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Suspected cyclopiazonic acid mycotoxicosis of quail in Indonesia
First Asia-Pacific Congress
39
A LAROE number of enterotoxins produced by different diarrhoeagenic bacteria have been described. It is essential to know if there exists any interrelationship amongst these toxins. Similarities relating to antigenic structure, receptor site, mode of action and gene composition have been well established in the case of heat-labile toxins of V. cholerae 01 and E. coll. Enterotoxins produced by V. cholerae non 01 and V. mimicus have been found to be similar in subunit structure and immunobiological properties with favourable indications of at least partial genetic homology. V. fluvialis, Aeromonas and Piesiomonas enterotoxins were found to activate cAMP. A new enterotoxin produced by V. cholerae 01 strains with or without the CT gene has been found to be antigenically similar to that of the V. fluvialis toxin. Campylobacter isolates from man and the environment have been shown to elaborate a diarrhoeagenic toxin that is immunobiologically similar to CT. Salmonella strains also have been found to produce a heat-labile toxin that is similar in certain properties to that of V. cholerae 01. Some strains of E.coli produce a Shiga-like toxin. All these observations indicate the existence of similarities in either one or more character(s) amongst the different enterotoxins or various diarrhoeagenic bacteria.
The effects o f apamin and crotamine on the EEG o f rats: a computer-assisted study. M. K. SIM, l C. K. TAN2and P. Gopalakrishnakone2 (~Department of Pharmacology and 2Department of Anatomy, Faculty of Medicine, National University of Singapore, Singapore 0511). ONE WAYof examining the effects of a toxin on the central nervous system is to study the electroencephalogram (EEG) before and immediately after intracerebroventricular administration of the toxin. In the past such studies were handicapped by the laborious task of analysing the EEG pattern manually. The employment of a computer to analyse the EEG data has made EEG studies more precise, reliable and attractive. In this poster presentation we display the computer-assisted analysed data of the effects of apamin, a bee toxin, on the EEG of rats. The analysed EEG data are presented in various forms of periodogram, power spectrum and per cent time occupied by certain frequency ranges. Statistical analysis of the data is also incorporated. Our data confirmed the general findings of JANICKIet al. (1985) in that the toxin caused an immediate drop in the amplitude and a shift of the mean frequency of the EEG. However, unlike Janicki and co-workers who employed the manual method of EEG analysis, we were able to decipher the actual frequency bands that were affected by the toxin and calculate the statistical significance of the changes observed. Preliminary data obtained with another toxin, crotamine (from the South American rattlesnake) showed that the snake toxin at higher dose (200 gg) produced effects opposite to those induced by apamin. JANICKI, P. et al. (1985) The effects of apamin in rats with trigeminal or high spinal transsection of the central nervous system. Toxicon 23, 9 9 3 - 996.
Renal pathogenesis o f snake bite: a review. V. SITPRIJA (Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand). THE SPECTRUMof renal changes in snake bite includes glomerulonephritis, vasculitis, tubular necrosis, interstitial nephritis, infarct and cortical necrosis. Through clinical and experimental studies by various investigators 3 mechanisms are believed to be involved in the pathogenesis of these renal lesions. (1) Non-specific effects of venoms. Chemical mediators released during envenomation can lead to hypotension, hemorrhage, hypovolemia, intravascular coagulation, intravascular hemolysis and myoglobinuria, leading to renal ischemia and tubular necrosis. Tubulo-interstitial lesions, infarct and cortical necrosis are usually caused by these non-specific effects. (2) Immunologic reactions. Immune complex glomerulonephritis may occur in snake bite, irrespective of antivenin therapy. This has been shown in renal biopsy studies when performed later in the course of the disease. (3) Direct nephrotoxicity. Vasculitis and various forms of glomerular changes can be seen in some viper bites. The direct role of the venom on tubular changes is less clear. However, in animal experiments depolarization of proximal tubules has been shown following Russell's viper envenomation and the effect was concentration dependent.
Suspected cyclopiazonic acid mycotoxicosis o f quail in Indonesia. D. R. STOLTZ, R. WIDIASTUTI, R. MARYAM, B. TRI AKOSO, AMANG and D. UNRUH (Balai Penelitian Veteriner, Bogor, and Balai Penyidikan Penyakit Hewan, Yogyakarta, Indonesia). LIVESTOCK feeds in Indonesia, especially those containing corn, are commonly contaminated with afiatoxins. Since cyclopiazonic acid (CPA) may be produced by aflatoxigenic Aspergillus flavus, and by other fungal species, the presence of CPA in local feeds was anticipated. Twenty-six batches of corn from a Bogor poultry feedmill were analyzed for aflatoxins, CPA, ochratoxin A and zearalenone by TLC. CPA was detected in 21 of the 26 corn samples at concentrations as high as 9 ppm. CPA was always accompanied by other mycotoxins, especially aflatoxins. Although this corn was not associated with any disease in poultry, we had the opportunity
40
First A s i a - Pacific Congress
to investigate an outbreak of disease in quail around Yogyakarta which had many characteristics of a mycotoxicosis, including reduction of disease after change of feed source. Clinically the quail exhibited opisthotonus and legs stretched fully backwards. One sample of feed was submitted at the time of the outbreak for analysis and found to contain 450 ppb aflatoxin BI, 15 ppb aflatoxin B2, 6000 ppb CPA and 500 ppb ochratoxin A. Zearalenone, T2 toxin and deoxynivalenol were not detected. We believe a diagnosis of CPA toxicosis may be warranted based upon the characteristic clinical signs, supported by histopathology and chemical analysis. The toxicological significance of CPA in poultry feeds and the interactive toxicity with afiatoxins should be further investigated.
Tick toxicoses and the causal toxins: tick paralysis. B. F. STONE and J. H. AYLWARD(CSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag 3, Post Office, Indooroopilly, Queensland 4068, Australia). PARALYISING ticks representing about 40 species occur in about 15 countries. As part of a study into the feasibility of producing vaccines to protect against paralysing toxicoses, notably the syndrome caused by the Australian paralysis tick, Ixodes holocyclus, the nature of the causal paralysing toxin ('holocyclotoxin') is being investigated. Extracts of whole ticks or of salivary glands have been subjected to chromatographic, electrophoretic and isoelectrofocussing separation methods to attempt isolation of holocyclotoxin. Salivary gland toxin, detoxified by glutaraldehyde, formed the active antigen in an experimental vaccine known from earlier work to stimulate immunity. A proteinaceous paralysing toxin, presumably identical to the original salivary gland toxin used in the successful vaccination trials, has been isolated in size-homogeneous form. Sequence data has also been obtained which should enable the construction of oligonucleotide probes, a copy DNA library from messenger RNA and finally expression of the protein by recombinant DNA techniques. This will enable the assessment of the antigenicity of any candidate protein(s) in detoxified form as a possible basis for a vaccine. An agreement has been negotiated with a manufacturer to produce a vaccine. Prospects for the production of this and other tick paralysis vaccines are reviewed.
Human toxic and allergic reactions due to the Australian paralysis tick Ixodes holocyclus. B. F. STONE,l M. GAUCIl and Y. H. THONG2 (tCSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag 3, Post Office, Indooroopilly, Queensland 4068, Australia, and 2Immunobiology Laboratory, University Department of Child Health, Mater Children's Hospital, South Brisbane, Queensland 4101, Australia). OF APPROXIMATELY60 species of ticks in Australia, 13 species are known to attack humans. Five species may produce serious toxic or allergic effects and those due to the Australian paralysis tick, Ixodes holocyclus, are being studied most intensively. This tick may produce a lethal paralysis or potentially fatal anaphylaxis and as part of research into human reactions to this tick, hypersensitivity is being studied. Skin prick tests are being carried out with whole body extracts prior to similar tests with the active allergens to be separated by SDS acrylamide gel electrophoresis, transferred by Western blotting and identified by immunochemical methods. Radioimmunoassays (RIAs), radioallergosorbent assays (RASTs) and enzyme immunoassays (EIAs) are used to measure IgE levels in serum. IgE specifically released in response to tick allergens has been detected and quantitated most effectively in serum from reactive humans by means of an RIA. Some reactions, previously thought to be allergic, may well be due to vasoactive and chemotactic components of tick salivary secretions. The role of 'holocyclotoxin', the paralysing toxin from I. holocyclus and other bioactive salivary components appearing at the time of early tick attachment, when 'allergic-type' symptoms appear, is being investigated.
Effects o f equinatoxin on single myelinated nerve fibres. D. SUPUT(Institute of Pathophysiology, Faculty of Medicine, U.E.K., Zalo~ka 4, 61105 Ljubljana, Yugoslavia). THREE lethal and cytolytic proteins, named equinatoxins (EqT) I, II and III have been isolated and purified from the sea anemone Actinia equina L. It has been shown that EqT I and EqT II block sodium currents in skeletal muscle fibres of frog (Rana esculenta) in 10-100 pM concentrations, and that EqT III increases the leakage conductance so much that its effects on other membrane conductances cannot be measured reliably. As the mechanism of action of EqT on other excitable membranes is poorly understood, it seemed reasonable to investigate its action on single myelinated nerve fibres. Frog sensory fibres were dissected, mounted to the perspex cell and nodes of Ranvier were voltage clamped at 12°C. One to two minutes after application of either EqT I or EqT II in 10-100 nM concentration a complete block of potassium conductance developed, without noticable changes of the leakage conductance and sodium current amplitude. The block of potassium conductance was reversible, as the potassium current amplitude recovered completely in 2 - 5 min of washout of
39
A LAROE number of enterotoxins produced by different diarrhoeagenic bacteria have been described. It is essential to know if there exists any interrelationship amongst these toxins. Similarities relating to antigenic structure, receptor site, mode of action and gene composition have been well established in the case of heat-labile toxins of V. cholerae 01 and E. coll. Enterotoxins produced by V. cholerae non 01 and V. mimicus have been found to be similar in subunit structure and immunobiological properties with favourable indications of at least partial genetic homology. V. fluvialis, Aeromonas and Piesiomonas enterotoxins were found to activate cAMP. A new enterotoxin produced by V. cholerae 01 strains with or without the CT gene has been found to be antigenically similar to that of the V. fluvialis toxin. Campylobacter isolates from man and the environment have been shown to elaborate a diarrhoeagenic toxin that is immunobiologically similar to CT. Salmonella strains also have been found to produce a heat-labile toxin that is similar in certain properties to that of V. cholerae 01. Some strains of E.coli produce a Shiga-like toxin. All these observations indicate the existence of similarities in either one or more character(s) amongst the different enterotoxins or various diarrhoeagenic bacteria.
The effects o f apamin and crotamine on the EEG o f rats: a computer-assisted study. M. K. SIM, l C. K. TAN2and P. Gopalakrishnakone2 (~Department of Pharmacology and 2Department of Anatomy, Faculty of Medicine, National University of Singapore, Singapore 0511). ONE WAYof examining the effects of a toxin on the central nervous system is to study the electroencephalogram (EEG) before and immediately after intracerebroventricular administration of the toxin. In the past such studies were handicapped by the laborious task of analysing the EEG pattern manually. The employment of a computer to analyse the EEG data has made EEG studies more precise, reliable and attractive. In this poster presentation we display the computer-assisted analysed data of the effects of apamin, a bee toxin, on the EEG of rats. The analysed EEG data are presented in various forms of periodogram, power spectrum and per cent time occupied by certain frequency ranges. Statistical analysis of the data is also incorporated. Our data confirmed the general findings of JANICKIet al. (1985) in that the toxin caused an immediate drop in the amplitude and a shift of the mean frequency of the EEG. However, unlike Janicki and co-workers who employed the manual method of EEG analysis, we were able to decipher the actual frequency bands that were affected by the toxin and calculate the statistical significance of the changes observed. Preliminary data obtained with another toxin, crotamine (from the South American rattlesnake) showed that the snake toxin at higher dose (200 gg) produced effects opposite to those induced by apamin. JANICKI, P. et al. (1985) The effects of apamin in rats with trigeminal or high spinal transsection of the central nervous system. Toxicon 23, 9 9 3 - 996.
Renal pathogenesis o f snake bite: a review. V. SITPRIJA (Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand). THE SPECTRUMof renal changes in snake bite includes glomerulonephritis, vasculitis, tubular necrosis, interstitial nephritis, infarct and cortical necrosis. Through clinical and experimental studies by various investigators 3 mechanisms are believed to be involved in the pathogenesis of these renal lesions. (1) Non-specific effects of venoms. Chemical mediators released during envenomation can lead to hypotension, hemorrhage, hypovolemia, intravascular coagulation, intravascular hemolysis and myoglobinuria, leading to renal ischemia and tubular necrosis. Tubulo-interstitial lesions, infarct and cortical necrosis are usually caused by these non-specific effects. (2) Immunologic reactions. Immune complex glomerulonephritis may occur in snake bite, irrespective of antivenin therapy. This has been shown in renal biopsy studies when performed later in the course of the disease. (3) Direct nephrotoxicity. Vasculitis and various forms of glomerular changes can be seen in some viper bites. The direct role of the venom on tubular changes is less clear. However, in animal experiments depolarization of proximal tubules has been shown following Russell's viper envenomation and the effect was concentration dependent.
Suspected cyclopiazonic acid mycotoxicosis o f quail in Indonesia. D. R. STOLTZ, R. WIDIASTUTI, R. MARYAM, B. TRI AKOSO, AMANG and D. UNRUH (Balai Penelitian Veteriner, Bogor, and Balai Penyidikan Penyakit Hewan, Yogyakarta, Indonesia). LIVESTOCK feeds in Indonesia, especially those containing corn, are commonly contaminated with afiatoxins. Since cyclopiazonic acid (CPA) may be produced by aflatoxigenic Aspergillus flavus, and by other fungal species, the presence of CPA in local feeds was anticipated. Twenty-six batches of corn from a Bogor poultry feedmill were analyzed for aflatoxins, CPA, ochratoxin A and zearalenone by TLC. CPA was detected in 21 of the 26 corn samples at concentrations as high as 9 ppm. CPA was always accompanied by other mycotoxins, especially aflatoxins. Although this corn was not associated with any disease in poultry, we had the opportunity
40
First A s i a - Pacific Congress
to investigate an outbreak of disease in quail around Yogyakarta which had many characteristics of a mycotoxicosis, including reduction of disease after change of feed source. Clinically the quail exhibited opisthotonus and legs stretched fully backwards. One sample of feed was submitted at the time of the outbreak for analysis and found to contain 450 ppb aflatoxin BI, 15 ppb aflatoxin B2, 6000 ppb CPA and 500 ppb ochratoxin A. Zearalenone, T2 toxin and deoxynivalenol were not detected. We believe a diagnosis of CPA toxicosis may be warranted based upon the characteristic clinical signs, supported by histopathology and chemical analysis. The toxicological significance of CPA in poultry feeds and the interactive toxicity with afiatoxins should be further investigated.
Tick toxicoses and the causal toxins: tick paralysis. B. F. STONE and J. H. AYLWARD(CSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag 3, Post Office, Indooroopilly, Queensland 4068, Australia). PARALYISING ticks representing about 40 species occur in about 15 countries. As part of a study into the feasibility of producing vaccines to protect against paralysing toxicoses, notably the syndrome caused by the Australian paralysis tick, Ixodes holocyclus, the nature of the causal paralysing toxin ('holocyclotoxin') is being investigated. Extracts of whole ticks or of salivary glands have been subjected to chromatographic, electrophoretic and isoelectrofocussing separation methods to attempt isolation of holocyclotoxin. Salivary gland toxin, detoxified by glutaraldehyde, formed the active antigen in an experimental vaccine known from earlier work to stimulate immunity. A proteinaceous paralysing toxin, presumably identical to the original salivary gland toxin used in the successful vaccination trials, has been isolated in size-homogeneous form. Sequence data has also been obtained which should enable the construction of oligonucleotide probes, a copy DNA library from messenger RNA and finally expression of the protein by recombinant DNA techniques. This will enable the assessment of the antigenicity of any candidate protein(s) in detoxified form as a possible basis for a vaccine. An agreement has been negotiated with a manufacturer to produce a vaccine. Prospects for the production of this and other tick paralysis vaccines are reviewed.
Human toxic and allergic reactions due to the Australian paralysis tick Ixodes holocyclus. B. F. STONE,l M. GAUCIl and Y. H. THONG2 (tCSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag 3, Post Office, Indooroopilly, Queensland 4068, Australia, and 2Immunobiology Laboratory, University Department of Child Health, Mater Children's Hospital, South Brisbane, Queensland 4101, Australia). OF APPROXIMATELY60 species of ticks in Australia, 13 species are known to attack humans. Five species may produce serious toxic or allergic effects and those due to the Australian paralysis tick, Ixodes holocyclus, are being studied most intensively. This tick may produce a lethal paralysis or potentially fatal anaphylaxis and as part of research into human reactions to this tick, hypersensitivity is being studied. Skin prick tests are being carried out with whole body extracts prior to similar tests with the active allergens to be separated by SDS acrylamide gel electrophoresis, transferred by Western blotting and identified by immunochemical methods. Radioimmunoassays (RIAs), radioallergosorbent assays (RASTs) and enzyme immunoassays (EIAs) are used to measure IgE levels in serum. IgE specifically released in response to tick allergens has been detected and quantitated most effectively in serum from reactive humans by means of an RIA. Some reactions, previously thought to be allergic, may well be due to vasoactive and chemotactic components of tick salivary secretions. The role of 'holocyclotoxin', the paralysing toxin from I. holocyclus and other bioactive salivary components appearing at the time of early tick attachment, when 'allergic-type' symptoms appear, is being investigated.
Effects o f equinatoxin on single myelinated nerve fibres. D. SUPUT(Institute of Pathophysiology, Faculty of Medicine, U.E.K., Zalo~ka 4, 61105 Ljubljana, Yugoslavia). THREE lethal and cytolytic proteins, named equinatoxins (EqT) I, II and III have been isolated and purified from the sea anemone Actinia equina L. It has been shown that EqT I and EqT II block sodium currents in skeletal muscle fibres of frog (Rana esculenta) in 10-100 pM concentrations, and that EqT III increases the leakage conductance so much that its effects on other membrane conductances cannot be measured reliably. As the mechanism of action of EqT on other excitable membranes is poorly understood, it seemed reasonable to investigate its action on single myelinated nerve fibres. Frog sensory fibres were dissected, mounted to the perspex cell and nodes of Ranvier were voltage clamped at 12°C. One to two minutes after application of either EqT I or EqT II in 10-100 nM concentration a complete block of potassium conductance developed, without noticable changes of the leakage conductance and sodium current amplitude. The block of potassium conductance was reversible, as the potassium current amplitude recovered completely in 2 - 5 min of washout of