The Intrapalpebral Mallein Test

The Intrapalpebral Mallein Test

GENERAL ARTICLES. THE INTRAPALPEBRAL MALLEIN 281 TEST. By Captain GOODALL, South African Veterinary Corps. SOME time ago I noticed a short abstr...

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GENERAL ARTICLES.

THE

INTRAPALPEBRAL

MALLEIN

281

TEST.

By Captain GOODALL, South African Veterinary Corps. SOME time ago I noticed a short abstract in Tlte Journal of Comparative Pathology and Therapeutics, for September 1914, of an article by Lanfranchi (II Moderno Zooiatro, Parte Scientifica, Vol. XXV., No. I, January 1914), in which he described the intrapalpebral mallein test. Having recently been placed in the unenviable position of having to attempt to pick out glandered animals from a large number of military horses under my care, I decided to give the method a trial, and the following notes are the result of the experience gained. I n the first place I was confronted with the problem of endeavouring to find the glandered animals amongst those suffering from septic pneumonia, strangles, catarrh, etc., whose temperatures were most irregular, and which could not be tested by the ordinary subcutaneous method. Repeated cases of clinical glanders were occurring nearly every day in animals which were apparently recovering from the complaints for which they were being treated. The fact that many of the animals were in a very debilitated state no doubt caused them to develop clinical symptoms earlier than they would have done otherwise, whilst many others contracted the disease ,"ery easily from previous clinical cases. Most of the animals had also just returned from an arduous campaign in South-West Africa. where they had to endure great hardships, and were suddenly called upon to face the damp, cold climate of the Cape Peninsula in winter. From the above facts it will be easily deduced that the bacillus mallei had a fruitful soil in which to develop, unhampered by much natural resistance. Those animals which had very slight lesions when they arrived probably soon developed the disease in a rapid form, II hilst many others were particularly susceptible to infection. The ordinary ophthalmic method was. at first tried (i.e., the instillation of undiluted mallein on to the conjunctiva), and it yielded fairly good results; but there were many grave drawbacks to its use, especially under the conditions encountered. The following are the principal ones : (a) The reaction obtained is often very transitory, and where large numbers of animals have to be dealt with it may be easily missed. (b) Where animals have to be tested in the open, or with very little shelter, a small amount of discharge may easily be encountered in the eye without the animal being glandered. This remark also applies to animals suffering from catarrh, etc. (c) Animals can easily rub the discharge away from the eye, or it may be removed by an attendant. I n this connection I may state that I have not seen the" gluing together" or thickening of the eyelids referred to by some observers in reactions obtained by the ophthalmic method, nor have I noticed any very marked conjunctivitis. My objection" to the ordinary ophthalmic method would therefore only apply to those cases where a diagnosis has to be based on a coJlection of muco-pus in the inner canthus, unaccompanied by other signs.

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M aZZein EmpZoyed.-In the original method described by Lanfranchi he used" crude mallein" diluted with normal saline solution ('25 cc. of the mallein to 2! CC. of saline solution). Being unable to obtain "crude mallein," I devised the plan of evaporating the ordinary mallein, prepared for the subcutaneous test by the Royal Veterinary College, London, down to one-fifth of its original volume in a water bath, and using '20 (one-fifth) cc. of this concentrated mallein as a dose. This is the dose and method of preparation employed by me throughout these tests. My objection to Lanfranchi's original method is that the dose, 2! cc., is too large, and likely to cause too much " mechanical" swelling when injected into the eyelid. Technique.- The skin over the lower eyelid is washed with ether, care being taken that none of the liquid comes into contact with the conjunctiva or eyeball. Carbolic or other strong disinfectants are

FIG. 1.

Very pronounceu (XXX or a) reaction.

avoided on account of the possibility of their irritating effect on the eye. The inoculation is made with the finest hypodermic needle obtainable, attached to a syringe of I cc. capacity, graduated in tenths, and fitted with a stop on the piston rod. The needle is inserted under the skin of the eyelid about a quarter inch below its edge, midway between the outer and inner canthi, and gently pushed about a half inch under the skin in a line parallel with the edge of the eyelid, towards the inner canthus. The dose is then introduced, the needle withdrawn, and the lid gently massaged in the direction of the inner canthu& The operation is extremely simple, and is very little resented by the animal. It is, of course, important to have the animal under complete control, and to avoid bruising or injuring the eyelid when injecting the mallein. For this reason also only the finest needles

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should be employed. For the convenience of a right-handed operator, the near eye is employed for the test, but if this is inflamed or injured the off eye is used. Character of Reaction.- The local reaction commences to appear, as a rule, four to six hours after inoculation-frequently earlier-and it goes on increasing.in size and intensity up to forty-eight hours after injection. In most animals it persists for seventy-two hours, and has generally disappeared entirely in four days. A perfectly certain diagnosis can generally be made during the first twenty-four hours, although there is a type of reaction which it is as well to leave until the second day before making a definite decision. This will be described later. It is always preferable, in dealing with large numbers of animals, to defer the final" passing out" until the second day, as then all animals can be dealt with at once.

:FIG. 2. Pronounced (XX or b) reaction.

The following is a useful classification of the local reactions according to their degree of intensity : -

Positive Glandered Reactions. XXX (a) Intense swelling of eyelid and infiltration of surrounding subcutaneous tissue, with complete or almost complete closing of eye, and a copious discharge of muco-pus from the inner canthus, accompanied by marked conjunctivitis (fig. I). XX (b) Marked swelling of eyelid, extending to cheek, with discharge of muco-pus, but only partial occlusion of the eye. Marked conjunctivitis (figs. 2 and 3). X (c) Swelling of eyelid persisting for forty-eight hours, with discharge of muco-pus, but only slight, if any, swelling of the surrounding tissue; marked conjunctivitis (fig. 4).

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Doubtful. ? Cd) Swelling of the eyelid persisting for forty-eight hours, with no other symptom, and no discharge of muco-pus, or a collection of muco-pus alone.

Negative. (e) No swelling of eyelid after forty-eight hours, and no muco-pus. Classes (a), (b), and Cc) positive reactions; Class Cd) doubtful reaction; Class (e) negative reaction. In describing swellings no measurements are given, as owing to the varying conformation of horses' heads these would be somewhat misleading; but the appearance of CI,!sses (a) and (b)-by far the most common-are very characteristic. Unfortunately this is most

FIG. 3.

Pronounced (XX or b) reaction.

difficult to reproduce by photography, but figs. I, 2, and 3 give a good idea of the condition. The hollow above the zygomatic ridge is filled in, and this gives the head a peculiar one-sided appearance when compared with the other side. The swelling also has the effect of making the affected eye appear smaller than its fellow. The swelling is hot and painful. The swelling of the eyelid in a reaction also makes the ocular orifice appear smaller on account of the lower eyelid having a marked tendency to protrude upwards at its free edge, particularly near the inner canthus. This is a most useful guide in doubtful cases, where there may be some slight swelling. It is this characteristic swelling of the free edge of the eyelid which is one of the most noticeable features of a positive reaction, and distinguishes it most markedly from any other condition.

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28 5

Swelling of the eyelid cause it to become firm, and makes it project upwards into the ocular orifice. If felt between the finger and thumb it will be found to be quite half an inch thick, often more. It may be pointed out that many horses with a natural "bagginess" under the eye show a slight soft swelling a few hours after the inoculation, but this generally disappears during the second twenty-four hours. It never protrudes upwards, nor does it cause the free edge of the eyelid to become thickened. It should not cause any confusion in diagnosis. In this method, as in the subcutaneous one, the violence of reaction is not much guide to the intensity of the infection, but I incline to the opinion that the presence of" new," growing, lesions gives rise to a larger local reaction than encapsuled or caseous lesions. The muco-purulent discharge is also characteristic, being of a

FIG. 4.

Slight (X or c) reaction.

dirty greenish-grey colour and of a sticky consistence, very similar to the discharge from a farcy bud. It collects at the inner canthus, and frequently overflows, when it adheres to the cheek (when dry it looks as if linseed oil had been applied), although in some very violent reactions the thickening of the eyelid seems to mechanically prevent the discharge from overflowing, and the eyelids become glued together. Lachrymation is sometimes observed during the early stages of a reaction, but, as the formation of muco-pus soon appears, this condition, in my opinion, is not important. In character the discharge is quite distinct from any other encountered by me in morbid conditions of the eye, or from those caused by systemic diseases (strangles, catarrh, etc.). I can only describe it as being" dirtier and stickier." A microscopic examination of the discharge shows that its

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elements are almost entirely composed of large neutrophile polynuclear leucocytes, and it differs very considerably in this respect from other ocular discharges examined. I have now examined a considerable number of specimens, and, whilst not wishing to claim that the presence of these elements in the discharge is positive evidence of a reaction in itself, I certainly think it would afford a considerable aid to a diagnosis in a doubtful case. The post-mortem appearances of the swelling are : (a) Marked engorgement of the capillaries in the eyelid, which gives a section a spotted appearance. (b) Infiltration of the subcutaneous connective tissue with a yellow <.edematous exudate. In Classes (a) and (b) this extends to the inferior extremity of th~ zygomatic ridge, sometimes lower, and fills up the natural hollow immediately above the ridge. In very violent reactions this infiltration can be traced to the external nares and to the lower border of the inferior maxilla. The following is the procedure which I now adopt in examining a batch of animals undergoing the intrapalpebral test : (a) Animals examined during first twenty-four hours after injection, preferably at the fifteenth to eighteenth hour. 'All definite reactions are then picked out. (b) Animals showing no swelling (other than a purely local swelling under the free edge of the eyelid) and no discharge at the above time can also be declared free. (e) All animals showing" doubtful" swellings, i.e., thickening of eyelid with a tendency to protrude upwards, or a tendency to extend to cheek, and also animals with any discharge from the eye, are put back for another inspection next day. (d) The animals kept back for reasons mentioned in par. (e) are examined during the second twenty-four hours after injection (up to the forty-eighth hour), and the reactions judged according to whether they have developed or diminished, and whether the discharge is still present. A persistent swelling without discharge is regarded as "doubtful," as is also a muco-purulent discharge unaccompanied by swelling, and the animal is put back for retest. (e) An animal showing muco-purulent discharge at any time during the reaction period is always called" doubtful," and retested if it shows no other symptoms. Temperature Reaetion.-This generally commences at the fourth to sixth hour after injection, and persists for twelve to eighteen hours. I regard temperature reaction, however, as of secondary importance, and I would always be prepared to base a diagnosis on the local reaction alone, and would never be deterred from applying the test on account of a high temperature, which so often delays the testing of animals by the subcutaneous method. If temperatures are taken as an adjunct to the test, it is advisable to record them at the sixth, ninth, twelfth, and eighteenth hours after injection, to ensure obtaining a proper curve, and it may be necessary to continue until the twenty-fourth hour.

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RECORD OF CASES.

Group I.-Clinical Cases. Inoculated with '20 cc. of concentrated mallein near lower eyelid. 1. Bay gelding. Badly affected with farcy of near hind and off fore leg. Slight ulceration of septum nasi and nasal discharge. Gave marked XX reaction. Temperature too high to be reliable. Post-mortem.-A few nodules in both lung5. 2. Brown gelding. An advanced clinical case. Ulcerated septum nasi, indurated submaxillary glands, nasal discharge. Gave good XX reaction. Temperature too high to be reliable. Post-mortem.-Both lungs crammed with nodules, evidently new and increasing. 3. Bay mare. An ordinary clinical case. Ulceration of septum nasi, indurated submaxillary glands, and nasal discharge. Gave XXX reaction. Post-mortem.- Two large "new" lesions in lungs, the size of marbles. 4. Bay gelding. A" suspicious" clinical case, having small farcy ulcer on the nose. Gave XX reaction. Post-11IOrtem.-Numerous lesions, old and new, in both lungs. 5. Brown mule mare. An acute case, with noisy breathing, confiuent ulceration of septum nasi, and profuse nasal discharge. Temperature 104° F. Gave definite XX reaction. Post-mortem.-Fairly numerous nodules in both lungs, septum nasi practically one mass of ulcers, almost occluding the nasal passage. 6. Bay gelding. Suspicious looking discharge from nose. Doubtful ulceration of septum nasi and thick hind leg. Gave negative reaction. Destroyed on account of debility and suspicious symptoms. Post-mortem.- Two large solidified cedematous areas in lungs, size of saucers (septic pneumonia). No glanders lesions discovered after prolonged search. Conclusions.-From the above it will be seen that the intrapalpebral test gives good reactions in cases where clinical symptoms exist. It may be argued that it should not be necessary to test clinical cases at all, but anyone who has had much experience of glanders will acknowledge that one often encounters cases which are highly suspiciou's, and even those in which symptoms of other diseases occur at the same time. Subcutaneous mallein is generally of very little assistance where clinical symptoms exist, as in my experience it can generally be accepted that" the more pronounced the clinical symptoms the less reaction." It is hoped that the intrapalpebral method will prove useful in diagnosing those extremely rapid cases of glanders so often encountered in South Africa in debilitated animals, and also in the acute form seen in the mule, both of which it is often extremely difficult to diagnose off-hand. Another point which should render the test of great service from a practical point of view is the fact that a veterinary surgeon should be able to prove more conclusively to the owner that his animal has glanders. I have often been placed in the unfortunate position of being asked to test an animal which I have declared to

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be suffering from glanders before testing the rest of the stud, the owner arguing from the perfectly sound point of view to the lay mind, that the more pronounced the disease the greater the reaction to be expected.

Group I f.-Animals wlticlt fwd previously given a Thermal and Local Reaction to the Subcutaneous Mallein Test. I njected near eyelid with '20 cc. of concentrated mallein. The main object in conducting these experiments was to ascertain whether an animal which had reacted to the subcutaneous test would give a reaction to the intrapalpebral method. I. Grey gelding. This animal gave a good double reaction to subcutaneous mallein seven days previously. Gave XXX reaction. Post-mortem.-Numerous nodules in both lungs, mostly old. 2. Chestnut gelding. Gave good double reaction to subcutaneous test seven days previously. Gave XX reaction. Also gave temperature reaction to intra pal pe bral test. Post-mortem.-A few small nodules in both lungs. 3. Bay gelding. Gave double reaction to subcutaneous test, and was tested by intrapalpebral method fifteen hours after subcutaneous injection (at height of temperature curve.) Gave XX reaction. The local reaction on the neck also developed during the second fortyeight hours. Post-mortem.-Numerous nodules in both lungs, all ages and sizes, some as large as marbles. One lesion in spleen. Three well-marked lesions in liver. 4. Grey gelding. Gave double reaction to subcutaneous test, and was tested by intrapalpebral method fifteen hours after subcutaneous injection (at height of temperature curve). Gave XXX reaction. Local swelling on neck also developed during second twenty-four hours. Post-J1lortem.- Two or three pinhead nodules in each lung. A piece of each lung solidified and cedematous, containing purulent centres, highly suspicious of glanders (acute ?). 5. Bay gelding.. Gave double reaction to subcutaneous test, and was tested by intrapalpebral method fifteen hours after subcutaneous injection. Gave XXX reaction. Local reaction on neck also developed during second twenty-four hours. Post-mortem.-A few very small but well-defined nodules in both lungs. 6. Bay gelding. Gave double reaction to subcutaneous test, and was tested by the intrapalpebral method fifteen hours after subcutaneous injection. Gave XXX reaction. Local swelling ~n neck also developed during second twenty-four hours. Post-mortem.-Both lungs crammed with nodules of all ages and sizes, from marbles downwards. One well-defined lesion in spleen. 7. Brown mare. Gave double reaction to subcutaneous test, and was tested by the intrapalpebral method fifteen hours after the subcutaneous injection. Gave XXX reaction. Local reaction on neck well marked. Post-mortem.- Two lesions as large as marbles, and a few small ones in lungs. Very numerous lesions in spleen, varying in size from

GENERAL ARTICLES.

marbles to peas. Two well-marked lesions in liver, size of marbles. Two lesions in mesenteric glands, probably glanders. Condusions.- The above cases show ( I) that the intrapalpebral method can be relied on to give a reaction when animals are reacting to the subcutaneous test, i.e., they can be injected when the subcutaneous thermal reaction is at its height, and still react to the intrapalpebral method in a marked manner; and (2) that it does not interfere with the local reaction to the subcutaneous method. A week after the subcutaneous test animals give a reaction to the intrapalpebral method, accompanied by a rise in temperature. (This statement has since been verified in many more cases). In practice I would prefer to allow at least a week to elapse before applying the intrapalpebral test to an animal which had given a subcutaneous reaction ; but it is shown that animals can and do react if injected at the height of the subcutaneous thermal reaction, and so far I have encountered no failures in this respect. Since writing the above I have had the opportunity of testing a number of animals-over 100-which have given reactions of different sorts to subcutaneous mallein, and I now feel perfectly justified in asserting that the intrapalpebral test can be applied to an animal directly after the subcutaneous test and give a definite reaction.

Group III .-A nilllals wlticlt Ilad given a previous" Doubtful Reaction" to tlte S l£bcl£taneol£s Test. (a) Temperature only, no local reaction :-1. Bay gelding. Gave temperature reaction to subcutaneous test fourteen days previous to applying intrapalpebral test. Gaye XX reacti on . Post-11l0rtem.- Two small calcareous lesions in lungs, and numerous doubtful lesions in liver. 2. Bay gelding. Gave temperature reaction to the subcutaneous test fourteen days previous to applying intrapalpebral test. Gaw XX reaction. Post-11lortem.-Both lungs crammed with miliary nodules, evidently a rapidly advancing case. 3. Chestnut gelding. Gave doubtful thermal reaction to subcutaneous test as follows: Before, 1000 to 99.20 Twelfth hour after, 104° ; fifteenth hour after, 102 c ; eighteenth hour after, 102°. No local reaction up to forty-eighth hour. Tested by intra palpebral method eighteen hours after subcutaneous injection. Gave X reaction. Post-11l0rte11l.-N umerous lesions in lungs, some the size of marbles, rapidly advancing. Several well-marked lesions in spleen, the largest the size of a hazel nut. 4. Black gelding. Gave thermal reaction t o subcutaneous test as follows: Before, 101 ·4° to IOI·S o • Twelfth hour after, I04·4° ; fifteenth hour after, 104.2°; eighteenth hour after, 104.4°. No local reaction up to forty-eighth hour. Tested by intrapalpebral method eighteen hours after subcutaneous injection. Gave XX reaction. Post-mortem.-A few small well-marked nodules in both lungs. 5. P. 529. Gave the followin g thermal reaction to the subcu-

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taneous test: Before, 100'6°. Twelfth hour after, 103° ; fifteenth hour, 100'6°; eighteenth hour, 101'4°. No local reaction. Tested by intrapalpebral method four days after subcutaneous injection. Gave Class Cd) doubtful reaction (swelling of eyelid persisting for fortyeight hours). Post-mortun.-One well-defined lesion, size of pea, in lung. 6. 22413. Gave the following thermal reaction to subcutaneous test: Before, 101'2°; twelfth hour after, 103°, fifteenth hour, 102'8° ; eighteenth hour, 100'2°. No local reaction. Tested by intrapalpebral method ten days after subcutaneous injection. Gave XX reaction. Post-mortem.-One (glanders?) lesion, size of marble, calcareous outside and caseous inside, and a few small nodules in lungs. 7. 3226. Gave the following reaction to the subcutaneous test: Before, 100°. Twelfth hour after, 105'4°; fifteenth hour, 102'4°; eighteenth hour, 100°. No local reaction. Gave XX reaction. Post-mortem.- Two or three pinhead nodules in lungs. Conclusz'ons,- The intrapalpebral method can verify a temperature reaction to the subcutaneous test even when it is of a very doubtful character-one degree rise only, and it can be applied immediately after the subcutaneous thermal reaction and still give definite results.

Group IV.-Anz'lllals which had given a "Doubtful Reaction" to the Subcutaneous Test. (b) Swelling only, no temperature : I. Grey mule gelding. Tested a month before by subcutaneous method, when it gave a small but painful swelling which persisted for forty-eight hours. No thermal reaction. Gave XX reaction. Post-mortem.-Several apparently recent nodules in lungs, size of peas downwards. . 2. Brown mule mare. Same reaction to subcutaneous test as previous case, Gave XX reaction. Post-11lortem.- Very numerous miliary lesions in lungs. 3. Brown mule gelding. Same reaction to subcutaneous test as previous cases. Gave XX reaction. Post-11lortem.-N umerous "new" lesions in both lungs, some the size of peas. 4. 2274· Bay gelding. Gave large typical swelling to subcutaneous test, and the following temperature reaction eighteen days previously: Before, 100'2°. Twelfth hour after, Ico'6°; fifteenth hour, 99°; eighteenth hour, 101·8 c • Gave XX reaction; also temperature reaction, with rise of 3°. Post-mortem.-Fairly numerous nodules in both lungs, size of peas downwards. Conclusions.- The intrapalpebral test is useful in verifying a doubtful reaction to the subcutaneous test-swelling only. This is most useful in the case of mules, which animals generally give very irregular temperature reactions, and also have a relatively very much lower normal temperature than the horse. The local swelling is also generally much less pronounced in the mule than in the horse, even in a definite reaction.

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Group V.-Animals wltich had jailed to react to tlte Subcutaneous Test. These animals have a most interesting history. A lot of seventynine came down from South- West Africa and were tested subcutaneously, when fourteen reacted, and one died of clinical glanders. The veterinary officer who conducted the test had the remainder of the batch isolated for another test six weeks later, when four more reacted. A month after this last subcutaneous test the survivors were tested by the intrapalpebral method, when the following nine animals gave reactions-eight positive and one doubtful. It will thus be seen that the animals described below had twice failed to react to the subcutaneous test : 1. No. 43. Gave XXX reaction. Post-1JlorteJ1t.-N umerous well-marked lesions in lungs, two small lesions in spleen. 2. NO.3 1. Gave XXX reaction. Post-mortem.- Two large" new" lesions, size of large marbles, and numerous smaller nodules in lungs; one doubtful lesion in liver. 3. No. 22. Gave XXX reaction. Post-mortem.-A few small nodules in lungs. Well-marked lesions in mesenteric glands. 4. No. 35. Gave XXX reaction. Post-mortem.-Both lungs crammed with nodules, old and new, from the size of marbles downwards. Several lesions in spleen, countless miliary lesions in liver (glanders ?), and calcareous nodules in mesenteric glands (parasitic ?). 5. No. I. Gave XX (Class (b)) reaction. Post·mortem.-Numerous nodules in lungs, all ages and sizes, from marbles downwards. Two large lesions in liver. 6. NO.4. Gave XX reaction. Post-mortem.-Fairly numerous nodules in both lungs, from size of peas downwards. 7. No. 28. Gave XX reaction. Post-mortem.- Two large" new" lesions, the size of marbles, and numerous smaller nodules in lungs. 8. No. 33. Gave XX reaction. Post-lIlortem.-Fairly numerous nodules in both lungs, size of peas downwards. 9. Roan mare. Gave doubtful, Class (d), reaction. (SwelIing of eyelid persisted three days.) Developed suspicious symptoms of clinical glanders fourteen days after test, after an attack of pneumonia with a temperature of 105°. Post-mortem.-Both lungs crammed with nodules of all ages and sizes, from marbles downwards. Two well-marked lesions in liver. Conclusiotls.- The intrapalpebral test can pick out cases which fail to respond to the subcutaneous test. It will be noticed that most of the above cases were very advanced, and the mesenteric lesions and those in the liver were also unusual. It has been stated by other veterinary officers that there was a peculiarly virulent type of glanders encountered in German South-West Africa, which some go as far as to assert had been, deliberately left behind in stables by the retiring Germans. Whether this is correct or not it is certain

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that the disease was of a most virulent type. The post-mortem findings appear to be very similar to those described by Hunting under the heading" Artificial Infection." The intrapalpebral test helped in no small measure in dealing with this particular form of the disease.

Group VI.-Attimals which had not been previously tested by any other Method. The following animals were in stables where cases of clinical glanders were constantly cropping up. The horses in these stables could not be tested by the subcutaneous method, as they were all either suffering or recovering from pneumonia, strangles, or catarrh. They were therefore tested by the intrapalpebral method, irrespective of whether they showed symptoms of other diseases or not. All the reactors were destroyed, and the following is a brief description of the post-mortems : 1. Grey gelding. Gave XXX reaction. Post~mortem.-Both lungs crammed with nodules, all ages and sIzes. 2. Black mare. Gave X reaction. Post-mortem.-A few small nodules in lungs. 3. Chestnut gelding. Gave XX reaction. Post-mortem.-A few nodules in lungs. 4. Brown gelding. Gave XXX reaction. Post-morte71l.-Fairly numerous nodules in both lungs, size of peas downwards. S. Bay gelding. Gave XX reaction. Post-mortem.-Both lungs crammed with nodules, all ages and sizes. 6. Black gelding. Gave XX reaction. Post-mortem. - A few nodules in both lungs, probably recent. Two small lesions in liver. 7. Chestnut mare. Gave XX reaction. Post-mortem.-One large lesion the size of an orange (probably glanders) and one or two pinhead nodules in lungs. 8. Grey gelding. Gave XXX reaction. Post-mortem.-Numerous nodules in both lungs. Two or three doubtful lesions in liver. 9. Bay gelding. Gave X reaction. Post-mortem.-Large lesions of pneumonia; numerous small nodules and one large lesion in lungs (size of florin). 10. Bay gelding. Gave XXX reaction. Post-mortem.-Lungs full of recent nodules, some the sIze of marbles. I I.-Grey gelding. Gave XX reaction. Post-mortem.-Both lungs crammed with nodules, all sizes from marbles downwards. 12. Chestnut gelding. Gave XX reaction. Post-mortem.-Fairly numerous small nodules in both lungs. Remarks. -Of 240 horses tested in these stables the above twelve reacted and were destroyed. Six gave doubtful, Class (d), reactions, and were retested when they failed to react. One animal which

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gave a negative reaction died of enteritis, and was found to have a few calcareous nodules in the lungs, probably of a parasitic nature. C01Zclusions.- The intra palpebral test can pick out animals affected with glanders even when they are suffering from other diseases, and when the subcutaneous test cannot be applied. I always adhere strictly to the rule never to apply the subcutaneous test to an animal with an elevated temperature, as I am confident, after a fairly extensive experience, tbat not only is the subcutaneous temperature reaction unreliable, but the local reaction is also frequently interfered with. The following case is inserted here to show that an X, Class (c), reaction (muco-purulent discharge and swelling of eyelid without much swelling of the surrounding parts) denotes that the animal is glandered. This type of reaction has so far only been encountered a few times. Grey gelding 642. This animal was handed over to me as a "doubtful reactor" to the subcutaneous test a month previous to the intra palpebral test, but no history as to the nature of the reaction could be obtained. Gave X reaction. Post-mortem.-One lesion size of pea, and two or three very small (pinhead) nodules in lungs.

Group VII. These seven animals have the following history. They had been tested by the subcutaneous method eighteen days previously, and were pronounced to have given reactions. When tested by the intrapalpebral method three gave positive reactions and four negative. Whilst undergoing the intra palpebral test they were kept under very careful observation, and temperatured every two hours for twentyfour hours. I. P. 1540. Gave the following reaction to the subcutaneous test: Before, 99°. Twelfth hour after, 102° ; fifteenth hour, 102° ; eighteenth hour, 103'5° ; twenty-fourth hour, IOI·So. Large local swelling which decreased on second day, Gave Class (e), negative, reaction to intrapalpebral test, and temperature reaction of 1'6° (99'So to 101'4°). Post-mortem.-Fairly numerous, very hard, calcified nodules in lungs, and a large cedematous patch the size of crown piece of very dou btful nature. No pus centres. 2, 2429. Gave the following reaction to subcutaneous mallein: Before, 101'6°, Twelfth hour after, 103'6°; fifteenth hour, 103'6°; eigltteenth hour, 100·So. Local swelling very distinct; increased much the second day. Gave XX reaction to intrapalpebral test, and temperature reaction of 3° (100'SO to 103·S·). Post-mortem.-Both lungs crammed with nodules of all ages and sizes from a pigeon's egg downwards. 3. 2274· Gave the following reaction to subcutaneous mallein: Before, 100'2°, Twelfth hour after, 100'6°; fifteenth hour, 99°; eighteenth hour, 101'8°. Swelling very large, and persisting for forty-eight hours. Gave XX reaction to intrapalpebral test, and temperature reaction of 2° (101'4° to 103'4°). Post-1llortem.-Fairly numerous nodules in both lungs, size of peas downwards. u

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N.B.- This animal gave a temperature reaction to the intrapalpebral but not to the subcutaneous test. 4. P. 1839. Gave the following reaction to the subcutaneous test: Before, 101'2°. Twelfth hour after, 101'6°; fifteenth hour, 105'6°; eighteenth hour, 104'6°; twenty-fourth hour, 104'2°. Swelling very large and painful. Gave Class (e), negative, reaction to intrapalpebral test, and temperature reaction of 1"6° (99'4° to 101 ° at second hour). Post-JJlortem.-N a lesions of glanders discovered. 5. 2398. Gave the following reaction to the subcutaneous test: Before, 100-6°. Twelfth hour after, 101'2°; fifteenth hour, 103'2°; eighteenth hour, 103'4°; twenty-fourth hour, 102'4°. Local swelling large, but disappeared on second day. Gave Class (e), negative, reaction to intra palpebral test and temperature reaction of 1° (100'2° to 101'8° at second hour). Post-mortem.-N a lesions of glanders discovered; signs of recent pleurisy. 6. 2037. Gave the following reaction to the subcutaneous test: Before, 100'6°. Twelfth hour after, 101 '6°; fifteenth hour, 103'4°; eighteenth hour, 103°; twenty-fourth hour, 103°. Swelling large and painful. Gave Class (e), negative, reaction to intrapalpebral test, and temperature reaction of 1'4 (100'4° to 101'8° at the fourth hour). Pdst-moltem.- Two completely calcified nodules in lungs. 7. 237· Gave the following reaction to the subcutaneous test: Before, 10::)"6°. Twelfth hour after, 103'8°; fifteenth hour, 101 '2° ; eighteenth hour, 101 '2°. Swelling very large and typical. Gave XX reaction to intrapalpebral test, and temperature reaction of 3'6° (100'4° to 104° at eighteenth hour). Post-1Jlorte11l.-Numerous nodules in both lungs, size from peas to marbles, mostly recent. An advancing case. Conclltsions.- The above cases are interesting, inasmuch as they seem to prove that the intrapalpebral method gives more accurate results than the subcutaneous test. These animals were selected in the hope that they would all give reactions to the intrapalpebral method for the purpose of recording temperatures. The postmortems were most carefully performed with the assistance of two other veterinary officers, and lesions were most diligently searched for. The calcified lesions alluded to in Nos. 1540 and 2037 were extremely hard and of a very doubtful nature; in all probability they were of parasitic origin, such are frequently encountered in South Africa. (I have found live nematodes encysted in the lungs on several occasions.) My colleagues concurred in this opinion. The absence of lesions in No. 1839 is remarkable after the reaction given to the subcutaneous test. If the animals which gave negative reactions to the intrapalpebral test were glandered, the previous subcutaneous test had removed what little power of reacting they ever possessed. 0

Group VIII.-Animals tested simultaneously bJ' Subcutaneous and Intrapalpebral Methods. I.

with

No. 24. This animal had not been previously tested. Injected 1 cc. of subcutaneous mallein, and '20 cc. of concentrated mallein

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in eyelid simultaneously. Gave temperature reaction of 2'8° (102'6° to 105'4° at the twelfth hour), XXX reaction intrapalpebral test, and small painful swelling on neck size of cricket ball, persisting fortyeight hours. Post-1IZortem.-Numerous nodules in both lungs, from a marble dowmvards, mostly recent. Two lesions in spleen. 2. No. 58. Injected as above. Not previously tested. Gave temperature reaction of 4'4° (101'6° to lOSe at the ninth hour), XXX reaction intrapalpebral test, and large 8 by 6 inches swelling on neck, persisting forty-eight hours. Post-mortem.-Fairly numerous nodules in both lungs, size of peas downwards, mostly recent. 3. No. 38. Injected as above. Had not been previously tested. Gave doubtful temperature reaction to simultaneous test (rise of I'6° at the ninth hour), X reaction intrapalpebral test, and small painful swelling on neck size of cricket ball, persisting forty-eight hours. Retested by intrapalpebral method after lapse of eight days, when gave definite XXX reaction off eye. Post-1Jlorte1ll.- Very numerous nodules both lungs, from marbles downwards, mostly" new." 4. No. 4983. Injected as above. Had given XX reaction to intrapalpebral test eight days previously. Gave temperature reaction of 5'6 (99'4° to 105° at twelfth hour), XX reaction to intrapalpebral test, and practically no swelling on neck. Post-mortem.-Both lungs crammed with nodules of all ages and sizes from marbles downwards; one lesion in spleen. 5. No. 5081. Injected as above. Had given XX reaction to intrapalpebra1 test eight days previously. Gave temperature reaction of 1 only ( 101 to 102° at ninth hour). No reaction on neck or to intrapalpebral test. Post-mortem.-A few small nodules in both lungs. 6. No. 8282. Injected as above. Had given XXX reaction to intrapa1pebral test eight days previously. Gave temperature reaction of 1'8 (102° to 103'8° at ninth hour). No swelling on neck. XXX intra pal pe bral. Post-mortem.-Numcrous nodules in both lungs, from pea downwards. 7. No. 1423. Injected as above. Had given previous XXX Temperature reaction to intrapalpebra1 test eight days before. reaction of 4'2 (99'8° to 104° at ninth hour). No local reaction on neck. XXX intrapa1pebral. Postcmortem.-Large cedematous lesion in lung with caseous nodules (glanders f). Both lungs crammed with nodules, c'!-seous and new. Conclztsio1Zs.-Animals can react to both tests at the same time, viz., with local reaction of eye and neck and rise of temperature; but this method is of no practical importance, especially in view of the fact that a doubtful reactor to the subcutaneous method can be retested by the intrapalpebral test directly afterwards. The local reaction on the neck seems to suffer at the expense of the ophthalmic reaction; and, as the intrapalpebral test is really a subcutaneous one, no good purpose is served by applying the two together. 0

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Group IX. Three animals which had reacted to the intrapalpebral test were injected in the other eyelid, twenty-four, forty-eight, and seventytwo hours after the first test, to ascertain how soon a reaction can be given after a previous one. I. Bay gelding (p). Had given a positive XXX reaction to intrapalpebral test, and was injected in the other eyelid twenty-four hours after the first injection, when it gave another definite XXX reaction to second test. Post-mortem.-Large cedematous patch in lung, size of orange, with caseous centres (glanders ?). Very numerous nodules in both lungs, size of peas downwards. Two definite lesions in spleen. 2. P. 1518. Same as above, injected in other eyelid forty-eight hours after first injection. Gave definite XXX reaction to second test. Post-mortem.--Fairly numerous nodules in both lungs, from the size of peas downwards, mostly" new." 3. P- 597· Same as above, injected in other eyelid seventy-two hours after first injection. Gave definite XXX reacticn to second test. Post-mortem.-Fairly numerous small nodules in both lungs. One large septic pneumonia" cavern." Conclusions.-Animals can react to another intrapalpebral test twenty-four hours after a previous reaction. This is an advantage over the subcutaneous method, and would probably be very useful where it is essential to retest as soon as possible.

Reacting Animals Destroyed. Fifty-five animals, besides those described above, have now been destroyed on the local intrapalpebral reaction alone, and in every case glanders nodules were found on making a post-mortem examination. In several of the cases other lung lesions, such as septic pneumonia, cedema, hepatised areas, and strangles abscesses were found; but the presence of these disorders seemed to have no effect on the reaction, and glanders lesions were always present as well. So far the writer cannot record any failure in finding lesions of glanders after a definite intrapalpebral reaction in some 130 cases. All the post-mortems were most carefully performed, and no doubtful lesions (e_g_, calcareous parasitic nodules ?) were allowed to pass muster as glanders nodules. In a few of the cases the nodules were extremely small, but I was perfectly satisfied that they were definite glanders lesions before declaring them as such.

General Conclusions. The intrapalpehral test is a safe and reliable method in the diagnosis of glanders, and its certain distinct advantages over the classical subcutaneous method are briefly summarised below : I. Clinical cases give constant reactions to this method. 2. Animals which had given double reactions to the subcutaneous

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test react to this method even during the height of the subcutaneous temperature curve. 3· Doubtful reactors to the subcutaneous test can be retested by this method, and give reactions immediately afterwards. 4. Certain animals which have failed to react to the subcutaneous test react to this method. 5. Animals which could not be tested by the subcutaneous method, on account of other diseases and high temperatures, can be tested by this method, and react if glandered. 6. A safe diagnosis can be made on the local reaction alone, which cannot be done in the subcutaneous method. 7. The local reaction is more delicate than the subcutaneous one, and more easily interpreted. Doubtful local reactions are extremely rare. 8. In ordinary cases one can obtain temperature, local, and ophthalmic reactions by one injection 9. Mules react to this test as readily as horses (which has certainly not been my experience with the subcutaneous method). As previously stated, I first employed the test to pick out glandered horses from stables where clinical cases were constantly cropping up, and where no other method could be safely adopted on account of high temperature, diseases, etc., and I decided to commence destroying animals which had reacted. As lesions were found in all cases, this. procedure was continued. Now, after the experience gained in some 130 cases, I feel quite justified in stating that an animal which reacts to the intrapalpebral test is glandered. I also feel justified in stating that an animal which does not react can be regarded as free from the disease. This latter statement has been verified by the subcutaneous test, and by the careful post-mortem examination of many animals which have died or been destroyed for other reasons after giving negative reactions. I desire to emphasise the fact that I started making experiments with the intrapalpebral test almost in a spirit of desperation, and that the results obtained were so satisfactory that it was gradually given a more extended trial. The method has now been subjected to a series of most severe and searching tests, ouf of which it has emerged with flying colours. Personally I consider it far superior to the ordinary subcutaneous method in every respect. In conclusion, I hope that the test will be tried by others, as I feel confident that it will be of the greatest help in the eradication of glanders, especially under circumstances where the application of the subcutaneous method is not practicable. Over 1400 animals have now been tested by the intra palpebral method as described above, and so far no failures can be recorded.