THE ROLE OF CLOSTRIDIUM NOVYI IN BOVINE DISEASE IN ALBERTA* L. Niilot, W. J. Dorwardt and R. J. Avery$ CLOSTRIDIAL DISEASES of cattle and sheep which nique over a period of four years. In addition, produce pathognomonic lesions in the liver are the following experimental work is reported: "black disease" or infectious necrotic hepatitis, (a) an attempt to demonstrate post mortem caused by Clostridium novyi (C. oedematiens) invasion of bovine liver by clostridia, and type B, and bacillary hemoglobinuria, caused (b) experimental infection of cattle with by C. novyi type D (C. hemolyticum). Both known pathogenic strains of C. novyi types A diseases are usually prevalent in geographic and B and comparison of the resultant disease areas where heavy liver fluke infestation exists. with the "sudden death" syndrome as seen in A primary liver damage by the fluke is thought the local feedlots. to be a predisposing cause. The laboratory confirmation of field diagnosis of these condi- MATERIALS AND METHODS tions has often been hampered by the difficulty experienced when attempting to isolate these Port Mortem Material fastidious anaerobes. In recent years, a method During a four-year period, from January of specific fluorescent-labelled antibody (FA) 1964 to December 1967, either tissues or air- staining has been developed for the identifica- dried impression smears of tissues from animals tion of several pathogenic species of clostridia suspected of having died from clostridial infec- including C. novyil (2, 3, 4). Although these tions were received at the Animal Diseases fluorescent-labelled antisera do not differen- Research Institute (Western), Lethbridge. tiate the types of C. novyi, the advantages of Most of the specimens were liver, but some- the ease of application and rapidity of the times spleen, muscle or lymph nodes were re- test have resulted in wide acceptance of the ceived. In addition, entire cadavers obtained technique by many diagnostic laboratories. from local feedlots in connection with another When the FA technique was first introduced study, formed a part of the source of the at this Institute in 1964, it was used to examine pathological material. smears made from material obtained post This material was examined for the presence mortem from cattle that died in local feed- of C. novyi, C. chauvei and C. septicum by lots. Initial examinations of smears made from means of the FA technique. livers of such carcasses indicated that a large percentage contained C, novyi organisms, often Killed Animals in a heavy concentration. Since many of these As a check on post mortem invasion of liver animals were so-called "sudden death" cases, by clostridia, three normal Hereford steers it was anticipated that the presence of the were killed by electrocution and not bled out. specific fluorescing organisms in these livers These carcasses were left unopened at prevail- might have some pathogenic significance. ing ambient temperatures for four to 24 hours. The primary purpose of this paper is to Necropsy was then conducted and liver sam- summarize and critically review the results of ples taken for smears and culture. Portions of examinations of livers and other post mortem the liver were enclosed aseptically in trays and material from cattle in Alberta for pathogenic held at ambient temperature for a further 24 clostridia by the use of the FA staining tech- hours followed by another series of examina- tions. 'In part presented as a paper to the C.V.M.A. Annual Meeting at Charlottetown, P.E.I., 1966. Fluorescent Antibody (FA) Staining tAnimal Pathology Division, Health of Animals Duplicate impression smears made from the Branch, Canada Department of Agriculture, Ani- liver or other tissues were stained with "Well- mal Diseases Research Institute (Western), Leth- come" brand of fluorescent anti-clostridial glo- bridge, Alberta. bulins2 employing methods developed for these tPresent Address: Animal Diseases Research In- products (2, 3). One smear was stained with stitute (Eastern), Hull, P.Q. a mixture of fluorescein isothiocyanate-labelled 1Differences of opinion may exist in the adoption anti-C. and lissamine rhodamine- of names and type designations of this group of septicum organisms. For the purposes of this paper C. novyi is assumed to be synonymous with C. oedematiens. 2Burroughs Wellcome & Co., London, England. L59 CAN. VET. JOUR., vol. 10, no. 6, June, 1969 CANADIAN VETENARY JOURNAL labelled anti-C. chauvei globulins. The second spores among the vegetative cells; those of smear was treated with fluorescein-labelled type B contained about 5% spores. A dose for anti-C. novyi (C. oedematiens) globulin. an inoculation was 10 ml per animal and con- The stained smears were examined with a tained the cells from 40 ml of culture. Sterile Zeiss miscroscope fitted with a U.V. light calcium chloride solution was added to the source (Osram Super Pressure Mercury Vapour inoculum for intramuscular injection (final con- Lamp, HBO, 200 W.). Excitation filter No. centration 1.0%); material for intravenous in- BG12 and barrier filter No. 47 were used. An oculation received no adjuvant. average relative concentration of fluorescing organisms in a smear was assessed arbitrarily, Experimental Animals ranging up to 3+. Hereford steers, about one year old and weighing approximately 700-800 lb were used Media and Cultivation for experimental infections. Four were injected A liquid medium was prepared, consisting intramuscularly with C. novyi and eleven intra- of equal volumes of Infusion Broth (BBL) and venously. These animals had been on range Bacto Liver Broth (Difco) to which the fol- most of the time and, while confined, were fed lowing ingredients were added: proteose pep- a maintenance ration of good quality alfalfa tone, 3%; yeast extract, 1%; Na2HPO4, 0.4%; hay and salt-mineral supplement. The animals K2HPO4, 0.1%; cysteine HC1, 0.1%; maltose, were not vaccinated. 1%; ground meat particles, 5%. This medium Guinea pigs (400 g weight) were used to was used to grow pathogenic cultures for ex- assess pathogenicity of the cultures and con- perimental inoculations and for primary isola- duct protection tests in the identification of the tion of C. novyi from tissues. For subcultures, isolates. Mice, weighing about 20 g were checking purity, growth and colony character- employed to determine toxicity of the culture istics, a modified blood agar medium, based on filtrates by the intravenous method. the above ingredients, was used. In addition, lactose egg-yolk agar (25) plates were used Experimental Infection in Cattle to compare the isolates. Intramuscular injections of C. novyi suspen- The solid media were prepared fresh each sions were made into the gluteal muscles. Only time and the plates were inoculated as soon as a single unilateral i.m. dose was given to each they were dry. The liquid medium was de- animal. The animals were kept under observa- aerated by heating immediately before use. tion. Necropsy was conducted as soon as an Anaerobic jars with low temperature catalysts animal was found dead and tissue samples (BBL) were used to incubate the cultures at were collected for bacteriological examination. 370C. Stock cultures to be stored under refri- Some of the animals that received C. novyi geration for days or weeks were also kept in intravenously were subjected to a liver biopsy. these jars. It was intended that this procedure would Procedure for isolation of C. novyi from tis- simulate the liver damage in natural cases of sues was essentially similar to a method for "black disease" associated with liver fluke in- recovering these organisms from contaminated festation. Biopsy of the liver was performed on soil (16). Chopped fragments of tissue (2-3 some animals two days after the administration g) were heated in 25 ml of de-aerated liquid of C. novyi, while in others the procedure was medium at 750C for 15 minutes and then delayed until 21 days post-inoculation. Liver incubated for 24 to 96 hours. Initial growths as biopsy was taken from 15 uninoculated control well as subcultures were examined in smears animals. The biopsies were performed using the stained by Gram's and by the FA method. method described by Hughes (11). Impression smears were made from all of the Inocula liver samples and examined after staining with C. novyi type A, strain No. CN2419 and C. Gram's and FA methods. Most of the samples novyi type B, strain No. CN2347 were sup- were also cultured. plied by the Wellcome Research Laboratories, Beckenham, England. For experimental infec- RESULTS tion in catfle, the strains of C. novyi were grown in the liquid medium for 72 hours at Post Mortem Material 37°C. The cultures were then filtered through Table I shows the results obtained on cheesecloth to remove meat particles; the cells examination by FA staining of impression were washed three times with 0.85% saline and smears of livers or other tissues from 308 cattle finally suspended in saline. C. novyi type A originating from Alberta, over a four-year suspensions so prepared contained about 25% period. Out of this total, 37 submissions were 160 Clostridium Novyi TABLE I F.A. STAINING. RESULTS ON EXAMINATION OF 308 BOVINE LIVERS AND OTHER TISSUES FOR THREE CLOSTRIDIAL SPECIES FA examined for: Number of cases 0 0 X X X *; > ot' 0 - - - 87 4 91 29.6 (8.4) + _ - 116 15 131 42.5 (9.1) + - + 44 11 55 17.9(2.3) + + - 4 1 5 1.6 + + + 0 1 1 0.3 - + + 3 0 3 1.0 - + - 11 2 13 4.2 - - + 6 3 9 2.9 (1.0) 62.3% 7.1% 21.2% 271 37 308 100.0 (20.8) FA results: -negative; + positive. Figures in brackets indicate the percentage of cases in which diagnoses other than clostridial infections were confirmed. tissues other than liver, mostly muscle tissue or untouched for 24, 20 and four hours, respec- spleen; no liver sample was included with tively, prior to necropsy.