Clostridial myocarditis in lambs JRW GLASTONBURY, JE SEARSON, IJ LINKS and LM TUCKETT

New South Wales Department of Agriculture, Regional Veterinary Laboratory, Wagga Wagga, New South Wales 2650

SUMMARY: Five outbreaks of myocarditis were investigated in young sheep. They occurred during late winter and spring when there was lush growth of pasture following a prolonged period of drought. Clinically the disease was characterised by sudden death and pathological findings were dominated by acute multifocal locally extensive necrotising and haemorrhagic myocarditis. A fluorescent antibody technique was used to demonstrate the presence of Closfridium chauvoeiin paraffin embedded sections of myocardium from 4 of the outbreaks. Aust Vet J 65: 208.209

Introduction Clostridial diseases, particularly enterotoxaemia, are of con-

siderable importance to the Australian sheep industry. How- ever blackleg caused by Clostridium chauvoei occurs less fre- quently in sheep than cattle (Seddon et a1 1931; Beveridge 1983) and is rarely seen in sheep in southern New South Wales. Myocarditis due to infection with C. chauvoei, “black- leg of the heart”, has been described in cattle (Beveridge 1983; Hulland 1985) but we were unable to find any specific reports of this disease in sheep.

The epidemiological and pathological features of 5 out- breaks of clostridial myocarditis in sheep form the basis of this paper. In addition the successful adaptation of a fluo- rescent antibody technique to confirm the presence of C. chauvoei in paraffin embedded tissue sections is described.

Epidemiology The outbreaks occurred during spring and late winter 1983

on 5 farms situated throughout the Murray-Riverina region of southern New South Wales. Three were investigated during August, and one each in September and November. Pastures during this period were extremely lush, contrasting sharply with severe drought which prevailed during the 12 months to Autumn 1983. At Wagga Wagga, which is situated centrally in the region, 298 mm of rain fell during 1982 and 771 mm in 1983.

Details regarding the age and breed of affected sheep and death rates are given in Table 1. Affected animals were 1 year old or less and the death rates varied from 3.4 to 7.0%.

Affected sheep had not been vaccinated against blackleg, but those in outbreak 4 had been vaccinated against entero- toxaemia and tetanus at the time of marking. Two to 4 days before the appearance of disease, the sheep in outbreak 1 were castrated and docked, those in outbreaks 2 and 3 were yarded and the yearlings in outbreak 4 were shorn. In outbreak 5, the mules operation had been performed on the weaners 6 weeks before the deaths occurred.

Clinical and Pathological Findings No clinical signs were observed with affected sheep being

found dead.

Gross Pathology All necropsies were performed on the farms by field veter-

inarians. In outbreaks 1 and 3 the sheep had died within the previous hour, whereas postmortem intervals were not given for the others. Myocardial congestion and interstitial hae- morrhage and large volumes of blood stained pericardial fluid containing copious fibrin were described for every case. Renal congestion and pulmonary congestion and oedema were seen less frequently. Appreciable haemorrhage and bruising around the base of the tail, adjacent to the docking wounds, were found in the lambs from outbreak 1. Lesions of blackleg in

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skeletal muscle were not specifically sought or found in any of the sheep.

Unfortunately clostridial myocarditis was not suspected in the field and no fresh myocardium was submitted to the laboratory for bacteriological examination. However a range of tissues, including myocardium but not skeletal muscle, was fixed in 10% neutral buffered formalin from at least 1 sheep in each outbreak.

Hislopathology At the laboratory, sections of the fixed tissues were prepared

by standard methods and stained with haematoxylin and eosin. In addition sections of myocardium were stained by the Gram method and with fluorescein isothiocyanate (F1TC)-labelled anti-C. chauvoei, C. septicum and C. oedematiens immuno- globulins*. The latter was achieved by clearing the sections to tapwater and fixing them in anhydrous acetone for I min. They were then stained with the appropriately labelled im- munoglobulin for 30 min at 37°C in a humidified chamber. Rinsing with phosphate buffered saline was performed for 10 min before mounting in buffered glycerine for immediate examination under ultraviolet light.

Histological examination of heart from each outbreak revealed large multifocal locally-extensive areas of acute coagulative myocardial necrosis. The affected areas contained moderate to large numbers of Gram-positive bacterial rods with straight sides and rounded ends, morphologically con- sistent with Clostridium sp, and were associated with consid- erable interstitial congestion, oedema and haemorrhage. Mod- erate numbers of degenerating neutrophils were associated with the necrotic myocardium in 4 cases. Occasional interstitial vessels in 2 cases contained cellular fibrin thrombi. In 3 cases, fibrinous, fibrinopurulent or fibrinohaemorrhagic epicarditis, mostly of diffuse distribution, was evident.

Except for outbreak 3, the bacteria associated with the lesions of necrotising myocarditis showed apple-green fluo- rescence in sections stained with FITC-labelled anti-C. chau- voei immunoglobulin (Figure 1) and no fluorescence in the C. septicum and C. oedematiens preparations. The bacteria in material from outbreak 3 failed to fluoresce.

Bacteriology Swabs taken from the castration and docking wounds in

outbreak 1 were submitted to the laboratory in Stuart’s trans- port medium. Smears were then prepared and stained with immunmoglobulins against the 3 species of clostridia. Positive fluorescence for C. chauvoei, only, was obtained for each swab.

Discussion On the basis of the history of sudden death, pathological

findings and demonstration of C. chauvoei in association with

Wellcome Diagnostics, Wellcome Australia Ltd, Concord, New South Wales

Australian Veterinary Journal, Vol. 65, No. 7, July, 1988

TABLE 1 Epidemiological details for 5 outbreaks of clostridial

myocarditis in sheep Outbreak Affected Sheep Deaths

Age Breed Number %

2 Weaners Merino 121350 3.4 1 Lambs' Merino 60/1200t 5.0

3 Lambs Crossbred 6 4 Yearlings Merino 201500 4.0 5 Weaners Merino 22013000 7.0

The lambs varied from 6 to 12 weeks of age. t Number deadlnumber in group.

the lesions, clostridial myocarditis was diagnosed in 4 of these outbreaks. The criteria for confirmation of clostridial disease proposed by Batty et a/ (1967) were fulfilled. Clostridia also were strongly suspected as being the aetiological agents in outbreak 3 because of the identical nature of the histological lesions and the consistent morphology of the organisms. They may have been C. sordellii or C. perfringens.

The fluorescent antibody staining procedure worked well on paraffin embedded histological sections. Variations from the procedure recommended by the manufacturer for the stain- ing of smears prepared directly from lesions were that our sections were fixed in anhydrous acetone for one instead of 10 min and following application of the labelled immuno- globulin the sections were placed in a moist chamber at 37°C instead of room temperature. This technique should have application in confirming diagnoses of black disease, blackleg involving skeletal muscle and malignant oedema in situations where fixed rather than fresh tissues are submitted to the laboratory.

The relative influence of various epidemiological factors in these mortalities is difficult to gauge. Clostridial myocarditis was not recorded in sheep from this region before these out- breaks and it has not been observed since. In addition classical blackleg in sheep also is relatively rare with only 7 cases being noted in the files of this laboratory since its establishment in 1974. This would suggest that the peculiar environmental conditions experienced during winter and spring of 1983 may have been of importance. It is unlikely that C. chauvoei multiplies in soil (Hulland 1985) but its survival is favoured by large quantities of organic matter (Smith 1957). The latter

Figure 1. C. chauvoei in section of myocardium from outbreak 5 showing apple-green fluorescence. (FITC - labelled anti-C. chauvoei immunoglobulin X 340).

Australian Veterinary Journal, Vol. 65, No. 7, July, 1988

would have been provided by the abundant growth of pasture which occurred following the breaking of the prolonged period of drought in 1982 and early 1983. The ample supply of feed also meant that the sheep on affected farms were gaining weight at the time of the outbreaks, a factor which is consid- ered to be most important in the epidemiology of blackleg in cattle (Smith 1975). Other epidemiological aspects of these outbreaks indicated that sheep may be predisposed to C~OS- tridial myocarditis by a lack of acquired immunity, age and stress associated with recent yarding. None of the sheep on any of the 5 properties had been vaccinated with C. chauvoei bacterin. In each instance young animals were affected indi- cating an age-related resistance because the equally unpro- tected dams were not affected. This has not been described for blackleg in sheep (Smith 1975). The sheep in 4 of the 5 outbreaks had been yarded for various management proce- dures 2 to 4 days previously.

Histologically the myocarditis in these sheep was compa- rable to the descriptions of blackleg in standard texts (Jones and Hunt 1983; Hulland 1985). Two variations were that interstitial emphysema was not a feature and there appeared to be a greater neutrophil response in 2 of the cases compared to that found in blackleg involving skeletal muscle.

Apart from outbreak 1, the portal of entry of C. chauvoei was not established in any of these sheep. Infection via wounds is thought to be the portal of entry for blackleg in sheep whereas endogenous infection is of most importance in cattle (Smith 1975). The myocarditis in lambs from outbreak 1 almost certainly resulted from bacteraemic spread from the malignant oedema associated with the wounds of castration and docking. Wounds from shearing and the mules operation may have provided sites of entry in outbreaks 4 and 5, re- spectively, while endogenous infection may have occurred in outbreaks 2 and 3. Apart from outbreak 1 it was not possible to determine whether the myocarditis was a primary lesion or the result of bacteraemic spread from skeletal muscles as the latter were not examined in detail.

This investigation added little to our understanding of the pathogenesis of clostridial myocarditis. Latent spores of C. chauvoei may be found in many tissues and in muscle (Kerry 1964; Hulland 1985). It is thought that they are stimulated to germinate by degeneration, intramuscular haemorrhage or low oxygen tension (Hulland 1985). The work of Fell et a1 (1985) would suggest that the yarding and management procedures to which the young sheep in outbreaks 1 to 4 were subjected would have significantly increased levels of plasma cortisol. This coupled with the subsequent release of catecholemines may have caused sufficient physiological change in the heart to stimulate germination of spores of C. chauvoei. The necrosis of muscle fibres associated with proliferation of the bacteria is due to release of exotoxins and ischaemia subsequent to injury to blood vessels (Hulland 1985). There was ample evidence of the latter in the present cases.

Acknowledgments We would like to sincerely thank D Harding, M Barry, N Buckley

and E Sergeant of Griffith, Albury, Wagga Wagga and Jerilderie, respectively, for carrying out the field investigations.

References Batty I, Kerry JB and Walker PD (1967) - Vet Rec %o: 32 Beveridge WIB (1983) - In Animal Health in Australia, Vol 4,

Bacterial Diseases of Cattle, Sheep and Goats; Australian Govern- ment Publishing Service Canberra, p 55

Fell LR, Shutt DA and Bentley CJ (1985) - Aust Vet J 62: 403 Hulland TJ (1985) - In Patholoo of Domestic Animals Jubb KVF,

Kennedy PC and Palmer N,-Voi 1, 3rd Edn, Academic Press, London, p 183

Kerry JB (1964) - Vet Rec 76: 396 Jones TC and Hunt RD (1983) - In Veterinary Pathology 5th Edn, Lea and Febiger, Philadelphia, p 579

Seddon HR, Belschner HG and Edgar G (1931) - Aust Vet J 7 : 2 Smith LDS (1957) - Adv Vet Sci 3: 463 Smith LDS (1975) - In The Pathogenic Anaerobic Bacteria, 2nd

(Accepted for publication 28 March 1988)

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Edn, Thomas, Illinois, p 231