ABSTRACTS AND REPORTS. 147

The small central bodies may be either central or peripheral in the rounded forms. In twin forms a connecting line between the two was clearly visible, producing an appearance resembling a dumb·bell.

The central bodies are especially clearly seen when stained with Loeffler's flagellum mordant for three to four hours in the cold, followed by anilin fuchsin. They appear deep black, surrounded by a clear zone, the blood corpuscles being tinted red. If as much as possible of the fuchsin be removed from the preparation by alcohol and a dilute Giemsa solution be introduced under the cover·glass with a capillary tube, the inner bodies can be clearly seen to be surrounded by a blue zone which becomes as large and as deeply stained as those seen in a Giemsa preparation after dry fixation.

Infect~d blood corpuscles which have been dissolved in distilled water and centrifuged contain in their stroma the somewhat swollen anaplasmata, which stain in a manner similar to well·differentiated specimens that have been treated by the Git:msa moist fixation method. In living preparations only small ill-defined refractile bodies can be seen in the erythrocytes. The parasites are somewhat more clearly seen with dark-ground illumination, appearing as clear projections. Addition of a weak solution of saponin or I per cent. sodium taurocholate solution causes solution of the red corpuscles and the outer zones of the parasite. With dark·ground illumina­tion the central bodies can be seen as small highly refractile points. If one adds some diluted chinese ink and examines the specimen with transmitted light the corpuscles appear as faint discs in a grey medium.

An attempt to set free the parasites by hremolysing the corpuscles with a hremolytic serum failed. Attempts to stain the living parasites with neutral red were unsuccessful. Bettcr results were obtained with a weak methylene-blue solution.

If one adds a weak solution of saponin or quinine to a drop of infected blood under a cover-glass a clear zone is formed around the rim of the parasites, which become somewhat swollen. Concentrated salt solution also produces swelling.

The addition of a weak solution of neutral red to a mixture of a 1 to 2

per cent. solution of quinine and blood immediately the parasites are swollen provokes a faint red colouration, especially around the outer rim, showing that the otganism is alive and that "vital" staining commences as the cell dies. No doubt the central bodies, rich in chromatin, are the actual parasites which obviously divide and multiply. They bear a loose morphological resemblance to the chlamydozoa of Prowazek in trachoma, vaccinia, scarlet fever, and fowl plague, and the bodies described by Babes in rabies, by Mayer and Kesselitz in variola and varicella, and which are familiar under the names Guarnier and Negri bodies. (Sieber, Bed. Tier. Woch., Vol. XXVI" p. 993, 1910.)

THE INFECTION OF BIRDS WITH ANTHRAX BY INGESTION.

THE author, after reviewing the lit~rature on the subject of the occurrence of anthrax in birds, concludes that, as the views expressed by the earlier authors as to the occurrence of anthrax in birds under natural circumstances' are in direct contradiction with thosc founded on present day observations, they should be accepted with very great reserve, and he thinks that the great

ABSTRACTS AND REPORTS.

majority of cases so diagnosed by those authors were in reality cases of either fowl cholera or fowl diphtheria. The author's experiments were carried out with the object of testing the possibility of infecting birds with anthrax by ingestion.

In view of the fact that starvation, insufficient nourishment, illness, etc., favour infection, account was taken in the experiments of the following factors: Fasting before and after feeding with anthrax material; feeding with powdered glass, chalk, vegetable foodstuff (bread); deprivation of water; plucking, cooling by means of cold baths; disease and immaturity. The animals used in the experiments were twenty fowls, four ducks, and five pigeons.

The infective material was derived from a surface agar culture made from the spleen of a bovine animal dead of the disease. The cultures used were in no case more than ten days old, and were very rich in ~pores. Growth was very luxuriant. The virulence of the material was tested from time to time by the inoculation of mice, and its purity by means of plate cultures.

The growth was scraped off the agar surfaces with a stout platinum needle and placed between two fragments of bread, which were then placed on the root of the tongue to ensure prompt swallowing. In the later experiments small gelatin capsules were used to contain the growth, as they were easier to manipulate, less of the growth was lost, and swallowing was quicker. On one occasion the capsule was smeared with a solution of keratin with the idea of delaying softe!1ing in the crop, but it was without effect, as the capsule was found destroyed in the crop at the post-mortem.

The following are the results of the first four of the author's experiments in detail.

Experiment I.-The subject was a cock that had been ailing for some weeks and was in a very weak state. On the morning of loth March 1909 the bird ate its food well and at the same time received three loopfuls of growth from a surface agar culture between two pieces of bread. A few hours later the bird's condition became markedly worse, it crouched with ruffled feathers and closed eyes, its comb hanging cold and amemic over one side. The follow­ing morning it was found dead.

Post-mortem.-Marked anremia of the extremities of the body and par­ticularly of the comb. Pasty offensive freces in the cloaca. No discharges from the natural orifices. Muscular tissue dark-coloured and opaque-looking. A sero·sanguineous fluid in the pericardium but none in the pleural or peri­toneal cavities. Congestion of the vessels of the mesentery of the duodenum. Slight swelling of the spleen and liver. With the exception of the dark red duodenum, the mucous membrane of which showed gelatinous swelling and large hremorrhages, the intestine was anremic to a marked degree. The intestinal contents were mixed with a large quantity of dark tarry blood. Congestion of the veins. Mucous membrane (except that of the gizzard) intact.

Microscopic examination showed anthrax bacilli. These were found in smears of the pericardial fluid and from the intestine in small numbers, and numerously present in smears frbm the spleen, liver, lungs, kidneys, and muscles. The bacilli varied considerably in form. In the preparations from the spleen, liver, kidneys, and muscles the bacilli were typical, either singly or in chains. In preparations from the intestine degeneration forms were found, disintegrating forms entwined in each other, with indistinct capsules and faintly stained. Faintly stained cap~ules were also found without central contents.

Mice inoculated from the spleen, liver, duodenum, and cloaca died. In plate cultures there were comparatively few but quite characteristic

anthrax colonies. Experiment 1I.-A well-nourished five-months-old fowl was starved from

ABSTRACTS AND REPORTS. I49

17th till 23rd March, and from the 23rd till the 26th received daily an agar culture between bread. Symptoms first appeared on the evening of the 23rd. The feathers were ruffled, the eyes were kept half closed, and the fowl moped in the corner of the cage. Further symptoms were not observed on the following day, and the bird died on the night of the 26th.

Post-mortem.-Discharge of greenish-yellow, pasty f<eces with an objection­able odour from the cloaca. An<emia of the comb. Increased quantity of serous fluid in the pericardium. Slight swelling of the spleen and liver. The usual alterations in the duodenum. Swelling and h<emorrhagic infiltra­tion of the congested mucous membrane. Extravasation of blood into the lumen of the bowel. Watery f<eces in the large intestine, with a fibrinous deposit on the mucous membrane.

Microscopic and cultural examinations were negative. Mice· inoculated from the spleen, liver, blood, and various parts of the

intestine remained healthy except for one inoculated from the large intestine, which died of septic<emia.

Experiment iII.-A well-nourished, healthy, six-months-old hen was starved from 17th till 23rd March and treated in the same way as NO.2. The dose of anthrax virus was without fatal effect. Only a few vague symptoms were observed for a couple of days, and disappeared shortlyafter­wards. The fowl remained healthy during the period of fourteen days for which it was fed, and it also resisted an inoculation (subcutaneous) with a large dose of anthrax. .

Experiment IV.-A lively six-months-old fowl was first fed with an anthrax culture spread upon bread on 22nd March and then starved. The first symptoms of illness were observed after the seventh feeding. The bird remained in a corner of its cage with its head tucked under its ruffled feathers, its eyes half closed, and quite indifferent to external stimuli. It offered no resistance to handling, and when placed in front of the cage returned immed­iately to its corner with a stag~ering gait. On the morning of 30th March the bird was found to be trembling, and after a few minutes it fell and died with convulsions.

Post-mortem.-Muco-sanguineous discharge from the nostrils. Slight dis­tension of the abdomen. Slight swelling of the liver, and softening of the spleen. Marked congestion of the mesenteric vessels and of the vessels of the serous covering of small intestine. The mucous membrane of the duo­denum was swollen and infiltrated with blood. Blood extravasations into the lumen of the small intestine. Contents of the large intestine yellowish-green and pasty in consistence. Diphtheritic deposit on the mucous membrane. Large quantities of gas in the intestines. Anthrax bacilli could not be found in smears from the various organs, and inoculation and cultural experiments were without result.

The following table shows the results of the experiments with the whole series of twenty fowls :-

[TABLE.

ABSTRACTS AND REPORTS.

Table I.

From this table it can be seen that it is extraordinarily dIfficult to ltlfect fowls with anthrax under ordinary circumstances.

It is difficult to produce an infection which can be proved by the demonstration of the bacilli, even by the administration of large doses under conditions which weaken the animal. Anthrax bacilli could be demonstrated in all the organs in two cases only-No. I, in which only a small quantity of infective material was given, and No. 20, in which a large quantity was administered.

ABSTRACTS AND REPORTS.

The author concludes from these experiments that anthrax may possibly occur in healthy fowls, but that it is extraordinarily rare. He is also of the opinion that the death of those animals that dIed (rom h~morrhagic enteritis, and in which anthrax bacilli could not be demonstrated, was due to an anthrax toxin.

The following table gives the results of the experiments carried out with ducks:-

Table II.

Feeding Experi· ments. Period 1

Elapsingl Cause of ~~-.-----~~ -

,. before I Death. Cause of , i ays. "Uatena. I ,eat ,.

No.1

E'xper'iment Animals.

Case.

i D 1"- 'I Death,. Number Dead. ]'\ I

~-I ____ ~_I __ I ___ -~--~'~---~---~-------

1 I Duck, starved 5 Ove culture f) days Haelllorrhagic five days before daily enteritis

2 Dnck, starved 7 One culture

I

feeding

3

4

six days after daily feeding Two cuI·

Duck, two months old, sta,rved after

the first feeding Duck, two months old

2

2

tures daily Two cuI·

tures daily

do.

3 days Anthrax

3" Infection with an organism

like the fowl cholera

bacillus

All the in· I Anthrax. oculated mice

do, The same infection as

the duck.

From this table it may be seen that ducks are also difficult to infect with anthrax, but that under suitable circumstances it is possible to produce infection by ingestion, especially in young birds.

Results of experiments carried out with pigeons are shown in Table III. :-

Table Iff. ------,---------------~----------- -----

Feeding Experi· ments. Period I

Elapsing Cause of ---- -- ~'--l ----------.-- befure I Death. I Cause oj D 1I,r t I Death. Number Dead'l' De th alJs. '.La, erlU . I a .

-1- Pigeon, t::- -4- One culture I~ays ~thrax~- ~the i=-I Anth::-months old daily ocnlated mice

2 Pigeon, two 10 Two cuI· 11" do. do, do. years old, 'I tures daily i

starved six days previously I

Pigeon, two 4 years old,

starved after ' feeding I

Pigeon, two 14 I do, 14 " years old, dt· I

prived of water I seven days I'

after feeding 5 Pigeon, three I. 8 Two cuI· }

I

years old, "tures daily i 19 sickly before j 4 \ Three cuI· ~ "

feeding ! tures daily I ---------~--

3

Case.

Experimental Animals.

No.

do. 5 " do. do. do.

4 do. do. do.

do. do. do.

ABSTRACTS AND REPORTS.

The results obtained provide proof of the assertion that among birds the pigeon possesses the highest degree of susceptibility to anthrax. Young birds are most easily infected. Thirst is more influential than starvation in causing infection by ingestion, and the period of starvation after the first administration of anthrax material is more important than a period of starvation of six days before the administration.

Conclusions.-From the above results the opinion may be arrived at that birds, and especially the fowl and the duck, possess a very slight degree of susceptibility to anthrax. Out of twenty-nine birds it was found possible to infect only seven by the ingestion of anthrax material. . In all these cases typical anthrax bacilli could be demonstrated in all the organs. Ten per cent. of the (owls, 25 per cent. of the ducks, and 100 per cent. of the pigeons, or an average of 24 '13 per cent. of the total, died of anthrax, Cases 7 and 19 (fowls) not being included. In addition a number of the birds died of h~morrhagic enteritis, which could not be proved to be due to anthrax, but the lesions found closely resembled those found in animals dead of the disease. Fifty per cent. of the fowls and 25 per cent. of the ducks fall into this group.

It is worthy of notice that clinically and post mortem these cases were all practically alike. and that in these respects the cases closely resembled those in which anthrax was proved by microscopic examination, cultivation, and experimental inoculation.

On these grounds the author thinks that the conclusion is justified that the birds died from anthrax.

At first he believed that the cause of the frequent occurrence of enteritis might either be hunger or the circumstance that after the period of starvation the animals ate excessively, thereby causing irritation of the intestine. However, the fact that enteritis occurred in animals that were not starved and that received no powdered glass, but were fed either on anthrax material with a vegetable diet, or on anthrax material with the ordinary food, shows that the fatal enteritis was not solely caused in this way.

The following experiment was devised in order to ascertain exactly the effects of the period of starvation :-

1. A well nourished six-months-old fowl was starved for eight days. It was then chloroformed to death and a post-mortem examination made. No abnormalities of any of the internal organs could be found, except for a slight diffuse reddening of the whole of the intestine, which was. in no way comparable to the condition found in the experimental animals that died.

2. A similar fowl was starved for ten days, and was then given a sufficiency of food. This was eaten greedily, and the bird remained alive and well.

It is therefore obvious that the enteritis which closely resembled that seen in animals dead of anthrax was not due to the technique of the experiments, but must have had some causal connection with the ingestion of the anthrax material. The lesions found at the post-mortem coincide with those found by Kienzle in birds suspected of having died of anthrax but in which only h~morrhagic enteritis could be found.

Precise investigations are necessary to determine whether the enteritis is due to the irritation of the intestine by anthrax toxin, and death due to intoxication.

The author does not look upon the enteritis as a localised intestinal anthrax, for in the immense majority of cases demonstration of the bacilli is impossible, either by the microscope or by experimental inoculation, and in the diagnosis of anthrax it is at least necessary to demonstrate either bacilli or spores at the seat of infection, in this case the intestine.

Frank and Lubarsch state that the invasion of the blood by the bacilli occurs relatively late, and in the pigeon may not occur at all.

ABSTRACTS AND REPORTS. 153

Possibly the lesion may be explained in this way. The inflammation of the small intestine and the pathological alterations occurring in the mucous membrane depend upon the virulence and quantity of the infective material, and the resistance offered depends upon the susceptibility and the bodily condition of the animal.

In those cases in which the animal died without having been infected with anthrax the blood was powerfully bactericidal, but possessed no power of neutralising toxin set free from the bacterial bodies (endotoxin). Death may occur indirectly as a result of the ingestion of anthrax material through intoxication with degeneration products (endotoxin) of the bacterial bodies, and with toxins elaborated by the bacilli.

It has already been proved that the injection of killed cultures may have serious effects, and may cause death in a longer or shorter time as a result of chronic intoxication. According to Arloing, it is possible to kill young lambs with filtered germ-free cultures of the anthrax bacillus.

In cases of severe illness in which the blood under normal conditions pos­sesses no bactericidal properties, in young animals, which, according to Trapeznikoff and Wagner have not the full capacity possessed by adult animals of opposing bacterial invasion, and in those animals which are specially susceptible to the disease, the body opposes the infection by means of an increased supply of blood to the part, but this possibly fails to prevent the bacterial invasion. The spores develop into bacilli, multiply, and pass through the inflamed intestinal mucous membrane, that is to say, localised anthrax becomes a general anthrax infection.

The cases in which the author's experimental animals were proved to have died of anthrax support this view.

With regard to the lesions found in the pigeons, it was to be noted, as a rule, that the alterations were more pronounced in the subcutaneous con· nect:ve tissue. in the mesentery of the small intestine, and particularly in the muco:JS membrane of the small intestine. The other tissues of the body were only slightly affected in every case. There was usually no marked swelling of the spleen, there being only a slight darkening in colour and softening, while the liver was frequently spotted and its edges rounded.

According to Oelmer, Spinola, Heusinger, Koch, and others, in naturally occurring cases of anthrax, as the result of ingestion, in birds and other animals the principal lesions are situated in the intestine. Koch makes special mention of the severe inflammatory changes and blood extravasa­tions into the lumen of the bowel in his experimental cases of infection by ingestion. The author's observations on birds agree with those of Koch, that in slightly susceptible animals the lesions are chiefly found at the seat of infection. The author also found that the longer the toxin acted upon the mucous membrane, that is to say, the more often anthrax material was administered, the more intense and extensive was the inflammation and the greater the blood extravasation. He also found that the longer death was delayed the further along the intestine was the most severe inflammation to be found.

The author's experiments also show the extraordinary rapidity and activity of the destructior. of the anthrax bacillus in the fowl. In the case of Fowl 7, the crop contents of which when inoculated into a mouse produced anthrax, no anthrax bacilli or spores could be found in smears made (rom the intestine, and inoculation and culture experiments with intestinal material were negative. The other lesions in animals that died of hremorrhagic enteritis confirmed these observations.

The experiments were not concerned with investigating the part played by the digestive juices, the blood serum, or the white corpuscles, either singly or together, in the destruction of the bacilli or spores. In the examination

ABSTRACTS AND REPORTS.

of the smears, however, no leucocytosis was observed, nor was any marked inclusion of bacilli or spores in the white corpuscles discovered. Only an odd bacillus was found in the body of a white corpuscle, the majority lying free between the cells.

The experiments show, in opposition to the conclusions of Koch, Lamer, Gaffky, and Feser, that even enormous quantities of spores may be fed to fowls and pigeons without effect, but that it is possible to produce anthrax if luge quantities of spores be used and conditions are favourable.

The Practical Effect of these Results.--Anthrax very seldom occurs under natural circumstances in birds, although opportunities for infection are great. For the production of infection large quantities of anthrax material must be taken, and also the circumstances must be favourable, such as disease, inanition, and immaturity. Under ordinary conditions anthrax in birds occurs as an acute general disturbance, and is sometimes apoplectiform.

The rapidly produced pathological changes seen in the experimental animals are rarely or never seen under natural circumstances. An animal would rarely get so large a quantity of spores. Under natural conditions, however, infection by the ingestion of anthrax flesh, which as is well known rapidly putrefies, and other infected materials, might be favoured by the decomposition products contained in them.

The rare occurrence of natural anthrax in birds is not opposed to the high percentage of fatal cases in the experiments, when one considers the favour­able moments chosen for infection and the large quantity of sporulating material used.

Under favourable circumstances anthrax may occur naturally in bird~, but it is only in the rarest of cases that the bacilli can be demonstrated. In these cases diagnosis must be based on the presence of haomorrhagic enteritis with more or less swelling of the organs, and upon tbe possibility tbat anthrax material may have been eaten in large quantities. (Hofherr, Centralbl. f. Bakt., Parasit. und Infektions., Bd. 55, Heft 6, 6th September 1910, PP·434-463.)

CANKER OF THE HORSE'S FOOT AND GREASE.

DEALING with canker first, tbe author reviews the literature on the subject and points out that comparatively little has been written regarding the histological alterations that occur in cases of canker. During the last four years about forty cases have come under his notice, and about twelve of these have been submitted to microscopical examination. His material was derived from animals that underwent operatiom, or from incurable cases that were destroyed. Pieces of diseased horn, with tbe epidermis attached, were fixed in 4 per cent. formalin-alcohol solution, and after hardening in alcobol of increasing strength they were prepared for embedding in paraffin and celloidin. In a few cases the paraffin method was found to be unsuitable on account of the hardness or brittleness of the material. Tbe method of staining that was principally employed was the haomatein and eosin method, methyl eosin being substituted in many cases for the ordinary solution. Gram's method alone, or associated with picrocarmine, safranin, toluidin, sudan, and orange G, was also used, and about 1500 sections were examihed. In the typical cases details of tbe history, clinical symptoms, and treatment were collected. Special attention was paid to the exact description of tbe histological alterations met with.

By way of introduction a brief description of tbe normal epithelium of the hoof, with special reference to the staining affinities of certain cells, is given, so tbat the pathological alterations may be better appreciated.