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Botulinum neurotoxin type B in milk from a cow affected by visceral botulism
The Veterinary Journal The Veterinary Journal 169 (2005) 124–125 www.elsevier.com/locate/tvjl
Short communication
Botulinum neurotoxin type B in milk from a cow affected by visceral botulism H. B€ ohnel a
a,*
, B. Neufeld b, F. Gessler
a
Institute for Tropical Animal Health, Georg-August-University, Kellnerweg 6, 37077 G€ottingen, Germany b Veterinary Clinic, Balterazhofer Strasse 45, 88299 Leutkirch, Germany Accepted 12 January 2004
Keywords: Clostridium botulinum; Neurotoxin; Milk; Bovine; Health
Two clinical forms of botulism may be distinguished in cattle: (1) muscular (classical) botulism (intoxication) (Smith and Sugiyama, 1988) and (2) visceral botulism (toxico-infection) (Schwagerick and B€ ohnel, 2001; B€ ohnel et al, 2001). The importance of botulism in cattle production as a cause of human food-borne disease is certainly underrated (Bell and Kyriakides, 2000) and the number of cases seems to have risen in the last decade in Germany (B€ ohnel and Gessler, 2003). Although there are no reports available that botulinum neurotoxin (BoNT) has been found in fresh cowÕs milk, some dairies refuse to accept milk from affected farms that have had cases of acute botulism (Galey et al., 2000; Cobb et al., 2002). Contamination of milk or meat by botulinum spores may lead to food contamination and pose a health risk for the consumer (Schocken-Iturrino et al., 1989; Jenzer et al., 1974; Popoff and Argente, 1996; Franciosa et al., 1999) and, at least theoretically, BoNT may be absorbed from the intestinal tract and could be excreted in milk (with low toxicity) before clinical symptoms became evident (Moberg and Sugiyama, 1980). An outbreak of visceral botulism in a dairy herd (about 40 cows with off-spring) in the late autumn of 2001 gave us the chance to gain a deeper insight into this disease. The farm is located in Southern Germany and has 56 ha of permanent pasture and 3 ha of arable land. Feed consisted of pasture and concentrates in the summer with hay and concentrates in winter. Silage was not
*
Corresponding author. Tel.: +49-551-39-33-96; fax: +49-551-39-34-
08. E-mail address: [email protected] (H. B€ ohnel). 1090-0233/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.01.006
used as a dairy for cheese production collected the milk. General clinical symptoms of the disease were reduced milk production, paralysis, parturient paresis and nonspecific digestive disorders. The presence of Clostridium botulinum types A, B, and C/D at the farm had been confirmed by the presence of BoNT and C. botulinum bacteria in the intestines and faeces. In the serum, high antibody titres against these types were found in several animals including the animal described below but no free BoNT was found. All animals had been vaccinated against C. botulinum type C/D in November 2001. In January 2002, a cow presented with crossed front legs when standing, a tucked-up abdomen, curved back, emaciation, positive venous pulsation and a chronic, antibiotic resistant mastitis in one quarter. The last veterinary antibiotic treatment had been about six weeks before sampling. After the withdrawal time, milk had been sent to the dairy up to the day of euthanasia. One day before the cow was euthanased, milk samples were taken from all quarters, immediately frozen to )15 °C, and kept until laboratory analysis. One animal showing a similar clinical picture, but without mastitis, served as control. Additional milk samples from other clinically sick animals were tested six weeks later (two cows/ quarter milk; and 12 cows/complete milk). A standard botulinum mouse bioassay was used (CDC, 1998). Antitoxins were obtained from CDC, Atlanta; NMRI mice were purchased from Harlan–Winkelmann, Borchen. The presence of BoNT type C/D in the small intestine, but not in the rumen, together with the clinical symptoms and the history of recent vaccination against muscular botulism confirmed the suspected diagnosis of ‘‘visceral botulism’’ (Neufeld and Belihart-Neufeld, 2004). Toxicity tests on the milk revealed C. botulinum type B toxin
H. B€ohnel et al. / The Veterinary Journal 169 (2005) 124–125
only in the quarter affected by mastitis. The toxin titre was estimated to be at least 10,000 LD50 i:p:; mouse /mL. No anaerobic growth was observed. The three other quarters and the control animals all showed negative results. To our knowledge, this is the first time that free BoNT has been demonstrated in fresh bovine milk. In the available literature, there is only one report where intestinal BoNT was found in the milk of rats (2.5 LD50 / mL) with no indication of the BoNT type, but presumably type A (Moberg and Sugiyama, 1980). In cows affected by acute botulism, milk has been reported to test negative (Pumukcu, 1954; Galey et al., 2000; Moeller et al., 2003). To estimate a possible health hazard for man, it is necessary to consider whether BoNT is secreted from the bacterial cell as progenitor-toxin causing (oral) food-borne botulism. In tests in mice, type B showed an extraordinarily high oral toxicity (Ohishi et al., 1977). A lethal dose for humans has been estimated to be about 3500 LD50 mouse (Meyer and Eddie, 1951). Considering 750 L as an average daily milk production from this farm, 250 mL of bulk milk out of the milk tank at the farm would thus have contained a lethal dose of BoNT for a single consumer. Of course, this must been seen in perspective as only the consumption of raw milk would pose a health risk, as BoNT is heat labile. Moreover, it is prohibited by law in Germany to sell milk from sick cows. The milk did not contain anaerobic bacteria, probably reflecting the preceding antibiotic treatment. However, some hidden C. botulinum must have been present in the damaged udder tissue and escaped the antibiotic treatment but still produced toxin. Unfortunately, tissue samples of the four quarters were not available for testing. So the origin of the toxin remains obscure. Many farms sell unprocessed milk to passers-by in Germany. Epidemiological data on botulism in farm animals are almost completely missing, due to a lack of awareness of public veterinary services, the difficult laboratory diagnosis, and a failure to accept that botulism might be more than just a flaccid paralysis of the locomotor muscles.
References Bell, C., Kyriakides, A., 2000. Clostridium botulinum. A practical approach to the organism and its control in foods. Blackwell Science, Oxford.
125
B€ ohnel, H., Gessler, F., 2003. Diagnosis of botulism since 1995. A report of experience (in German). Berliner und M€ unchener Tier€arztliche Wochenschrift 116, 269–273. B€ ohnel, H., Schwagerick, B., Gessler, F., 2001. Visceral botulism – A new form of bovine Clostridium botulinum toxication. Journal of Veterinary Medicine A 48, 373–383. CDC, 1998. Botulism in the US, 1899–1996. In: Handbook for Epidemiologists, Clinicians, and Laboratory Workers. Centers for Disease Control and Prevention, Atlanta. Cobb, S.P., Hogg, R.A., Challoner, D.J., Brett, M.M., Livesey, C.T., Sharpe, R.T., Jones, T.O., 2002. Suspected botulism in dairy cows and its implications for the safety of human foods. Veterinary Record 150, 5–8. Franciosa, G., Pourshaban, M., Franceschi, M., Gattuso, A., Fenicia, L., Ferrini, A.M., Mannoni, V., De Luca, G., Aureli, P., 1999. Clostridium botulinum spores and toxin in mascarpone cheese and other milk products. Journal of Food Protection 62, 867–871. Galey, F.D., Terra, R., Walker, R., Adaska, J., Etchebarne, M.A., Puschner, B., Fisher, E., Whitlock, R.H., Rocke, T., Willoughby, D., Tor, E., 2000. Type C botulism in dairy cattle from feed contaminated with a dead cat. Journal of Veterinary Diagnosis and Investigation 12, 204–209. Jenzer, G., Mumenthaler, M., Ludin, H.P., Robert, F., 1974. Botulism type B. Report on a mild course with disturbances mainly of the autonomous innervation (in German). Schweizer Medizinische Wochenschrift 104, 685–693. Meyer, K.F., Eddie, B., 1951. Perspectives concerning botulism. Zeitschrift f€ ur Hygiene und Infektionskrankheiten 133, 255– 263. Moberg, L.J., Sugiyama, H., 1980. The rat as animal model for infant botulism. Infection and Immunity 29, 819–821. Moeller, R.B., Puschner, B., Walker, R.L., Rocke, T., Galey, F.D., Cullor, J.S., Ardans, A.A., 2003. Determination of the median toxic dose of type C botulinum toxin in lactating dairy cows. Journal of Veterinary Diagnosis and Investigation 15, 523–526. Neufeld, B., Belihart-Neufeld, A., 2004. Chronic visceral botulism in a dairy farm in the German region of Allg€au (in German). Tier€arztl. Umschau 59, 19–24. Ohishi, I., Sugii, S., Sakaguchi, G., 1977. Oral toxicities of Clostridium botulinum toxins in response to molecular size. Infection and Immunity 16, 107–109. Popoff, M.R., Argente, G., 1996. Botulism in animals – Is it a danger for man (in French). Bulletin de l’ Academie Veterinaire Francßaise 69, 373–382. Pumukcu, A.M., 1954. Hemorrhagic encephalomyelitis due to botulism in cattle in Turkey. Zentralblatt f€ ur Veterin€ar-Medizin 1, 707– 722. Schocken-Iturrino, R.P., Avila, F.A., Berchielli, S.C.P., Nader Filho, A., 1989. First case of type A botulism in zebu (Bos indicus). Veterinary Record 126, 217–218. Schwagerick, B., B€ ohnel, H., 2001. A chronic disease in dairy cattle connected with the proof of botulinum toxin – a case study (in German). Der Praktische Tierarzt 82, 516– 524. Smith, L.D., Sugiyama, H., 1988. Botulism. The organism, its toxins, the disease, second ed. Charles C. Thomas, Springfield.
Short communication
Botulinum neurotoxin type B in milk from a cow affected by visceral botulism H. B€ ohnel a
a,*
, B. Neufeld b, F. Gessler
a
Institute for Tropical Animal Health, Georg-August-University, Kellnerweg 6, 37077 G€ottingen, Germany b Veterinary Clinic, Balterazhofer Strasse 45, 88299 Leutkirch, Germany Accepted 12 January 2004
Keywords: Clostridium botulinum; Neurotoxin; Milk; Bovine; Health
Two clinical forms of botulism may be distinguished in cattle: (1) muscular (classical) botulism (intoxication) (Smith and Sugiyama, 1988) and (2) visceral botulism (toxico-infection) (Schwagerick and B€ ohnel, 2001; B€ ohnel et al, 2001). The importance of botulism in cattle production as a cause of human food-borne disease is certainly underrated (Bell and Kyriakides, 2000) and the number of cases seems to have risen in the last decade in Germany (B€ ohnel and Gessler, 2003). Although there are no reports available that botulinum neurotoxin (BoNT) has been found in fresh cowÕs milk, some dairies refuse to accept milk from affected farms that have had cases of acute botulism (Galey et al., 2000; Cobb et al., 2002). Contamination of milk or meat by botulinum spores may lead to food contamination and pose a health risk for the consumer (Schocken-Iturrino et al., 1989; Jenzer et al., 1974; Popoff and Argente, 1996; Franciosa et al., 1999) and, at least theoretically, BoNT may be absorbed from the intestinal tract and could be excreted in milk (with low toxicity) before clinical symptoms became evident (Moberg and Sugiyama, 1980). An outbreak of visceral botulism in a dairy herd (about 40 cows with off-spring) in the late autumn of 2001 gave us the chance to gain a deeper insight into this disease. The farm is located in Southern Germany and has 56 ha of permanent pasture and 3 ha of arable land. Feed consisted of pasture and concentrates in the summer with hay and concentrates in winter. Silage was not
*
Corresponding author. Tel.: +49-551-39-33-96; fax: +49-551-39-34-
08. E-mail address: [email protected] (H. B€ ohnel). 1090-0233/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.01.006
used as a dairy for cheese production collected the milk. General clinical symptoms of the disease were reduced milk production, paralysis, parturient paresis and nonspecific digestive disorders. The presence of Clostridium botulinum types A, B, and C/D at the farm had been confirmed by the presence of BoNT and C. botulinum bacteria in the intestines and faeces. In the serum, high antibody titres against these types were found in several animals including the animal described below but no free BoNT was found. All animals had been vaccinated against C. botulinum type C/D in November 2001. In January 2002, a cow presented with crossed front legs when standing, a tucked-up abdomen, curved back, emaciation, positive venous pulsation and a chronic, antibiotic resistant mastitis in one quarter. The last veterinary antibiotic treatment had been about six weeks before sampling. After the withdrawal time, milk had been sent to the dairy up to the day of euthanasia. One day before the cow was euthanased, milk samples were taken from all quarters, immediately frozen to )15 °C, and kept until laboratory analysis. One animal showing a similar clinical picture, but without mastitis, served as control. Additional milk samples from other clinically sick animals were tested six weeks later (two cows/ quarter milk; and 12 cows/complete milk). A standard botulinum mouse bioassay was used (CDC, 1998). Antitoxins were obtained from CDC, Atlanta; NMRI mice were purchased from Harlan–Winkelmann, Borchen. The presence of BoNT type C/D in the small intestine, but not in the rumen, together with the clinical symptoms and the history of recent vaccination against muscular botulism confirmed the suspected diagnosis of ‘‘visceral botulism’’ (Neufeld and Belihart-Neufeld, 2004). Toxicity tests on the milk revealed C. botulinum type B toxin
H. B€ohnel et al. / The Veterinary Journal 169 (2005) 124–125
only in the quarter affected by mastitis. The toxin titre was estimated to be at least 10,000 LD50 i:p:; mouse /mL. No anaerobic growth was observed. The three other quarters and the control animals all showed negative results. To our knowledge, this is the first time that free BoNT has been demonstrated in fresh bovine milk. In the available literature, there is only one report where intestinal BoNT was found in the milk of rats (2.5 LD50 / mL) with no indication of the BoNT type, but presumably type A (Moberg and Sugiyama, 1980). In cows affected by acute botulism, milk has been reported to test negative (Pumukcu, 1954; Galey et al., 2000; Moeller et al., 2003). To estimate a possible health hazard for man, it is necessary to consider whether BoNT is secreted from the bacterial cell as progenitor-toxin causing (oral) food-borne botulism. In tests in mice, type B showed an extraordinarily high oral toxicity (Ohishi et al., 1977). A lethal dose for humans has been estimated to be about 3500 LD50 mouse (Meyer and Eddie, 1951). Considering 750 L as an average daily milk production from this farm, 250 mL of bulk milk out of the milk tank at the farm would thus have contained a lethal dose of BoNT for a single consumer. Of course, this must been seen in perspective as only the consumption of raw milk would pose a health risk, as BoNT is heat labile. Moreover, it is prohibited by law in Germany to sell milk from sick cows. The milk did not contain anaerobic bacteria, probably reflecting the preceding antibiotic treatment. However, some hidden C. botulinum must have been present in the damaged udder tissue and escaped the antibiotic treatment but still produced toxin. Unfortunately, tissue samples of the four quarters were not available for testing. So the origin of the toxin remains obscure. Many farms sell unprocessed milk to passers-by in Germany. Epidemiological data on botulism in farm animals are almost completely missing, due to a lack of awareness of public veterinary services, the difficult laboratory diagnosis, and a failure to accept that botulism might be more than just a flaccid paralysis of the locomotor muscles.
References Bell, C., Kyriakides, A., 2000. Clostridium botulinum. A practical approach to the organism and its control in foods. Blackwell Science, Oxford.
125
B€ ohnel, H., Gessler, F., 2003. Diagnosis of botulism since 1995. A report of experience (in German). Berliner und M€ unchener Tier€arztliche Wochenschrift 116, 269–273. B€ ohnel, H., Schwagerick, B., Gessler, F., 2001. Visceral botulism – A new form of bovine Clostridium botulinum toxication. Journal of Veterinary Medicine A 48, 373–383. CDC, 1998. Botulism in the US, 1899–1996. In: Handbook for Epidemiologists, Clinicians, and Laboratory Workers. Centers for Disease Control and Prevention, Atlanta. Cobb, S.P., Hogg, R.A., Challoner, D.J., Brett, M.M., Livesey, C.T., Sharpe, R.T., Jones, T.O., 2002. Suspected botulism in dairy cows and its implications for the safety of human foods. Veterinary Record 150, 5–8. Franciosa, G., Pourshaban, M., Franceschi, M., Gattuso, A., Fenicia, L., Ferrini, A.M., Mannoni, V., De Luca, G., Aureli, P., 1999. Clostridium botulinum spores and toxin in mascarpone cheese and other milk products. Journal of Food Protection 62, 867–871. Galey, F.D., Terra, R., Walker, R., Adaska, J., Etchebarne, M.A., Puschner, B., Fisher, E., Whitlock, R.H., Rocke, T., Willoughby, D., Tor, E., 2000. Type C botulism in dairy cattle from feed contaminated with a dead cat. Journal of Veterinary Diagnosis and Investigation 12, 204–209. Jenzer, G., Mumenthaler, M., Ludin, H.P., Robert, F., 1974. Botulism type B. Report on a mild course with disturbances mainly of the autonomous innervation (in German). Schweizer Medizinische Wochenschrift 104, 685–693. Meyer, K.F., Eddie, B., 1951. Perspectives concerning botulism. Zeitschrift f€ ur Hygiene und Infektionskrankheiten 133, 255– 263. Moberg, L.J., Sugiyama, H., 1980. The rat as animal model for infant botulism. Infection and Immunity 29, 819–821. Moeller, R.B., Puschner, B., Walker, R.L., Rocke, T., Galey, F.D., Cullor, J.S., Ardans, A.A., 2003. Determination of the median toxic dose of type C botulinum toxin in lactating dairy cows. Journal of Veterinary Diagnosis and Investigation 15, 523–526. Neufeld, B., Belihart-Neufeld, A., 2004. Chronic visceral botulism in a dairy farm in the German region of Allg€au (in German). Tier€arztl. Umschau 59, 19–24. Ohishi, I., Sugii, S., Sakaguchi, G., 1977. Oral toxicities of Clostridium botulinum toxins in response to molecular size. Infection and Immunity 16, 107–109. Popoff, M.R., Argente, G., 1996. Botulism in animals – Is it a danger for man (in French). Bulletin de l’ Academie Veterinaire Francßaise 69, 373–382. Pumukcu, A.M., 1954. Hemorrhagic encephalomyelitis due to botulism in cattle in Turkey. Zentralblatt f€ ur Veterin€ar-Medizin 1, 707– 722. Schocken-Iturrino, R.P., Avila, F.A., Berchielli, S.C.P., Nader Filho, A., 1989. First case of type A botulism in zebu (Bos indicus). Veterinary Record 126, 217–218. Schwagerick, B., B€ ohnel, H., 2001. A chronic disease in dairy cattle connected with the proof of botulinum toxin – a case study (in German). Der Praktische Tierarzt 82, 516– 524. Smith, L.D., Sugiyama, H., 1988. Botulism. The organism, its toxins, the disease, second ed. Charles C. Thomas, Springfield.