Infectious Necrotic Hepatitis (Black Diseuse) Among Sudanese Sheep

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Rev. Elev. Méd. vét. Pays trop., 1984, 37 (4) : 422-429.

Infectious necrotic hepatitis (black diseuse) among Sudanese sheep

par M. T. ABU-SAMRA (l), S. M. EL SANOUSI (2), S. 0. IDRIS (l), H. 0. BAGADI (3), I.S.A. SALAM (3), B. H. AL1 (l), B. E. MUSA (4)

(1) Department of Medicine, Pharmacology and Toxicology. (2) Department of Microbiology and Parasitology. (3) Department of Preventive Medicine. (4) Department of Surgery, Obstetrics and Gynaecology, Faculty of Veterinary Science, University of Khartoum, Sudan.

RÉSUMÉ SUMMARY

ABU-SAMRA (M. T.), EL SANOUSI (S. M.), IDRIS ABU-SAMRA (M. T.), EL SANOUSI (S. M.), IDRIS (S. O.), BAGADI (H. 0.) SALAM (1. S. A.), AL1 (S. 0.). BAGADI (H. 0.). SALAM (1. S. A.). AL1 (B. H.), MUSA (B. E.). - Hépatite infectieuse nécrosante (B. H.1, MUSA (B. ‘E.). -’ Tnfectious necrotic hepatitis (black disease) chez des moutons soudanais. Rev. Elev. (black disease) among Sudanese sheep. Rev. Elev. Méd. Méd. v&. Pays trop., 1984, 37 (4) : 422-429. vét. Pays trop., 1984, 37 (4) : 422-429.

Dans un troupeau de 75 moutons du désert de la pro- In a flock of 75 desert sheep in Khartoum Province, vince de Khartoum, 12 animaux ont subitement trouvé la 12 animals died suddenly showing no clinical signs. On mort sans svmptômes cliniques évidents. A l’autopsie, les post-mortem, the’ subcutaneous blood vessels were vaisseaux sanguins sous-cutanés étaient congestionnés avec engorged and showed petechial haemorrhages, and there des pétéchies et de grandes quantités de sérosité mêlée de were large volumes of blood-stained serous fluid in the sang dans le péricarde, la plèvre et le péritoine. Le foie pericardial, pleural and peritoneal cavities. The livers were était foncé, congestionné et montrait des zones nécrosées dark, congested and had yellow necrotic areas of de 0,5 à 3,5 cm de diamètre, entourées d’une zone d’hype- 0.5-3.5 cm in diameter, surrounded by a zone of hyper- rhémie. Des douves (Fasciola gigantica) adultes ont été aemia. Adult Fasciola gigantica were found in the livers of trouvées dans le foie de tous les moutons morts, et 5 a11 dead sheep and in five of them Cysticercus tenuicollis d’entre eux étaient porteurs de kystes de Cysticercus tenui- cysts were found attached to the liver which showed severe collis. Les foies révélaient une nécrose sévère et des chan- necrotic and marked histopathological changes. Bacterio- gements histopathologiques intenses. logical examination of the liver necrotic areas revealed the L’analyse bactériologique des zones nécrosées a égale- presence of Clostridium novyi type B in a11 the dead sheep, ment révélé la présence de Clostridium novyi type B chez and in ten of them C. sordelli was also isolated. The tous les moutons morts. Enfin, sur 10 organes, Clostri- strains of C. novyi isolated was highly pathogenic and dium sordelli a été isolé et cette bactérie était hautement toxigenic to rabbits and guinea pigs in which they pathogène et toxique pour le lapin et le cobaye, dont elle a produced rapid death and severe histopathological provoqué la mort rapide avec des changements histopatho- changes. logiques sévères.

Mots-clés : Hépatite infectieuse nécrosante - Moutons - Key words : Infectious necrotic heptatitis - Sheep - Sudan. Soudan.

1. INTRODUCTION tiens) type B, in necrotic anoxic liver tissue (3). DODD (9, 10) reported that infection of Infectious necrotic hepatitis (black disease) animals with liver flukes is necessary for the is an acute toxaemic disease of sheep and production of the disease, and ALBISTON cattle and rarely pigs. The disease is caused by (1927) suggested that the disease is caused by a the alpha toxin of Clostridium novyi (oedema- bacterium., However, the association of the

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bacterium C. novyi type B and the liver fluke hydrogen and carbon dioxide, generated by a Fasciola hepatica in the causation of the gas generating kit (Oxoid Ltd., London). disease was established by TURNER (17), After checking for purity thrqugh several JAMIESON (15) and BAGADI and SEWELL subcultures, two types of colonies were (4). recognized on the blood agar and were In Africa, there has been only one report of designated A and B. They were further the disease in Mali (8) and in the Sudan no subcultured on sheep blood agar prepared as publication on black disease in sheep was described above and were incubated encountered (2). However, the occurrence of anaerobically. liver fascioliasis and cysticercosis in sheep in The isolated organisms were tested for their the Sudan were reported since 1947 and 1955, ability to ferment arabinose, glucose, inositol, respectively (2, 12). inulin, lactose, maltose, manitol, raffinose, This paper describes an outbreak of asymp- rhamnose, salicin, sorbitol, sucrose, trehalose tomatic sudden deaths among Sudanese desert and xylose. The isolates were also tested for sheep. The investigations conducted are herein their ability to release ammonia from urea, described. produce indole and hydrogen sulphide and reduce nitrates to nitrites. They were also tested for their proteolytic activity on cooked meat medium, gelatin liquefaction and II. MATERIALS AND METHODS digestion of litmus milk. The isolates were also tested for lecithinase activity in half-antitoxin- 2.1. History egg yolk-lactose medium (20). The inhibition of lecithinase activity was also tested by the The outbreak occurred among 67 male and addition of a mixture of antisera of Cl. 8 female desert sheep in Halfaya area (Khar- perfringens type A and Cl. novyi type A. The toum Province). The sheep were 3-4 years old. lipase activity of the isolates was demonstrated The owner reported that, over a period of two by the presence or absence of a pearly layer days, ten male and two female sheep were (Nagler’s reaction). found dead over night without clinical signs. Blood films were stained with polychrome 2.2.2. Pathogenicity and toxigenicity of the methylene blue and anthrax was excluded. The isolates to laboratory anima& carcasses were opened and subjected to a thorough post-mortem examination. The Five rabbits and four guinea pigs were used whole livers of the dead sheep were removed, for each isolate. kept in sterile polythene bags and immediately The isolates were each suspended in phos- transported in an ice box to the laboratory. phate buffered saline to give a concentration of 106 organisms/ml. Cell-free filtrates of each 2.2. Laboratory investigations isolate in cooked meat medium was prepared by centrifugation of cultures at 4 000 r.p.m. The following investigations were carried for 15 minutes at 4 “C and then passing it out in the livers of the dead sheep. through a millipore filter (0.22 y). One rabbit was inoculated intravenously 2.2.1. Bacteriological examination with 0.5 ml of the above suspension. The second rabbit was inoculated intramuscularly Impression smears were made from freshly with a similar dose of the suspension to which tut surfaces of the necrotic areas fixed with 0.2 ml of a 5 p. 100 sterile calcium chloride heat and stained with Gram’s technique. was added. The third rabbit was inoculated Freshly prepared cooked meat medium was intravenously with 1 ml of ce11 free filtrates. inoculated with a portion of the necrotic liver The fourth ,and fifth rabbits were used as and incubated at 37 “C for 48 hours. Another controls. portion was streaked onto freshly prepared One guinea pig was inoculated intramuscu- 10 p. 100 sheep blood agar to which cysteine larly with 0.5 ml of the suspension to which hydrochloride was added to a final concen- 0.2 ml of 5 p. 100 sterile calcium chloride was tration of 0.05 p. 100. The plates were then added. The second guinea pig was inoculated incubated in BTL jars in an atmosphere of intradermally with 0.1 ml of the cell-free

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filtrate to test for dermonecrotic reactions. peritoneum on the rumen, reticulum and The third and fourth guinea pigs were used as omasum was slightly congested. No other controls. significant macroscopical changes were obser- The inoculated rabbits and guinea pigs were ved. observed closely. Dead animals were subjected to a thorough postmortem examination, and necropsy specimens were subjected to bacte- 3.2. Laboratory investigkions riological and histopathological examination. 3.2.1. Bactfriological findings 2.2.3. Parasitological examinaiion Gram’s ‘stains revealed gram-positive rods, 0.8 x 5 um occurring singly (Photo l), in pairs The livers of dead sheep were examined for and rarely in short chains. The spores were the presence of parasites which were isolated ovoid and placed subterminally but were and identified. occasionally placed centrally. On blood agar, colonies (A) were small and 2.2.4. Histopathological examination lozenge-shaped. Their long axis was following the direction of streaking. The colonies were Necropsy specimens from the livers of a11 raised with ridged crenated and tentacular dead sheep and different organs from the margins and with a great tendency for experimentally inoculated laboratory animals swarming. They were initially transparent, but were fixed in 10 p. 100 formol saline, on aging became opaque and yellowish. The embedded in wax, tut at 5 um and stained with colonies were surrounded with a narrow zone Haematoxylin and Eosin and Gram’s stain. of haemolysis. On egg yolk agar, the colonies were small to medium in size with slightly 2.2.5. Vaccination raised rough or amoeboid edges and were surrounded by a wide zone of opalescente The remaining 63 sheep were vaccinated without luster. Culture of colonies (A) on half- with Heptavac vaccine (Hoechst Pharmaceu- antitoxin gave opalescente which was inhibited ticals UK Ltd., England), using a Phillips by anti-sera. automatic vaccinator (N.J. Phillips PTY Ltd., Acid and gas was produced from’ glucose. Australia). Each sheep was inoculated sub- Arabinose and raffinose were weakly fer- cutaneously on the side of the neck with 2 ml mented. The organism (A) did not ferment of the vaccine. inositol, inulin, lactose, maltose, manitol, rhamnose, salicin, sorbitol, sucrose, trehalose or xylose. Ammonia was not released. Indole III. RESULTS and hydrogen sulphide were produced and nitrates were reduced to nitrites. The organism 3.1. Postmortem findings (A) was lipase negative. It was proteolytic to cooked meat medium. It liquified gelatin and Al1 dead sheep showed engorgement and curdled, digested and blackened litmus milk. petechial haemorrhages of the subcutaneous On blood agar, colonies (B) were 2-6 mm in blood vessels. Blood stained serous fluid was diameter. They were flat, swarming, fluffy, present in abnormally large amounts in the feather-like, thin and difficult to see. The pericardial, thoracic and peritoneal cavities. presence of the colonies was betrayed by the The liver was dark and congested and showed marked haemolysis beneath the filmy delicate on its diaphragmatic surface, yellow necrotic growth. There was complete haemolysis areas about 0.5-3.5 cm in diameter. Those beneath the colonies and 1-2 mm around necrotic areas were surrounded by a zone of them. This zone of haemolysis was again hyperaemia. The liver showed migratory tracts surrounded by an intense cherry-red zone of of liver flukes and the bile ducts were fibrosed. 4 mm in width. On egg yolk agar, the colonies In eight sheep the gall bladder was distended were small, irregular, transparent and and in five sheep parasitic cysts were seen produced a wide regular and sharply defined attached to the liver. In nine sheep the zone of precipitation beneath and beyond the duodenal mucosa showed patchy areas of colonies without a pearly layer. The antitoxin congestion and the parietal surface of the did not inhibit this opalescente.

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Fig. 1. - Gram positive rods in impression smears from the necrotic areas of the livers of sheep. Gram’s stain x 1000.

Acid and gas was produced in glucose and skin at the site of inoculation was intact but trehalose. Inositol was invariably fermented, the area was swollen with extensive oedema while maltose, salicin and xylose were weakly involving the legs, flanks, abdomen and fermented. The organism (B) did not ferment thorax. Cutting through the skin at the site of arabinose, inulin, lactose, manitol, raffinose, inoculation, extensive oedema was ‘evident and rhamnose, sorbitol and sucrose. Ammonia was the muscles showed gas gangrene. They were not released. Indole and hydrogen sulphide black, surging in gas bubbles and were were not produced and nitrates were not surrounded by a wide zone of hyperaemia. reduced. The organism (B) was not proteolytic The lungs were slightly congested and the to cooked meat medium. It liquified gelatin hearts were engorged and hyperaemic. The and produced acid without curdling in litmus spleens were black and double their normal milk. size. The intestines were severely inflamed. Colonies of isolate (A) were identified as The rabbit inoculated intravenously died Clostridium sordellii while colonies of isolate after 48 hours showing generalized septi- (B) were identified as Clostridium novyi caemia. type B. Identification was in accordance to The guinea pig inoculated intradermally died COWAN and STEEL (7) and STERNE and after 48 hours showing severe dermonecrotic BATTY (16). reactions. Clostridium sordellii and C. novyi type B Control animais survived and ‘showed no were isolated from the necrotic areas in the detectable abnormality. livers of ten and twelve sheep, respectively. Smears made from the livers and muscles of the experimentally inoculated laboratory ani- 3.2.2. Pathogenicity and toxigenicity of the mals revealed gram positive rods. The orga- isolates to laboratory animals nism was isolated in pure culture and was typical in morphology and biochemical cha- Only the rabbit which was inoculated intra- racters to those orginally isolated from the venously with the suspension of Cl. sordellii necrotic areas of the livers of the sheep. died 72 hours post-inoculation. No ‘dermonecrotic reactions were produced in the skin of the guinea pig. 3.2.3. Parasitological findings The rabbit and guinea pig inoculated intramuscularly with the suspension of Cl. novyi In five sheep bladder worms of Cysticercus type B. died 24 hours after inoculation. The tenuicollis, measuring 5-8 cm in diameter were

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found attached to the liver. In a11 twelve sheep immature and mature ‘(Photo 2) Fasciola gigantica were extracted from the excised bile ducts.

3.2.4. Histopathological findings

Sections from the livers of sheep revealed an extensive zone of necrosis (Photo 3) surrounded by cellular infiltrate composed of neutrophils and lymphocytes. There was extensive damage to the liver parenchym and marked haemorrhage. There was liver cirrhosis and the bile ducts were thickened and fibrosed, and contained white and red cells and ce11 debris. The muscles of the rabbit and guinea pig which were inoculated with Cl. novyi were markedly distorted and showed extensive oedema and gis separating the muscle bundles (Photo 4). There was severe haemorrhage and marked infiltration with neutrophils and lymphocytes. In gram stained sections, numerous gram-positive bacilli were seen. The kidneys revealed severe haemorrhage and degenerative changes, and 6ere infiltrated with neutrophils and lymphocytes. The liver was congested and showed numerous necrotic foci infiltrated with neutrophils and lymphocytes and in sections Fig. 2. - Liver Fluke (F, gigantica), extracted stained with gram’s stain many bacilli were from the excised bile ducts of the sheep’s livers. x 1.5. seen. The ‘lungs were markedly emphyse-

Fig. 3. .- Extensive areas of liver necrosis surrounded by neutrophils and lymphocytes. H & E x 200.

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Fig. 4. - Muscle of guinea pig inoculated with suspension of CI. novyi type B. Note separation of muscle bundles with oedema and gas. H & E x 200.

matous, with severe haemorrhages and the that one of the outstanding features of the alveoli were filled with exudate. The spleen disease is the rarity with which one observes a was congested and revealed marked tracts of sick sheep ; and BLOOD et al. (6) mentioned haemorrhage and extensive proliferation of that affected sheep were commonly found cells . dead without exhibiting any previ:ous’ signs of Skin sections from the guinea pig which was illness. inoculated intradermally revealed marked The current outbreak occurred among both dermal necrosis. The epidermis was ulcerated male and female sheep. This agrees with and the surface was covered with a serous JAMIESON et al. (15) and BAGADI (3) who cruts. There was marked dilatation of the reported that differing breed susceptibility and blood capillaries and the dermis showed sex among sheep appeared to have no severe haemorrhages. There was marked influence upon the disease rate. epidermal and dermal infiltration with neutro- Black disease in sheep is caused !y the alpha phils and lymphocytes. toxin of Clostridium novyi type B in necrotic liver tissue (3, 16). In the current investigation 3.2.5. Vaccination the extensive liver damage seen was probably produced by Fasciola gigantica in a11 sheep, in Until the preparation of this manuscript addition to Cysticercus tenuico& in five (2 months after the outbreak), no new mor- sheep. This supports the reports of TURNER talities were reported among the 63 vaccinated (17), JAMIESON (14), HRECZKQ (13), WIL- sheep . LIAMS (18, 19), BAGADI (3, 4), DUNN (ll), and BLOOD et al. (6). It was interesting to extract mature E. gigan- IV. DISCUSSION AND CONCLUSIONS tica from the distended bile ducts of sheep’s livers. This was in contrast to the reports of Black disease has been reported in’ many WILLIAMS (18), DUNN (11) and BLOOD et parts of the world (3). However, this is the al. (6) who mentioned that immature or first report of the disease in the Sudan (2). mature flukes are not ordinarily found. Sudden death occurred among sheep which It was interesting to isolate CI. sordellii showed no sign of the disease. This finding from the livers of ten sheep. This organism supported that of BAGADI (3), who showed probably aggravated and complicated the

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infection of sheep with Cl. novyi as was in Khartoum particularly those infested with F. reported by STERNE and BATTY (16). gigantica and/or Cysticercus tenuicollis. In The strain of Cl. novyi type B isolated in this respect work is in progress in the labora- this investigation was highly pathogenic and tory, according to which massive and routine toxigenic to laboratory animals, and were vaccination of sheep against black disease more toxigenic and pathogenic than the strains might be suggested. recovered from the soi1 and normal livers of sheep by BAGADI and SEWELL (6). The occurrence of the disease in Khartoum Province is not surprising because the presence ACKNOWLEDGEMENTS of F. gigantica and Cysticercus tenuicollis which predispose to this disease were pre- The authors wish to thank Dr. KHALID viously recorded in this Province (2, 12). ABDEL MAGEED, Department of French, While this is warranted it is suggested that a Faculty of Arts, University of Khartoum, for thorough bacteriological investigation should participation in the French translation of the be carried out in the livers of sheep slaughtered summary of the manuscript.

RESUMEN

ABU-SAMRA (M. T.), EL SANOUSI (S. M.), IDRIS zona de hiperhemia. Se encontraron Fasciola gigantica (S. O.), BAGADI (H. O.), SALAM (1. S. A.), AL1 adultas en el higado de todos 10s carneros muertos y 5 de (B. H.), MUSA (B. E.). - Hepatitis infecciosa con ellos tenian quistes de Cysticercus tenuicollis. Los higados necrosis (Black diseuse) en carneros del Sudan. Rev. Elev. mostraban una necrosis grave y modificaciones histo- Méd. vét. Pays trop., 1984, 37 (4) : 422-429. patologicas intensas. El analisis bacteriologico de zonas con necrosis En un rebano de 75 carneros del desierto de la provincia demostro también la presencia de Clostridium novyi tipo B de Khartoum, 12 animales murieron subitamente sin en todos 10s carneros muertos. De 10 oraanos. se aislo sintomas clinicos evidentes. Al autopsia, se observaron 10s Clostridium sordelli que era muy pat6geno ; t6xico para el vasos sanguineos subcutaneos congestionados con conejo y el conejillo de Indias que murieron rapidamente petequias e importantes cantidades de serosidad mezclada con modificaciones histopatological graves. con sangre en el pericardio, la pleura y el peritoneo. El higado era oscuro, congestionado y mostraba zonas con Palabras claves : Hepatitis infecciosa con necrosis - Car- necrosis de 0,5 a 3,5 cm de diametro, rodeadas por una neros - Sbdan.

REFERENCES

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