with 0.3mg bovine P P D until at least 60 days after the last caudal fold test with this reagent.

Acknowledgments We are pleased to acknowledge the assistance and co-operation

provided by Mr J . Cooper , Manager of the Struan Research Centre, and his staff in the performance of thi, experiment. We are grateful to Mr P. McCloud, Mathematical and Computing Services Unit of the Department of .4griculture, Adelaide for his assistance in the statistical analysis.

References Anon. 1982(a) - Bovine Brucellosis a n d Tuberculosis National

Eradication Campaign S tanda rd Definit ions and Rules. Vol. 1 : 21, Aust . Bur. An im. Hlth, Dept of Prim. Indust. , Canberra .

Anon. 1982(b) - Borine Brucellosis a n d Tuberculosis National Eradication Campaign S tanda rd Definitions a n d Rules. Vol. 2: 41, Aust. Bur. An im. Hlth, Dept of Prim. Indust. , Canberra .

Buxton, J . B. and Glover, R. E . (1939) - Tuberculin Tests in Cattle, HMSO, London , p , 35. ( A R C Rept. Ser. N o . 4 . )

Choi, C . S. (1972) - Studies on the Imrnunoserological Diagnosk o f Mycobacrerial Infections, MVSC Thesis, Univ. of Qld, Brisbane.

deJong, H . and Ekdahl , M O . (1969) - N.Z. vet. J . 17: 213. C o w , G . M. (1948) - Proc. 52nd U.S. live stk sanil. Ass., p 21. Huitema, H . (1973) - Tijdschr. Diergeneesk. 98: 396. Kerr, W. R . , Larnont , H . G . and McGirr , 3 . L. (1946) - Vet. Rec.

Kleeberg, H . H . (1960) - J l S. Afr . vet. med. Ass. 31: 312. Lepper, A . W . D. , McKay, D. J . , Lord, V . (1979) - Aust. vet. J .

McLaughlin, A . R . et al, (1973) - Proc. 17th Ann. Conf . ,4111.

Ritchie, J . N . , Robertson, A, , hluir , R . 0. (1952) - Stare vet. J . ,

Robertson, T. G . (1963) - N.Z. vet J . 1 1 : 6 . RosHurm, J . D. and Konyha, L . D. (1974) - Proc. 77th Ann . U S .

Rushford, B. H . (1964a) - Aust. vet. J . 40: 406. Rushford, B. H . (1964b) - Aust. vet. J . 40: 412. Suther, D. E . and Franti , C. E. (1974) - Am. J . vet. Res. 35: 379. Swindle, B. C . e t a/, (1950) - Proc. 54th U.S . livestk sanit . Ass.,

Van Waveren, G . M . (1953) - Proc. lSrh int. vet. Congr., p 140. Worthington, R . W . (1967) - Onderstepoort ./. vct. Res. 34: 345.

(Accepted f o r publication I I December 1984)

58: 443.

55: 507.

Ass. vet. lab. Diagn., p 391.

London. 7: 28.

An im. Hlth. Ass., p 368.

p 110.

CASE REPORT

Haemorrhagic necrotising enteritis in foals associated with Clostridium perfringens

L . D. SIMS*, S. TZIPORI*, G . H . H A Z A R D t and C. L . CARROLL*

SUMMARY: Two foals aged 35 and 48 h from 2 Thoroughbred studs died several hours after developing clinical signs of depression, severe haemorrhagic diarrhoea and dehydration. Both foals had an acute haemorrhagic enteritis extending from the anterior jejunum to the terminal ileum which was characterised histologically by villus necrosis. Necrotic villi were surrounded by large numbers of rod-shaped Gram positive bacteria. CIosfridium perfringens was recovered from the intestines of both foals and the isolates were considered to be C. perfringens type C. Other cases of diarrhoea were also observed in foals of the same age on these 2 studs, but the aetiology of these was not determined. Aust. vef. J. 62: 194-196

Introduction Clostridiurn perfringens is a known cause of an often fatal

haemorrhagic, necrotising enteritis in neonates of a number of species, including calves (Griner and Bracken 1953), lambs (Griner and Johnson 1954), and piglets (Barnes and Moon 1964). Enteric disease associated with C . perfringens has also been described in foals, albeit rarely. I t was first reported by Montgomerie and Rowlands (1937) in Wales and C. perfringens type B was incriminated as the causative agent. Subsequently, an outbreak associated with this organism was described by Mason and Robinson (1938) in South Africa. Leader (1952) described 2 outbreaks of acute enteritis attrib- uted to C. perfringens in the United Kingdom but the type of the organism was not determined. More recently, hae- morrhagic necrotising enteritis has been described in the United States of America (Dickie e f a/ 1978) and in Canada (Niilo and Chalmers 1982). In these 2 reports C. perfringens type C was isolated, and considered to be causing the disease.

This report describes 2 fatal cases of necrotising enteritis i n foals from which C. perfringens was isolated and which occurred on separate farms in southern Victoria.

* “Attwood” Institute for Veterinary Research, Department of Agriculture, Mickleharn Road, Westmeadows, Victoria 3047 529 B u r k e Road, Camberwell, Victoria 3124 t

I94

Case Histories and Clinical Findings Case I The first case occurred on a Thoroughbred stud north of

Melbourne in 1981. The foal was one of 29 born on the farm, 5 of which developed severe diarrhoea in the first 48 h of life. I t commenced scouring when 48 h old and displayed signs of severe colic and profuse yellow-brown watery diarrhoea. Over a period of 3 to 4 h it became depressed, anorectic and dehydrated and the passage of dark haemor- rhagic fluid faeces was noted. Oral treatment, with a mixture of intestinal protectants and absorbants, antibiotics and spasmolytics (kaolin, chalk, catechu$, 1375 mg neomycin sulphate, 1500 rng sulfathiazole, 1500 mg sulfadimidine, 15 mg diphenoxylate hydrochloride and atropine sulphate (15 ug)§), and intravenous fluid therapy (one litre of normal saline) were initiated but the foal failed to respond and died within 6 h.

Two weeks prior to the case described above, a foal had died on the stud from a disease with similar clinical signs but tissue samples were not submitted for histopathology or bacteriology. During the following year 3 further cases of diarrhoea were seen on this stud in foals of the same age.

$ Parnell’s diarrhoea powder, Parnell Laboratories Pty Ltd, Kirrawee, N e w South Wales.

5 Lornotil. lntervet (Australia) Pty Ltd, 34 Hotharn Pde, Artarrnon. New South Wales.

Australian Veteririary Journal, Vol. 62, No. 6, June, 1985

Affected foals were normal until they developed a yellow- brown foetid scour when about 30 to 40 h old. Two hours later the foals were disinclined to feed and became depressed. The severity of depression varied but all foals recovered with treatment in 24 to 48 h. Faecal samples from 2 of these were collected prior to treatment.

Case 2 This case occurred in 1982 on a Thoroughbred stud 20

km from the flrst farm and was one of 17 foals born on the farm during that year. The affected foal was clinically normal until 35 h after birth when it began to scour profusely, passing bloody and foul-smelling faeces. Despite therapy using a regime identical to that used in case 1, with the addition of one litre of pooled horse serum intravenously, the foal rapidly became depressed and died approximately 15 h after it commenced scouring.

Four other foals on the farm also developed diarrhoea at about 30 to 40 h of age and faecal samples from 2 of these were collected for microbiological examination. These foals all survived with treatment and displayed clinical signs similar to the non-fatal cases on the first farm.

Foalings were observed on both studs and the foals that subsequently developed diarrhoea were strong a t birth and were observed to suck from their dams.

Gross Findings Pal hologg

Lesions were similar in both cases. Significant changes were restricted to the gastro-intestinal tract. There was congestion of the serosal surface of the distal two-thirds of the small intestine with mild gaseous distention of mesenteric blood vessels. The mucosa of the small intestine was coated with a pasty grey material, considered to be kaolin, and clots of this were present in the stomach and anterior duodenum. The contents of the small intestine from the anterior jejunum to the ileo-caecal junction were red. The stomach was only partly filled and contained little milk. The large intestinal contents were bloody and fluid but the mucosa appeared normal. In the first case approximately 4 litres of fluid were present in the large bowel, in the second, about one litre.

Histopa thology In both cases there was a severe necrotising enteritis

extending from the anterior jejunum to the terminal ileum. The villi in affected areas were necrotic and surrounded by massive numbers of rod-shaped Gram-positive bacteria 2 to 3 p in length. Most villi were devoid of internal structure, but some contained occasional neutrophils and macrophages. Some of the necrotic villi were sloughing into the intestinal lumen and the remaining intestinal mucosa was composed of truncated, denuded villi to which bacteria were adherent (Figure 1). There was evidence of a diffuse, mild, acute inflammatory reaction in the lamina propria and submucosa of affected areas. Changes were not detected in the anterior small intestine, stomach and large intestine. Other organs from the second case, including brain, heart, mesenteric lymph node, lung, liver, kidney and spleen, were also normal.

Micro biology Aerobic and anaerobic cultures of mesenteric lymph node

and multiple sites of the small and large intestine from both foals were made on sheep blood agar1 and MacConkey agar1 plates. Liver, spleen and lung from case 2 were also cultured.

A mixed growth of C. perfringens and non-haemolytic Escherichia coli was obtained from the large intestine and mesenteric lymph node of case 1. The only significant findings from case 2 were a very heavy, pure growth of C. perfringens from the distal small intestine and a mixed growth of C. perfringens and alpha-haemolytic streptococci from the large colon.

Bacteria were identified as C . perfringens according to the methods described by Holdeman et al (1977). Identification

Figure 1. Microscopic section of terminal i leum from foal (case 1) which died of necrotising enteritis; note rod-shaped Gram positive bacteria attached to necrotic villi (arrow) (Giemsa X350).

was based on their Gram reaction, colonial morphology and haemolytic activity, as well as their metabolic products as determined by gas liquid chromatography and their carbo- hydrate fermentation reactions. The ability of C. perfringens type A diagnostic antiserum* to inhibit the lecithinase (alpha toxin) activity of the organisms, using egg yolk agar as the substrate, was also used as an aid in identification.

Attempts were made to type 2 isolates of C. perfringens from case 2 by intradermal guinea pig inoculation (Carter 1978). A control strain of C. perfringens type C (CN 3714) of unknown origin was tested concurrently. Zones of der- monecrosis, approximately 0.5 to 1 .O cm in diameter, were produced using supernatants of test cultures grown in a peptone medium (Sakurai and Duncan 1977), but not by those of cultures grown in cooked meat medium. Dermo- necrosis was prevented by incubation of supernatants with a mixture of commercial C. perfringens types A and C antiserums prior to inoculation, and also by trypsinisation. Treatment with other combinations of C. perfringens anti- serums as described by Carter (1978), or with horse serum free of antibodies to major C. perfringens toxins, had no effect on the reactions. Zones of dermonecrosis produced by the control strain were 3 to 4 times larger than those of the test strains. Supernatant fluids from cultures of one of the test isolates producing dermonecrosis had no apparent effect on 2 mice when inoculated intravenously.

Toxicity tests in mice (Carter 1978) were also carried out on filtrates of intestinal contents from case 2, but n o deaths resulted. Toxin was, however, demonstrable by intradermal guinea pig inoculation (Carter 1978). Three pairs of guinea pigs were inoculated with intestinal contents clarified by centrifugation a t 10,000 g. As with the bacterial isolates from this foal, only a combination of C. perfringens type A and C antiserums prevented (3 guinea pigs) or markedly reduced (2 guinea pigs) dermal necrosis. Trypsinisation had little effect on the reaction.

Dr L . Niilo (Lethbridge, Alberta, Canada) kindly examined an isolate of C . perfringens from case 1 for toxin production. Supernatants of cultures grown in both a meat mash medium and in a peptone medium (Hauschild et a1 1968) were not lethal to mice when injected intravenously, indicating that major toxins were not produced. H e also examined filtrates of 6-hour cultures for production of haemolytic toxins using sheep and rabbit erythrocytes. Production of all 3 major haemolytic toxins (alpha, gamma and delta) was detected. Alpha and gamma toxin were produced in amounts compa- rable to that produced by 2 “classical” type C strains grown concurrently as controls. However, the amount of delta toxin produced (measured as the reciprocal of the lowest dilution

1 Oxoid Australia Pty Ltd, West Heidelberg, Victoria

Australian Veterinary Journal, Vol. 62, N o . 6 , June, 1985

Wellcome Reagents Ltd. Beckenham, England

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at which haemolysis occurred) was 4 to 16 times lower than that produced by control strains, and diminished to barely detectable levels on repeated sub-culture.

Faecal samples obtained from 4 other foals with diarrhoea, described earlier, from the same farms as the fatal cases were cultured aerobically and anaerobically. From 2 of these, C. perfringens was isolated as well as alpha- and non- haemolytic streptococci and non-haemolytic and haemolytic E. cofi, the 2 latter organisms being present in all samples. Isolates of C. perfringens were not typed.

Samples from all foals were also examined for viruses by electron microscopy using techniques described previously (Tzipori and Williams 1978). No viruses were detected in intestinal contents from the dead foals or faeces from non- fatal cases of diarrhoea.

Discussion

The disease described in these 2 foals was characterised by a severe necrotising enteritis associated with massive numbers of C. perfringens in the small intestine. It is similar to the disease reported recently in the United States of America and Canada (Dickie et a1 1978; Niilo and Chalrners 1982), referred to a s enterotoxaemia in foals and associated with C. perfringens type C .

Typing tests suggested that the organisms isolated from the 2 foals were probably C. perfringens type C . The isolate from case 1 was tentatively classified as this type on the basis of detection of the minor delta toxin in culture filtrates. This toxin is produced only by certain strains of C. perfringens type C (Niilo 1980). An explanation for the inability to detect major toxins by in v ivo testing of this strain is lacking, but may be related to the relative sensitivities of the techniques used. Production of alpha toxin by this isolate was detected by in vitro techniques but not by in vivo methods, lending support to this possibility. Strains of C. perfringens type C which produce very small amounts of beta toxin are recog- nised (Taylor 1940, Buddle 1954), and results of tests on isolates from the second case suggest these organisms were poor toxin producers. These isolates produced some beta toxin, as recognised by the production of small zones of derrnonecrosis in guinea pigs when supernatants of cultures were inoculated intradermally, but insufficient to kill mice inoculated intravenously.

Previous reports of necrotising enteritis associated with C. perfringens in foals have usually described single cases of the disease, although Mason and Robinson (1938) described one outbreak which affected more than one foal. In 2 successive years on the one stud they reported losses of 7 and 9 out of 43 foals as a result of severe dysentery. The outbreaks described by us resulted in lower mortality rates than this, but on both affected studs more than one animal was affected with a severe enteric disease, and, on the first farm, 2 foals died.

Outbreaks of diarrhoea in foals less than 48 h of age were, until 1981 rarely seen in Victoria (G. Hazard unpublished data), although Bain (1954) regarded diarrhoea as one of the commonest conditions encountered in young foals elsewhere in Australia. O n the 2 farms reported in this paper on which foals died with necrotising enteritis, other foals of the same age developed a clinical syndrome with many similarities to

that seen in the fatal cases. These foals developed profuse watery diarrhoea and became depressed, but ultimately recovered with treatment. The possibility of a relationship between the fatal and non-fatal conditions was therefore considered. Bacteriological examination of faeces from the non-fatal cases was unrewarding and the aetiology of the diarrhoea was not determined. C. perfringens was isolated from 2 of these foals but this was considered to be of little significance. The prevalence of this organism in the faeces of young foals has not been reported but it is highly likely that, a s in other species, high numbers of C. perfringens become established in the small intestine of normal foals in the first 24 to 48 h of life (Smith and Jones 1963). Normal adult horses excrete low numbers of this organism in faeces, but numbers may increase in horses with diarrhoea (Wierup and Di Pietro 1981). In addition to the 2 cases of necrotising enteritis in neonatal foals associated with C. perfringens described in this report, a t least 2 other cases of a similar condition have been diagnosed in Victoria recently (G.B. Smyth personal com- munication; R.J. Graydon personal communication). O n one of the affected studs, severe scouring in other neonatal foals was observed concurrently.

The reason for the appearance of necrotising enteritis due to C. perfringens in foals in Victoria is not known.

Acknowledgments

We wish to thank Dr L,. Niilo for typing organisms isolated from case 1 , Dr P. Coloe for assistance in biochemical examination of isolates of C. perfringens, J . Hayes for the photography and the Australian Equine Research Foundation for financial assistance.

References Bain, A . M. (1954) - Aus t . vet. J . 30: 9. Barnes, D. M. and Moon, H . W . (1964) - J . Am. vet. nied. Ass.

Buddle, M . B. (1954) - J. conip. Path. Therap. 64: 217. Carter, G . R . (1978) - Diagnostic Procedures in Vcterinary

Microbiology, 3rd edn, Charles Thomas, Illinois. Dickie, C . W . , Klinkerman, D. L . and Petrie, R . J . (1978) ~ J.

A m . vet. med . Ass. 173: 306. Griner, L. A . and Bracken, F. K . (1953) - J . A m . vel . med . Ass.

122: 99. Griner. L . A . and Johnson, H . W. (1954) - J. Am. vet. nied. Ass.

125: 125. Hauschild, A . H . W . , Niilo, L. and Dorward, W . J . (1968) -

AppI. Microbiol. 16: 1235. Holdeman, L . V . , Cato, E. P. and Moore, W . E. C. (1977) -

Anaerobe Laboratory Manual , 4th edn, Virginia Polytechnic Institute, Virginia.

Leader, G. H . (1952) - Vet . Rec. 64: 241. Mason, J . H . and Robinson, E . M . (1938) - Onderstepoort J . vet.

sci. 11: 333. Montgomerie, R. F. and Rowlands, W . T . (1937) - Vet . Rec. 49:

398. Niilo, L . (1980) - Can. vet. J . 21: 141. Niilo, L . and Chalmers, G. A . (1982) - Can. vet. J. 23: 299. Sakurai, J . and Duncan, C. L . (1977) - Infect . Immun. 18: 741. Smith, H . W. and Jones, J . E. T. (1963) - J . P a f h . Bact. 82: 387. Taylor. A . W. (1940) - J . cornp. Path. Therap. 53: 50. Tzipori, S. and Williams, I . (1978) - Aust . vet. J . 54: 188. Wierup, M. and Di Pietro, J . A . (1981) - Am. J. vet. Res. 42:

(Accepted for publication 12 December 1984)

144: 1391.

2167.

I96 Australian Veterinary Journal, Vol . 62, N o . 6, June, 1985