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Toxin-types of Clostridium perfringens strains isolated from sheep, cattle and paddock soils in Nigeria
Veterinary Microbiology, 12 {1986) 93--96 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
93
Short Communication TOXIN-TYPES OF CLOSTRIDIUM PERFRINGENS STRAINS ISOLATED F R O M S H E E P , C A T T L E A N D P A D D O C K S O I L S IN N I G E R I A
A.E. ITODO 1., A.A. ADESIYUN1, J.O. ADEKEYE 2 and J.U. UMOH 1 Departments of Veterinary Public Health and Preventive Medicine ~ and Veterinary Pathology and Microbiology 2, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria (Nigeria) {Accepted for publication 14 October 1985)
ABSTRACT Itodo, A.E., Adesiyun, A.A., Adekeye, J.O. and Umoh, J.U., 1986. Toxin-types of Clostridium perfringens strains isolated from sheep, cattle and paddock soils in Nigeria. Vet. Microbiol., 12: 93--96. Two-hundred and forty-five strains of Clostridium perfringens isolated from the faeces of apparently healthy sheep and cattle and from their environments {paddock soils) in Kano and Kaduna States of Nigeria were studied. The isolates were examined by the toxin--antitoxin neutralisation tests performed intradermally in depilated albino guineapigs. One-hundred and twenty-seven {53.1%) of the isolates were type A, 17 (7.1%) were type B, 14 (4.9%) were type C, 44 {18.4%) were type D and 19 (7.9%) were type E. Eighteen {7.5%) were not typable while six were lost during storage. The significance of this distribution and the potential danger to animal health, especially as regards enterotoxaemias, cannot be over-emphasized.
INTRODUCTION It is n o w k n o w n t h a t specific t y p e s o f Clostridium perfringens are imp o r t a n t aetiological agents in certain diseases in animals o f e c o n o m i c imp o r t a n c e . A m o n g s t t h e five m a j o r t y p e s (A, B, C, D a n d E), t y p e s B, C a n d D in particular have been i m p l i c a t e d in sheep, goat a n d cattle disease worldwide (Russel, 1 9 7 0 ; Sterne, 1 9 8 1 ; H o g h and Muller, 1982). In Nigeria, Princewill and A g b a ( 1 9 8 2 ) first r e p o r t e d t h e prevalence o f Clostridium spp. in bovine faeces, b u t the t o x i n - t y p e s o f t h e isolates were n o t d e t e r m i n e d . C. p e r f r i n g e n s was r e c e n t l y r e p o r t e d to be responsible f o r a sporadic o u t b r e a k o f e n t e r o t o x a e m i a in three a d u l t Friesian cattle with a case fatality rate o f 100% ( I t o d o et al., 1983). With t h e a p p a r e n t absence o f r e p o r t i n g o f C. perfringens e n t e r o t o x a e m i a in Nigerian livestock c o u p l e d *Present address and correspondence to: Dr. A.E. Itodo, Bacteriology Division, National Veterinary Research Institute, Vom via Jos, Nigeria.
0378-1135/86/$03.50
© 1986 Elsevier Science Publishers B.V.
94 with the potential danger this organism may pose to the livestock industry, this study was conducted to specifically type C. perfringens strains isolated from the faeces of sheep and cattle and their environments (paddock soils) and to determine their toxicity. MATERIALS
AND
METHODS
Two-hundred and forty-five strains of C. perfringens isolated from the faeces of apparently healthy sheep and cattle and from their environments (paddock soils) in Kano and Kaduna States of Nigeria were studied. All isolation work was done according to standard methods on sulfitepolymyxin-sulfadiazine agar (Angelotti et al., 1962). The isolates were confirmed as C. perfringens by the methods of Nagler (1939) and Labbe (1983). Formolized antigens (toxins) of five toxigenic types of C. perfringens type A (CN 3692), type B (CN 2003), type C (CN 3706), type D (CN 4529) and type E (CN 1493) kindly supplied by the Wellcome Research Laboratories, U.K., were used to produce type-specific antisera in rabbits using the procedure described by Yamagishi et al. (1971). The principal types of C. perfringens strains were determined using prepared type-specific antisera and culture filtrates (toxins) of isolates for neutralisation tests in depilated skin of albino guinea-pigs as previously described (Oakley and Warrack, 1953; Warrack, 1963). RESULTS
AND
DISCUSSION
The distribution of C. perfringens toxin-types amongst faecal and soil samples obtained from cattle and sheep farms in Kaduna and Kano States of Nigeria is shown in Table I. The percent distribution of toxin-types was 53.1 (127) type A, 7.1 (17) type B, 5.9 (14) type C, 18.4 (44) type D and 7.9 (19) type E. Eighteen (7.5%) strains were not typable while six (2.4%) were TABLE
I
Distribution of C. p e r f r i n g e n s t o x i n - t y p e s a m o n g s t faecal and soil samples o b t a i n e d s h e e p f a r m s i n K a d u n a a n d K a n o States o f Nigeria Toxintypes
A B C D E NT Lo~
Faecal samples
Soil samples
Total
f r o m cattle and
Percent distribution
Kaduna S t a t e
Kano State
Kaduna State
Kano State
Sheep
Cattle
Sheep
Cattle
Sheep
Cattle
Sheep
Cattle
28 5 4 20 -6 --
22 3 3 3 3 1 1
21 2 4 17 3 6 1
21 2 2 4 9 3 --
10 2 -. ----
9 ---
7 -1 . 2 -3
9 2 --
NT, not typable;--,
n o t detected.
.
. 1 1 --
1 1 1
127 17 14 44 19 18 6
53.1 7;1 5.9 18.4 7.9 7.5 2.4
95 lost during storage. C. perfringens t y p e A was most prevalent in soil and faecal samples in both states. No C. perfringens type D was isolated from the soil samples collected in both states. This study has demonstrated that the various types of C. perfringens are present, to varying degrees, in the alimentary tracts of cattle and sheep and in paddock soils in Nigeria. It appears that t y p e D is more prevalent in sheep flocks than in cattle herds {Table I). The frequency of isolation of C. perfringens type D from sheep faeces was significantly higher (P < 0.05; paired t-test) than from cattle herds in both states. It is noteworthy, however, that those cattle faeces that were positive for C. perfringens t y p e D originated from cattle kept together with sheep under a mixed husbandry system. It is obvious that these herds of cattle are perpetually exposed to herbage and feeds contaminated by sheep faeces. The failure to isolate C. perfringens type D from any of the soil samples agrees with the findings of Smith {1975). Smith {1975) had observed that cultures of C. perfringens that are intestinal parasites {types B, C, D and E) die within a few months when added to soil, being unable to compete with type A strains that are native to soil. The non-typability of some of the isolates, even following repeated tests, in this study may be due to reduction or loss of toxi~enicity during storage and subcultures in laboratory media, as was observed by Lozano et al. {1970). The finding in the present study that C. perfringens type D appears to pose the greatest danger to the livestock industry in Nigeria, particularly to sheep farms, agrees with findings in other countries {Sterne, 1981; Hogh and Muller, 1982). However, the ability of other serotypes (B, C, and E) to cause disease under favourable conditions can n o t be ignored. C. perfringens type A, which was most frequently isolated in this study, is primarily of human health importance from the viewpoint of f o o d b o r n e infections. It is, however, known to cause 'the yellows', a rare disease in lambs in the U.S.A. {Sterne, 1981). An enterotoxaemia caused by C. perfringens type A was also reported in captive wild goats (Russel, 1970). Although this is the first report of the toxin-types of C. perfringens associated with cattle and sheep farms in Nigeria, a design for preventive measures against C. perfringens enterotoxaemia will only be advisable after a thorough study on the aetiology, the disease and its economic impact has been documented nationwide. ACKNOWLEDGEMENTS This study was supported financially by the National Veterinary Research Institute, Vom, Nigeria. The laboratory assistance of Martin Mgbegha and Joseph Damsa is appreciated. We are also grateful to the government sheep and cattle farm managers and private owners who allowed us to sample their animals and farms.
96 REFERENCES Angelotti, R., Hall, H.E., Foter, M.J. and Lewis, H., 1962. Quantitation of Clostridium perfringens in foods. Appl. Microbiol., 10: 193--199. Hogh, P. and Muller, J., 1982. The present epizootiology of Hemorrhagic Necrotising Enteritis and Botulism. In: IVth Symposium of the Commission for the Study of Animal Diseases caused by Anaerobes, Paris. 16--18 Nov. 1982. Itodo, A.E., Agba, M.I. and Okewole, P.A., 1983. Clostridiumperfringens type D enterotoxaemia in cattle. Bull. Anita. Health Prod. Aft., 31 : 1 9 3 - 1 9 4 . Labbe, R.G., 1983. Enumeration and confirmation of C. perfringens. J. Food Prot., 46: 68--73. Lozano, E.A., Catler, J.E. and Hawkins, W.W., 1970. Incidence of C. perfringens in neonatal enteritis of Montana calves. Cornell Vet., 60: 347--357. Nagler, F.P., 1939. Observations on a reaction between the lethal toxin of C. welchii (type A) and human serum. Br. J. Exp. Pathol., 20: 473. Oakley, C.L. and Warrack, G.H., 1953. Routine typing of C. welchii. J. Hyg. (Lond.), 51: 102. Princewill, T.J. and Agba, M.I., 1982. Examination of bovine faeces for isolation and identification of Clostridium species. J. Appl. Bacteriol., 52: 97--102. Russel, W.C., 1970. Type A enterotoxaemia in captive wild goats. J. Am. Vet. Med. Assoc., 157: 643--649. Smith, L.D.S., 1975. The Pathogenic Anaerobic Bacteria. C.C. Thomas, Springfield, IL, pp. 115--175. Sterne, M., 1981. Clostridial infections. Br. Vet. J., 137: 443--454. Warrack, G.H., 1963. Some observations on the typing of C. perfringens. Bull. Off. Int. Epiz., 59: 1363--1400. Yamagishi, T., Yoshizawa, J., Kawai, M., Seo, N. and Nishida, S., 1971. Identification of isolates of C. perfringens type C and D by agglutination and fluorescent antibody methods. Appl. Microbiol., 21: 787--793.
93
Short Communication TOXIN-TYPES OF CLOSTRIDIUM PERFRINGENS STRAINS ISOLATED F R O M S H E E P , C A T T L E A N D P A D D O C K S O I L S IN N I G E R I A
A.E. ITODO 1., A.A. ADESIYUN1, J.O. ADEKEYE 2 and J.U. UMOH 1 Departments of Veterinary Public Health and Preventive Medicine ~ and Veterinary Pathology and Microbiology 2, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria (Nigeria) {Accepted for publication 14 October 1985)
ABSTRACT Itodo, A.E., Adesiyun, A.A., Adekeye, J.O. and Umoh, J.U., 1986. Toxin-types of Clostridium perfringens strains isolated from sheep, cattle and paddock soils in Nigeria. Vet. Microbiol., 12: 93--96. Two-hundred and forty-five strains of Clostridium perfringens isolated from the faeces of apparently healthy sheep and cattle and from their environments {paddock soils) in Kano and Kaduna States of Nigeria were studied. The isolates were examined by the toxin--antitoxin neutralisation tests performed intradermally in depilated albino guineapigs. One-hundred and twenty-seven {53.1%) of the isolates were type A, 17 (7.1%) were type B, 14 (4.9%) were type C, 44 {18.4%) were type D and 19 (7.9%) were type E. Eighteen {7.5%) were not typable while six were lost during storage. The significance of this distribution and the potential danger to animal health, especially as regards enterotoxaemias, cannot be over-emphasized.
INTRODUCTION It is n o w k n o w n t h a t specific t y p e s o f Clostridium perfringens are imp o r t a n t aetiological agents in certain diseases in animals o f e c o n o m i c imp o r t a n c e . A m o n g s t t h e five m a j o r t y p e s (A, B, C, D a n d E), t y p e s B, C a n d D in particular have been i m p l i c a t e d in sheep, goat a n d cattle disease worldwide (Russel, 1 9 7 0 ; Sterne, 1 9 8 1 ; H o g h and Muller, 1982). In Nigeria, Princewill and A g b a ( 1 9 8 2 ) first r e p o r t e d t h e prevalence o f Clostridium spp. in bovine faeces, b u t the t o x i n - t y p e s o f t h e isolates were n o t d e t e r m i n e d . C. p e r f r i n g e n s was r e c e n t l y r e p o r t e d to be responsible f o r a sporadic o u t b r e a k o f e n t e r o t o x a e m i a in three a d u l t Friesian cattle with a case fatality rate o f 100% ( I t o d o et al., 1983). With t h e a p p a r e n t absence o f r e p o r t i n g o f C. perfringens e n t e r o t o x a e m i a in Nigerian livestock c o u p l e d *Present address and correspondence to: Dr. A.E. Itodo, Bacteriology Division, National Veterinary Research Institute, Vom via Jos, Nigeria.
0378-1135/86/$03.50
© 1986 Elsevier Science Publishers B.V.
94 with the potential danger this organism may pose to the livestock industry, this study was conducted to specifically type C. perfringens strains isolated from the faeces of sheep and cattle and their environments (paddock soils) and to determine their toxicity. MATERIALS
AND
METHODS
Two-hundred and forty-five strains of C. perfringens isolated from the faeces of apparently healthy sheep and cattle and from their environments (paddock soils) in Kano and Kaduna States of Nigeria were studied. All isolation work was done according to standard methods on sulfitepolymyxin-sulfadiazine agar (Angelotti et al., 1962). The isolates were confirmed as C. perfringens by the methods of Nagler (1939) and Labbe (1983). Formolized antigens (toxins) of five toxigenic types of C. perfringens type A (CN 3692), type B (CN 2003), type C (CN 3706), type D (CN 4529) and type E (CN 1493) kindly supplied by the Wellcome Research Laboratories, U.K., were used to produce type-specific antisera in rabbits using the procedure described by Yamagishi et al. (1971). The principal types of C. perfringens strains were determined using prepared type-specific antisera and culture filtrates (toxins) of isolates for neutralisation tests in depilated skin of albino guinea-pigs as previously described (Oakley and Warrack, 1953; Warrack, 1963). RESULTS
AND
DISCUSSION
The distribution of C. perfringens toxin-types amongst faecal and soil samples obtained from cattle and sheep farms in Kaduna and Kano States of Nigeria is shown in Table I. The percent distribution of toxin-types was 53.1 (127) type A, 7.1 (17) type B, 5.9 (14) type C, 18.4 (44) type D and 7.9 (19) type E. Eighteen (7.5%) strains were not typable while six (2.4%) were TABLE
I
Distribution of C. p e r f r i n g e n s t o x i n - t y p e s a m o n g s t faecal and soil samples o b t a i n e d s h e e p f a r m s i n K a d u n a a n d K a n o States o f Nigeria Toxintypes
A B C D E NT Lo~
Faecal samples
Soil samples
Total
f r o m cattle and
Percent distribution
Kaduna S t a t e
Kano State
Kaduna State
Kano State
Sheep
Cattle
Sheep
Cattle
Sheep
Cattle
Sheep
Cattle
28 5 4 20 -6 --
22 3 3 3 3 1 1
21 2 4 17 3 6 1
21 2 2 4 9 3 --
10 2 -. ----
9 ---
7 -1 . 2 -3
9 2 --
NT, not typable;--,
n o t detected.
.
. 1 1 --
1 1 1
127 17 14 44 19 18 6
53.1 7;1 5.9 18.4 7.9 7.5 2.4
95 lost during storage. C. perfringens t y p e A was most prevalent in soil and faecal samples in both states. No C. perfringens type D was isolated from the soil samples collected in both states. This study has demonstrated that the various types of C. perfringens are present, to varying degrees, in the alimentary tracts of cattle and sheep and in paddock soils in Nigeria. It appears that t y p e D is more prevalent in sheep flocks than in cattle herds {Table I). The frequency of isolation of C. perfringens type D from sheep faeces was significantly higher (P < 0.05; paired t-test) than from cattle herds in both states. It is noteworthy, however, that those cattle faeces that were positive for C. perfringens t y p e D originated from cattle kept together with sheep under a mixed husbandry system. It is obvious that these herds of cattle are perpetually exposed to herbage and feeds contaminated by sheep faeces. The failure to isolate C. perfringens type D from any of the soil samples agrees with the findings of Smith {1975). Smith {1975) had observed that cultures of C. perfringens that are intestinal parasites {types B, C, D and E) die within a few months when added to soil, being unable to compete with type A strains that are native to soil. The non-typability of some of the isolates, even following repeated tests, in this study may be due to reduction or loss of toxi~enicity during storage and subcultures in laboratory media, as was observed by Lozano et al. {1970). The finding in the present study that C. perfringens type D appears to pose the greatest danger to the livestock industry in Nigeria, particularly to sheep farms, agrees with findings in other countries {Sterne, 1981; Hogh and Muller, 1982). However, the ability of other serotypes (B, C, and E) to cause disease under favourable conditions can n o t be ignored. C. perfringens type A, which was most frequently isolated in this study, is primarily of human health importance from the viewpoint of f o o d b o r n e infections. It is, however, known to cause 'the yellows', a rare disease in lambs in the U.S.A. {Sterne, 1981). An enterotoxaemia caused by C. perfringens type A was also reported in captive wild goats (Russel, 1970). Although this is the first report of the toxin-types of C. perfringens associated with cattle and sheep farms in Nigeria, a design for preventive measures against C. perfringens enterotoxaemia will only be advisable after a thorough study on the aetiology, the disease and its economic impact has been documented nationwide. ACKNOWLEDGEMENTS This study was supported financially by the National Veterinary Research Institute, Vom, Nigeria. The laboratory assistance of Martin Mgbegha and Joseph Damsa is appreciated. We are also grateful to the government sheep and cattle farm managers and private owners who allowed us to sample their animals and farms.
96 REFERENCES Angelotti, R., Hall, H.E., Foter, M.J. and Lewis, H., 1962. Quantitation of Clostridium perfringens in foods. Appl. Microbiol., 10: 193--199. Hogh, P. and Muller, J., 1982. The present epizootiology of Hemorrhagic Necrotising Enteritis and Botulism. In: IVth Symposium of the Commission for the Study of Animal Diseases caused by Anaerobes, Paris. 16--18 Nov. 1982. Itodo, A.E., Agba, M.I. and Okewole, P.A., 1983. Clostridiumperfringens type D enterotoxaemia in cattle. Bull. Anita. Health Prod. Aft., 31 : 1 9 3 - 1 9 4 . Labbe, R.G., 1983. Enumeration and confirmation of C. perfringens. J. Food Prot., 46: 68--73. Lozano, E.A., Catler, J.E. and Hawkins, W.W., 1970. Incidence of C. perfringens in neonatal enteritis of Montana calves. Cornell Vet., 60: 347--357. Nagler, F.P., 1939. Observations on a reaction between the lethal toxin of C. welchii (type A) and human serum. Br. J. Exp. Pathol., 20: 473. Oakley, C.L. and Warrack, G.H., 1953. Routine typing of C. welchii. J. Hyg. (Lond.), 51: 102. Princewill, T.J. and Agba, M.I., 1982. Examination of bovine faeces for isolation and identification of Clostridium species. J. Appl. Bacteriol., 52: 97--102. Russel, W.C., 1970. Type A enterotoxaemia in captive wild goats. J. Am. Vet. Med. Assoc., 157: 643--649. Smith, L.D.S., 1975. The Pathogenic Anaerobic Bacteria. C.C. Thomas, Springfield, IL, pp. 115--175. Sterne, M., 1981. Clostridial infections. Br. Vet. J., 137: 443--454. Warrack, G.H., 1963. Some observations on the typing of C. perfringens. Bull. Off. Int. Epiz., 59: 1363--1400. Yamagishi, T., Yoshizawa, J., Kawai, M., Seo, N. and Nishida, S., 1971. Identification of isolates of C. perfringens type C and D by agglutination and fluorescent antibody methods. Appl. Microbiol., 21: 787--793.