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. The range of ambient specimens in tbis column. temperature during this period was 710 to In 64 cases (20.8% of total) shown in 820F. When carcasses No. 1 and No. 2 were brackets in Table I, the specimens were either opened, a considerable amount of post mortem entire carcasses or a good representative sam- change, including gas formation in muscula- pling of tissues, exudates, etc., with a detailed ture and subcutaneous tissues, was noticeable. clinical history. This allowed thorough labora- The liver appeared soft. Impression smears tory examinations to be made, using necropsy, made from the liver of these carcasses and conventional bacteriological and histological stained by the FA technique demonstrated the methods which confirmed diagnoses other than presence of C. novyi in both livers (Fig. 1). clostridial infections as the cause of death. Most of these deaths were due to either one or a combination of several of the following con- ditions: pneumonia, peritonitis, nephritis, trau- matic pericarditis, septicemia, and rhinotrachei- tis. Pasteurella and Corynebacterium were commonly involved in such cases. Of the total number of liver or other tissues examined by the FA staining method, 29.6% were negative for all of the three clostridial species examined. C. novyi alone was found in 42.5% of the cases and C. novyi together with one or both of the other two species accounted for 19.8%, the overall incidence of C. novyi being 62.3%. C. chauvei was found in four specimens of muscle tissue, all from clinical FIGURE 1. Fluorescent photomicrograph of liver cases of blackleg. About 80% of the tissues that impression smear of a normal killed animal (No. 2) were positive for C. novyi contained the orga- 20 hours post mortem. Stained with anti-C. novyi nism in a moderate to heavy concentration globulin. Field of view under 40X objective; con- (five or more organisms per field) and only centration of organisms +++. 20% showed light concentration (one plus). Samples from No. 1 showed also C. septicum Killed Animals in low numbers. Clostridial rods were also seen Carcasses of the three normal healthy ani- in Gram-stained smears (Table II, Figs. 2 and mals that were killed by electrocution were left 3). Smears from No. 3 were negative. 161 t-o-Z + I.j
C.) t. >>I i16 II 1)
>
sK-1 C) 9 i
.11.4 L) U, 1) U) j ~z0
¢4I I 04 L.. _ex Cld n1 ¢9 0 o 0 ++ z ++ I1
_.4 S . Is 0 ++ " ++II1 "n
0 0 xI- U, 0- Q * 0 0 U C,.. r.* C.) 0 D~bb X0bO3-
u L OI II ++ + :1~ cN1.4 cqi0 1.14
r-f cqI CC
162 Clostridium Novyi agar, but did not produce opalescence on lac- tose egg-yolk agar. "Pearly layer" formation was weak or almost indistinct. Toxigenicity. Sterile filtrates prepared from cultures grown for 48 hours to five days, were diluted serially and injected into 48 mice in volumes of 0.3 ml. The toxicity of these fl- trates ranged from five to 12 MLD/ml. As a control, filtrates from known C. navyi types A and B pure cultures, grown under similar con- ditions, showed toxicity up to 1000 MLD/ml and 1400 MLD/ml, respectively. Pathogenicity. Washed cells were pathogenic for guinea pigs, causing death within 24 to 96 ,jAfiA;k. .: .. _4E-r:;B::' 5i_' hours. The minimal lethal dose was 0.1 ml of FIGuRE 2. Conventional photomicrograph of the the bacterial suspension and approximately ten same liver as in Fig. 1. Smear stained with Gram's; times that of the known C. novyi types A and B. In protection tests all guinea pigs passively immunized with both C. septicum and C. novyi type A anti-sera survived. Other clostridial antisera, singly or in various combinations, con- sistently failed to protect the animals against challenge. A total of 62 guinea pigs were used for these trials. Two calves were inoculated intramuscularly with 10 ml of the bacterial suspension. Both remained unaffected. Experimental Infection in Cattle Intramuscular. Three of the four animals used for experimental infection by the intra- muscular route received C. novyi type B and one received type A preparation. Tenderness in the inoculated hind quarter and visible evi- FIGURE 3. As in Fig. 2, under 10OX objective. dence of swelling, accompanied by a slight elevation of body temperature, occurred in two About one-half of each liver was placed animals 24 hours post-inoculation (Nos. 4 and aseptically into trays and left standing at the 7). In the. other two cases, swelling became same temperature for another 24 hours. Then, evident two days after inoculation. External a series of FA stained smears showed the signs of the infected quarter were most pro- presence of C. novyi in all of the liver samples, nounced on the third day; the swollen area including No. 3, in a concentration of three exhibited moderate crepitation in response to plus. No additional appearance of C. septicum manual pressure and it extended to perineum, or C. chauvei was seen in these smears. inguinal region, posterior flank and half way Isolates. Both C. novyi and C. septicum down to the hock joint. The perineal region were demonstrated in cultures of all three liver appeared oedematous rather than crepitating. samples taken at the time of necropsy. Al- At this stage the animals were dull and ano- though repeated attempts were made to isolate rexic, but showed only moderately elevated C. novyi in pure culture, these attempts failed. body temperature. The time of death varied The organism grew relatively well if the inocu- from 54 to 84 hours post-inoculation. The re- lum was sufficiently heavy, but single colonies sults are summarized in Table III. picked from solid media did not support Necropsy of all four carcasses revealed se- growth. Laboratory animal inoculations, there- vere gas gangrene in the affected quarter (Fig. fore, were done with mixed cultures containing 4). Hemorrhagic and edematous infiltration both C. novyi and C. septicum. However, the with finely dispersed gas pockets was seen mixed cultures contained between 50% and 70% throughout subcutaneous and muscle tissue. of C. novyi as determined by identification of Some areas of the lesion appeared black, but colonies on solid media. no abscessation was present. No gross patho- The colonies resembled those of known C. logic lesions were found in the visceral organs. novyi type A. They were hemolytic on blood The opposite hind quarter appeared unaffected. 163 CANADIAN VETERINARY JOURNAL
TABLE III INTRAMUSCULAR INOCULATION OF C. novyi-CLINICAL AND NECROPSY FINDINGS C. novyi Animal type Clinical observations Necropsy findings 4 B 24 hrs: moderate swelling of 2 hrs. post mortem. injected quarter, temp. 1030 F, Tissues fresh, no P.M. change. appetite slightly reduced. Swelling and gas gangrene in 48 hrs: extreme swelling and crep- hind quarter (Fig. 4). Viscera grossly itation of quarter, extending to normal. perineum flank. Depression, eating a little, temp. 1040 F. 72 hrs: swelling increasing and extending further, unable to bear weight. Depression, off feed, temp. 103.5. Died at 76 hours postinoculation. 5 B 24 hrs: grossly normal 10-13 hrs. post mortem. 48 hrs: tenderness and moderate swell- Marked autolysis but no gas ing at site of injection. Appetite and in organs. Swelling and gas temperature normal. gangrene in hind quarter. 72 hrs: severe swelling and crepitation in hind quarter. Depression, very little appetite, temp. 1040 F. Died at about 81 hours postinoculation. 6 B Course of disease similar to No. 2. 10-12 hrs. post mortem. Died at about 84 hours postinoculation. Some autolysis in tissues. Swelling and gas gangrene in hind quarter. 7 A 24 hrs: Slight swelling and tenderness, 10-14 hrs. post mortem. not supporting weight. Carcass bloated. Gas in Appetite and temperature normal. most tissues. Gas gangrene in 48 hrs: Pronounced swelling, reluctant hind quarter extending through to move, depression, temp. 101.50 F. musculature into parietal Died at about 54 hours postinoculation. peritoneum on the affected side of the pelvic cavity.
cropsy are listed in Table Illa. The colony characteristics of C. novyi isolated on cultures from tissues of animals 4 and 6 appeared sirni- lar to type B on lactose egg-yolk agar. Cultures from animal 5 contained a mixture of anae- robic bacteria. No attempt was made to purify and type these cultures. Intravenous. The clinical and necropsy re- sults of the intravenous inoculation of C. novyi types A and B followed by liver biopsy at two or 21 days post-inoculation, or no liver biopsy, are tabulated in Table IV. The three animals on which liver biopsy was performed 48 hours after the intravenous administration of C. novyi washed cells died within three to seven days. FIGURE 4. Ventral view of inguinal region of Clinical signs were not dramatic. Animals ap- carcass No. 4, two hours post mortem. Incisions made into the muscles. Gas gangrene in the right peared normal at the time the biopsy was Inoculum: C. B. performed. One animal (No. 8) developed a quarter. novyi type brief period of fever of 105.50F. and depres- Variable post mortem autolysis of tissues, par- sion and died 29 hours after the biopsy. In the ticularly in the liver, was present in the three other two cases (Nos. 9 and 10) fever of animals which died during the night. An esti- 1050 to 106°F., accompanied by marked de- mate placed their deaths 10 to 14 hours prior pression, anorexia and laboured breathing, de- to necropsy. The ambient temperature was veloped 30 to 40 hours prior to death. 770F. At necropsy, animal 8 had a moderately Results of the bacteriological examination of swollen liver with a diffuse indistinct area of .the liver and muscle lesions obtained at ne- necrosis surrounding the biopsy site. The 164 Clostridium Novyi
TABLE Illa INTRAMUSCULAR INOCULATION OF C. novyi-BACTERIOLOGICAL RESULTS Smear: Gram Smear: FA Culture Tissue Animal examined Clost. rods C. novyi C. chauv. C. sept. C. novyi 4 I Liver -ve + -ve -ve +ve Muscle lesion +++ +++ -ve -ve +ve 5 I Liver +++ ++ -ve + +ve Muscle lesion +++ +++ -ve -ve +ve 6 f Liver +++ + -ve -ve +ve 6 Muscle lesion +++ +++ -ve -ve +ve 7 J Liver +++ +++ -ve -ve ND M uscle lesion +++ +++ -ve -ve ND = 1-4 organisms per field (40 X objective). ++ = 5-10 organisms per field. = > 10 organisms per field. ND = not done. TABLE IV INTRAVENOUS INOCULATION OF C. novyi AND LIVER BIOPSY-CLINICAL AND NECROPSY RESULTS C. novyi Liver biopsy, Death, days Necropsy Animal type days post-inoc. post-inoc. hrs. p.m. Necropsy findings 8 B 2 3 4 Tissues fresh. Liver swollen, necrotic area around biopsy site. Spleen enlarged, severe enteritis. 9 B 2 7 9-12 Some P.M. change. Hemorrhagic and edematous lungs. Liver slightly swollen, necrosis around biopsy site. 10 A 2 5 5-8 Slight P.M. change. Generalized edema; petechial hemorrhages on serosa. Blood clot, necrosis and congestion in liver at biopsy site. 11 A 21 Survived, no illness 12 B 21 Survived, no illness 13 B 21 Survived, noillness 14 A - 4 2 Tissues fresh. Gas gangrene in neck muscles (accidental). Some congestion of liver. 15 A - Survived, no illness 16 B Survived, no illness 17 B Survived, no illness 18 A -Survived, no illness Controls (15) - Biopsy only Survived, no illness spleen was also moderately enlarged. Severe site appeared necrotic, but no distinct lines of hemorrhagic enteritis, involving most of the demarcation were detectable. small intestine, was evident. The lungs, heart, Animals 11, 12 and 13, in which the liver kidneys, pleura and peritoneum appeared nor- biopsy was performed 21 days post-inoculation, mal. Hemorrhagic-edematous lungs were ob- did not show any signs of illness either after served in animal 9 while animal 10 showed the inoculation of the washed cells or following edema in most tissues accompanied by pete- the biopsy. chia on serous surfaces. The liver in these two One of the three animals on which no liver appeared dark, somewhat soft and slightly biopsy was intended (No. 14) developed typi- swollen. Some of this appearance was thought cal gas gangrene in the side of the neck chosen to be due to post mortem change. Areas in for the intravenous inoculation. The swelling the liver immediately surrounding the biopsy began two days after the inoculation and 165 CANADLAN VETERINARY JOURNAL gradually increased in size so that the diameter the presence of C. novyi in a high percentage of the neck appeared about twice the normal (62.3%) of cases. These results are generally size at the time of death on the fourth day. similar to those of Wallace (22) who reported This infection was considered to be an acci- an incidence of C. novyi by FA technique in dental sequel to intravenous inoculation al- 70% of post mortem material from sheep in though care was taken to avoid extravasation New Zealand. The predominance of this orga- of the inoculum during the injection. Necropsy nism, in tissues from sheep, cattle, pigs, and of this animal, which had received C. novyi dogs in Australia was reported by Corbould type A, showed tissue lesions in the neck (7) who found the ratio of C. novyi to C. sep- similar to those following intramuscular inocu- ticum to be approximately 3:1 and noted that lations. All visceral organs had a normal the former was found most frequently in the appearance. liver. Our results on cattle tissues from Alberta Four animals which received C. novyi type showed the same 3:1 ratio (Table I). A and B intravenously (Nos. 15-18) without While some workers have commented on the subsequent liver biopsy, remained healthy. uncertainty of pathogenic significance of C. Similarly, none of the 15 control animals novyi in post mortem material (7, 10), a few biopsied developed signs of illness during the earlier reports tended to interpret the finding following observation period of six months. of this organism in FA stained smears as a Results of the bacteriological examination cause of death (1, 5, 8, 9). In our 64 cases are shown in Table IVa. Colonies and cells of where diagnosis by conventional means was some of the C. novyi cultures grown from the established beyond reasonable doubt, the find- tissues appeared morphologically uniform, but ing of C. novyi in 35 of the FA stained smears about half of the isolates had mixed morpho- appears to show no relation to the cause of logical characteristics. However, these cultures death. were neither purified nor typed. The density of Corbould (7) reported that normal sheep organisms seen in the smears was variable and livers did not reveal clostridia in FA stained tended to be relatively low in tissues not con- smears after slaughter, but when incubated for taining actual lesions. Even in the liver, the 20 to 24 hours, C. novyi was demonstrated in fluorescing C. novyi cells were concentrated in 75% and C. septicum in 17% of the livers the necrotic tissue surrounding the biopsy site. examined. In our three normal cattle killed by electrocution, similar liver invasion occurred. DIsCUSSION This clearly illustrates the prevalence and ra- pidity with which C. novyi appears in liver The results of examination by means of the post mortem. The conditions for bacterial FA staining technique of bovine tissue smears growth may not be the same in incubated liver from post mortem material in Alberta revealed taken from a slaughtered bled-out carcass and TABLE IVa INTRAVENOUS INOCULATION OF C. nOVyi AND LIVER BIOPSY-BACTERIOLOGICAL RESULTS Smears: Gram Smears: FA Ctulture Tissue Animal examined Clost. rods C. novyi C. chauv. C. sept. C. novyi Liver biopsy -ve -ve -ve -ve -ve 8