ABSTRACTS.

The rhipicephalus annulatus is known to go through all its developmental stages on the same animal. The first change of the larv::e is complete on the fourth day. As the parasites were only found in the blood on the twelfth to fourteenth day, the larv::e could not have caused the infection. Thi~ is proved by Motas's experiments on sheep and Lounsbury's on dQgs, which sbowed that the sexually mature. ticks alone are capable of transmitting infection. (Motas, ArhilJa Veterinaria, No. I, Feb. 19°5; ex. Deutsche Tierdrztl. Woclzen., No. 40, 7th Oct. 1905.)

THE IDENTIFICATION OF THE PRINCIPAL SUGARS FOUND IN ANIMAL URINES.

Two sugars have most commonly been found in animal urines, viz., glucose and lactose or milk sugar.

Glucose appears in the urine of animals of both sexes, and from very varying causes; its pas~age is generally due to some special morbid condition. Lactose, which is elaborated by the tissue of the udder, is found in the urine of female animals whose udders are functionally active whenever milk is retained or secreted in undue proportion. It can also be found in the urine of individuals that have been kept over long on milk diet. In both cases its passage in the urine is a purely physiological phenomenon: in the first, it indicates the activity of a gland whose products of secretion, not being eliminated by the usual pa!hs, are reabsorbed, pass into the blood, and, in certain cases at least, reach the kidney; in the second, it suggests that an excess of milk has been taken, and that, a portion having escaped digestion, it is not directly absorbable by the tissues and is accordingly eliminated by the kidneys.

The varying conditions under which glucose and lactose appear in the urine cause glycosuria (in the broadest sense of the term) and lactosuria'to assume a very different significance from the diagnostic and prognostic points of view. The former is almost always closely allied to some diseased condition, of which it is one of the essential phenomena; it is therefore of great importance. The latter when appearing during the course of any disease seldom has more than a very distant connection with it, and is directly due to suspension of milking; it seldom calls for attention.

The above remarks show why, when testing the urine of a female yielding milk, it is not sufficient merely to detect the presence of sugar, but that, in order to be able to draw any useful conclusion from the discovery, it is necessary to determine the variety of sugar present.

Two tests are required for this object: A, a test for the presence of sugar, and B, a test for the variety of sugar (glucose or lactose). Except when dealing with a female animal in process of lactation, the former is sufficient.

(,1) The commonest method of detecting sugar in the urine is based on the reduction (abstraction of oxygen) by glucose and lactose of certain metallic salts dissolved in alkaline media, particularly of oxide of copper. The fluid usually employed is Fehling's solution,l a blue liquid, really a solution in

1 Fehling's liquid should be used fresh. It is prepared by mixing the two following solutions :-

Blue solution {Sulphate of copper .

• 'Yater • . . • • •

{

Potash . . . . . • • Double tartra.e of potash and soda.

'Yater . . . . . • Colourless solution

35 grammes 500 cc. 125 grammes 173 500 cc.

The solutions should be kept in separate bottles and an equal volume of each be mixed at the moment of use; the mixture should be shaken. Hydrated oxide of copper forms and is dissolved by the double tartrate.

ABSTRACTS. 53

tartaric acid of oxide of copper (Cu 0), which the above-mentioned sugars reduce to Cu, 0, a red insoluble precipitate.! This test is very convenient and sensitive, but to give certain results it calls for careful manipulation. The following method gives good results.

One test-tube is half filled with urine, which. if turbid (as is the rule in the horse). should previously have been filtered; a second test tube is half filled with Fehling's liquid. The contents of both tubes are brought to boiling poirrt' j a funnel, furnished with a filter paper, is placed in the tube containing the Fehling's liquid (the bottom of the stem of the funnel should touch the inside of the tube) j and then the urine is cautiously poured on to the filter. The above precautions are for the purpose of preventing substances like earthy salts and coagulated albuminoids, which are rendered insoluble by boiling, from passing into the Fehling's liquid, and to favour the urine and the test fluid coming into contact without mixing. When the manipulation is carefully carried out the two liquiqs only mix for a short distance; the reaction is con· fined to a, space of a few millimetres in height, and is much more clearly marked. Once the urine has filtered through, the liquids are allowed to cool.

When the urine is normal the zone of contact of the two liquids retains its limpid appearance even for a considerable time, only assuming a greenish tint as the blue solution and yellowish urine mix. There is no suggestion of deposit, either of a yellow, orange, or red colour; the appearance of greyish or dirty white flocculi has no significance.

When the urine contains sugar reduction occurs at the plane of contact of the two liquids, producing a change of colour, a tendency to turbidity, and later a coloured deposit. These changes occur more or less rapidly, and are subject to trifling modifications depending on the quantity of sugar present.

When the proportion of sugar is high (3 grammes or more per litre), the reaction is so clearly marked as to leave no room for uncertainty. Immediately the liquids come in contact, or very soon thereafter, the common surface appears of an orange· yellow, and a certain amount of turbidity is caused by precipitation of oxide of copper; this substance produces an orange-coloured layer at the bottom of the tube.2 If the liquids become mixed to a con· siderable depth other tints appear later, the upper layers being yellowish, shading off gradually into green towards the bottom of the tube.

When the urine contains less than 3 grammes per litre of sugar the reaction only occurs after cooling and the appearances are somewhat different.

The area of contact becomes more or less turbid and assumes a green tint when examined by reflected light; it is still semi-transparent, however, and appears reddish-orange by transmitted light.3 The green tint and the turbidity are indications of reductIon and prove the presence of sugar. If, however, in spite of their appearance, the operator is still'in doubt, it is only needful to await the formation of the precipitate of oxide of copper.

The above described method of carrying out the test is not difficult. It has the advantage of avoiding the uncertainty which sometimes follows the

1 The formation of this substance is mq)lained by tbe simple formula, 2 Cu 0=Cu2 0+0. 2 Oxide of copper precipitated by a pure solution of sugar is of a fine vermilion·red;

precipitated by sugar·containing urine, it is reddish·orange, orange. or yellow. This change of colour is in part due to krea.tinin; urea also has some influence, probably on account of the ammonia which it disengages when heated in presence of potash, which is in excess in Fehling's solution. Ammonia itself, if added to a saccharated solution in insufficient quantity to dissolve all the oxide of copper, modifies the colour of the precipitate in the manner suggested.

3 These appea.ra.nces are due to the fact that the liquid dissolves the oxide of copper a.lready formed, and a.gain gives it up in part on cooling (which explains the slowness of reduction), in the form of a very delica.te, orange·red precipita.te, dispersed uniformly through the liquid. The oxide of copper is dissolve,\ to a considerable extent by the kreatinin, proba.bly as a consequence of the ammonia produced by that substance when heated with Fehling's solution. Urea, which gives off ammonia under similar conditions, may playa similar part.

54 ABSTRACTS.

common method, viz., that of mixing equal quantities of the urine and Fehling s liquid and bringing the mixed liquids to boiling point.!

(B) The existence of sugar being proved, it becomes necessary, in the case of females giving milk, to determine whether the sugar is glucose or lactose or a mixture of the two. Strictly speaking, it is sufficient to ascertain the presence or absence of glucose, this being the only sugar of any real pathological importance.

Phenylhydrazine is the test employed. Its use is based on the fact that it gives in warm solutions containing acetic acid, (I) with glucose a crystalline compound (glucosazone) which is only slightly soluble in boiling water, and which produces a crystalline deposit on the surface of the liquid in which it forms even when warm; (2) with lactose a crystalline compound (lactosazone) which is very soluble in boiling water, and only deposits when the containing liquid becomes cold.

The test is carried out in the following way: If possible the urine is boiled to remove coagulable albuminoids and is filtered; 10 cc. of this urine are placed in a test-tube, and to it are added three pinches of acetate of soda and two of hydrochlorate of phenylhydrazine. The tube is placed for one hour in boiling water, at the end of which time it is examined. If the urine contains glucose a more or less abundant yellow precipitate is found dispersed throughout it; the substance appears in the form of light flocculi, in which it is generally possible to detect fine silky needles matted together, which remain for a long time in suspension and only precipitate slowly.

If the sugar present was lactose no precipitate whatever is visible on removal from the boiling water; the precipitate (which is also yellow in colour) only forms as the liquid cools. It appears if the tube is quickly cooled in a current of cold water, rapidly disappearing again on boiling, and once more appearing as the temperature falls. This behaviour is character­Istic of the presence of lactose.

When both forms of sugar are present the appearances are mixed; the liquid when withdrawn from the boiling water already shows a precipitate which increases as the temperature falls, but does not entirely disappear on again heating.

The differentiation between glucose and lactose by phenylhydrazine is based on the variations in solubility of the compounds produced by this substance with the sugars in question, and becomes more difficult when the proportion of sugar falls to a low point. This -difficulty is in part due to the fact that glucc,sazone, which is slightly soluble in boiling water, is still more soluble in urine; if formed only in small quantities it may remain dissolved in the hot urine, from which it is only precipitated by cooling,2 hence the possibility of confusion. Microscopic examination in the manner following may then clear up an otherwise doubtful point :-

When a positive but feeble reaction has been obtained with Fehling's solution the phenylhydrazine test is applied as above described, and the tube is allowed to cool very ~lowly, immersed either in the hot water bath or in a large quantity of warm water. The precipitate is then examined under the microscope. The compound formed by glucose always appears in the shape of bundles, brooms, or lines radiating from a common centre, and formed of

1 The normal urine of animals contain. reducing substances, among which may be mentioned uric acid (in carnivora and omnivora), kreatinin (in all domestic animals), certain phenol com­pounds: pyrocatechin and hydrochinon (especially in herbivora). Some of these only exert a reducing effect after long boiling in Fehling's solution; others, like kreatinin, reduce Fehling's solution immediately if at boiling point. but the oxide of copper so formed is again dissolved, and is not usually precipitated unless boiling is continued for some minutes ..

" Certain constituents of urine have the power of dissolving osazone equally well whether in hot or cold solutions; others dissolve it when warm, but yield it up again when cold. It is therefore easy to understand that when the urine contains little sugar, the glucosazone may either fail to be precipitated or only precipitate as the liquid grows cold.

ABSTRACTS. 55

needles, sometimes very long, always perfectly straight, rigid, and of the colour of picric acid. This torm of crystal is characteristic; when present it infallibly indicates the existence of glucose.

Microscopic examination need not be reserved exclusively for such cases as are above mentioned; it may be employed in all cases where it is necessary to di~tinguish between glucose and lactose, and serves to confirm the con­clusions arrived at by other means. (Nicholas, Rev. Generale de Med. Vet., No. 69, 1st November 1905.)

THE HISTOLOGICAL DIAGNOSIS OF RABIES.

IN March 1903 Negri announced that in many nerve cells obtained from rabid animals It was possible to detect corpuscles having special staining properties. These Negri described as being the parasite of rabie~.

Whether obtained from fresh cells or dissociated after twenty-four hours' maceration in 30 per cent. alcohol, they appear as oval, rounded, or kidney­shaped bodies varying from 5IL to 27 or 30IL in diameter, lodged in the proto­plasm close to the nucleus.

These corpuscles are strongly refractile and appear to contain granules in their interior. They vary in number in different cells, from ten to fifteen being present in one nerve cell. In general the most numerous and the largest are found in the ox, sheep, and goat. The corpuscles can be found in all parts of the nervous system, but are mo~t numerous and constant in the large pyramidal cells of the hippocampus. They are also numerous in Purktnje's cells in the cerebellar cortex.

These corpuscles exhibit special staining properties, taking eosin, acid fuchsin, etc., strongly.

The best method of staining is Mann's: A little fragment ofthe hippocampus is fixed in a saturated solution of sublimate, washed slightly, and the excess of sublimate is removed by iodised alcohol. It is next embedded in paraffin, the sections are fixed to the slide, treated with toluene and absolute alcohol, and hydrated and stained for ahout ten hours in the following solution: I per cent. aqueous solution of methylene blue, 14 parts; I per cent. aqueous solution of eosin, 17 parts; distilled water, 40 parts.

The sections are washed very rapidly in water, then in absolute alcohol, and afterwards treated with absolute alcohol containing a few drops of I per cent. solution of caustic soda in absolute alcohol.

The sections, which become red, are washed in absolute alcohol and passed into water acidified with a few drops of acetic acid. They again become bluf>. They are then dehydrated and mounted in balsam. The examination is made with an immersion lens.

Methylene blue may advantageously be replaced by toluidine blue. When stained by Mann's process the corpuscles of Negn appear like red

blood corpuscles of a bright red tint, while the other parts of the preparation are blue.

Sometimes the nucleoli of the nerve cells preserve a violet tint. The granules contained within the corpuscles can be rendered evident by other methods of staining, and appear as brighter point~, sometimes coloured blue.

In his first note Negri drew attention to the fact that, as a result of the constant presence of the above-described corpuscles in certain well-defined portions of the nervous ~ystem of animals suffering from rabies, a method of rapid diagnosii' had become available.

In a second note he showed that examination of the hippocampus is sufficient for diagnostic purposes, but, nevertheless, that in rare cases these