540 D . A . KINCH

FOERSTER . . . . . . . . LANG, E., AND DE NUNNO, R. . . LIRA, V., AND MARAWO, E. . . LIVINGSTONE, D. J., AND SANDISON,

PINSTON, M. . . . . . . . WIUIS, R. A. . . . . . . .

A. T.

RUDDUCK, H. B., AND WILLIS, R. A.

. . . . . . . ,,

1860. 1963. 1965. 1962-63.

1958. 1938. 1960.

1962.

Wurzb. med. Z., 1, 24. Minerva chir., 18, 141. This Journal, 89, 317. Br. J. Surg., 50, 291.

M.D. Thesis, Univ. Lyons. Amer, J. Cancer, 33, 205. Pathology of tumours, 3rd ed.,

London, p. 616. The borderland of embryology

and pathology, 2nd ed., London, p. 557.

A RAPIDLY FATAL INFECTION CAUSED BY NOCARDIA CAVIAE IN A DOG

D. A. KINCH* Veterinary Investigation Centre, Cambridge

PLATE CLXXXVI

INFECTION with members of the Nocardia group of organisms causes a wide variety of lesions in dogs. In many reported cases, identification of the causal agent has been inadequate. Frost (1959) drew attention to the lack of precise identification of the causal agent and to the variation in lesions; these points are illustrated in some of the cases published during the last 19 yr (see the table). McGaughey (1952) reviewed the literature extensively.

Short illnesses associated with actinomycotic infection are relatively uncommon in dogs, but in a series reported by Mansi (1952) deaths usually occurred within 3 to 5 days of the onset of clinical signs. The present paper records a rapidly fatal infection with Nocardia caviae in an 18-mth-old male Corgi. Possible predisposing factors are discussed.

REPORT Clinical history

On 11 Sept. 1964, the dog was exercised for a prolonged period over fields; it was not accustomed to such exercise. On the next day, a veterinary surgeon examined the animal and attributed severe muscular stiffness in the dog's hind- quarters to the excessive exercise of the previous day. A 4-day course of the corti- costeroid preparation, betamethasone (Betsolan, Glaxo Laboratories Ltd), was prescribed. The dog had recovered fully after treatment for 1 day, but on the day after completion of the course, 17 Sept., it became ill and would not eat. It was listless and its rectal temperature was 105.2"F (40.7"C). There was a painful sub- cutaneous swelling just medial to the angle of the left mandible with enlargement of the submaxillary lymph-glands. Although there was also a severe pharyngitis, the dog could swallow normally at that time. Treatment with parenteral chlor- amphenicol was instituted.

The dog's clinical condition deteriorated during the next 24 hr, so that it could drink only with difficulty and choked after swallowing. The temperature had risen to 106°F (41-1°C) and the submandibular swelling had enlarged. Treatment with

* Present address: I.C.I., Ltd, Alderley Park, near Macclesfield, Cheshire.

CANINE NOCARDlOSlS 541

intramuscular chloramphenicol was continued, antihistamines were administered and the application of hot fomentations to the swelling was advised. On the next day, 19 Sept., the dog was unable to stand and would no longer drink anything; the temperature was 104.8"F (40.4"C) and though the throat swelling was reduced, the submaxillary lymph-glands were still enlarged; the pharyngitis was unimproved. Treatment with antihistamine and chloramphenicol was continued and, in addition, dextrose saline was injected subcutaneously. The dog continued to deteriorate and it died early the next day, 8 days after showing muscular stiffness, but less than 4 days after the onset of acute illness.

Gross pathology

The dog was in only fair bodily condition; the subcutaneous tissues were dry and the carcass musculature was deep red. The conjunctival mucous membranes were very congested, but the appearance of other visible membranes was normal. Medial to the angle of the left mandible was a circumscribed abscess surrounded by oedema fluid; it was approximately 2.5 cm. in diameter and contained foul-smelling greyish- white fluid pus. The liver was normal in size but very dark reddish-purple; under the capsule and throughout its substance were numerous grey nodular lesions of varying size up to 4 mm. in diameter. Most of these nodules were solid, but small amounts of thick greyish-white pus could be expressed from some of the larger ones.

The kidneys were dark reddish-brown and contained a few small greyish-white nodular lesions up to 5 mm. in diameter in the cortex; no nodular lesions were found in the medulla.

The lungs were collapsed, congested and oedematous and the airways were full of thin mucus. Many nodular lesions similar to those described above were present throughout the lung substance. The smallest nodules were quite firm, but the larger ones contained a small amount of thick greyish-white pus.

There was a small amount of blood-tinged fluid in the thoracic cavity; ecchy- motic haemorrhages were present on the parietal pleura and smaller haemorrhages on the epicardium. The meningeal vessels were engorged with blood, but no gross lesions were seen in the brain.

Histopathology Pieces of liver, lung and kidney were fixed in 4 per cent. neutral formaldehyde.

Paraffin sections were cut and stained with (i) Ehrlich's haematoxylin and eosin (HE), (ii) Masson's trichrome, (iii) Gram's stain, and (iv) the Ziehl-Neelsen stain, and by (v) Gordon and Sweets' silver method for reticulin, and (vi) the periodic acid-Schiff (PAS) procedure.

Liver. The sinusoids, central and portal veins are engorged with blood. Many hepatic cells are distorted into triangular or rectangular forms; their nuclei are eccentric but otherwise they appear normal in =-sections. The cytoplasm of many hepatocytes contains vacuoles up to 5 p in diameter. In other cells much larger vacuoles with clear centres are present; these resemble fat vacuoles. Many of the non-vacuolated hepatocytes have yellowish-brown bile pigment granules in their cytoplasm; these granules are also present extracellularly. Surrounding the blood vessels in the larger portal tracts are lakes of strongly acidophilic exudate containing neutrophil leucocytes and branching filamentous organisms.

Small granulomatous abscesses are present throughout the liver, often close to blood vessels (fig. 1). The smallest abscesses are composed of cores of lymphocytes and macrophages surrounded by a few epithelioid cells and fibroblasts (fig. 2). Small amounts of collagen and reticulin are present round the periphery of these lesions. In larger lesions the cores are composed of neutrophil leucocytes and free red cells and these are surrounded by a typical granulomatous type of reaction. In some lesions, however, there is no fibrosis; though macrophages, lymphocytes and

542 D. A. KlNCH

leucocytes can be seen, neither fibroblasts, collagen nor reticulin can be demonstrated round the periphery of these lesions.

Kidney. Venous and capillary congestion is present throughout the cortex and medulla. Very many glomeruli contain foamy acidophilic exudate within their capsules. The cytoplasm of the tubules is strongly acidophiljc and often hyalinised; cell outline in the lumen of the ducts is indistinct. The nuclei of the tubular epithelium show no obvious change. Two abscesses in the cortex are mainly composed of neutrophil leucocytes and small numbers of macrophages and epithelioid cells. There is no evidence of encapsulation by fibrous tissue in either of these abscesses (fig. 3).

Lung. There is venous and capillary congestion with intra-alveolar haemorrhage in many areas. The alveoli and smaller bronchi are full of homogeneous acido- philic exudate containing large numbers of cells, both polymorphonuclear leuco- cytes and alveolar macrophages. Numerous granulomatous lesions up to 1 mm. in diameter are seen in the alveolar regions of the lung, often adjacent to blood vessels or bronchi. The structure of the lesions is similar to that of the granulo- matous nodules in the liver.

Bacteriology Microscopic examination of direct smears from the mandibular abscess and

lesions of the liver, lung and kidney, revealed large numbers of branching fila- mentous Gram-positive organisms with beaded staining (fig. 4). The organisms in stained sections of the liver, lung and kidney were seen to be Gram-negative, PAS- negative and non-acid-fast, and could be seen ramifying throughout most abscesses. The organisms were particularly numerous in the exudate round the portal vessels in the liver.

Material from the mandibular abscess and from representative lesions in the liver, lung and kidney was cultured aerobically on 5 per cent. sheep blood agar at 37°C. After 18 hr, profuse pure growths of fine grey colonies were obtained from each tissue and after incubation for 48 hr the colonies were approximately 1 mm. in diameter with slightty raised, fluffy white surfaces surrounded by narrow grey haloes. The colonies were firmly embedded into the surface of the medium. They continued to grow at room temperature and, after several days, aerial hyphae could be demonstrated with the low-power objective of the microscope. The cultures were composed of Gram-positive acid-fast filamentous organisms with beaded staining. Subcultures of the organism were examined by Dr R. J. Olds of the Department of Pathology, University of Cambridge. He identified the organism as Nocurdiu cuviae on the criteria of Gordon and Mihm (196%).

DISCUSSION The actinomycetes have long presented problems of accurate identification.

Wilson and Miles (1964) divided the family into an anaerobic genus, Actinomyces, and an aerobic genus, Nocardia. Nocardia is subdivided on the basis of acid- fastness into two subgroups. N . asteroides and N. brasiliensis fall into the acid- fast group. Such a subdivision no longer seems valid, for Gordon and Mihm (19620 and b) showed that 11 of 44 strains of N. asteroides and 12 of 62 strains of N. brasiliensis were not acid-fast. These workers suggested that once the criteria for the separation of Nocardia and Streptomyces had been met (Gordon and Smith, 1955), the patterns of decomposition of casein, tyrosine and xanthine provided a fairly reliable basis for the presumptive recognition of N. asteroides and N. caviue and, to a lesser extent, of N. brasiliensis (Gordon and Mihm, 19626). In a series of 219 cultures of actinomycetes, Gordon and Mihm (19626) found only 15 cultures of N. caviae; 9 of these had not been identified previously and a further 5 had been identified as N. asteroides.

There can be little doubt that the Nocardia species isolated from the present

KINCH PLATE CLXXXVI

CANINE NOCARDIOSIS

FIG. 1 .-Distribution of granulomata FIG. 2.- Liver. Early granuloma. HE. x 525. in theliver. Haematoxylin and eosin

(HE). X35.

FIG. 3.-Kidney. Cortical abscess and FIG. rl.--Gram-positive filaments in glomerular and tubular damage. purulent exudate. Gram. x 910. HE. X60.

CANINE NOCARDIOSIS 543

case was the cause of the generalised lesions described. It was easily seen in all lesions and was readily isolated from them, though the variation in its staining characteristics in tissue sections, in direct smears from lesions and in smears from cultures was notable. The gross appearance of the mandibular abscess suggested that it had been present for at least 2-3 wk, and as no other chronic lesions were found elsewhere in the body it seems likely that this was the primary focus of infection. Submandibular abscesses are not unusual in nocardia infections in the dog; Ginsberg and Little (1948), Mansi (1952), Bohl et al. (1953) and Johnston (1956) describe dogs with such lesions. These authors make no particular comment about infection of the throat. It may be that infection gains entry at the time of change of dentition in a young dog so that a small encapsulated focus of infection occurs in a local lymph-gland where it may remain quiescent until activated by some stress factor.

Two factors may have precipitated a flare-up of latent infection in the present case. The dog was subjected to unusually vigorous exercise and it was treated with an anti-inflammatory drug. Muscular stiffness as a prelude to nocardia infection is recorded by Sautter, Rowsell and Hohn (1953). In one of their cases, the lameness of a 6-yr-old German pointer was associated with an abscess in the lumbar region that eventually discharged. Lameness in association with nocardia infection is not recorded by other authors and in the writer's opinion the stiffness in the present case was associated with the excessive exercise rather than the infection.

Modern corticosteroids in veterinary use have a marked anti-inflammatory action in addition to their glucogenic activity. The susceptibility of animals to the effects of corticosteroid drugs varies with the species (Long and Shewell, 1954). In susceptible species these drugs may reduce vascular dilatation and permeability brought about by inflammation. Leucocytes may emigrate less freely into tissues, and phagocytic activity of polymorphs and macrophages is reduced (Florey, 1962). Florey further states that cortisone may depress the development of granulation tissue in some species but not in others; the depression is independent of vascular changes. Hydrocortisone and other substances interfere with the formation of ground-substance and appear to cause fibroblasts to withdraw their cytoplasmic processes. If the host's reaction against micro-organisms is thus suppressed, the organisms may be able to invade tissues much more readily. It is notable that, in mice, low-grade infections with Actinomyces muris (Long and Shewell, unpublished, cited by Long, 1955) and Corynebacterium muris (Watanabe et al., 1960) flare up to produce serious disease under the influence of cortisone.

In the present case it is suggested that the stress and disturbance of exercise together with the administration of a highly anti-inflammatory corticosteroid created a situation analogous to actinomycotic infection in mice. Thus, organisms in a primary focus of infection in the cervico-facial region of the Corgi dog were probably released and spread haematogenously throughout the body. The associa- tion of many of the lesions with blood vessels supports the theory of haematogenous spread. There is no direct evidence that the corticosteroids encouraged dissemina- tion, but there is indirect evidence to suggest that spread of infection was accelerated. The miliary lesions in the present case were very active and of very recent origin; only the submandibular abscess was of long standing. Although miliary lesions occur in canine nocardiosis, they are usually accompanied by a variety of chronic lesions in various parts of the body. Moreover, the course of the disease was unusually short if one accepts that it lasted for only 4 days; most of the reported fatal cases lasted at least 2-3 wk (see the table). In Mansi's series in which the duration of illness was only 3-5 days, the description of the lesions suggests that systemic infection had been active for a rather longer period. It seems possible therefore that, in the present case, spread of infection was encouraged by some factor. The absence of early fibrosis in some lesions may suggest that the anti- inflammatory mechanisms were suppressed. I t may be pertinent that the lesions in the kidney were completely unencapsulated ; the corticosteroids are excreted

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546 D. A. KINCH

through the kidney and this organ is more likely to be subjected to higher concentra- tions of the hormone than other tissues.

It therefore seems reasonable to suggest that the cortiwsteroids probably affected the dog’s capacity to contain a focus of nocardia infection within the cervico-facial region.

SUMMARY A rapidly disseminating fatal infection caused by Nocardia caviae in a dog is

described and some of the recent literature on related conditions in dogs is reviewed. The possibility that rapid spread of infection in the present case may have been facilitated by therapy with a corticosteroid preparation is discussed.

I wish to express my thanks to Mr J. C. Davies of Royston who brought this case to my attention, and to Mr A. H. Chooi for helping with the preliminary examinations.

REFERENCES 1953. J. Amer. Vet. Med. Assoc., 122, 81. BOHL, E. H., JONES, D. O., FARRELL,

R. L., CHAMBERLAIN, D. M., COLE, C. R., AND FERGUSON, L. C.

BROWN, A. R., AND OSBORNE, A. D. FAW, M. T., OBEID, H. M., AND 1963. Sudan J. Vet. Sci., 4, 12.

FLOREY, H. W. . . . . . . 1962. General pathology, 3rd ed., London,

FORSYTH, H. . . . . . . . 1949. Vet. Rec., 61, 586. FROST, A. J. . . . . . . . 1959. Austral. Vet. J., 35, 22. GINSBERG, A., AND LIITLE, A. C. W. GORDON, RUTH E., AND MIHM, JOAN

1962. Vet. Rec., 74, 371.

HASSANEIN, A. 0.

chap. 43.

1948. This Journal, 60, 563. 1962a. J . Gen. Microbiol., 27, 1.

M. 9, Y Y 1, ,, 196%. Ann. N.Y. Acad. Sci., 98, 618.

GORDON, RUTH E., AND SMITH, 1955. J. Bact., 69, 147.

JOHNSTON, K. G. . . . . . . 1956. This Journal, 71, 7. LONG, D. A. . . . . . . . 1955. Int. Archs Allergy Appl. Itiimitn., 6,

MILDRED M.

337. LONG, D. A., AND SHEWELL, JENNIFER LOVEDAY, R. K. . . . . . . MCGAUGHEY, C. A. . . . . . MCGAUGHEY, C. A., BATEMAN, J. K.,

MANSI, W. . . . . . . . . MORANT.KATHLEEN M. . . . . SAUTTER, J. H., ROWSELL, H. C., AND

THORDAL-CHRISTENSEN, A., AND

WATANABE, M., SUZUKJ, K., FUJIKI,

WILSON, G. S., AND MILES, A. A. .

AND MACKENZIE, P. Z.

HOHN, R. B.

CLIFFORD, D. H.

M., AND ABE, N.

1954. 1963. 1952. 1951.

1952. 1951. 1953.

1953.

1960.

1964.

Br. J. Exp. Path., 35, 503. J. S. Afr. Vet. Med. Assoc., 34, 273. Br. Vet. J., 108, 89. Ibid., 107, 428.

Ibid., 108, 14. Ver. Rec., 63, 82. N . Amer. Vet., 34, 341.

Amer. J. Vet. Res., 14, 298.

Bull. Natn. Inst. Anim. Hlth, Tokyo, no. 38, p. 23.

Topley and Wilson’s Principles of bacteriology and immunity, 5th ed., London, vol. I, p. 508.