Presence of Clostridium botulinum and botulinum toxin in milk and udder tissue of dairy cows with suspected botulism.

Clostridium botulinum is an anaerobic spore-forming bacterium prevalent in the environment, and causes botulism in man and animals via toxins. Dairy cattle may be contaminated or infected by feed, water or other environmental factors. Milk may also carry the pathogen. Hence, milk and udder samples need to be tested. The number of clinical cases of bovine botulism in Germany has been increasing since the mid-1990s. Besides routine samples, additional 99 milk samples from 37 farms, and 51 udder samples from 51 farms from sick animals presumably affected by botulism were tested microbiologically by the mouse bioassay. Milk from three farms (8.1 per cent) contained botulinum toxin, and from two (5.4 per cent) bacterial states of C botulinum. Ten udder samples (19.6 per cent) contained toxin, and 7 (13.7 per cent) bacterial forms, including one case where both toxin and bacteria were found. The findings are discussed. Positive milk samples containing botulinum toxin or bacteria raise concern of food safety for the human consumer. Pathological udder samples may show either infection prior to, or contamination after death.

The catalase gene differentiates between some strains of Staphylococcus aureus ssp. anaerobius.

Staphylococcus aureus ssp anaerobius strain S10 was isolated from an outbreak of sheep abscess disease. Sequence of the catalase gene of this strain showed 99% identity to the catalase gene (katB) sequence of the reference strain (S. aureus ssp. anaerobius strain MVF213) with mismatching of three base pairs. An important substitution located 1036 nucleotides upstream of the initiation codon from "C" in katB to "T" in the catalase gene of strain S10 originated a stop codon. The deduced protein (345 amino acids) is 105 amino acids shorter than that of katB. Partial sequence of the catalase gene of other 8 local isolates in addition to another reference strain (DSM 20714/ATCC 35844) revealed the same mutations in all local (African) strains, whereas the sequence of the reference (European) strain was typical to that of katB. Sequence of the catalase gene of S. aureus ssp. anaerobius strain S10 was deposited in GenBank under accession no. EU281993.

Tonsils--place of botulinum toxin production: results of routine laboratory diagnosis in farm animals.

Since a case of a veterinarian was reported, who was likely to be infected/intoxicated by Clostridium botulinum during the handling of a diseased animal, tonsils in animals were tested for botulinum neurotoxin and bacterial forms of neurotoxic Clostridium botulinum during routine botulism laboratory examinations including standard samples (intestinal tract and liver) from 48 cattle, 11 horses, and 14 goats. Ten out of 60 samples from tonsils contained free botulinum toxin, and 12 out of 59 were positive for live toxin producing bacteria. In 32 out of 162 intestinal samples toxin was detected. Toxin producing bacteria were found in 37 samples. Eight of 56 liver samples contained free toxin, and 15 out of 43 toxigenic bacteria. Samples from 10 slaughter pigs were all negative, whereas from slaughter cattle tonsils had a high incidence of toxin (7 of 10) or toxigenic bacteria (2 of 8). The results are discussed in the context of effects on animal health and botulism as zoonosis.

Botulinum toxins--cause of botulism and systemic diseases?

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.

Two cases of equine grass sickness with evidence for soil-borne origin involving botulinum neurotoxin.

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.

Is there a link between infant botulism and sudden infant death? Bacteriological results obtained in central Germany.

UNLABELLED: 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.

Visceral botulism--a new form of bovine Clostridium botulinum toxication.

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'.

Clostridium botulinum and bio-compost. A contribution to the analysis of potential health hazards caused by bio-waste recycling.

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 bio-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.

Active immunisation of black tiger prawn (Penaeus monodon) against vibriosis in Thailand.

Mechanisms of host defence in Crustaceae and vibriosis in shrimp and methods for its prevention are discussed as introduction. The own work deals with the development of a site-specific multivalent anti-vibrio vaccine and its application in Thailand. The vaccine was produced in a continuous bioreactor system from field strains of Vibrio alginolyticus, V. harveyi, V. parahaemolyticus and V. vulnificus, purified through ultrafiltration, and inactivated by formalin. It was applied to Penaeus monodon PL prior to release into the ponds via artemia larvae. As mean result from numerous trials carried out under commercial field conditions in Eastern Thailand it was shown that the PL tolerated the vaccine perfectly. Due to technical problems only few of the field trials could be monitored until harvest. The data available allow claiming an obvious protective effect of the vaccine.

Production and purification of Clostridium botulinum type C and D neurotoxin.

Neurotoxins of Clostridium botulinum are needed in basic neurologic research, but as therapeutic agent for certain neuromuscular disorders like strabism as well. A method for the production and purification of botulinum neurotoxins C and D is reported using a two-step hollow-fiber cross flow filtration and a newly developed chromatographic purification procedure. Hollow-fiber filtration proved to be a rapid and safe concentration and pre-purification step, which can easily be scaled up. The chromatographic purification included hydrophobic interaction, anion exchange and size exclusion chromatography runs. Botulinum neurotoxins C and D could be recovered with an overall yield of 12.6% and 10.6%, respectively. A specific toxicity of 1.86 x 10(7) minimal lethal dose mg(-1) (type C) and 5.26 x 10(7) minimal lethal dose mg(-1) (type D) was determined in the mouse bioassay.

[Botulism--a forgotten disease?]. / Botulismus--eine vergessene Erkrankung?

The laboratory diagnosis of C. botulinum is described for cattle and horses in the years 1995-1998. Out of 122 cases 66 were positive. All types of toxins were identified; in cattle mainly types C and D. In 9 cases typing was not conclusive. The results of an enquiry of afflicated animal owners showed, that modern agricultural technology has an important impact on toxinogenesis in feed stuff. Possibilities to prevent the disease and to reduce economic losses are discussed; a solution cannot be presented.

Equine intestinal clostridiosis in a group of polo ponies in Dubai, U.A.E.

An acute outbreak of a C. perfringens Type C enterotoxemia in a polo club killed 8 polo ponies in less than 24 hours. Hay with a massive fungal contamination (Aspergillus and Fusarium sp.) is suspected to have triggered the enterotoxemia. Our cases show numerous similarities with the previously reported cases of EIC. However, acute toxic tubulonephrosis in the necropsied horses and longstanding elevation of AST and gamma GT-levels in survivors is unique in our cases.

[The importance of tropical animal hygiene for indigenous animal production]. / Die Bedeutung der Tropentierhygiene für die heimische Tierproduktion.

Tropical animal hygiene firstly supports the improvement of animal production in tropical countries. Examples show the different disease complexes and the possible ways of transmission. The possibility of an importation of ¿tropical flues¿ means an important danger for our indigenous animal production. Therefore it is necessary to improve both the hygienic situation in the tropics as well as the knowledge on these diseases here, to protect our own animals.

[Preliminary trials of oral immunization of wild animals against anthrax]. / Vorversuche zur oralen Immunoisierung von Wildtieren gegen Milzbrand.

As a pilot trial for the vaccination of game in African game parks against anthrax, trials with guinea-pigs were undertaken to vaccinate the animals orally against anthrax. The vaccine has been prepared with the Göettingen Bioreactor Technology obtaining sporulation in suspension. Guinea-pigs vaccinated orally and subcutaneously with the vaccine resisted a challenge of 1000 spores with a pathogen field strain isolated from elephants in Zambia. With a challenge dosis of 2500 spores orally and subcutaneously immunized animals died. A technique has been developed to identify anthrax organisms excreted with the faeces by means of gas chromatography.

Real continuous biomass production ofBradyrhizobium japonicum in a veterinary vaccine production unit.

In a real continuous culture,Bradyrhizobium japonicum strain 1755 was maintained in the 'Göttingen bioreactor unit' developed to produce veterinary bacterial vaccines for a period of 110 days without contamination or mutations. The average yield of viable bacteria over the whole period was more than 10(10)/ml.

[Bacteriological studies of the problem "Epidemic losses in hares and deer in habitats with high cultivation of 00-rapeseed"]. / Bakteriologische Untersuchungen zur Frage "Seuchenhafte Verluste bei Hasen und Rehen an Standorten mit Hoher Anbaudichte von 00-Raps".

Within the scope of the research work for possible reasons of the so-called "wildlife mortality" more than 900 specimens of 440 animals (183 hares, 153 deer, 14 rabbits found dead, and 98 hares and 6 deer shot by hunters) were checked for the presence of Clostridia in inner organs. The results show that in 40.3% of the animals found dead but only in 13.3% of the hunted hares Clostridia were to be found. Out of the isolated Clostridia strains 24.4% could not be identified; potentially pathogenic species were found in 78.3% of isolates of hares, 81.7% of those of deer and in 9.2% of those of hunted hares. 23.9% of the isolates were C. perfringens. Considering the known epizootiology of clostridioses it can be concluded that clostridia certainly play a role in losses of wildlife, as well in hares as in deer. Triggering factors could lie in the kind of feed which is available for animals in modern agrarian landscape. During the critical period in autumn and winter there are mainly crop plants like rapeseed, wheat and barley only available for wildlife.

[The seasonal toxigenicity of different cultured plants for clostridia in relation to so-called wildlife mortality]. / Untersuchungen zur saisonalen Toxigenität verschiedener Kulturpflanzen für Clostridien im Zusammenhang mit dem sog. Wildsterben.

In the juice of plants which could be eaten by hares different amounts of toxins (haemolysin, lecithinase) could be found after the partly addition of a C. perfringens field strain and subsequent anaerobic incubation. Sterile filtrates showed a very pronounced toxigenicity. The presented results proof in tendency that oilseed-rape (00-rape seed), wheat, and barley as green plants can contribute in clostridial toxicosis in hares, whereas grass and beets are involved only partially, and clover is practically completely atoxigenic.