Contagious Pustular Dermatitis of the Sheep

318

GENERAL ARTICLES.

CONTAGIOUS PUSTULAR DERMATITIS OF THE SHEEP.

By R. E. GLOVER, Department of Animal Pathology, Cambridge. (Received for publication 29th August, 1928).

CONTAGIOUS Pustular Dermatitis or Stomatitis (Contagious Ecthyma) of the sheep is a malady with which flockownets in many parts of this country are well acquainted, and several excellent clinical descriptions of the disease have appeared in the literature from time to time. The pustular or ulcerative lesions which characterise this disease may show considerable variations in their severity, and all gradations may be encountered from the mild form in which the pustules are strictly localised to the lips and the surrounding tissues, to the. acute form in which severe lesions occur on the mucous membrane of the mouth, the vulva, and the cornea, and on various parts of the body, particularly the regions of the coronet and the tail. Sheep of any age may be attacked, but the disease is most frequent amongst weaned lambs up to one year old. It is not unlikely that this Contagious Ecthyma of the lips of older lambs is closely related to Contagious or Malignant Aphtha, a disease which affects ewes and sucking lambs. In these outbreaks the lesions in the lambs take the form of an extensive ulceration of the nose and lips, whilst a similar eruption is seen in the ewes on and around the teats. The mammary gland frequently becomes the seat of an acute inflammation. The earlier investigators clearly established the contagious nature of the disease under both natural and experimental conditions. They recognised that once the disease arose in a flock it usually spread with great rapidity, and that the recovered animals acquired a considerable degree of immunity against further attacks. Apparently they were of the opinion that the probable cause of the disease was the B. necrophorus, but there is no direct evidence to lead one to suppose that this organism was other than a secondary invader. It is beyond the scope of the present paper to consider the views of these early observers, details of whose work can be found in the concise article on . Contagious Pustular Dermatitis written by Wallis Hoare.1 During the past decade, however, the study of these eruptive diseases has been undertaken from a fresh point of view. It has been shown that they fall within the class of virus diseases which have a special affinity for epithelial tissue, and that, in this respect, they are closely allied to the vaccinia-variola group. It would appear that the credit for this conception of the disease should be given to Aynaud,2 who has furnished a very complete description of a condition of sheep which he terms "Contagious Pustular Stomatitis." It is the cause of considerable losses in sheep in France, particularly amongst fat lambs which are being prepared for the butcher. In his article Aynaud mentions a benign and a severe form of the disease. In the former the lesions,

GENERAL ARTICLES.

319

whic~ take the form of thick crusts, are essentially confined to the lips and adjacent tissues, whereas in the latter they invade the buccal cavity, producing an extensive stomatitis. He is of the opinion that these two conditions are different manifestations of one disease caused by a single virus which evokes a vesico-pustular. eruption presenting a close analogy with vaccinia. The buccal lesions are inoculable on the skin, where they lead to the production of lesions that have a highly characteristic. appearance, passing through the stages of macule, papule, vesicle, pustule, and scab. This investigator furnishes definite proof that a filterable virus exists in great abundance in the pustules and that this virus has marked powers of resistance to desiccation, the dried crusts remaining fully virulent for at least twelve months. A method of vaccination is indicated whereby an emulsion in glycerine of the powdered dried crusts, attenuated by chloroform, is applied to the scarified skin of the thigh. The local lesion which subsequently develops induces a solid immunity. Under the designation "Contagious Ecthyma of the Lips of Sheep," Moussu 3 gives a description of a seasonal disease which occurs chiefly in summer. This condition is usually moderately benign although a considerable mortality sometimes follows through secondary complications. Moussu is of the opinion that the name " Contagious Pustular Stomatitis" applied to the disease by Aynaud is a misnomer because the lesions can be strictly localised to the lips without the slightest evidence of a stomatitis. Aynaud points out, however, that the severe and the benign form are due to a single specific virus, and that, under certain circumstances, the benign form of the disease may be transformed into the more severe type. Lanfranchi4 and his co-workers have reported the existence of a disease in Italy which appears to correspond with the condition occurring in France. They describe experiments dealing with the filtrability of the virus and the immunity which follows the natural and the experimental disease. They affirm that the immunity is not always of long duration and may disappear in from five to eight months after infection. Contrary to the findings of other investigators, they claim that young rabbits can be infected with the virus. In 1921 Blanc, Melanidi, and Caminopetros5 carried out an investigation into an analagous condition amongst goats in Greece. This eruptive disease of the lips, which is caused by a filterable virus, can be reproduced experimentally in both the goat and the sheep. Following the procedure of Aynaud, they reproduced the condition by the application of fresh or dried crusts to the scarified skin of the thigh. The development of the characteristic vesico-pustular eruption led them to believe that the disease should be included in the vaccinia group. They noted that sheep that were immune to sheep-pox gave a typical reaction to the inoculation of this goat virus and vice versa. Blanc, in the course of some experiments with the virus of Aynaud, concluded that the disease of the sheep is probably identical with that of the goat. Confirmation of these observations is afforded by Jacatot,6 who has found that" Maladie du Chancre," or Contagious Ecthyma of the lips, is comparatively common amongst goats in Annam, attacking chiefly the young stock and sometimes producing a high mortality. Whilst the local breed of goat is particularly susceptible, cases are not encountered amongst the sheep. Jacatot has investigated this disease and considers that it corresponds in every respect with that of the sheep describep by Aynaud. Both Blanc and Aynaud are of the opinion that a form of variola in the goat described by Zeller 7 in South-west Africa is probahly identical with pustular stomatitis of sheep and goats.

820

GENERAL ARTICLES.

The present investigation was undertaken in order to obtain information on the occurrence of eruptive diseases of this type amongst sheep and lambs in this country and to determine, if possible, whether such outbreaks bore any resemblance to the virus disease described by Aynaud. The occurrence of three outbreaks in different localities has provided the opportunity of making this comparison, and it will be seen that the experimental evidence is strongly suggestive of a close analogy between the disease in this country and that which occurs on the Continent.

The Natural Disease. Outbreaks of the disease are not confined to any particular period of the year, but seem to be more prevalent during the spring and the summer months. Lambs are sometimes attacked within a few weeks of birth, but in many cases outbreaks occur at a later period amongst older lambs under one year of age. The advent of the disease in a flock does not lead to a high mortality, but there is a considerable loss in condition which may extend over a period of five or six weeks, a loss which is all the more serious in that the lambs which are being fattened seem to be particularly prone to infection. The lesions are usually localised to the nostrils and the external aspect of the lips, particularly to the commissures. The eruptions, which commence as discrete vesicles or pustules surrounded by a marked inflammatory zone, are succeeded by small erosions which gradually spread in a circular manner. These ulcers, which occasionally exhibit a tendency to persistent hremorrhage, are covered by an abundant exudate which later becomes desiccated to form thick yellowish-black crusts. In most cases these scabs are shed and the underlying ulcerating surface undergoes cicatrisation until no trace of a lesion can be detected. Sometimes, however, there appears to be a continued proliferation of the epithelium, leading to the formation of a dense wart-like outgrowth having a superficial resemblance to a papilloma. These growths, which may attain the size of a filbert nut, persist for several weeks but are eventually resorbed. The lesions show little or no tendency to extend to the mucous membrane of the buccal cavity, but they may spread to the tissues immediately adjacent to the lips. In these benign outbreaks the lesions are confined to the lips, but occasionally the disease assumes a more serious character. The lesions then spread to other parts of the body, such as the coronet, the under surface of the tail, the thighs, and the axillre. In these positions, the lesions, which at the outset are small pustules, rapidly increase in size to form large fungoid masses which bleed very rapidly. The disease is, without doubt, spread by means of the crusts; which are highly infective, and once the malady has made its appearance in a flock few of the lambs are likely to escape. The absence of lesions in the ewes is, in all probability, dependent upon an immunity which has been acquired during early life, for there is no evidence to show that adult animals are naturally less susceptible to the disease than the younger stock.

GENERAL AR'rICLES.

S21

The Experimental Disease. It has been shown by the investigators to whom reference has been

made that the crusts which occur around the lips are inoculable on the skin, where they produce a highly typical eruption. The lesions can be produced on any part of the body, but the region where the skin is smooth and devoid of wool is the most suitable site for this purpose. In the first outbreak dealt with in this investigation a large number of lambs were affected with fungus-like growths around the lips and the regions of the tail. An attempt was made to reproduce the disease in experimental animals, but in no case was it possible to obtain the characteristic vesico-pustular eruption owing to the rapid formation of a crop of pustules within twenty-four to forty-eight hours. These pustules, which yielded a pure culture of Staphylococcus albus, undoubtedly arose from the presence in the original lesion of this organism as a secondary invader. The following experiments illustrate the nature of these lesions.

Experiment 1. May 5th, 1926. About 0·2 gm. of crusts from a natural case was emulsified in 15·0 C.c. of normal saline solution. The mixture was set aside for one hour in order to allow the coarse particles to settle, after which the supernatant fluid was withdrawn. Lamb No. 63.-The skin over the inguinal region of both thighs was cleansed , several superficial scarifications were made with a scalpel, and a few drops of the supernatant fluid were applied with a swab. The following day a confluent pustular eruption had formed along the lines of scarification. By the fourth day these pustules had opened, liberating a thick tenacious yellowish exudate, which dried to form a thick yellowish-brown crust. Experiment 2. May 26th, 1926. An emulsion of crusts from a natural case of the disease was prepared as in the previous experiment. Lamb No. 64.-The skin over the inguinal region was scarified, and the supernatant fluid from the emulsion was applied. The following day an almost continuous pustular eruption appeared along the lines of scarification. On the third day it was noted that between the pustules, which had now attained the size of a pea, there were areas in which the skin was slightly raised and reddened. A scraping which was taken from one of these points, avoiding, as far as possible, the surrounding pustules, was emulsified in normal saline solution. Applied to the scarified thigh of a fresh Iamb (No. 65), this material gave rise to a violent pustular eruption at the twentyfourth hour. As a further supply of fresh tissue was not available, these experiments had to be abandoned until a later date when another and more benign outbreak furnished an opportunity for the continuation of the investigation. Material was obtained from a flock in which the disease appears regularly each year during the summer months. The lesions, which were mainly confined to the fat lambs and were always comparatively benign, took the form of wart-like growths around the lips, and did not show the slightest tendency to spread to the mouth or to other parts of the body.

822

GENERAL ARTICLES.

A portion of one of these lesions was emulsified in normal saline solution and applied to the scarified thigh of a lamb, but, as in the previous experiments, a violent pustular eruption was produced in less than forty-eight hours. The inoculation of fresh material, having proved unsatisfactory, was abandoned and the effect of the application of dried crusts was investigated. The resulting vesico-pustular eruption was so characteristic that in subsequent experiments this method has been employed almost exclusively. In order to obtain information on the relative potency of the crusts and to check the degree of the immunity which follows the experimental disease, known dilutions of the powdered crusts have always been utilised. The method adopted, which was based on that suggested by Gordon 8 for the titration of vaccine virus in the rabbit. was as follows : The scabs from a fourteen to eighteen days old lesion were finely minced and placed over sulphuric acid in a desiccator for twenty-four hours. The partially desiccated tissue was then ground to a fine powder and dried for a further period of forty-eight hours. This powder, when stored in sealed tubes in the cold room, remained consistently virulent over a long period of time. When required for use, a weighed amount-usually 0·05 gm.-was transferred to a mortar and made into a fine paste with distilled water. Sufficient water was added to make a dilution of 1/100 and the emulsion was allowed to stand for two hours. This suspension was then further diluted to 1/500 with normal saline solution and Hghtly centrifuged for five minutes. From the slightly opalescent supernatant fluid, which was free from coarse particles, dilutions of 1/1,000, 1/5,000, 1/10,000, 1/50,000 and 1/100,000 were made in normal saline solution. The requisite number of scarifications having been made on the cleansed and dried skin of the thigh by means of a vaccino-style, the dilutions were applied by means of small sterile swabs. Since in a large series of experiments the lesions which follow these inoculations have shown a marked uniformity, one example will serve to illustrate the very characteristic eruption which is produced on the thigh. Lamb No. 23. November 11th, 1927.-Dilutions of 1/1,000 to 1/100,000 prepared in the manner indicated above were inoculated on the thigh of this lamb. During the first three days following the inoculation the lines of scarification showed no change. On the fourth day the lines were slightly raised and reddened. On the fifth day they became very distinctly raised above the surface of the skin and deeply congested. On the seventh day this papular stage was followed by the formation of small vesicles containing a trace of a clear exudate. These vesicles were whitish in colour and possessed umbilicated centres. The vesicular stage was very transient, as on the following day there was a marked increase in the exudate, which became purulent. On the twelfth day the pustules ruptured, liberating a yellowish discharge, which dried to form thick brownish-black crusts. These scabs were adherent to the skin for several days, but by the twenty-fourth day they had fallen, leaving a slight cicatrix. This reaction was obtained with all the dilutions up to 1/50,000 but not in the 1/100,000 dilution.

Gf
323

The negative phase during the three days following the inoculation is in great contrast to the reaction following the cutaneous inoculation of the pyogenic bacteria, which, as the preliminary experiment indicated, gave an inflammatory reaction that was most marked during the first three days. The dried crusts have usually given a reaction in a dilution of 1/50,000 and occasionally in 1/100,000. Isolated lesions can be obtained bv the intradermal inoculation of the virus, but thevare rather less chiracteristic than the cutaneous lesions. The virus 'can be transmitted to the lips by the inoculation of the dried crusts from the lip or thigh lesions, but in this position scarification is not always successful, owing, no doubt, to the ease with which the virus may be carried away in the saliva, but intradermal inoculation is always followed by a lesion, even in a dilution of 1/50,000. As in the case of the thigh, there is a latent period of about four days. Commencing on the fifth day, the lip at the site of inoculation becomes swollen, and by the seventh day a hard nodule about the size of a pea can be felt at this point, whilst the surrounding tissue is distinctly cedematous. This nodule develops into a pustule, which ruptures and is succeeded by a small ulcer covered with a tenacious yellowish exudate. In some cases the exudate dries in the form of a thick crust beneath which healing occurs fairly rapidly, but sometimes there is a continued proliferation of the epithelium which leads to the production of a wart-like outgrowth. After several passage inoculations on the thigh the virus can bere-transmitted to the lip, giving rise to an eruption which resembles the natural disease in every respect. The lesions, at least in the experimental disease, remain localised to the point of inoculation. Moreover, the virus does not appear to pass into the blood stream. The following experiment shows that over a period of ten days the blood was consistently non-infective. Lamb No. 16S. May 14th, 1927.-This lamb was inoculated on the scari-

fied thigh with a virus emulsion. A typical eruption resulted, with the formation of pustules on the ninth day. On the day following the inoculation and every day subsequently until the tenth day, 5·0 C.c. of blood were withdrawn from the jugular vein, defibrinated, and 0·5 c.c. inoculated intradermally in the thigh of Lamb No. 105. These inoculations were all negative. This lamb, re-tested at a later date, reacted to a 1/50,000 dilution of dried virus.

Contrary to the experience of Aynaud, lesions have never been obtained on the scarified cornea with this particular strain of virus. The subcutaneous or intravenous inoculation of relatively large doses of the virus does not set up the disease but appears to confer a considerable degree of immunity. Thus, two lambs which received 5·0 C.c. of a 1/100 dilution of dried virus subcutaneously and intravenously respectively failed to react.' They showed no rise in tempera. ture and no sign of a local lesion. Re-tested on the skin one month later, both lambs were immune to a 1/1,000 dilution of virus. Since a naturally immune animal has not yet been found amongst the experimental stock, it is reasonable to suppose that the immunity resulted from these inoculations.

324

GENERAL ARTICLES.

Apart from the sheep, the goat appears to be the only animal which can be infected with this virus. Goat 1. September 22nd, 1927.-This goat was inoculated on the thighs with varying dilutions· from 1/100 to 1/100,000, and intradermally in the lip with 1/1,000 dilution of dried crusts from Lamb No. 161. A typical vesicopustular eruption was produced on the thigh with the 1/10,000 dilution, but not with the 1/50,000. On the lip, a small pustule formed on the fifth day and ruptured on the seventh day. The lesions, although somewhat less severe than those seen in the sheep, were, nevertheless, quite typical. The virus appears to be strictly specific to the sheep and the goat. All attempts to transmit the virus to laboratory animals have failed. The rabbit, guinea-pig, mouse, fowl, and pigeon are entirely refractory to the cutaneous inoculation of the virus. Intratesticular, subdural, corneal, and intravenous inoculation of the virus with a concurrent scarification of the skin have led to negative results in the rabbit and guinea-pig. It is fortunate that, as in the case of vaccinia infection in the rabbit, it)s possible to make multiple inoculations on the skin of a sheep. The lesions which develop do not interfere with one another and it is thus possible to test a large number of dilutions of virus on the one animal. HISTOLOGY.

The natural lesions can scarcely be held to represent a pure virus infection on account of their frequent invasion by various microorganisms. On the other hand, the experimental lesions which follow the inoculation of the thigh with an emulsion of the dried scabs are relatively free from this complication and are, therefore, more suitable for a study of the histo-pathological changes which are caused by this virus. Accordingly, in order to determine the tissue response at the various stages, a series of sections were prepared from thigh lesions excised under a local anresthetic at different phases of the disease. Mention has already been made of the analogy between the lesions of this disease and those of the vaccinia-variola group. This similarity is clearly shown in sections of the different stages up to and including that of the scab formation. The papillomatous growths, which occur on the lip and in the region of the tail, represent a further phase and are of a different nature. In considering the experimental lesions it is convenient to recognise three stages, corresponding approximately with the papulo-vesicle, the vesico-pustule, and the scab.

First Stage.

(Papulo-vesicle.)

The changes which are characteristic of this phase can be seen in Fig. 1, which shows a section from a thigh lesion excised at the fifth day of the eruption. The epidermis in this region is composed of a stratified epithelium which is normally three to four cells in thick.

325

GENERAL ARTICLES.

l. H:emalum and eosin.

FIG.

Lesion on thigh, five a.ays.

X

60.

ness. A portion of normal skin is seen in the right hand corner of Fig. 1. The earliest change to be noted is a commencing proliferation of the cells of the rete Malpighii. In the more superficial zone, which seems to correspond with the stratum granulosum, the cells, instead of appearing elongated, become distinctly swollen and rounded. The nucleus shrinks, forming a dense mass of chromatin which stains intensely with h~malum, and the cytoplasm shows a number of vacuoles of irregular size and shape. The vesicles arise between the more superficial cells immediately beneath the stratum lucidum and are represented by collections of polynuclear leucocytes. A small vesicle can be seen at the top of Fig. 1. It is evident that, even at this stage, the dermis is also involved. There is a commencing infiltration with polyneuclear leucocytes which is most marked in the proximity of the vesicles.

Second Stage. (Vesico-pustule.) During this phase there is a continued proliferation of the cells of the rete Malpighii, resulting in the production of an extensive zone of vacuolated cells, as shown in Fig. 2. The cells in this area are undergoing still further degeneration, forming an irregular network which bears a strong resemblance to the " ballonisante " stage seen in the lesions of vaccinia. The nucleus becomes entirely disrupted into a collection of small, deeply-stained granules, whilst the cytoplasm is reduced to a mere cell outline. The line of demarcation between the zone in which the cells are in the early stage of this degeneration and the zone showing complete disintegration of the cells is extremely sharply defined (Fig. 3). There is no evidence of intracellular particles of the nature of the inclusion bodies of Guarnieri. The vesicles I.'.

326

GENERAL ARTICLES.

FIG. 2. Lesion on thigh, nine days. Hiremalum and eosina.

FIG. 3. Lesion on thigh, nine days. Ha!malum and eosin.

X

X

65.

350.

327

GENERAL ARTICLES.

FIG. 4. Lip lesion, eighteen days.

Normal lip.

Hremalum and eosin.

FIG. 5. Hremalum and eosin.

X

X

60.

60.

BIZ

828

GENERAL ARTICLES.

have now become considerably larger, and, as the lesions become older, the migration of leucocytes continues until the vesicle changes to a pustule containing a debris of epithelial cells and leucocytes with a few micrococci. During this process, provided there is no gross bacterial invasion, no breach is formed in the Malpighian layer, the cells of which are still in an active stage of mitosis. The invasion of the corium by polynuclear leucocytes becomes more intense. These cells are not only found in the connective tissue immediately adjacent to the rete Malpighii but also infiltrate the muscular layer.

Third Stage. (Scab.) During this phase the pustule enlarges and finally breaks through the stratum lucidum. The crusts are formed from the cellular debris which is thus liberated, fibrin, and the remains of the stratum lucidum. In some places, owing no doubt to the more intensive invasion of the breach thus formed by the various bacteria, the epidermis is completely disorganised. The crusts then cover a mass of epithelial cells in various stages of degeneration, intermixed with polynuclear leucocytes and young fibroblasts. Healing now takes place beneath the crusts by regeneration of the epithelium and gradual resorption of the cell debris. A comparative examination of the early stages of the thigh lesions with those. of the lip shows that, up to about the fifteenth day, there is a substantial similarity in the tissue changes. In the case of the lip, however, there is a further phase which may be termed the stage of epithelial proliferation and which terminates in the formation of the papillomatous growths. The commencement of this process can be seen in Fig. 4, which represents a section from an eighteen day lesion. This should be compared with Fig. 5, a section of the normal lip. Beneath the "scab," which is composed of a superficial layer of fibrin and a deeper layer of cellular debris derived from the putsule, the cells of the rete mucosum have sent down finger-like processes into the corium. At the same time the cells of the glands and the papillre, which up to this point have not taken part in the process, have begun to multiply and have united with the downgrowing epithelium from the rete. The papillomatous growths seem to arise from the fusion of these epithelial masses, which in the course of their extension cut off groups of leucocytes and young fibroblasts. Finally, the leucocytes are entirely absorbed, leaving either a fibro-cellular stroma invaded by irregular masses of epithelial cells or islands of connective tissue entirely surrounded by an epithelial layer. Fig. 6, which represents a section from a dense wart-like growth on the lip from a natural case, exhibits the latter phase. This production of a connective tissue stroma apparently embedded in a ring of epithelial tissue is not the result of a transverse section of the lesion but is due to the fusion of groups of proliferating epithelial cells. The appearance of the warty excrescences at the commissure of the lip is shown in Fig. 7, which is a photograph of a natural case.

329

GENERAL ARTICLES.

Resistance. The virus shows a very marked resistance to desiccation. The crusts from the lesions, powdered and dried over sulphuric acid and stored in sealed tubes, remain fully virulent over a long period. This method of preservation seems to be preferable to any other for the maintenance of a supply of virus of constant potency. A few experiments on the action of glycerine, liquid paraffin, chloroform, and ether demonstrated that when stored in sealed tubes of 25 per cent. glycerine in normal saline solution the crusts are still fully virulent after six weeks, but that after four months there is a considerable decrease in potency. This loss in infectivity is still more marked when 50 per cent. glycerine in saline is used. The virus persists unaltered in liquid paraffin for at least six weeks. Chloroform and ether slowly destroy the virus, as shown by the fact that a suspension of crusts in a 10 per cent. solution of these reagents, which originally gave a reaction in a dilution of 1/50,000, only reacted slightly in a 1/1,000 dilution after eight days.

FIG .

Lip lesion, natural case.

6.

Hremalum and eosin .

X

65.

Experiment 1.-Resistance to desiccation.

Lamb 161. August 5th,·1927.-This lamb was inoculated on both thighs with a 1/1,000 dilution of dried crusts . A typical vesico-pustular reaction followed, with the formation of scabs on the fourteenth day. These crusts, dried over sulphuric acid and powdered, were stored in sealed tubes in the cold room. One unsealed tube was left at laboratory temperature.

I

22-6-28

30-8-27 11-11-27 21-2-2R 21-2-28

Date.

I

---._--

145

Stored at 0° C.

194 23 110 left side 110 rt. side

temperature Stored at 0° C.

" " Stor~d at L~b.

Virus 161

Lamb No.

+ + + + +

1/1,000

+ + + +? +

1/5 ,000

[

i

!

I

I I

+

-

+ + +

1/10,000

+

-

I

!

'

I

I

!

_

-

-

-

-

+ + +

lntmal.

0 days 73 days 175 days

175 d ays ! 296 days

I II

i I

11/100,000;

1/50,000

-----------------

The potency of the virus was tested at intervals as follows :-

I

I

I

~

~ en

is

~

~

~

~

331

GENERAL ARTICLES.

Glycerine and Liquid Paraffin. Experiment 2. November 2nd, 1927. The powdered and dried scabs from Lamb 161 were made up in a 1 (500 dilution in 25 per cent. glycerine, 50 per cent. glycerine, and liquid paraffin respectively. These emulsions were stored in sealed tubes at 0° C. and tested on the skins of lambs at various intervals with the following results ;Date.

Lamb No.

16-11-27

I

Virus 161.

Dilution.

107 (rt. side)

In 25% glycerine

107 (left side)

Dried virus

1/50,000 1/100,000 1/50,00 1jlOO,000

.. 16-11-27

18-12-27

----. 7-3-28

106 (rt. side)

In 50% glycerine

106 (left side)

In liquid paraffin

122 (rt. side)

In 25% glycerine

122 (left side)

In liquid paraffin

109 Crt. side)

In 25% glycerine

109 (left side)

In liquid paraffin

1/1,000 1/5,000 1,'50,000 1/100,000 1/5,000 1/10,000 1/50,000 1/100,000 1/500 1/500 1/1,000

Interval in days.

Reaction.

+ + -

14

+ + -

14

+ + -

46

-

-

-

-

+

126

-

Chloroform and Ether. Experiment 3. A 1/500 emulsion of dried crusts in normal saline was prepared. 10 per cent. chloroform was added to the emulsion and the tube was sealed and allowed to remain at laboratory temperature for twenty-four hours. The chloroform was driven off in the incubator and Lamb 103 inoculated with the emulsion. Three emulsions of dried crusts in saline (1(500) were prepared. 10 per cent. chloroform was added to one, 10 per cent. ether to the second, and the third served as a control. After eight days in the cold room the tubes were placed in the incubator for 2 hours, and the emulsions tested on Lambs 127 and 128. \

Date.

Lamb No.

11-10-27

103

11-10-27

101

20-12-27

127 rt. side 127 left side

20-12-27

128 rt. side 128 left side

~.----~~.--.-

Virus. Virus

+ CHela

Virus only Virus

+ Ether

Virus only Virus

+ CHela

Virus only

Dilution. 1/10,000 1/50,000 1/50,000 1/100,000 1/1,000 1/5,000

1/50,000 1/100,000 1/1,000 115,000 1/50,000 1/100,000

Re-action.\

+ + + + + + -

Interval. 24 hours

-

24 hours

.8 days

8 days \

332

GENERAL ARTICLES,

Filtration Experiments, The marked similarity between the papulo-vesicular lesions in this disease and the eruptions in vaccinia suggest the action of a filterable virus, and it is not surprising that Aynaud was able to establish the presence of a filterable agent in the lesions. He states that, as in the case of vaccinia, the virus passes through bacterial candles only with difficulty; moreover, he found that the filtrates from dried or glycerine preserved crusts were invariably non-infective. The fresh exudate from the lesion, well diluted with water, autolysed at 37° C. and then passed through candles of the types Chamberland L, and Ll bis, and Berkefeld V, yielded infective filtrates. Jacotot succeeded in filtering the virus of contagious ecthyma of the goat through Chamberland candles types F and L2 after digesting the crusts for two hours at 37° C. He does not mention any particular difficulty in the preparation of virulent filtrates, but his protocols show that the inoculation of the filtrate led to a delayed eruption at the end of the second week. Blanc and his co-workers also maintain that the virus passes the Chamberland candle type L2 quite readily, the filtrate giving rise to a chacteristic eruption on the thigh and the lip. The experiments which follow show that it has been possible to demonstrate the presence of a filterable virus both in the fresh lesions and in the dried crusts. Infective filtrates, however, were only obtainable with difficulty, and it seems highly probable that although the virus was capable of passing the coarser gracJ.es of filters only a small portion came through. the bulk being retained by the candle. Experiments w#h Fresh Material. A dilute emulsion of the scrapings from the fresh lesions was autolysed at 37° C. for three hours and then passed through different types of candles. It was found that the virus passed the Berkefeld V but was retained by the Berkefeld N and the Chamberland L 2 • Experiment 1. August 5th, 1927. Lamb 161.-This lamb was inoculated on the left thigh with a 1/1,000 dilution of dried crusts. On the ninth day, when the vesicular eruption was at its height and was passing on to the pustular stage, the lesions were scraped with a scalpel and the material which was obtained was ground with sterile sand in a mortar. 10·0 C.c. of distilled water were added to the mixture, which was then allowed to remain at 37° C. for three hours. The emulsion, made up to 100·0 c.c.with normal saline solution, was lightly centrifuged for five minutes, and the supernatant fluid was removed and inoculated as follows ; . Lamb 163 (Control) was inoculated on the thigh with dilutions of 1/1,000 1/10,000, 1/50,000, and 1/100,000. A typical eruption resulted from the 1/50,000, but not from the 1/100,000 dilutions. To 20·0 c.c. of the supernatant fluid were added 2·0 c.c. of a 24 hour broth culture of B. prodigiosus and the mixture was passed through a small Berkefeld V candle under a negative pressure of 15 cm. Hg. in twelve minutes. 10·0 C.c. of the filtrate, sown into broth, showed no visible growth after 48 hours. Lamb 162.-The skin over both thighs was scarified and a part of the rem;!inder of the filtrate was applied. No changes were observed during the

GENERAL ARTICLES.

3SS

following seven days, but on the eighth day the lines of scarification became slightly raised. On the tenth day six small papules were noted, and these were succeeded by a typical vesico-pustular eruption, reaching its maximum about the seventeenth day. On the fourteenth day a scraping from one of the vesicles transferred to fresh lamb (No. 174) gave rise to a typical eruption. Lambs 162 and 163 re-inoculated one month later, and No. 174 after the lapse of eight months, were found to be immune. This experiment was repeated with a very similar result. Experiment 2. December 20th, 1927. Scrapings from the vesico-pustular lesions on the thigh of lamb 112 were used in this experiment. 0·2 grms. emulsified in 20·0 c.c. of distilled water were autolysed at 37° C. for three hours. A 1/2,500 dilution was then prepared by the addition of 480 c.c. of normal saline. 50·0 c.c. of this emulsion were lightly centrifuged for five minutes and the supernatant fluid was withdrawn. Retaining 1·0 c.c. for the inoculation of the control lamb, the remainder was divided into two parts after the addition of 5·0 c.c. of a twenty-four hour broth culture of B. prodigiosus. One portion was then filtered through a Chamberland L 2 , under a negative pressure of 20·0 cm. Hg. in thirteen minutes; the other portion was filtered through a Berkefeld N under the same pressure in nine minutes. 10·0 c.c. from each filtrate sown into broth showed no visible growth after 48 hours' incubation. Lamb 108 was inoculated on the right thigh with the Chamberland L2 filtrate and on the left thigh with the Berkefeld N filtrate. No reaction had occurred on either thigh after three weeks. The lamb was then re-inoculated with a 1/10,000 dilution of dried crusts and gave a typical reaction. Lamb 128 (Control) inoculated with dilutions of the unfiltered fluid re-acted to a dilution of 1(50,000 but failed to re-act to 1/100,000.

Experiments with Drif?d Crusts. The virus in the dried crusts, after dilution with normal saline, failed to pass through a Berkefeld V candle, but a similar emulsion acted upon by trypsin yielded an infective filtrate with the same type of candle. This trypsinised emulsion, when suspended in a buffered phosphate solution with a reaction of pH 7 '6, failed to give an infective filtrate after passag through a Mandler candle, but when the reaction was raised to pH 8'2 the filtrate was infective. Experiment 3. August 30 th, 1927. 0·02 gm. of desiccated stored crusts from Lamb 161 was emulsified in 20·0 c.c. normal saline solution and left over night in the cold room. The following morning the mixture was placed in the 37° C. incubator for three hours and then centrifuged at a low speed for five minutes. To the supernatant portion, after the withdrawal of 1·0 c.c. for the inoculation of control lamb, were added 2·0 c.c. of a twenty-four hour culture of B. prodigiosus. The fluid was passed through a Berkefeld V candle under a negative pressure of 15 cm of Hg in ten minutes. 10·0 C.c. of the filtrate sown into broth showed no visible growth after 48 hours' incubation. Lamb No. 78 was inoculated on the scarified skin of both thighs with 2·0 C.c. of the filtrate. On the fifteenth day a slight papule-like eruption was noted at a few points along the lines of scarification, but these lesions did not progress to the vesicular stage. When transferred to the thigh of a fresh lamb a scraping from these eruptions failed to give a reaction.

384

GENERAL ARTICLES.

Lamb No. 194 (Control) was inoculated with 1/1,000,1/10,000,1/50/000, and 1/100,000 dilutions of the supernatant fluid and reacted to the 1/50,000 dilution. The immunity of these lambs was tested after one month. Lamb No. 194 was found to be immune, whereas No. 78 gave a typical reaction. Experiment 4. October 11th, 1927. 0·05 gm. dried stored crusts from Lamb 161 was emulsified in 5·0 c.c. distilled water. 0·1 c.c. of liq. trypsini Co. and 0·1 c.c. of 1 per cent. sodium carbonate solution were added and the mixture was incubated at 37° C. for five hours. At the end of this period the suspension had undergone almost complete digesdon, yielding a slightly opalescent fluid free from coarse particles. The reaction of the liquid was now adjusted by the addition of N/20 HCl. to pH 7·6 and 20·0 c .c. of normal saline and 2·5 c.c. of a twenty-four hour broth culture of B prodigiosus were added after the withdrawal of 1·0 c.c. for the inoculation of the control lamb. This autolysate was now Fassed through a Berkefeld V candle under a negative pressure of 15 cm. Hg. in seven minutes. 10·0 c.c. of the filtrate sown into broth showed no visible growth after 48 hours incubation. Lamb No. 102 was scarified on both thighs and 2·0 c.c. of the filtrate applied by means of a swab. The lines of scarification remained unchanged for six days, but on the seventh a slight reddening was observed at a few points. Two days later definite papules appeared and were followed by a typical though mild vesiculo-pustular eruption reaching its maximum on the sixteenthday. Lamb No. 101 (Control) was inoculated with dilutions of 1/5,000, 1/10,000, 1/20,000,1/50,000, and 1/100,000. Severe reactions resulted from the 1/20,000 dilution with the production of vesicles on the eighth day. In the 1/50,000 dilution the reaction was less marked and the appearance of vesicles delayed until the twelfth day. The 1/100,000 dilution was negative . Re-tested at a subsequent date, both lambs were found to be immune. Experiment No.5. November 11th, 1927. 0·05 gm. desiccated crusts from Lamb 161 was made into a 1/500 dilution with normal saline solution. The emulsion was placed in the 37° C. incubator for three hours and then lightly centrifuged for five minutes. The supernatant portion, after the addition of 2·0 c.c.of a twenty-four hour culture of B. prodigiosus, was passed through a Mandler candle under a negative pressure (6-9Ibs.) of15 cm. Hg. in fourteen minutes. 10·0 c.c . of the filtrate was sown into broth and showed no visible growth after 48 hours incubation. Lamb No. 104 was scarified on both thighs and 2·0 c.c. of the filtrate was applied. There was no evidence of any reaction during the following three weeks. Lamb No. 23 (Control) was inoculated on the thigh with a dilution of 1/50,000 of the fluid before filtration, and gave a typical reaction. Re-inoculated one month later, Lamb No.. 23 was immune whereas Lamb No. 104 gave a typical reaction with a 1/50,000 dtlution. Experiment No.6 . December 19th, 1927 . 0·05 gm. of dried crusts from Lamb 107 was emulsified in 3·0 c.c. of dl~tilled water and the suspension left in the cold room over night. The tollowing morning solutions of trypsin and sodium carbonate were added as in Experiment 4. The emulsion was then neutralised by the addition of N /20 Hel and divided intonvoparts. Two solutions of a definite reaction were then prepared as follows :-

GENERAL ARTICLES.

335

A.

Emulsion. 1·4 c.c. M/50 NaH 2P04H 20 24·0 c.c. 16·0 c.c. M/50 Na2HP04 6Hp The reaction of this fluid was pH 7·6. 1·4 c.c. B. Emulsion. 2·0 c.c. M/50 NaH 2P04 H 20 38·0 c.c. M/50 Na2 HP04 6H 20 The reaction of this fluid was pH 8.2 . Each part, after the addition of 2·0 c.c. of a twenty-four hour culture of B. prodigiosus, was passed through a Mandler candle. 10·0 c.c . of each filtrate sown into broth remained sterile. Lamb 120 was inoculated on the right thigh with filtrate B (0· 5 c.c.). Mter four days the line of scarification became slightly reddened and during the succeeding days progressively more prominent. On the ninth day papules commenced to form at various points and were succeeded by a typical vesicopustular eruption on the thirteenth day. This reaction, although delayed, was as severe as that which was set up in the control lamb. (No. 121.) The same lamb was inoculated on the left thigh with 0·5 c.c. of filtrate A. There was no reaction. Lamb No. 121 (Control) was inoculated with the emulsion before filtration in dilution of 1/100, 1/1,000,1 /10,000, and 1/100,000. A typical reaction, reaching its maximum on the ninth day, was produced by the 1/10,000 dilution, whilst the 1/100,000 dilution was negative.

Immunity. Sheep which have passed through a natural attack of the disease possess a solid immunity against re-infection. Moreover, recovery from the experimental disease, which is much less severe, confers a high degree of resistance. By the ninth day of the disease there is some resistance to reinfection and this immunity becomes progressively more solid until by the fifteenth day, when the lesions have advanced to the scab stage , it is absolute. The following experiment suggests this gradual acquisition of an immune state. Experiment 1.

August 5th, 1927.

Lamb 161 was inoculated on the scarified left thigh with a 1/1,000 dilution of dried crusts. On the seventh, ninth, eleventh, thirteenth, and fifteenth days, corresponding with the papular, vesicular, vesico-pustular, pustular, and scab stage respectively, a 1/100 dilution of dried crusts was applied to scarified areas on the opposite thigh. The inoculation on the seventh day gave rise to a typical eruption which was as severe as the original lesion. The ninth day inoculation caused a typical but slightly milder vesico-pustular lesion, which commenced to develop three days later and reached the scab stage by the twenty-first day. It was less severe than the original eruption. The inoculations on the eleventh and thirteenth days were followed by a slight and abortive pustular eruption which lasted two days, whilst the fifteenth day inoculation was entirely negative. This progressive development of an immunity between the ninth and fifteenth days was confirmed in another manner :-

336

GENERAL ARTICLES.

Experiment 2. June 7th, 1928. Four young lambs (136, 138, 141, 144) were inoculated on the under surface of the tail with a 1/100 dilution of virus 161. The tails of these lambs were removed at a point at least two inches above the lesions at the following intervals. . June 11th, 1928. Lamb 141.-Papular stage. June 14th, 1928. Lamb 138.-Commencing vesicular stage. June 18th, 1928. Lamb 136.-Well marked pustular stage. June 21st, 1928. Lamb 144.-Scab stage. On the June 22nd, 1928, these lambs together with a control lamb (No. 145) were tested with virus 161. Lamb No. 1/100

- - -- - 145 141 138 136 144

1/ 500

1/1,000

1/5,000

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ + ++ + + + + + = Severe reaction. +

++

=

-

Moderate reaction.

I 1110,000 +++ +++ ++ -

=

=

1/50,000

1/100,000

++ +

-

-

-

I

Slight reaction. No reaction.

After the fifteenth day all attempts at re-infection have failed. The immunity of recovered lambs is still solid after a period of many months. Seven lambs which had been experimentally infected on the thigh and had given typical reactions were re-tested at intervals of one and a half, four, six, and eight months, and were found to possess a resistance against many infective skin doses of virus. In order to ascertain the degree of this immunity, falling dilutions of virus from 1/10 to 1/10,000 were inoculated on the thigh. The following table shows the results of these inoculations : -

Experiment 3. Date of 1st Inoc.

Lamb No. 163 162 101 102 197 198 174

I I

I

Material.

Reinoc. dried virus 1/101/10,000

Result.

I

Period.

14-8-27 14-8-27

Dried Virus Filtered

30-9-27 30-9-27

Neg. 1/10 Neg. 1/10

47 days

11-10-27 11-10-27

Dried Virus Filtered

24-2-28 24-2-28

Neg. 1/10 Neg. 1/10

138 days

5-9-27

Dried Virus

15-3-28

5-9-27

Dried Virus

15-3-28

Pos. 1/500 Neg. 1/1,000 Neg. 1/10

192 days

28-8-27

Fresh Virus

27-4-28

Pos.l/100 Neg. 1/500

243 days

"

"

Control lambs which were included in each group reacted to a

1/50,000 dilution of dried virus. In Lambs 197 and 174 the reactions, which were not severe, con-

sisted of a mild vesico-pustular eruption which attained its maximum at about the fifth day and then rapidly disappeared.

GENERAL ARTICLES.

SS7

It will be seen from this table that after a lapse of four months the immunity is sufficient to protect against 5,000 minimal infective skin doses. After six and eight months there is a slight decrease in the degree of immunity, although this is still remarkably high and undoubtedly sufficiently solid to afford protection against a natural infection. Recovery from an inoculation of the thigh leads to an immunity not only of the original seat of the eruption but also of other parts of the skin. For example, a re-inoculation of the opposite thigh or of the lips show that in these positions the skin has acquired a considerable resistance against the virus. Experiment 4. May 11th, 1927. Lamb No. 101 was inoculated on the thigh with falling dilutions of virus and gave a typical reaction. On the February 24th, 1928 (136 days later), the lamb was re-inoculated on the opposite thigh with a 1/10 dilution of dried crusts and 0·4 c.c. of the same dilution was inoculated intradermally into the upper lip in four places. No reaction followed either the lip or the thigh inoculation.

Experiments with Materialfrom other Outbreaks. With the exception of the two preliminary inoculations detailed on page 321, a single strain of virus, which will be referred to as Strain U .F " has been used in all the preceding experiments. In order to determine whether other outbreaks of this disease were caused by the same or a similar virus, material was obtained from two flocks widely separated from the first outbreak and from one another. Normal lambs inoculated with dried and powdered crusts reacted in a characteristic manner to these strains.

Strain A.-In this outbreak the lambs when about 14-21 days old were affected with small vesicles around the lips. These vesicles were succeeded by pustules which ruptured. liberating a thick exudate. Beneath the scab which formed from the dried exudate the epithelium commenced to proliferate very rapidly until a dense papilloma-like growth was formed which sometimes reached the size of a pigeon's egg. In each case where a lamb was affected the ewe also bore a lesion of a similar nature on the udder. Experiment 5. April 15th, 1928. A portion of a lesion from one of these lambs was finely divided and dried over sulphuric acid for 48 hours. Dilutions from 1/500 to 1/100,000 were made in normal saline solution and inoculated on the scarified thigh of Lamb No . 179. No change occurred during the three days following the inoculation . On the fourth day there was s distinct reddening and slight raising of the lines of scarification, which by the fifth day had become very pronounced. On the seventh day several vesicles commenced to develop and reached their maximum by the eighth day. The lesions then passed on to the usual pustular and scab stages. A marked reaction was obtained to the 1/50,000 dilution and a slight reaction to the 1/100,000.

338

GENERAL ARTICLES.

Experiment 6. April 24th, 1928. 0·02 gm. of dried powdered lesion was emulsified in 2·0 c.c. distilled water. To the emulsion were added 0·053 c.c. of liquor trypsini and o· 5 c.c. of 1 per cent. sodium carbonate solution. The mixture was incubated at 37° C. for three hours after which 80 c.c. of normal saline solution and 2·0 c.c. of a twenty-four hour broth culture of B. prodigiosus were added and the mixture filtered through a small Berkefeld V candle in three minutes under a negative pressure of 15 cm. Hg. 5·0 c.c. of the filtrate sown in EO broth showed no visible growth after 48 hours' incubation. Lamb 181 was scarified on both thighs and the remainder of the filtrate applied with a swab. No change was noted until the seventh day, when several distinct papules formed. On the ninth day there were five small but distinct vesicles which later passed on to the pustular and scah stage.

It was evident from these experiments that a filterable virus showing a marked similarity to the one which had already been studied was present in the lesions in this outbreak. The resemblance of the type of eruption produced by this strain to the preceding was very striking. Strain F.-In this outbreak about seventy lambs were affected with small papilloma-like growths around the lips. The very young lambs were not affected. Experiment 7. February 17th, 1928. A portion of the scabs from one of the lesions was powdered and dried over sulphuric acid for 48 hours. Dilutions of 1/5,000 to 1/100,000 were made in normal saline solution and applied to the scarified thigh of Lamb 129. A. typical vesicular eruption developed on the sixth to seventh day and was followed by a pustular formation on the tenth day. A reaction was obtained to the 1/50,000 dilution but not to the 1/100,000. Filtration experiments were not carried out with this strain, but the type of lesion produced by the dried crusts conformed exactly with that following the inoculation of the previous strain.

Experiment 8. February 24th, 1928. A scraping from the vesicular lesions on the thigh of lamb 129 (Experiment 7) was emulsified in a few drops of saline and inoculated into the upper lip of lamb 130. There was no apparent reaction until the fourth day, when a pea-like nodule was noted at the point of inoculation. The nodule developed into a pustule which ruptured about the ninth day and was succeeded by a shallow ulcer covered by a thin layer of necrotic tissue. The epithelium at the edge of this ulcer then appeared to proliferate, until a dense papillomalike growth about the size of a bean and dependent from the lip was formed. The lesion tlten resembled in every way the growths which are seen in natural outbreaks. These strains were next proved by the inoculation of lambs recovered from strain U.F. to be immunologically identical. In each case a fresh lamb was inoculated with dilutions of 1/500-1/100,000 of A and F strains respectively, together with a lamb immune to the U.F. strain. I.-February 17th, 1928. A normal lamb (No. 129) was inoculated on the thigh with Strain F and gave a reaction to a 1/50,000' dilution, attaining its

GENERAL ARTICLES.

3139

maximum on the tenth day, At the same time Lamb 108, which had reacted to U.F. seven weeks previously, was inoculated on the right thigh with U.F. and on the left with F. On each side a very slight and abortive pustular eruption developed quickly and had disappeared by the fifth day. Lamb 129 re-tested one month later with U.F. failed to show the slightest trace of a reaction. 2.-ApriI15th, 1928. A normal lamb (No. 179) which was inoculated with Strain A gave a marked reaction to a 1/50,000 dilution and a slight reaction to a 1/100,000 dilution, the lesions reaching their maximum on the ninth day. At the same time Lamb 107, which had reacted to U.F. five months previously, was inoculated on the left thigh with A and on the right thigh with U.F. On each thigh a slight reaction occurred to the 1/500 dilution while other dilutions were negative. Lamb 179 was re-tested one month later with U.F. and failed to react to a 1/500 dilution.

The preceding experiments having clearly shown that a lasting immunity of a high order can be readily produced, the question arises as to the possibility of devising a practical method of vaccination against the disease. Aynaud (Zoe. eft.) vaccinated 10,000 sheep with complete success. In his experience, if the thigh is inoculated with a suitable vaccine which provokes a comparatively benign local lesion there need be no apprehension of a generalisation of the disease. The vaccine which he recommends is prepared from the scabs on the thigh collected at the fifteenth to twentieth day of the disease, dried over sulphuric acid, exposed to the action of chloroform for twenty-four hours, and again dried. A 1 per cent. emulsion of this powder is made in a 50 per cent. solution of glycerine in normal saline and a few drops are applied to the scarified thigh. The vaccin~ should be used within six days of its preparation. Jacatot, Moussu, Blanc, and others have also reported successful vaccination on similar lines. Up to the present no opportunity has arisen of testing the effect of a vaccine in flocks in which the disease is known to occur, but numerous experiments on our own Iambs suggest that a slight modification of the method of Aynaud will prove satisfactory in the field. The vaccine is prepared from crusts taken from the thigh at the fifteenth day of the eruption. These are powdered and dried in the manner described in an earlier part of the paper. A weighed amount of the powder is emulsified in a 25 per cent. solution of glycerine in normal saline to make a final dilution of lil,OOO. The thigh is scarified with a single superficial scratch about an inch long and two or three drops of the vaccine are applied. A typical reaction is produced and remains strictly localised to the inoculated area. In a series of Iambs inoculated in this manner the vaccine has never caused an excessive reaction, whilst the subsequent immunity has been remarkably solid. At the commencement of the investigation a chloroformtreated vaccine, prepared according to the recommendation of. Aynaud, was tested, but no appreciable alteration in the potency of the virus could be detected.

340

GENERAL ARTICLES.

In a 1/1,000 dilution in 25 per cent. glycerine in normal saline solution the vaccine will still be potent after six weeks at ordinary temperatures. SUMMARY AND CONCLUSIONS.

1. Contagious Pustular Dermatitis is frequently seen in sheep in this country, chiefly as a disease of lambs. 2. In three outbreaks which have been studied it has been possible to demonstrate the presence of a filterable virus in the lesions. 3. Sheep and goats alone appear to be susceptible to this virus, which has a marked affinity for epithelial tissue. 4. Under experimental conditions the lesions show a marked resemblance to those of the vaccinia-variola group in that they pass through the stages of papule, vesicle, pustule, and scab. In natural cases, since the lesions are confined to certain positions such as the lip, the coronet, and the region of the tail, they are liable to pass unnoticed until they have reached the later stages, when they appear either as shallow ulcers covered by a purulent exudate or as papillomatous outgrowths. 5. In so far as the present investigation has been persued, the strains of virus studied conform to a single type.· . 6.-The virus, which exists in great abundance in the lesions, is highly resistant to desiccation. 7. It can be shown that, under experimental conditions, recovered animals possess a high degree of immunity, sufficient to protect them against re-infection for a period of at least eight months. It is suggested therefore, that the preventive inoculation of flocks by means of a vaccine prepared from the dried and powdered crusts would be of value in the control of the disease in the field. I desire to thank Mr. F. T. Harvey, F.R.C.V.S., and Mr. A. S. Adams, M.R.C.V.S., for providing material for this investigation. REFERENCES. lWallis Hoare, E. "Contagious Pustular Dermatitis of Sheep." System of Vet. Med., 1913, Vol. 3, p. 300. 2Aynaud, M. "La Stomatite Pustuleuse Contagieuse des ovines." Ann. lnst. Past., 1923. T. xxxvii, p. 498. 3Moussu, G. "L'Ecthyma Contagieux des levres chez Ie Mouton." Rec. de Med. Vet., 1923, Jan. 15th, p. 5. 4Lanfranchi, A. "Di recerche sperimentale su la stomatite pustulocontagiosa degli ovini." Nuova Veter., 1925, Jan. 15, p. 1. 5Blanc, Melanidi et Caminopetros. "R~cherches Experimentales sur une maladie eruptive de la chevre observee en Grece." Ann. lnst. Past., 1922 T. xxxvi, p. 614. 6Jacatot, H. "L'Ecthyma Contagieux des levres en Annam." Ann. lnst. Past., 1926, T. xl, p. 49. 7Zeller, H. "Ueber Pocken bei Zeigen Sudwest Mricas." Arbeiten aus dem Reich., 1920, Oct., Vol. 52, p. 501. 8Gordon, M. H. "Studies of the Viruses of Vaccinia and Variola." Med. Res. Council, Spec. Rep. Series, No. 98.