First report of infectious necrotic hepatitis (black disease) among Nubian goats in Sudan.

In a flock of 425 female and male Nubian goats in the Khartoum Province, an outbreak of a disease causing sudden death of 18 apparently healthy goats occurred (11 females and 7 males, 3-6 years old). Adult Fasciola gigantica were found in the livers of all goats and in seven of them Cysticercus tenuicollis cysts. These organs showed necrotic and severe histopathological changes. Clostridium novyi type B was isolated from necrotic areas of all livers and found to be highly pathogenic and toxigenic to laboratory animals. The disease was diagnosed as infectious necrotic hepatitis (black disease). Faecal examination revealed the presence of F. gigantica eggs. Lymnaea natalensis snails were found to be prevalent in the water canals. As the Khartoum Province is regarded as an endemic area for black disease, routine vaccination is highly recommended for its control in goats and sheep.


Introduction and case histoty
An outbreak occurred among 425 female and male Nubian goats of the Khartoum province in the El Kabashi area. The goats were 3-6 years old. The owners reported that over a period of 60 hours, 18 healthy goats (11 females and 7 males) were found dead overnight without clinical signs. They added that a similar acute disease with sudden death of 64 goats had occurred among this flock between late Autumn and early Winter over the last two years (198811989).
The area was repeatedly visited and blood films were stained with polychrome methylene blue and anthrax was excluded. The carcasses were opened and subjected to a thorough post mortem examination. The whole liver of each dead goats was removed, kept in a sterile polythene bag and immediately transported in an ice box to the laboratory. 1

Materials and Methods
Bacteriological examination Impression smears were made from freshly tut surfaces of the necrotic areas, fixed with heat and stained with Gram's stain. Freshly prepared cooked meat medium was inoculated with a portion of the necrotic liver and incubated at 37 "C for 48 h. Another portion was streaked onto freshly prepared 10 % sheep blood agar to which cysteine hydrochloride was added to a, final concentration of 0.05 %. The plates were then incubated in BTL jars in an atmosphere of hydrogen and carbon dioxide, obtained by a gas generating kit (Oxoid Ltd., London).
After checking for purity through several subcultures, thk isolates were further subcultured on shbep blood agar plates and incubated under anaerobic conditions for routine tests.

Pathogenicity and toxigenicity
A randomly selected isolate was sqspended in phosphate buffered saline to give a concentration of 106 organisms/ml. A cell-free filtrate of the isolate in cooked meat medium was prepared by centrifugation of the culture at 4.000 rpm for 15 min at 4 "C and then passed through a millipore filter (0.22 cl).
Six rabbits and four guinea pigs were used. One rabbit was inoculated intravenously with 0.5 ml of the above suspension. The second rabbit was inoculated intramuscularly with a similar dose of the suspension to which 0.2 ml of 5 % sterile calcium chloride was added. The third rabbit was inoculated intravenously with 1 ml of a cell-free filtrate. The fourth, fifth and sixth rabbits were used as controls and inoculated intravenously with sterile phosphate buffered saline, intraniuscularly with sterile phosphate buffered saline to which sterile calcium chloride was added, and intradermally with a sterile filtrate of cooked meat medium.
One guinea pig was inoculated intramuscularly with 0.5 ml of the suspension to which 0.2 ml of 5 % sterile calcium choride was added. The second guinea pig was inoculated intradermally with 0.1 ml of the cell-free filtrate to test for dermo-necrotic reations. The third and fourth guinea pigs were used as controls and were inoculated intramuscularly with a sterile phosphate buffered saline, to which a sterile calcium chloride solution was added, and with a sterile filtrate of cooked meat medium, respectively.

Parasitological examination
Livers and faecal samples were examined and snails were collected from the water canais in the grazing areas.

His topa thological examination
All the livers and different organs from the experimentally inoculated laboratory animals were fixed in 10 % formol saline, processed, embedded in wax, tut at 5 prn and stained with haematoxylin and eosin and Gram's stain.

Results
Post mortem examination of all dead goats showed engorgement and petechial haemorrhages of the subcutaneous blood vessels. A blood stained serous fluid was present in abnormally large amounts in the perîcardial, thoracic and peritoneal cavities. The liver was dark and congested and showed yellow necrotic areas ranging from 0.5 to 6.0 cm in diameter (photo 1). These necrotic areas were surrounded by a zone of hyperaemia. Migratory tracts and holes made by liver flukes were also seen. The bile ducts were fibrosed and the gall bladders were distended. In seven goats many parasitic cysts of C. tenuicollis were found attached to the livers, and in five goats many stomach flukes of Paramphistomum cervi were found attached to the rumen.
According to the method of COWAN and STEEL (5) and STERNE and BATTY (8), the organism (photo 2) isolated from the necrotic livers was identififed as C. novyj type B. Rabbits and guinea pigs inoculated intramuscularly with the suspension of the organism (0.5 ml of 106 cells/ml) died 24 h after inoculation. Rabbits inoculated intravenously with either the cell suspension or the cell-free extract died after 48 h showing generalized speticaemia. This was also the case for the guinea pigs inoculated intradermally with the cell-free extract showing severe dermo-necrotic reactions. C. novyj type B was isolated in pure culture from the livers and muscles of the rabbits and guinea pigs with the cell suspension.
In all 18 goats immature and mature F. gigantica were extracted from the excised bile ducts. Twenty faecal samples out of 50 were found positive for Fasciola eggs. The snails collected from water canais were identified as L. natalensis.
Sections from the livers of goats revealed an extensive zone of necrosis (photo 3) surrounded by a cellular infiltrate composed of neutrophils and lymphocytes. There was a marked disfiguration and extensive damage of the liver parenchyma and severe haemorrhage. There was cirrhosis and the bile ducts were thickened, fibrosed and contained white and red cells and debris.

Treatment -Control
Ranide (rafoxanide, MSD, Agavet) in combination with antibiotics (Oxytetracycline or Amoxicillin) were not successful in the treatment and control of black disease in goats, but markedly reduced the mortality rate among the flock.

Comparative discussion
Black disease has been reported from many parts of the world (2) including the Sudan (1). However, this is the first report of the disease in goats. The disease among goats was found to be quite similar to that described in sheep (1, 2, 4). The outbreak occurred among both male and female goats. This finding is in keeping with those of BAGADI (2) and JAMIESON et a/. (7).
Black disease is caused by the a-toxin of C. novyi type B in necrotic liver tissue (1,2,8). In the present investigation the extensive liver damage seen was probably produced by F. gigantica in all goats in addition to C. tenuicollis cysts in seven goats.
Mature f. gigantica were easily extracted from the distended bile ducts of goats livers. This result supported the finding of ABU-SAMRA et a/. (l), who extracted mature F. gigantica from the livers of affected sheep. The same workers reported the isolation of both C. novyi type B and C. sordelli from the livers of affected sheep in the Khartoum province. In the current outbreak only C. novyi type B was isolated from the livers of goats. This strain was found to be more pathogenic and toxigenic to laboratory animals than the strains recovered from the soi1 and normal livers of sheep by BAGDI and SEWELL (3).

Conclusion
The presence of Fasciola gigantica and C. tenuicollis has been reported in the Khartoum province (1,6). In the present work F. gigantica eggs in faecal samples and the prevalence of L. natalensis snails were demonstrated. These facts highly suggest that the Khartoum province is an endemic area for black disease.
It is strongly suggested that massive and routine vaccinations of goats and sheep should be conducted in the Khartoum province using the Heptavac vaccine (Hoechst pharmaceuticals UK Ltd, England) which has proven to be efficient in controlling the disease among sheep (1). This treatment should be applied rather than dosing the animals with fasciolicides and antibiotics which have limited effects in the current outbreaks and are only useful in reducing the mortality rate rather than in controlling the disease. However, treating the animals with fasciolicides is also recommended to control fascioliasis which predisposes to the disease. were found in the livers of a11 goats and in seven of them Cysticercus tenuicollis cysts. These organs showed necrotic and severe histopathological changes. Clos@idium novyi type B was isolated from necrotic areas of a11 livers and found to be highly pathogenic and toxigenic to laboratory animals. The disease was diagnosed as infectious necrotic hepatitis (black disease). Faecal examination revealed the presence of F. gigantica eggs. Lymnaea natalensis snails were found to be prevalent in the water canais. As the Khartoum Province is regarded as an endemic area for black disease, routine vaccination is highly recommended for its control in goats and sheep. Key words : Goat -Infectious necrotic hepatitis -Closiridium novyi type B -Fasciola gigantica Sudan.