61 6 . I 26-02 2 . 7 (Streptococcus fcemlis) : 6 I 9 . 3 I

STREPTOCOCCAL ENDOCARDITIS IN LAMBS

S. JAMIESON and JEAN STUART Veterinary Laboratories, Mil l of Craibstone, and Department of

Bacteriology, University of Aberdeen

(PLATES L AND LI)

APART from the specific type associated with Erysipelothrix rhusio- pathim infection, endocarditis is seldom reported in animals. Blakemore, Elliott and Hart-Mercer (1941) described endocarditis in lambs, but only as part of the disease " Joint-ill '' caused by infection with group-C streptococci.

The purpose of this paper is to record in young lambs an outbreak of primary endocarditis unaccompanied by arthritic or other lesions. In our opinion the causal organism was Streptococcus fmmlis, a view supported by the successful experimental reproduction of endocarditis in rabbits with strains isolated from the affected lambs.

CLINICAL HISTORY

In a group of thirty ewes with their lambs, part of a flock of five hundred ewes and lambs grazing on hill pasture, five lambs died suddenly within eleven days. The group of thirty ewes had been lambed in a small enclosed pasture which had sustained sheep for this purpose for many years. The remainder of the flock were lambed under different conditions.

The lambs were 63, 64, 71, 72 and 80 days old when the first sign of illness was noted. This showed itself as a loss of playfulness and dislike of sporting with the rest of the flock. The respiratory and pulse rates were obviously increased. The onset of symptoms was sudden and the affected lamb was usually dead within twelve hours. One lamb which died while suckling had shown no symptoms of respiratory or cardiac embarrasspent.

The only lesions observed were large excrescences on the tricuspid and bicuspid valves of the heart (fig. 1). These vegeta- tions, which were greyish yellow in colour, had obliterated the valvular structures and involved the adjacent ventricular and auricular endocardium. They were irregularly infiltrated with blood. No changes were observed in the aortic or pulmonary valves.

Autopsy.

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236 S. JAMIESON AND J . STUART

BACTERIOLOGICAL INVESTIGATIONS

A streptococcus was obtained from the cardiac vegetations of The organism was isolated from the spleen and four of the lambs.

liver of one lamb but never from the joints.

Methods

The strains isolated from the lambs were designated S1, S2, S3 and 54. Fermentation reactions. The medium was one per cent. peptone water

containing one per cent. of fermentable substrate and Andrade's indicator. The tubes were incubated for 14 days at 37" C.

Hydrolysis of sodium hippurate. The method of Slavin (1948) was employed. The strains were grown for 2 and 5 days in arginine monohydrochloride medium containing 2 per cent. sodium hippurate and 0-1 per cent. aesculin. From these cultures 1.0 ml. of the clear supernatant was added to 0.8 ml. of the following solution :-ferric chloride 12 g., concentrated hydrochloric acid 2.5 ml., distilled water 1 litre. Hydrolysis of the sodium hippurate was indicated by the formation of a precipitate.

Heat resistance. Three drops of a 24-hour broth culture were added to 5 ml. of glucose broth in tubes of uniform thickness. These were evenly heated in a closed water-bath for 30 minutes at 60' C. The tubes, together with McConkey and blood-agar plates inoculated from them, were incubated for two days at 37O C.

Growth on bile-blood agar. Sheep blood-agar plates containing 10 and 40 per cent. of ox bile and control blood-agar plates without bile were simultaneously inoculated from 48-hour glucose-broth cultures and incubated for 5 days at 37' C.

Final pH. The final p H was determined in 1 per cent. dextrose broth. This medium was inoculated with a young actively growing culture and after five days' incubation the p H was determined colorimetrically with bromo-cresol purple against a range of standard buffer solutions of known p H .

One per cent. glucose broth containing 6.5 per cent. NaCl was inoculated with actively growing cultures and incubated for five days at 37" C.

Growth at pH 9-6. Lactose agar was adjusted immediately before use by the glass-electrode method. The medium was made up in slopes and inoculated with an actively growing broth culture. The tubes were incubated with un- inoculated controls in an air-tight jar containing soda lime.

Swpens iom for rabbit inoculation. Successive subcultures were made in 0.5 per cent. Hiss's plasma broth. The most active culture was grown for 18 hours at 37" C., when 10 ml. of the culture were centrifuged and the deposit re-suspended in 0-2 ml. of the supernatant. This was injected into the ear vein of rabbits.

In the single-massive-dose experiments the suspension used was obtained in the same way but was made up with normal saline in place of supernatant. The desired density was secured by matching against Brown's opacity tuba.

Penicillin sensitivity. Sensitivity to penicillin was estimated by the impregnated-disc method described by Morley ( 1945).

Serological tests. These were carried out with standard Burroughs Wellcome antisera.

Growth in 6.5 per cent. NaCl.

Results Smears from the vegetations stained by Gram's method showed

numerous Gram-positive cocci, arranged in pairs and small chains.

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STREPTOCOCCAL ENDOCARDITIS IN LAMBS

PLATE L

FIG. 1.-Heart of lamb, showing typical vegetations on both cusps of the mitral valve. x c.l .8.

PIG. 2.-Histology of vegetation from heart of lamb, showing matrix of fibrin, peri- pheral bacterial foci with lenaoaytia infiltmtion, and surface blood-clot. HEematoxylin and eosin. x 50.

STREPTOCOCCAL ENDOCARDITIS I N LAMBS 237

Good growth was obtained in 1 per cent. glucose broth. The growth was not noticeably granular except with strain S4. On ox, sheep and human blood-agar plates hBmolysis was not observed after cultivation a t 37'C. for 48 hours. Strain S4 gave a narrow green zone on blood agar which was not present with chocolate agar. Growth in broth containing 5 per cent. horse or ox blood did not produce hzmolysis after 18 hours' incubation.

All strains fermented glucose, lactose, sucrose, mannitol, inulin, zsculin and trehalose without gas production. In addition, strain S1 fermented sorbitol and salicin but not raffinose ; strain S3 fermented salicin and raffinose but not sorbitol, and strain S4 fermented raffinose but neither sorbitol nor salicin.

Sodium hippurate was irregularly hydrolysed. Strains S1 and S2 failed to grow in the presence of 6.5 per cent. NaCl but strain S4 consistently yielded a good growth in this medium. All the strains readily grew on McConkey's medium and in the presence of 10 per cent. ox bile (irregularly in 40 per cent.).

Exposure to 60" C. for thirty minutes failed to destroy any of the strains. In methylene-blue milk good growth with decolorisation of the dye was observed in dilutions from 1 in 1000 to 1 in 20,000. In a controlled water-bath all strains grew a t 45" C. A feeble reaction with group-D serum was obtained with all the strains. Gelatin was not liquefied and all strains were quite insensitive to penicillin. The final pH ranged from 4.8 to 5.

The morphology, general cultural characters, and subsidiary characters such as resistance to 60" C . for 30 minutes, growth a t 45" C. and at p H 9-6 and sometimes in the presence of 6.5 per cent. NaCl, insensitivity to penicillin and type-D serological grouping, all justify defining the organism as Str. fcecalis.

HISTOLOGY

In lambs the vegetations consisted of a matrix of structureless faintly eosinophilic material, best demonstrated by Gram's method. For this reason the material was considered to be degenerated fibrin. On the surface of the vegetations numerous bacterial foci were present (fig. 2 ) . These were demarcated by a zone of leucocytic infiltration. Bacterial foci were not observed deep in the matrix of the vegetation. The leucocytic zone was composed principally of polymorphonuclear leucocytes in various stages of activity and degeneration, frequently with large numbers of ingested cocci. Macrophages crowded with Gram-positive cocci, red cells and blood pigment were also present. Elastic tissue was not demonstrated by Weigert's method nor calcium by von Kossa's method. A variable amount of recent blood clot was present on the surface of the vegetations and at their valvular base. Myocardial changes as reported by Ellakemore et al. were not observed, but a few capillaries showed perivascular cuffing by leucocytes.

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238 8. JAMIESON AND J . STUART

EXPERIMENTAL The development of endocarditis in rabbits after inoculation with

non-hamolytic streptococci is a well-recognised event, but we thought it of interest to find if our strains of Str. fcecalis could produce lesions on the heart valves of rabbits and if these lesions were similar to those found in the lambs.

Effect of repeated doses

After this animal had received a total of 18 intravenous injections of strain S3 suspension it showed signs of cerebral irritation and died. The only changes observed post mortem were in the heart. The joints were normal. There was dilatation of the right ventricle. The mitral valve had extensive vegetations, involving the whole of that structure and its chorda tendinex! (fig. 3). Examination of films from the vegetations showed numerous Gram-positive cocci arranged in pairs or short chains. Sections of the lesions showed a similar structure to that of the affected lambs (fig. 4). Cultures from the lesions yielded Str. fcecalis of similar reactions to strain S3.

This rabbit was given 19 intravenous injections of a suspension of strain S4, after which it showed signs of debility and lassitude. It was killed by chloroform. Visible changes were confined to the heart. The myocardium was soft and the left ventricle dilated. On the mitral valve there was a circumscribed haemorrhagic patch (fig. 5). From this a streptococcus was isolated which gave reactions similar to those of our strain of Str. fcecalis S4.

This animal received 17 intravenous injections of a suspension of strain S4, when it became ill and was killed by chloroform. Vegetations similar to but not so extensive as those seen in rabbit 1 were found on the mitral valve. From these lesions a streptococcus identical in cultural reactions with strain S4 was recovered.

Rabbit 1.

Rabbit 2.

Rabbit 3.

The effect of a single massive dose

This animal received a single intravenous injection of 1 ml. of a saline suspension of live Str. fcecalis standardised to Brown’s tube no. 1 0 x 2 . The animal showed no clinical disturbance after the injection and six weeks later it was killed by chloroform. No lesions were observed post mortem and Str. fcecalis was not isolated from the heart valves, spleen, liver, kidneys or intestinal contents.

One ml. of a saline suspension of live Str. fcecahk standardised to Brown’s tube 10x4 was given intravenously to this rabbit. I ts temperature was elevated 24 hours after injection and the animal appeared dull and drowsy, but 48 hours later it had become normal. Twelve weeks later the animal was killed with chloroform. No lesions were observed post mortem and Str. fcecalis was not isolated from the heart valves, liver, spleen, kidneys or intestinal contents.

Rabbit 4.

Rabbit 5.

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STREPTOCOCCAL ENDOCARDITIS IN LAMBS

PLATE LI

FIG. 3.-Heart of rabbit 1, showing vegetations on mitral valve follow- ing repeated intravenous injections of a suspen- sion of Str. foecalis. x c.4.

FIG. 4.-Histology of lesion in rabbit 1. Hrematoxylin and eosin. x 50.

FIG. 5.-Heart of rabbit 2, showing an early sub- endocardia1 hiemorr- hagic lesion x c.4.

STREPTOCOCCAL ENDOCARDITIS I N LAMBS 239

DISCUSSION

The chief point of interest is the natural occurrence of endocarditis in lambs caused by Streptococcus fcecalis and unaccompanied by joint changes. No evidence was obtained about the mode of infection, but we think it probable that the primary infection was umbilical, as the conditions under which the ewes were lambed are well known to produce such infections. We think it significant also that in rabbits the same strains of Str. fcecalis produced no joint lesions, although such lesions are commonly produced by other hsmolytic and non- hzemolytic streptococci (Cowan, 1924 ; Wright, 1926).

While we hesitate to state, on the evidence, that the strains of Str. fcecalis isolated from the valvular vegetations of the lambs have a predilection for the heart valves, we think it is suggestive that no other lesions were found, either in the lambs or in the experimental animals.

The gross nature of the lesion in the affected lambs and in rabbit 1 reduced the value of microscopic examination in assessing the origin of the lesion. The single hamorrhagic lesion in the mitral valve of rabbit 2, from which Str. fcecalis was also recovered, suggests that the onset of the lesion is embolic by way of the small capillaries of supply to the endocardium. The failure to reproduce the condition by single massive doses of Str. fcecalis suggests that additional factors are involved in the genesis of the lesion and that the pathogenicity of the infecting organism is of low grade.

SUMMARY 1. An outbreak of endocarditis in young lambs without joint

lesions is reported. S t y . fcecalis was isolated from the cardiac lesions and appeared to be the causal organism.

2. Repeated intravenous doses of suspensions of the organism reproduced similar endocardia1 lesions in rabbits ; single massive intravenous doses did not.

We are indebted to Professor J. Cruickshank, Department of Bacteriology, University of Aberdeen, for his helpful encouragement and advice.

REFERENCES

BLAREMORE, F., ELLIOTT, 8. D., 1941. This Journal, lii, 57.

Cowm, MARY L. . . . . . 1924. Brit. J . Exp. Path., v, 226. MORLEY, D. C. . . . . . . 1945. This Journal, lvii, 379. SLAVIN, D. . . . . . . . 1948. J . Camp. Path. and Therap., lviii,

WRIGHT, H. D. . . . . . . 1926. This Journal, xxix, 5.

AND HART-MERCER, J.

161.