Browsing by Author "Bohnel, H."

In March 1999 the Bovine Health Service was asked to investigate an unknown disease in a larger dairy cattlestock. Problems had developed since 1995, until in December 1998 milk production had to be given up. Main symptoms were lameness (noninfectious chronic laminitis), digesting disturbances and unsatisfactory feed utilisation, circulation disturbances, decreases in weight and inexplainable death after fixation. Since 1997 extensive feed and metabolic investigations, clinical, microbiological and pathological investigations took place via different institutions. They did not furnish a note to the cause of the loss happening. Renewed laboratory tests regarding an intoxication resulted in the suspicion of a mycotoxicosis. Particularly mycotoxins from Fusarium species could be found in the feed and the bile acid from the cows. The valuation, proved however, very difficult. Practically no valuation could be given to the proof of certain mycotoxins. Sick calves were investigated in the Veterinary Faculty of the University of Leipzig - clinical, immunological, microbiological and pathological examinations took place. In this context, samples of faeces were investigated in the Institute for Tropical Animal Health, University of Gottingen. There was found free botulinum neurotoxin (BoNT) in the faeces of calves. In other dairy cattlestock with similar problems BoNT in the faeces could be found likewise. Information for identical illness-found with finding of BoNT came also from other Lands of the Federal Republic. Mycobacterium paratuberculosis, other Clostridium and Bacillus species were also discovered as reason for different diseases. A dysbacteriosis of intestinal tract with owergrowth of spore-forming organisms was observed. There are many questions about the reason of changed intestinal flora, the formation of BoNT, the role of Clostridium species and other reasons for the illness mentioned above. Scientific investigations must be carried out urgently.

Toxins of Clostridium botulinum (types A-G) are known as 'neurotoxins', causing the clinically well-known picture of flaccid muscular paralysis. The molecular biological background is the blocking of acetylcholine secretion in neuromuscular junctions by enzymatic cleavage of molecules forming the machinery of exocytosis. Two 'non-neurotoxins' (types C2, C3) are produced by some strains of C. botulinum types C and D. These affect the cytoskeleton by ribosylating actin filaments. All these toxins are used as cell biological tools for the study of specific actions and effects in different eukaryotic cells. Pharmaceutical and molecular biological research has shown their influence on several crucial organs (or cell cultures thereof) of humans and animals (brain and spinal cord, cerebellum, hippocampus, hypophysis, pancreas, adrenal glands, salivary glands and others). Under natural conditions, botulinum toxins may pass the intestinal barrier and circulate in the bloodstream for a certain time. Carriers occurring naturally in food, such as wheat germ agglutinin, digitonin or saponin, and bacterial toxins such as streptolysin O, perfringolysins, C2 toxin or botulinolysin may also form pores in cell walls. They facilitate the entry of botulinum toxins into cells that may not have natural binding receptors. It is concluded that in vivo actions of different botulinum toxins after their entry into the organism may contribute to the onset of different diseases of hitherto cryptogenic origin. Some examples are given and future problems are discussed.

Bio-waste recycling and the production and use of bio-compost are politically encouraged in Europe. Quality control takes no consideration of pathogenic anaerobic spore formers, e.g. Clostridium botulinum. A protocol for health hazard analysis concerning this pathogen has been developed. Samples of marketed bio-compost were tested and results showed that about 50% of the tested samples contained C: botulinum. For the first time it has been shown that the use of bio-compost represents a health hazard to humans and animals, especially in the future when spores will have accumulated in the environment. The use of household bic-waste collected in bio-bins' is apparently one factor involved in the production of contaminated compost end-products. Environmental factors in the propagation of C botulinum are discussed. The improvement of bio-waste recycling technology and management should be encouraged in order to minimize the health hazard caused by contaminated bio-compost.

Laboratory methods for the detection of botulinum toxins are insufficient as indicated in recent discussions,on bio-terrorism. Diagnostic veterinary laboratories experience problems with the absence of suitable methods with an increasing number of samples. The mouse bioassay remains the diagnostic method of choice. Alternative methods have, however, been developed, which could replace the use of animals in some instances. Data on test performance with complex sample material is only available for the mouse assay. All other assays have been tested with purified neurotoxins or culture supernatants. This short and incomplete review lists several suitable methods which are recommended for routine laboratory use.

The disease >>botulism<< occurs in two clinically distinct forms. Classical muscular botulism is an intoxication presenting most commonly as a flaccid muscular paralysis. Visceral botulism appears to be increasing in incidence in recent years as an intoxication and/or infection and which affects a number of organ systems. There is no uniformity to the clinical picture. This review describes the development cascade >>botulinomics<< starting with Clostridium botulinum in the environment resulting in contamination of feed, followed by toxinogenesis, toxic effects and finally to clinical disease. The authors' experience of the disease over the last few years together with a review of the published literature is discussed.

In previously conducted research, Clostridium botulinum spores were found in bio-waste compost. Household bio-waste, collected in 'bio-bins', was suspected to be one of the reasons for contamination. Maggots of Calliphoridae were collected inside and outside of bio-bins from 8 different locations in central Germany and examined for C. botulinum toxin and/or bacterial forms using standard mouse bioassay. Young maggots (instar 1) contained neither C. botulinum nor toxin, but these were found in instar 2 and 3 larvae. For the first time these results show that fly larvae out of bio-bins are not only a nuisance, but may be vectors of the potentially lethal C. botulinum.

Despite the fact that botulism was described in Germany for the first time by Kerner in 1820, the disease is almost forgotten in this country. Only about 10-20 cases of classical botulism (intoxication) are recorded every year, including 1-2 cases of clinical infant botulism. As we assumed a high incidence of botulism to be connected with cases of sudden infant death (SID), we undertook the research work presented here. From every case of unexpected infant death up to 12 months of age, standardised specimens (blood, liver and intestine) were taken at autopsy. They were tested for the presence of botulinum neurotoxin (BoNT) and/or bacterial forms of Clostridium botulinum with subsequent BoNT neutralisation tests by the international standard mouse bioassay. Age, sex, pathological findings and season were recorded. Over a 5-year period, 75 samples including 57 SID cases were tested. Free toxin was found in nine and bacterial forms were detected in six samples. Toxin neutralisation revealed the definite presence of BoNT/BoNT producing bacteria (mainly type E), whereas another 11 toxin tests were inconclusive. According to international literature, these 15 cases are to be interpreted as infant botulism. Conclusion: the results show a remarkable incidence of infant botulism without any known previous medical history, partly hidden as sudden infant death. We propose to systematically search for botulism in connection with sudden infant death.

In a study to isolate Clostridium botulinum from field samples, 53 C. botulinum-like isolates were found neutralizable by botulinum antitoxins using the standard mouse bioassay. The neutralization results of 14 of them were found false-positive due to contamination with C. tetani which was separated from these isolates and found neutralizable by the same botulinum antitoxin types. Due to this result, C. tetani was isolated from other five field samples to test their toxins by botulinum antitoxins. They were also found neutralizable by certain botulinum antitoxins. Supernatants of C. tetani isolates were confirmed by being neutralized by tetanus antitoxin. Tetani toxins were found neutralizable by different botulinum antitoxins (A-F) but mainly by polyvalent type ABE (Aventis Behring, Marburg) and type CD mixture (Onderstepoort Veterinary Institute, Onderstepoort). These results were mainly attributed to the non-specificity of the proteins of some (at least ABE and CD) botulinum antitoxin hyperimmune sera which may contain antibodies against C. tetani as well.

Botulism is caused by different types of Clostridium botulinum , a soil bacterium. Equine grass sickness (equine dysautonomia) is suspected of being a clinical form of this disease. On a stud where this disease occurred twice within 8 months, grass and soil samples and necropsy specimens of one horse were tested for the presence of bacterial forms and toxin of C. botulinum . Different types and type mixtures (A-E) of C. botulinum and botulinum neurotoxin were found. For the first time, it has been shown that green grass blades contain botulinum toxin. The results support the hypothesis that equine grass sickness is a clinical form of botulism, a soil-borne disease.

There are reports of a hitherto unknown bovine disease in Germany. The symptoms are, in general, indigestion (constipation alternating with diarrhoea), non-infectious chronic laminitis, engorged veins, oedemas, retracted abdomen, emaciation and apathy. Most cases occur during the peripartal period and often result in unexpected death. In addition, there are findings of delayed growth and wasting in heifers, as well as decreasing milk yield. Clinical and standard laboratory examinations leave the origin undisclosed. Bioassays for Clostridium botulinum, its spores and toxins in animals of affected farms revealed the presence of free botulinum, toxin in the contents of the lower sections of the intestine, In two control farms without signs of the disease, the tests remained negative. This seems to support our hypothesis that long-lasting absorption of low quantities of botulinum toxin may interfere with the neurological control of intestinal physiology. The authors propose to name this disease complex 'visceral botulism'.