 3 experiments. 11. By ingestion, in 1 experiment. 111. By subcutaneous inoculation, in 1 experiment. iv. By intravenous inoculation, in 2 experiments. (b) Directly to the new born lamb by ingestion, in 1 experiment. 2. Negative results were obtained in attempts to infect lambs before birth by drenching pregnant ewes with freces and intestinal ulcers derived from natural cases of the disease in 4 experiments. 3. Negative results were obtained in attempts to infect newborn lambs with dysentery by soiling the umbilicus with freces from a natural case of the disease in 2 experiments. 4. Lamb dysentery can definitely be produced in new-born lambs by ingestion of freces from natural cases of the disease. Positive results were obtained in four out of seven experiments.
JOURNAL OF
COMPARATIVE PATHOLOGY AND
THERAPEUTICS. Vol. XLI.-No. 3.
SEPTEMBER 29, 1928.
STUDIES IN LAMB DYSENTERY.
PRICE 3s. 6d.
*
By
W. A. POOL, H. PRESTON,
A. BROWNLEE,
Animal Diseases Research Association of Scotland.
THE opmlOns held by different investigators concerning the nature of the dysentery of new-born lambs prevalent in the counties bordering Scotland and England have been very varied. The earliest view was that infection is acquired after birth by the ingestion of the causal agent, but whether infection occurs before or after birth, and in the latter event whether the path of entry is via the mouth or otherwise, are questions which have been disputed. Pathologists have even doubted if the disease is a specific infectious condition and capable of being transmitted directly from lamb to lamb. This uncertainty concerning the nature of the disease has led to revision of available data and has shown the need for further investigation. Different authors have obtained curiously varying results from transmission experiments.
* Received for publication,
14th March, 1928. A2
174
GENERAL ARTICLES.
When using material which might be expected to be most virulent, some have failed to set up the disease in experimental conditions even when the most likely paths of infection have been followed. The results now described are part of an investigation planned to review the whole question from the beginning. Much of the work at present in progress is still incomplete. It is deemed advisable, however, to publish certain parts which are complete as far as the material available in the 1927 lambing season is concerned. A subsequent paper will record the continuation and amplification of the inquiry in the next season's field work.
I. A
SEARCH FOR PATHOGENIC MICRO-ORGANISMS IN THE GENITAL ORGANS OF EWES WHOSE LAMBS HAD DIED FROM DYSENTERY.
For this work ewes were obtained from farms on which lamb dysentery had caused considerable losses. Ewes were obtained in the winter and spring of 1926-27, while in lamb or barren, which had lost their lambs from the disease in the 1926 lambing season. The barren ones were received at the laboratory before the rams were put out on the farms in the winter of 1920. Recently lambed ewes were dealt with as soon as they could be obtained after their lambs died from the disease in the 1927 lambing season.
Abbreviations used in the text.
Technique.
Culture Media Used. Robertson's bullock's heart medium with a BHM. liquid paraffin seal. pH 7·4 Ditto, plus 10 per cent. sterile unheated sheep serum, plus a piece of sterile unheated rabbit kidney, the depth of the column of liquid being about 7·5 ems. and a vaseline seal being provided. pH 7·4 Hall's glucose (0·2 per cent.) agar (1 per cent.) medium used in deep tubes for shake cultures. HSM. pH 7·4 Plain agar slopes. Blood agar slopes. About 30 per cent. of sterile sheep blood added to 3 per cent. agar when at 45 C., mixed and set • Incubated aerobically BAA. Incubated anrerobically BAN. Anrerobic conditions for the surface cultures were provided by M'Intosh and Fildes jars. The tubes of BHM and HSM were always boiled for 15 minutes and cooled immediately before they were inoculated. In each case at least three tubes of each type of culture medium mentioned above were inoculated from each tissue studied. 0
GENERAL ARTICLES.
175
The fleece over the whole of the ventral surface of the body was clipped. The ewe was fixed to a table with the ventral surface upwards and bled to death under chloroform anresthesia. The fleece was wetted with 2 per cent. lysol and a doth which had been soaked for 24 hours in 1 : 1,000 perchloride of mercury was spread over the body. A hole of such a size as to expose the operation area had been cut out of the centre of the cloth before it was disinfected. Wide lines were seared with red hot firing irons around the line of proposed incision. The abdominal wall was removed in one piece, the incisions being made with sterile instruments along the seared lines. In the case of barren ewes the vagina was clamped close to the os uteri and cut through distally with sterile scissors. The attachments of the uterus were similarly freed, and it was transferred immediately to a sterile glass dish with a cover. . The subsequent manipulations were carried out in an inoculating hood. The examination of ewes which had lambed recently and of ewes due to lamb was carried out at a field laboratory established on a farm in Roxburghshire. In the former case, after exposing the uterus as described above, it was seared with a hot firing iron and an incision was made with a sterile scalpel over the seared area. In the latter case the amniotic fluid was first aspirated with precautions to avoid contamination, so that after exposure of the lumen of the uterus there would be no fluctuation of the liquid and consequent possible infection from back wash. In all cases when uterine cotyledons were examined culturally pieces were removed and crushed with sterile sand in the improvised pestle and mortar composed of two modified test tubes described by Hagan:l A separate sterile improvised pestle and mortar was used for each manipulation. The Direct Microscopical Examination of Films Prepared from Uterine Exudate. In all cases the films were prepared within at least an hour of the completion of the autopsy. They were always prepared immedi~tely after the cultural work had been completed. The moist films were prepared by placing a drop of a suitable suspension on a thin slide, placing a cover glass in position, and sealing it with hard paraffin. They were examined under an oil immersion lens by dark ground illumination. Dry films were stained with warm 10 per cent. carbol-fuchsin and by the methods of Gram-Jensen & Leishman The results of the microscopical and cultural examinations carried out are given in Table 1. 1
J I. Exp. Med., Vol. xxxvi,
1922, p. 711.
176
GENERAL ARTICLES. CONCLUSIONS.
Direct observation of films from 11 ewes has given negative results. Observations by cultural methods were also negative except in two instances. As the growths in each of these were mixed and they were from ewes which had lambed a few days previously, it is considered that they were due to contamination or were of accidental occurrence in the tissues from which they were obtained.
II. TRANSMISSION EXPERIMENTS TO DETERMINE THE PATH BY WHICH LAMBS BECOME INFECTED WITH LAMB DYSENTERY.
The delay in acquiring accurate knowledge concerning Iamb dysentery has been caused by the short interval in each year in which natural infective material is available, and by the fact that in normal circumstances the only satisfactory test animal, the new-born lamb, is unobtainable throughout most of the year. Thus; by the end of the lambing season, when investigators have studied and selected the material available, it is not possible to buy lambs for experimental purposes. The alternative of maintaining a special flock of ewes of suitable breed at headquarters and arranging for them to lamb later than the normal season and over a more prolonged period, is only possible in special circumstances. To meet the requirements of the present investigation, Scotch Blackface ewes and a ram were obtained from an area remote from any history of Iamb dysentery infection and kept on clean ground until the lambs were born. The writers are indebted to Mr. T. M. Macdonald, of Barguillean, TaynuiIt, Argyllshire, for purchasing thirty (30) ewes from the neighbourhood of Oban and for presenting them with a reliable ram of the same breed. The ewes and ram arrived in Edinburgh in the autumn of 1926, and the ram was put with the ewes at intervals to provide a sequence of about two lambings a week from the middle of March to the end of June. The arrangement worked fairly well. Several of the ewes, as was expected, took the ram more than once. It was not always possible to forecast accurately when Iambs would be available to test particular materials, and in one or two instances when they were available the material required was unobtainable. When the transmission experiments were about half finished it was decided that every effort must be made to get more pregnant Scotch Blackface ewes, as there were insufficient lambs for the work which had been planned. Blackface ewes due to lamb early in May were accordingly purchased. They were said to have been put to a Blackface ram, but all the Iambs were Greyfaces (progeny of Border Leicester ram). The writers consider that the more robust Greyface Iambs were much more difficult to infect. The healing ulcers found in two of them, which would almost undoubtedly have recovered if they had been
GENERAL ARTICL ES
177
allowed to live, supports this view. The experiments with these lambs are included for the sake of completion.
Preparation of Material. Uterus.-C'otyledons were collected with pre<:autions to avoid
contaIIfination, ground with sterile sand, and suspended in sterile normal saline solution. Scrapings from the lumen of the uterus were mixed with this suspension. Intestinal ulcers.-Ulcers were separated from the healthy surrounding tissue with sterile instruments, washed in five changes of sterile normal salt solution, ground up with sterile sand, and suspended in sterile normal saline solution. Fa'ces.-F
(a)
Through the agency of the ewe. 1. With material from the, uterus of ewes whose lambs had died from dysentery. i. By Introduction into the vagina. Test Animals. Ewes 11, 14 and 17. Dosage. 20 c.c. of a turbid suspension. Age of material tested. Used immediately. Source of material. Ewe 100. Result. All three ewes lambed normally and the lambs remained healthy. 11. By Ingestion. Test animal. Ewe 27. Dosage. 250 c.c. of a turbid suspension. Source of material. Ewe B1. Age of material. 6 hours. It was sent by passenger train to Edinburgh from the field laboratory. Result. 1 lamb born. Remained healthy. 111. By Subcutaneous Inoculation. Test animal. Ewe 19. Source of material. Ewe B1. Age of Material. 6 hours. Dosage. 20 c.c. of a turbid suspension. Result. 1 lamb born. Remained healthy. IV. By Intravenous Inoculation.· Test animals. Ewes 20 and 26. Source of material. Ewe 101. Age of material. 18 hours (kept in cold store). Dosage. 20 c.c. of supernatant fluid after standing for 1 hour. Result. 1 lamb born to each ewe. Remained healthy. 2. With intestinal ulcers and with freces from lambs affected with the disease. By Ingestion. Using intestinal ulcers as test material. Test animals. Ewes 12 and 15. Age of material. Half an hour.
178
(b)
GENERAL ARTJCLFS.
Dosage. 100 C.c. of a turbid suspension. Result. One lamb born to each ewe. Both lambs remained healthy. Using freces as test material. Test animal. Ewe 28. Age of material. Under 18 hours. Dosage. 100 c.c. Result. One lamb born. Remained healthy. By direct infection of the lamb. 1. With material from the uterus of ewes whose lambs had died from dysentery. By Ingestion. Test animal. Lamb 49. Source of Material. Ewe Bl. Age of material. Two days (in cold storage at about 5° C.). Dosage. 5 c.c. of a turbid suspension. Result. Remained healthy. 2. With intestinal ulcers and with freces from lambs affected with the disease. 1. Through the umbilicus. Blackface Lamb 53. Dosage. Freces smeared over the umbilical cord. Age of material. Under 18 hours. Result. Remained healthy and was sold fat at 4 months old. Blackface Lamb 56. Dosage. 2 c.c. of frecal matter was inserted into umbilicus and a ligature was applied to prevent escape. The ligature was removed 6 hours later. Age of material. About 40 hours (last 24 hours in cold store). Result. The umbilicus became swollen but healed and the lamb was sold fat at 4 months old. 11. By Ingestion. (a) With Freces. Blackface Lamb 54. Dosage. 3 c.c. of undiluted freces. Age of material. Under 18 hours. Result. Well up to 4th day, when diarrhrea started and lamb was visibly ill. At times it lay on its side and appeared to be almost in a state of collapse. Its condition gradually improved and it finally recovered. Blackface Lamb 57. Dosage. 3 c.c. of freces. Material from the same sample was also smeared over the udder and teats of the ewe. Age of material. About 40 hours. Result. Well up to second day, when a yellowish-brown diarrhrea began. It died on the third day. Lesions. Typical L.D. ulcers were found in the small intestine. Greyface Lamb 63. Dosage. 3 c.c. per os, and udder and teats of ewe smeared. The dosage was repeated at the 24th and 48th hour. Age of material. Under 10 hours for first dose. Result. Well up to 3rd day, when it was dull, lying in a corner, unwilling to move and showing diarrhrea.
GENERAL ARTICLES.
179
There was steady improvement up to the 11th day, when diarrhrea and straining were still present. It was considered that the lamb might recover if allowed to live. As in that event there would be no knowledge about the result of the experiment, it was decided that the lamb should. be killed. Lesions. A few dozen healing ulcers were present along the whole length of the small intestines. Four small discrete healing ulcers were present in the large colon. Blackface Lamb 72. Dosage. 5 C.c. of freces per os, and teats and udder of ewe smeared. Repeated at the 24th and 48th hours. Age of material. About 6 hours for first dose. Result. Well up to the second day, when slight diarrhrea started. This continued till death occurred on the 4th day. Lesions. About the junction of the middle and lower thirds of the small intestine there was an ulcer 4 m.m. in diam. In the succeeding 5-ft. of intestine there was continuous ulceration. The remainder of the small intestine to the ileo-caecal valve showed diffuse inflammation. There were patches of inflammation in the large intestine. The kidneys were very congested. Greyface Lamb 68. Dosage. 5 c.c. per os, repeated at the 24th and 48th hours. The Ildder and teats of the ewe were not smeared. Age of material. From a lamb found dead. Date of death unknown . . Result. There was diarrhrea on the 2nd, 3rd, and 4th days. On the 6th day appeared to be healthy. Was killed and examined. Lesions. All organs healthy. Greyface Lamb 82. Dosage. 5 c.c. per os, and udder and teats of ewe smeared. Repeated at the 24th and 48th hours. Age of Material. Under 6 hours for first dose. Result. Diarrhrea from 2nd to 5th days. Killed on 7th day as appeared to be healthy. Lesions. There were 3 healing ulcers in the small intestine. Greyface Lamb 85. Dosage. 3 C.c. of freces per os, and udder and teats of ewe smeared. Repeated at 24th and 48th hours. Age of Material. From a lamb found dead. Date of death unknown. Result. There was slight diarrhrea on the second and third days. Remained healthy.
(b)
With washed ulcers. Greyface Lamb 71. Dosage. 5 C.c. of a turbid suspension per os, and udder and teats of ewe smeared also. This was not repeated. Age of material. 6 hours. Result. Lamb remained healthy.
180
GENERAL ARTICLES. TABLE
I
I
'I,,
No. of Ewe.
Dale 0/ Autopsy.
Breed.
!
·1
I.
1
History .
i
i
i
-100-1- Scotch
1- - ;;;1/2"7.- ,
_ _ _ ' _ _ Blackface ___ _ _
I
I
101
I, I
Scotch Blackface
I '
I
i
Lamb said to have died I Ba-r-ren-.-S-m-o-n-th-s i -N-o-r-m-al-.-N-o-c-hoc-,-o-Ia-te-,from L.D. in 1926. I since lamb died coloured exudate. !
id. t,'on lh9a2v6e. died , Due to lamb. Lfarmomb Lsa.D
- -B-2-.-II-SB-C01-atcc-hk-fa-cel
12/4/27.
1
- Lamb said to have died : Due to lamb. from L.D. in 1926.
1
1,'- - I 0 3I' s ,
-1'--7/4/27·
cotch Blackface
,i
I "
Lamb said to have died from L.D. in 1926.
- --1--- - ' - - - - ' - - - -- B1.
:
Cheviot
'
!
I, I \
B3.
I
-23/4/27.
Cheviot.
i
1I--1 i- --- ----iI '
!
B4.
Cheviot.
B5.
Cheviot.
23/4/27.
I
I
2/5/27.
I
1
!
!
Her lamb was found to be affected with L.D. on the 22/4/27 when said to be 5 days old.
: One lamb in uterus. No ' abnormality. There was no chocolate. coloured exudate.
i Now !:>3rren. Her \'
Normal. No chocolatecoloured exudate.
lamb had died
II
' 12 months pre· viously.
i
--~--,-------
Lamb said!o have di~d 1 from L.U. III 1925. Th,s , year her lamb died from ! L.D. when said to be 4 : days old. She was killed I the day after i.t died.
12/4/27. :
:1
I
One lamb in uterus. There was a chocolate.coloured exudate around some of i the cotyledons . The lamb was healthy.
, __________ i______ :
I
, I
'
7/4/27.
Appearance Of lumen 0/ ulerlls.
Present state.
I Some of the cotyledons of °bnroewnho,'nncowloeurre anddarka
1
I
yellowish.grey mucus covered some others. The cotyledons of the , other horn were dar k red , in colour and free from any coating of mucus. There was a chocolate. coloured exudate around the cotyledons.
I'
1_
_
_ _ _ _ _ _ _ _ _ __ __
Her lamb died on the 30/4/27 from L.D. when said to be 4 days old.
I
I !
I The cotyledons o f the right horn were dark
'
~~~b~~t~e~~o~~ ~~vecr~
or surrounded by any exudate. They were rather easily detached from the uterus. The cotyledons of the left horn were dark red in colour and there was no exudate.
II
1 1
_
Said to have lost twin I Normal. lambs in 1926 from L.D. i She was killed as soon as I her lamb was born. ' i _____.__________
I
[ 1- -B-7!" ,'
•
' Ii
Cheviot.
I 1
!
- II- La-m-b-w-as-sa-id-t-O-h-a-ve-:i I All the cotyledons showed died from L.D. when a brownish colouration. about 7 days old.. This , was four days before the I ewe was killed. I- - - - - -I- - - - - - - - -! 7/5/27. I' Lamb, died from L.D. [ The cotyledons were eov- ! when about 4 days 0 d. ~~"i:o~~~e~~u~~n:;~~ ! I or I date. This involved the b~~eh~:n:~e IUmen of
, I,'
I' ,
- -I 88.
-1·--7-/5~/2-7~.
-Ch-e-v-io-t
86.
~fe~ ili~s d~~:~ I:b~
I
I----~·II -----------------1 I,
Cheviot.
14/5127.
I I
Lamb died from L.D .• said to be about 4 days I old. Ewe was killed the day arter the lamb died. !
[I
There was a chocolate. coloured exudate arounj the cotyledons.
181
GENERAL ARTICLES. TABLE
L
Micr'!!..~~m... Exami':!!!ion. _______ '
Cultural Examinatio'!:. ____ Foet us. Ewe. I Foet us. , S~;;g;j;:;;;;;-i;;n-;;;-;;-;i;;;·-1 Scrapings of sto-;;'ach , - - - - - - - - - contents by dark, Crushed cotyledon Stomach: Dark RTDund ground illumination. , from uterus. :, contents. Stained. illumination. I I Neg. I Neg-.- - - - j - - - - - - - - - I "N-;g-. '1 _ _ _ _ _ _
1
Ewe.
I
--I
I
I
---------,\'-----1
,----~--- ·--~---i--~------II------------':-----Neg. i Neg. Neg. , Neg.
1
:
'
I Neg.
I,-------,----,----------,-----_·_---,---1 I· I
Neg.
!
I
INeg. ;
'
' Neg.
I
I
'
Neg.
'
----I Neg~--.--1-------1
---I
I
i
iN-;g-.
I
Neg.
I
1
I
Neg- : - - - - - - - - - -
:
I
rN~--I- ---
I
N~
;
I
I
----!
I I
I i i
I
'
1
I I ' I I
Neg.
I
Neg~-----i-------
Neg.
i
---I ""~---- --I--
I
[!
I
.
' - _ · __ ·_------1-----', : \ - - - - - - - -" - - - - - - - , !- - - - - - - - _ · _
i
I Neg.
I
'I,
I Neg.
Neg.!
!N-;g-:-----rN-;g~---I,--------1 Two
,
I
' I ''
'
,
I
I
,
I
!
, I
i
I
'
I
'-·----I------__________ ,
Neg
I
,
I
---I N~
Neg.
i,
'
other media rem~n.:d~~rile·l----, Neg. :
1
I
• _ _ _ _ _ .1
'-N-eg-.
out -.;J;;;;-SR.M.';-j'----: One con" tained a bacillus like B. wel- i chii, a coliform bacillus, and I a streptococus. The other ' tube showed a slender,Gram- I positive, non-motile bacillus, I' a short stout, Gram-positive non-motile bacillus, and 1 cocci. One tube out of 6 H.S.M.' s showed growth. There was a bacillus like B. welchii, a smaller and more I slender Gram-positive bacillus, and a coliform bacilluo. I No growth occurred on the B.A.N.tubes till the third day, j when a greyish thick, white film appeared in one out of 6 , tubes. These cultures were I not studied further. All the
, showed growth.
I
Neg~------ ----I ---
I ' -N-e-g-.----! Neg. I
----I
•
'
I I,
I
----------
Lefthorn.-l out of 6 B.H.M.s
I·
1------1
showed a bacillus like B. welchii. Right horn.-2 out of 6 B.H.M.'s showed growth I consisting of a long, slender, Gram-positive,gas-producing bacillus in one and a mediumsized motile bacillus in the , other. These cultures were not studied further. All the other media remained sterile.
I
._-
_.
'I'
' I , '[
!
I
I
II
20/4/27.
19/4/27.
26/4/27.
13/6/27.
7/5/27.
16/6/27.
6/5127.
21/4/27.
23/4/27.
1516127.
6
4
10--12
17
II
16
20
20
10--11
6/1/27.
6/1/27.
14/4/27.
12/4/27.
8/4/27.
12/4/27.
18/4/27.
18/4/27.
11/4/27.
14
17
27
19
26
20
12
15
28
~--.
Dateo of Lambing.
6-8
Week of Gestation at the time of dosing.
6/1/27.
Date of Dosing.
11
Number of Ewe.
TABLE
II.
+ + +
By introduction into the Vagina.
+
By Ingestion.
+
Subcut.
+ +
Intraven.
By Inoculation.
Through the Agency of the Ewe.
I
I
I
I
Ewe by Ingestion of
+ +
Ulcers.
I
-'-
Faeces.
I Through the Agency of the
A Mixture of ground cotyledons and scrapings from the lumen I of the uterus. I
Table Showing Results of attempts to Infect Lambs with Dysentery through the Agency of the Ewe.
Result.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
Scotch Blackface.
Breed.
r.j
rn
rl r
~
~
~
...... ~
Date of Dosing.
S.B.F.
S.B.F.
G.F.
S.B.F.
G.F.
G.F.
G.F.
G.F.
G.F.
S.B.F.
Under 8
Under 8
Under 8
4
Under 8
Under 8
Under 8
Under 8
4
20/4/27.
21/4/27.
30/4/27.
6/5/27.
2/5/27.
12'5/27.
21/5/27.
5/5/27.
11/5/27.
6/5/27.
15/5/27.
54
57
63
72
68
82
85
71
80
75
83
G.F.
S.B.F.
I
+ +
'I
-I-
+
+ +
' . 1 LD UnfIltered Faeces OJ • • Lamb. B Through Y. I Umbilicus. IngestIOn.
+ +
of L.D. Lambs I by Ingestion.
I Filtered Faeces
+ +
Tri/ urated I1lcers.
I
I
.
Results.
+
+
I I
I
+
+
+ +
+
+
+ +
+ + +
+
Died. Killed. ljtm'f/;ed ea Y, _ _ _ _ _ _.
GF=Greyface (Border Leicester-Scotch Blackface).
Suspension of cotyledon of Ewe .by IngestIOn.
SBF=Scotch Blackface.
2
21/4/27.
56
S.B.F.
20/4/27.
53
, S.B.F.
I
I
14/4127.
Under 8
I
Breed.
49
----~-
I .
i
l
I
4 e of Lamb n
I' g HOUTS.
'I
----- -------
1
ljuLmbebr OJ am.
I-~--~-------
III.
Table Showing Results of attempts to Infect Lambs with Dysentery.
TABLE
+
+ + +
Lesions of L.D.
______
I
00 <:0
......
~
t"'
(")
i§ C)
184
GENERAL ARTICLES.
Greyface Lamb 80. Dosage. 5 C.c. of a turbid suspension per oSi,lld udder and teats of ewe smeared. Repeated at the 24th, 48th, and 72nd hour. Age of material. 1 hour for first dose. Result. Killed at tenth day, as was healthy. Organs all normal. (c) With sterile filtrates of fresh freces. The freces were passed through sand and pulp and then through a Seitz filter. About 1 C.c. of the filtrate was inoculated into B.H.M. and failed to grow. Greyface Lamb 75. Dosage. 5 c.c. per as, and smeared over udder and teats of ewe. This was not repeated. Result. Remained healthy. Blackface Lamb 83. Dosage. 5 c.c. per as, and smeared over udder and teats of ewe. This was not repeated. Result. Remained healthy. These results are shown in Tables 2 and 3. In the course of the work the evidence obtained always appeared to negative the possibility that infection occurs in utero. The only factor suggestive of any abnormality within the lumen of the uterus was the presence of the chocolate-coloured exudate around some of the cotyledons in a few of the cases. The positive results obtained from introducing infected freces into the alimentary tract of new born lambs are very significant. They prove beyond all possible doubt that infection can occur from ingestion and suggest that in lamb dysentery infection occurs after birth and through the alimentary tract.
1.
CONCLUSIONS.
Attempts to demonstrate by means of transmISSIOn experiments an infective agent in the uterus of ewes whose lambs had died from dysentery gave negative results. In the course of these experiments materials under test were introduced by the following paths. (a) Through the agency of the pregnant ewe i. By introduction into the vagina, in 3 experiments. 11. By ingestion, in 1 experiment. 111. By subcutaneous inoculation, in 1 experiment. iv. By intravenous inoculation, in 2 experiments. (b) Directly to the new born lamb by ingestion, in 1 experiment. 2. Negative results were obtained in attempts to infect lambs before birth by drenching pregnant ewes with freces and intestinal ulcers derived from natural cases of the disease in 4 experiments. 3. Negative results were obtained in attempts to infect newborn lambs with dysentery by soiling the umbilicus with freces from a natural case of the disease in 2 experiments. 4. Lamb dysentery can definitely be produced in new-born lambs by ingestion of freces from natural cases of the disease. Positive results were obtained in four out of seven experiments.