CA2212718A1 - Diagnosis of toxicoinfectious clostridiosis - Google Patents
Diagnosis of toxicoinfectious clostridiosisInfo
- Publication number
- CA2212718A1 CA2212718A1 CA 2212718 CA2212718A CA2212718A1 CA 2212718 A1 CA2212718 A1 CA 2212718A1 CA 2212718 CA2212718 CA 2212718 CA 2212718 A CA2212718 A CA 2212718A CA 2212718 A1 CA2212718 A1 CA 2212718A1
- Authority
- CA
- Canada
- Prior art keywords
- botulinum
- antibody
- horses
- diagnosis
- egs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
A method and a kit for the diagnosis of equine toxicoinfectious clostridiosis which comprises detecting the presence of an antibody to a C. botulinum antigen or a phenotype thereof and/or an antibody to a clostridial toxin in a biological sample. The method is preferably used for the detection of equine grass sickness.
Description
DIAGNOSIS OF TOXICOINFECTIOUS CLOSTRIDIOSIS
The invention relates to the diagnosis of equine toxicoinfectious clostridiosis, particularly to the diagnosis of grass sickness (also known as equine dysautonomia).
Equine Grass Sickness (EGS), also known as Mal Seco in Argentina, is an unpredictable disease which characteristically affects younger individual horses in successive generations grazing contaminated pastures and over a time-scale measured in decades. EGS is one manifestation of toxicoinfectious clostridiosis. Clinical EGS cases may be seen as manifestations of a complex of equine gastro-intestinal and/or neurologic disorders.
The present inventor has found that horses grazing in endemic EGS areas are invariably seroconverted to a Clostridium no wi phenotype. The evidence of sporadic disease in such populations demonstrates the limitations of natural immunoprotection, which is also dependent upon effective operation of the immune response at the individual horse level. Clostridial toxicoinfectious disease may present a variety o~ non-specific gastro-intestinal and/or neurologic signs which are not pathognomic, especially in older animals, thus exacerbating the problem of diagnosis of EGS.
Previously, diagnosis of EGS could only be confirmed by ileal biopsy (laparotomy) or at autopsy.
In 192~ Tocher and his colleagues presented evidence PCT/CB9''~C323 from which they concluded that equine Grass Sickness (EGS) is a form of botulism. The contention was immediately discredited on the basis of inadequate microbiological corroboration and for its failure to explain notable clinical features of EGS which are domina~ed by dysautonomia (i.e. disturbance of the autonomic nervous system) anc which are patently different from those of classical equine botulism or forage poisoning. The botulinum toxin theory as it related to EGS was further undermined by apparent failure of a prototype vaccine (which was prepared by combining Clostridium botulinum Type A toxin with homologous antitoxin, in vitro) to control disease in susceptible populations of horses.
In the intervening years research interest has focused on the neuronal lesions of EGS which were believed to be the essential ~eature o~ the disease and to be evidence o~ a neurotoxic influence but apparently irreconcilable with botulinum toxin involvement.
Furthermore, in a recent review Pollin and Grif~iths concluded that there were no literature re~erences to infectious agents or toxins capable or likely to be capable of causing the type and distribution o~ neuronopathy which con~irms the autopsy (or biopsy) diagnosis of EGS
Studies which led to the present invention were based on two ~undamental assumptions. Firstly that EGS is a toxicoinfectious disease process which depends upon prior colonization of the bowel (or an intercurrent disease W 096/25669 PCT/GB95/~323 lesion) with toxin-producing clostridia and secondly, that the strain of Clostridium botulinum responsible for EGS
invariably produces two neuroactive toxins. Thus the ~ concerted toxic insult could account for the dysautonomia and for the classical gastro-enteric and neuroloyic manifestations of EGS so providing a satisfactory explanation for the most controversial aspect of the disputed botulinum theory.
It is known that the majority of Group III strains of Clostridium botulinum (Types C and D) produce a variable yield of secondary toxins in association with their type specific neurotoxins (C1 and D1). Clostridium botulinum Type C2 binary toxin was recognized to be of particular relevance in the context of EGS in view of its well documented role as a potent ADP ribosyltransferase: thus C2 toxin has the capacity to activate a broad spectrum o~ cell membrane receptors which have been characterised by in vitro studies and in laboratory ~n; m~ 1 models. C.
botulinum types C and D are the only serotypes which have the capacity to elaborate the combination of neuroactive and enterotoxic components necessary to account for the dramatic clinical signs of EGS.
A number of bacterial protein toxins interfere with the processes of cellular communication, or signal transduction, by acting as ~reelance ADP
ribosyltransferases. Additional ribose groups are added to the ADP molecule to ~orm a polyribose complex which cannot W 096/25669 P~l/~b,"00323 be phosphorylated to ATP.
Individual ADP ribosyltrans~erases have specific substrates whlch are activated to disrupt membrane function operating systems and their coupled intercellular processes: in the case o~ botulinum C2 toxin the substrate is monomeric G-actin, a guanylate protein, which forms the cytoskeletal structure of all secretory and motile cells derived from the embryonic neural crest.
The precisely targeted consequences o~ botulinum C2 toxin-induced ADP ribosylation are considered to be responsible for the neuronal lesions associated with EGS, and for their pattern of peripheral distribution from an enteric toxicoinfectious ~ocus by the process of retrograde axonal transmission. Studies carried out in vitro have demonstrated that neuronal exposure to C2 binary toxin leads to uncontrolled neurochemical discharge and similar exposure inhibits the activity of motile cells. These events are accompanied by ultrastructural changes, defined as chromatolysis, which indicate extreme physiological stress: more prolonged or more concentrated exposure to C2 toxin leads to cell lysis. These are the neuronal lesions associated with equine dysautonomia. However, prior to the present implication of C. botulinum Type Cl and C2 toxins in the neurotoxicology of EGS, there had been no previous recorded indictment of the two toxins in any human or animal disease process.
Type C organisms present in carrion are responsible W O 96/25669 PCT/GB9~ 323 for most epizootic botulism but such outbreaks (associated with the ingestion of preformed toxin) which may involve horses, or other species, are not associated with any - clinical evidence of C2 toxin involvement.
Type Cl neurotoxin inhibits the release of acetyl choline from its storage vesicles by a specific zinc dependent cleavage of syntaxin, a neuronal endopeptide.
The present inventor has demonstrated a relationship between a primitive organism and a highly specific G-protein mediated signal transduction system, the phosphotidylinositol cascade, which mediates all calcium-sensitive processes such as secretion and chemotaxis.
Toxicoinfectious manifestations of botulism are frequently encountered on lower animals: significantly, in the context of EGS, all avian infections (which predominantly involve Type C organisms) are toxicoinfectious reflecting the evolutionary significance of this intimate host-parasite relationship. However, it was not until 1976 that a human infant disease was associated with the spontaneous elaboration o~ botulinum toxins in vivo: so called infant botulism is a well recognised cause of morbldity and mortality in the U.S.
where it results from ingestion of botulinum spores (Types A and B) predominantly by susceptible in~ants in the 3-6 month age range.
Classical toxicoinfectious botulism of horses (the Shaker Foal Syndrome) which is prevalent in Kentucky, W 096/25669 PCT/~b9GJ'~~323 elsewhere in the eastern US and which occurs in Australia is associated with C. botulinum Type B. However, this condition is more akin to human wound botulism since it can only be reproduced in the presence of pre-existing anaerobic lesions which may be clinically insignificant but which nevertheless provide a focus for localization of the toxigenic agent.
As the name suggests Grass Sickness is almost invariably associated with access to grazing and the seasonal pattern of disease is generally assumed to result from exposure to a transient toxic factor in herbage.
However, the present inventor has postulated that contagion results from clostridial spores in soil ingested during grazing: such spores would be expected to vegetate in the bowel to initiate a speci~ic immune response which will signify the carrier state.
Horses in enzootic EGS areas develop antibodies to a phenotype of Clostridium botulinum (Group III), demonstrating the toxicoinfectious nature of the disease process. A meaningful pattern of serological (i.e.
antibody related) responses indicates the degree of previous exposure and provides a measure of susceptibility of disease.
However, the serodiagnostic approach is complicated by the fact that all horses harbour vast enteric populations of non-pathogenic clostridia. Therefore testing methods leading to the present invention took W 096/2~669 PCT/GB96/00323 account of between-group and within-group comparisons of clostridial seroconversion in clinically normal horses and those in specific disease categories as demonstrated in the Examples set out below.
The invention provides a method for the diagnosis of equine toxicoinfectious clostridiosis which comprises detecting the presence of an antibody to a C. botulinum antigen or a phenotype thereof or an antibody to a clostridial toxin in a biological sample. The method of the invention is useful for the diagnosis of manifestations of equine toxicoinfectious clostridiosis such as anterior enteritis or colitis X which are associated with toxinogenic strains of C. ~erfrinqens.
More preferably, the invention relates to a method for diagnosis of equine grass sickness which comprises detecting the presence of an antibody to C. botulinum Group III antigen or a phenotype thereof, and also detecting the presence of an antibody to a botulinum type C toxin.
By phenotype is meant herein a clostridial-derived antigen provoking a similar immunological reaction to types of C. botulinum. In the case of C. botulinum Group III
(types C and D), ~he phenotype is preferably derived from Clostridium novvi Type A. The metnod of the invention is preferably an ELISA method.
2s The invention ~urther provides a kit for diagnosis of equine toxicoin~ectious clostridiosis, which comprises means for detecting an antibody to a C. botulinum antigen W 096/25669 PCT/~9G/~323 or a phenotype thereof and/or means for detecting an antibody to a clostridial toxin. The kit may be adapted for field use.
Preferably, the inventicn relates to a kit for diagnosis of equine grass sickness, which comprises means for detecting the presence of an antibody to C. botulinum Group III antigen or a phenotype thereof and means for detecting an antibody to a botulinum type C toxin.
The test method according to the invention permits identification of apparently normal horses which have developed antibody to cell wall antigens of the indicator organism Clostridium novYi; individual clinical cases of suspected EGS can be identified as such by the detection of antibodies (antitoxins) to the EGS-specific neurotoxin.
Hitherto, confirmation of EGS diagnosis in vivo required stressful and costly laparotomy and ileal biopsy in equine hospitalisation facilities.
The demonstration of a rising serological (i.e.
antibody) response to C. novyi cell surface antigens in parallel with a decreasing response to botulinum Cl toxin in EGS affected horses typifies the toxicoinfectious nature of the disease. The bowel has been identified as the site of toxin elaboration at least in classical manifestations of EGS.
Monitoring of the results of the test of the invention has confirmed that many "carrier" horses are exposed to the toxicoinfectious agent of EGS and that W 096/25669 PCT/~'0~323 evidence of such exposure, at the individual horse level, can be determined by titration of the antibody response to C. novyi, the indicator phenotype for C. botulinum Types C/D. The fact that very few carrier horses succomb to classical or atypical EGS attests to the general effectiveness of immunosuppressive mechanisms and/or detoxification processes. In suspect clinical cases evidence of a serological response (i.e. antitoxin titre) to a primary EGS toxin, now identified as botulinum Type C
neurotoxin, denotes failure of the immune or protective response. These tests can be used separately to identify infected animals, or preferably, will be used in combination to provide a reliable method for the diagnosis of EGS.
Cell surface preparations of Clostridium sporoqenes, deposited at the National Collection of Type Cultures at Colindale as NCTC 8594, Clostridium ~erfrinaens deposited as NCTC 8237 and Clostridium novyi deposited as NCTC 538 provide antigens which are phenotypically representative of Clostridium botulinum Group I (types A, proteolytic B and F), Group II (types non-proteolytic B and E) and Group III
~types C and D) t-espectively. The inventor has Eound that Group III of Clostridium botulinum, and the phenotypically representative antigen derived from C. novyi are mainly responsible for toxicoinfectious clostridiosis.
Biological samples, preferably serum samples are tested according to the method of the invention by an adaptation o~ a standard ELISA technique (enzyme-linked immunosorbant assay). Suitably, a non-competitive ELISA
method is used according to the invention.
Enzyme-linked immunosorbent assay (ELISA) The diagnostic value o~ an ELlSA technique is dependent upon purity of antigens and especially so ~or the characterization of anaerobic in~ections where the antigenic structures are uncertain and variable.
Hence in the present application screening versatility o~ the test procedure may be provided by strategic use of somatic antigens derived ~rom appropriate clostridial phenotypes viz. Clostridium novyi, C.
s~oroqenes and C. per~rinaens which combine to indicate responses to a broad spectrum o~ toxinogenic clostridia including all known strains o~ Clostridium botulinum.
Therea~ter speci~ic "diagnostic" analysis o~ the serological response is ensured by the use o~ puri~ied toxin antigens precisely to categorize individual antitoxin components o~ the host reaction to toxicoin~ectious discharge.
An ELISA protocol, suitable ~or use in the diagnostic method of ~he invention is as ~ollows:
1. Dilutions o~ antigen are made in coating bu~er (0.05M sodium carbonate bu~er pH 9.6 containing 0.02 2S sodium azide) and 100 ~l volumes, containing 10-100 ~g antigen are added to wells o~ microtitre plates. Plates are covered and incubated at 37~C ~or 4 h and then at 4~C
overnight.
The invention relates to the diagnosis of equine toxicoinfectious clostridiosis, particularly to the diagnosis of grass sickness (also known as equine dysautonomia).
Equine Grass Sickness (EGS), also known as Mal Seco in Argentina, is an unpredictable disease which characteristically affects younger individual horses in successive generations grazing contaminated pastures and over a time-scale measured in decades. EGS is one manifestation of toxicoinfectious clostridiosis. Clinical EGS cases may be seen as manifestations of a complex of equine gastro-intestinal and/or neurologic disorders.
The present inventor has found that horses grazing in endemic EGS areas are invariably seroconverted to a Clostridium no wi phenotype. The evidence of sporadic disease in such populations demonstrates the limitations of natural immunoprotection, which is also dependent upon effective operation of the immune response at the individual horse level. Clostridial toxicoinfectious disease may present a variety o~ non-specific gastro-intestinal and/or neurologic signs which are not pathognomic, especially in older animals, thus exacerbating the problem of diagnosis of EGS.
Previously, diagnosis of EGS could only be confirmed by ileal biopsy (laparotomy) or at autopsy.
In 192~ Tocher and his colleagues presented evidence PCT/CB9''~C323 from which they concluded that equine Grass Sickness (EGS) is a form of botulism. The contention was immediately discredited on the basis of inadequate microbiological corroboration and for its failure to explain notable clinical features of EGS which are domina~ed by dysautonomia (i.e. disturbance of the autonomic nervous system) anc which are patently different from those of classical equine botulism or forage poisoning. The botulinum toxin theory as it related to EGS was further undermined by apparent failure of a prototype vaccine (which was prepared by combining Clostridium botulinum Type A toxin with homologous antitoxin, in vitro) to control disease in susceptible populations of horses.
In the intervening years research interest has focused on the neuronal lesions of EGS which were believed to be the essential ~eature o~ the disease and to be evidence o~ a neurotoxic influence but apparently irreconcilable with botulinum toxin involvement.
Furthermore, in a recent review Pollin and Grif~iths concluded that there were no literature re~erences to infectious agents or toxins capable or likely to be capable of causing the type and distribution o~ neuronopathy which con~irms the autopsy (or biopsy) diagnosis of EGS
Studies which led to the present invention were based on two ~undamental assumptions. Firstly that EGS is a toxicoinfectious disease process which depends upon prior colonization of the bowel (or an intercurrent disease W 096/25669 PCT/GB95/~323 lesion) with toxin-producing clostridia and secondly, that the strain of Clostridium botulinum responsible for EGS
invariably produces two neuroactive toxins. Thus the ~ concerted toxic insult could account for the dysautonomia and for the classical gastro-enteric and neuroloyic manifestations of EGS so providing a satisfactory explanation for the most controversial aspect of the disputed botulinum theory.
It is known that the majority of Group III strains of Clostridium botulinum (Types C and D) produce a variable yield of secondary toxins in association with their type specific neurotoxins (C1 and D1). Clostridium botulinum Type C2 binary toxin was recognized to be of particular relevance in the context of EGS in view of its well documented role as a potent ADP ribosyltransferase: thus C2 toxin has the capacity to activate a broad spectrum o~ cell membrane receptors which have been characterised by in vitro studies and in laboratory ~n; m~ 1 models. C.
botulinum types C and D are the only serotypes which have the capacity to elaborate the combination of neuroactive and enterotoxic components necessary to account for the dramatic clinical signs of EGS.
A number of bacterial protein toxins interfere with the processes of cellular communication, or signal transduction, by acting as ~reelance ADP
ribosyltransferases. Additional ribose groups are added to the ADP molecule to ~orm a polyribose complex which cannot W 096/25669 P~l/~b,"00323 be phosphorylated to ATP.
Individual ADP ribosyltrans~erases have specific substrates whlch are activated to disrupt membrane function operating systems and their coupled intercellular processes: in the case o~ botulinum C2 toxin the substrate is monomeric G-actin, a guanylate protein, which forms the cytoskeletal structure of all secretory and motile cells derived from the embryonic neural crest.
The precisely targeted consequences o~ botulinum C2 toxin-induced ADP ribosylation are considered to be responsible for the neuronal lesions associated with EGS, and for their pattern of peripheral distribution from an enteric toxicoinfectious ~ocus by the process of retrograde axonal transmission. Studies carried out in vitro have demonstrated that neuronal exposure to C2 binary toxin leads to uncontrolled neurochemical discharge and similar exposure inhibits the activity of motile cells. These events are accompanied by ultrastructural changes, defined as chromatolysis, which indicate extreme physiological stress: more prolonged or more concentrated exposure to C2 toxin leads to cell lysis. These are the neuronal lesions associated with equine dysautonomia. However, prior to the present implication of C. botulinum Type Cl and C2 toxins in the neurotoxicology of EGS, there had been no previous recorded indictment of the two toxins in any human or animal disease process.
Type C organisms present in carrion are responsible W O 96/25669 PCT/GB9~ 323 for most epizootic botulism but such outbreaks (associated with the ingestion of preformed toxin) which may involve horses, or other species, are not associated with any - clinical evidence of C2 toxin involvement.
Type Cl neurotoxin inhibits the release of acetyl choline from its storage vesicles by a specific zinc dependent cleavage of syntaxin, a neuronal endopeptide.
The present inventor has demonstrated a relationship between a primitive organism and a highly specific G-protein mediated signal transduction system, the phosphotidylinositol cascade, which mediates all calcium-sensitive processes such as secretion and chemotaxis.
Toxicoinfectious manifestations of botulism are frequently encountered on lower animals: significantly, in the context of EGS, all avian infections (which predominantly involve Type C organisms) are toxicoinfectious reflecting the evolutionary significance of this intimate host-parasite relationship. However, it was not until 1976 that a human infant disease was associated with the spontaneous elaboration o~ botulinum toxins in vivo: so called infant botulism is a well recognised cause of morbldity and mortality in the U.S.
where it results from ingestion of botulinum spores (Types A and B) predominantly by susceptible in~ants in the 3-6 month age range.
Classical toxicoinfectious botulism of horses (the Shaker Foal Syndrome) which is prevalent in Kentucky, W 096/25669 PCT/~b9GJ'~~323 elsewhere in the eastern US and which occurs in Australia is associated with C. botulinum Type B. However, this condition is more akin to human wound botulism since it can only be reproduced in the presence of pre-existing anaerobic lesions which may be clinically insignificant but which nevertheless provide a focus for localization of the toxigenic agent.
As the name suggests Grass Sickness is almost invariably associated with access to grazing and the seasonal pattern of disease is generally assumed to result from exposure to a transient toxic factor in herbage.
However, the present inventor has postulated that contagion results from clostridial spores in soil ingested during grazing: such spores would be expected to vegetate in the bowel to initiate a speci~ic immune response which will signify the carrier state.
Horses in enzootic EGS areas develop antibodies to a phenotype of Clostridium botulinum (Group III), demonstrating the toxicoinfectious nature of the disease process. A meaningful pattern of serological (i.e.
antibody related) responses indicates the degree of previous exposure and provides a measure of susceptibility of disease.
However, the serodiagnostic approach is complicated by the fact that all horses harbour vast enteric populations of non-pathogenic clostridia. Therefore testing methods leading to the present invention took W 096/2~669 PCT/GB96/00323 account of between-group and within-group comparisons of clostridial seroconversion in clinically normal horses and those in specific disease categories as demonstrated in the Examples set out below.
The invention provides a method for the diagnosis of equine toxicoinfectious clostridiosis which comprises detecting the presence of an antibody to a C. botulinum antigen or a phenotype thereof or an antibody to a clostridial toxin in a biological sample. The method of the invention is useful for the diagnosis of manifestations of equine toxicoinfectious clostridiosis such as anterior enteritis or colitis X which are associated with toxinogenic strains of C. ~erfrinqens.
More preferably, the invention relates to a method for diagnosis of equine grass sickness which comprises detecting the presence of an antibody to C. botulinum Group III antigen or a phenotype thereof, and also detecting the presence of an antibody to a botulinum type C toxin.
By phenotype is meant herein a clostridial-derived antigen provoking a similar immunological reaction to types of C. botulinum. In the case of C. botulinum Group III
(types C and D), ~he phenotype is preferably derived from Clostridium novvi Type A. The metnod of the invention is preferably an ELISA method.
2s The invention ~urther provides a kit for diagnosis of equine toxicoin~ectious clostridiosis, which comprises means for detecting an antibody to a C. botulinum antigen W 096/25669 PCT/~9G/~323 or a phenotype thereof and/or means for detecting an antibody to a clostridial toxin. The kit may be adapted for field use.
Preferably, the inventicn relates to a kit for diagnosis of equine grass sickness, which comprises means for detecting the presence of an antibody to C. botulinum Group III antigen or a phenotype thereof and means for detecting an antibody to a botulinum type C toxin.
The test method according to the invention permits identification of apparently normal horses which have developed antibody to cell wall antigens of the indicator organism Clostridium novYi; individual clinical cases of suspected EGS can be identified as such by the detection of antibodies (antitoxins) to the EGS-specific neurotoxin.
Hitherto, confirmation of EGS diagnosis in vivo required stressful and costly laparotomy and ileal biopsy in equine hospitalisation facilities.
The demonstration of a rising serological (i.e.
antibody) response to C. novyi cell surface antigens in parallel with a decreasing response to botulinum Cl toxin in EGS affected horses typifies the toxicoinfectious nature of the disease. The bowel has been identified as the site of toxin elaboration at least in classical manifestations of EGS.
Monitoring of the results of the test of the invention has confirmed that many "carrier" horses are exposed to the toxicoinfectious agent of EGS and that W 096/25669 PCT/~'0~323 evidence of such exposure, at the individual horse level, can be determined by titration of the antibody response to C. novyi, the indicator phenotype for C. botulinum Types C/D. The fact that very few carrier horses succomb to classical or atypical EGS attests to the general effectiveness of immunosuppressive mechanisms and/or detoxification processes. In suspect clinical cases evidence of a serological response (i.e. antitoxin titre) to a primary EGS toxin, now identified as botulinum Type C
neurotoxin, denotes failure of the immune or protective response. These tests can be used separately to identify infected animals, or preferably, will be used in combination to provide a reliable method for the diagnosis of EGS.
Cell surface preparations of Clostridium sporoqenes, deposited at the National Collection of Type Cultures at Colindale as NCTC 8594, Clostridium ~erfrinaens deposited as NCTC 8237 and Clostridium novyi deposited as NCTC 538 provide antigens which are phenotypically representative of Clostridium botulinum Group I (types A, proteolytic B and F), Group II (types non-proteolytic B and E) and Group III
~types C and D) t-espectively. The inventor has Eound that Group III of Clostridium botulinum, and the phenotypically representative antigen derived from C. novyi are mainly responsible for toxicoinfectious clostridiosis.
Biological samples, preferably serum samples are tested according to the method of the invention by an adaptation o~ a standard ELISA technique (enzyme-linked immunosorbant assay). Suitably, a non-competitive ELISA
method is used according to the invention.
Enzyme-linked immunosorbent assay (ELISA) The diagnostic value o~ an ELlSA technique is dependent upon purity of antigens and especially so ~or the characterization of anaerobic in~ections where the antigenic structures are uncertain and variable.
Hence in the present application screening versatility o~ the test procedure may be provided by strategic use of somatic antigens derived ~rom appropriate clostridial phenotypes viz. Clostridium novyi, C.
s~oroqenes and C. per~rinaens which combine to indicate responses to a broad spectrum o~ toxinogenic clostridia including all known strains o~ Clostridium botulinum.
Therea~ter speci~ic "diagnostic" analysis o~ the serological response is ensured by the use o~ puri~ied toxin antigens precisely to categorize individual antitoxin components o~ the host reaction to toxicoin~ectious discharge.
An ELISA protocol, suitable ~or use in the diagnostic method of ~he invention is as ~ollows:
1. Dilutions o~ antigen are made in coating bu~er (0.05M sodium carbonate bu~er pH 9.6 containing 0.02 2S sodium azide) and 100 ~l volumes, containing 10-100 ~g antigen are added to wells o~ microtitre plates. Plates are covered and incubated at 37~C ~or 4 h and then at 4~C
overnight.
2. The plates are washed three or four times with 0.9 Na Cl containing 0.05~ Tween 20.
- 3. Dilutions of antibody in antibody conjugate buffer are made (0.05M phosphate bu~fer, pH 7.4, containing 0.85~
Na Cl, 0.05~ Tween 20 and 0.02~ sodium azide) and added to wells. The wells are incubated for up to 4 h at room temperature.
Na Cl, 0.05~ Tween 20 and 0.02~ sodium azide) and added to wells. The wells are incubated for up to 4 h at room temperature.
4. Washing Step 2 is repeated.
5. 100~1 volumes of suitably diluted anti first species antibody - enzyme conjugate (doubling dilutions from l in 500) are added to the wells which are incubated overnight at room temperature (enzyme conjugate used horseradish peroxidase).
6. Washing Step 2 is repeated.
7. Diluted enzyme substrate is added and the wells are incubated at room temperature for 1 h. Results are read spectrophotometrically.
The sample containing the Group III antigen or analogue thereof may be a biological ~luid or tissue sample, and is pre~erably serum.
The diagnostic method according to the invention provides a rapid, non-invasive and conclusive method ~or confirming the diagnosis of equine dysautonomia, o~herwise known as grass sickness. The method allows for the differential diagnosis of clostridial involvement in non-speci~ic gastro intestinal and/or neurologic equine W 096t25669 PCT/GB96100323 disorders.
As a ~urther feature, the method of the invention may be used as a facility for monitoring responses to treatment o~ clinical toxicoinfectious clostridiosis, including Grass Sickness, in individual horses. Host responses to individual clostridial toxins involved in the toxinology o~
equine toxicoinfectious clostridiosis may also be identified and quantified by performing the method o~ the invention and interpreting the results obtained.
The diagnostic method of the invention may also be used as the basis of a technique for the parallel assessment of bacterial virulence ~actors or toxins which combine to determine the pathogenesis of field strains o~
toxinogenic clostridia.
Identification and quantification of host responses to individual clostridial toxins together with the parallel assessment of bacterial virulence ~actors or toxins responsible for the pathogenesis of specific toxinogenic clostridia are prerequisites ~or the epidemiological study of equine toxicoinfectious disease.
The invention is further illustrated by the ~ollowing hxamples. In the Examples, four categories of horses showlng clinical signs were identified. The groups are as follows:
Group I - Acute Grass Sickness (AGS) Group II - Chronic Grass Sickness (CGS) Group III - Clinical gastroenteric (GE) including acute W 096/25669 PCT/GB~Gi'~-~23 abdominal crises.
Group IV - Clinical miscellaneous including specimens submitted ~rom a broad spectrum o~ diagnosed cases or with - key presenting signs Example 1 - Determination o~ indicator antiaens A group o~ 34 clinical specimens was tested by the (IgG) ELISA technique at two serum dilutions i.e. 1/25 and 1/50. Each specimen was tested in duplicate and ELISA OD
values were presented as a mean o~ the two results.
Table 1 shows the mean OD values (with ranges and variance) ~or the four clinical groups, against the three indicator antigens.
I II III IV
AGS CGS GE nonGE
Antigen n 9 23 12 25 C novvimean 0 302 0 634 0.677 0 399 range0.001-0.6350.195-1.100 0 064-1 238 0.087-0 925 variance 0.029 0 066 0 121 0 057 C ~er~rinqens n 8 8 6 12 mean 0 480 0_622 0 502 0.596 range-0 043-0 9S4 0 173-0.866 0 184-1 007 0 337-0.966 variance 0 088 0 057 0.088 0.048 C s~oroqenes ~ ~ 3 ~ 12 ~ean 0 7710.996 Q 696 0 819 range 0 38S-1 165 0 479-1.634 0 251-1 067 0 511-1.499 variance 0.066 0.145 O.lOS 0 064 The representative data presented in Table 1 indicate that the overall range o~ ELISA OD values was broadly - 14 -=
similar for each of the three antigens. However, significant between-group variation was only evident for the C.novyi antigen.
The summarised clinical data provided in Table 1 support the assumptions that all Group I (AGS) and the majority of Group IV (miscellaneous clinical) horses have low levels of antibody to C. novyi and are therefore susceptible to EGS. Conversely horses in Group II (CGS) are expected to be variably seroconverted according to stage of 'incubation' at the time of sampling; inconclusive or positive results from the individual horses in the non-specific gastro-intestinal (Group III) ~nim~l S may indicate involvement of a toxicoinfectious clostridial component in the disease process. Group I (AGS) and Group IV
(miscellaneous clinical, non GE) may therefore be amalgamated to form a combined 'seronegative/inconclusive' category; similarly Group II (CGS) and III (clinical, GE) were statistically indistinguishable and may be combined to ~orm an 'inconclusive/positive' category.
Comparison o~ these two combined groups I + IV and II
III provides a basis for meaningful analyses of variance.
l~ABLE 2 Analyses of variance o~ ELISA OD values for the three antigens based on amalgamated clinical grouping CA 022l27l8 l997-08-08 W 096125669 PCT/~h,''~~323 Seronegative/ InConclusive/ VR Sig.
inconclusive Seroposi~ive 1:34 Groups I + IV Groups II ~ III
n 20 15 C. nov~ri 1:50 Q.358 0.554 8.03 p<0.01 1:25 0.514 0.616 2.03 NS
C . Derf rinqens 1:50 0.550 0.553 0.00 NS
1:25 0.492 0.535 0.21 NS
C sPoroqeneS
1:50 0.800 0.850 0.23 NS
1:25 0.852 0.816 0.09 NS
The individuality of the serological response to the three indicator antigens was further substantiated by the absence of significant correlation between the data sets.
TABLE 2A Correlation matrix ~or ELISA OD at 1:50 serum dilution C.novyi 1.000 C.Per~rinqes 0.476 * 1.000 20 C.s~oroqenes 0.429* 0.530** 1.000 C.novyi C.per~rinqens C.s~oroqenes EXAMPLE 3 ELISA of Clinical qrouDs (a) Group I Acute Grass Sickness (AGS) Nine specimens were submitted ~rom con~irmed cases of AGS and with ~he exception of one specimen all were seronegative to the C.novyi antigen (i.e. OD <0.4). The single inconclusive result (OD 0.635) was recorded ~or a 12 year old horse in Group I which was also characterised by above group average seroconversion to the two non-specific antigens (Table 3).
ELISA OD Values Horse 5 (D20.91) 0.635 0.539 1.165 Group I min 0.001 0.043 0.385 mean 0.302 0.480 0.771 max 0.635 0.954 1.165 variance 0.029 0.088 0.066 (b) Group II Chronic Grass Sickness (CGS) By definition horses susceptible to CGS are likely to be in the seronegative range (i.e. OD<0.4) at the outset o~
disease; clinical progression will be accompanied by a variable rate o~ seroconversion modi~ied according to individual animal responses to the toxicoin~ectious challenge. The majority of specimens included in Group II
(con~irmed CGS) were sampled at a relatively early stage in the disease process Mean duration at sampling 19 days range 11-35 days n = 23 (c) Group III Clinical GE
Table 4 provides a summary o~ the diagnoses or clinical signs recorded ~or the 12 horses included in this category Clinical GE - summary o~ diagnoses (or clinical signs) expressed in descenaing order oi~ ELISA OD ~or C novvi antigen at 1:50 serum dilution for Group III horses No. N50 OD RefDuration Diagnosis Seropositive days (signs) i.e. OD > 0.8 1 1.238 F456 LS ND
2 1.076 E50 ~S ND
3 1.052 F462 LS ND
4 0.806 86/13 ~ Pharyngeal paralysis Inconclusive i.e. OD 0.4 - 5 0.744 E55 ~S ND
0.8 6 0.718 926 * Colitis X
7 0.706 86/17 6.0 'colic' 8 0.660 967 ~ 'diarrhoea' 9 0.426 86.10 14.0 stenosis Sero-negative i.e. OD <0.4 10 0.350 92/64 1 0 Epiploic entrapment 11 0.292 Nl ~ Intussuscep-tion 12 0.064 939 * 'diarrhoea' Key Duration - stage of disease at time o~ sampling LS - long standing i.e. > 28 days ND - not diagnosed ~ - missing data Nine horses were either positive (OD ~0 8) or inconclusive (OD 0 4-0 8) to the ELISA N50 diagnostic test and were therefore serologically indistinguishable from the majority of chronic Grass Sickness cases (Group II) The remaining three listed horses were seronegative (OD < 0 4) and there~ore indistinguishable ~rom con~irmed cases o~
acute Grass Sickness (Group I); it is noteworthy that cases 10 and ll were acute abdominal crises and that the lowest OD value (0.064) was recorded from an ~undiagnosed~ case o~
diarrhoea in a foal.
Horses 1, 2, 3 and 5 were all from endemic Grass Sickness areas and presented signs consistent with a 'field diagnosis of chronic Grass Sickness i.e. sporadic inappetance, mild colic and weight loss. However they could not be di~ferentiated serologically from clinically normal animals in the same populations (Group V, mean OD
0.781).
Horse 6 (Colitis X) presented serological results which were indicative of C.perfrinqens involvement (OD
1.007) possibly associated with nonspecific seroconversion to the other two antigens (Table 5): conversely horse 4 (Pharyngeal paralysis) and horses 7 and 8 which were not diagnosed, did not show any evidence of non-speci~ic seroconversion. The pyloric stenosis case (horse 9) had been a~fected 14 days prior to samplingi the N50 reaction was borderline (OD 0.426) but specific.
TABLE 5 Group III Pattern of responses to three indicator antigens in four clinical cases with gastro-enteric diagnoses/signs ~ Hors~ Diagnosis/ N53 P50 s~O
'sisr.s~
4 Pharyngeal 0.806 0.560 0.751 paralysis 6 colitis X 0.718 1.007 0.875 7 'colic' 0.706 0.562 0.882 9 Pyloric stenosis 0.426 0.226 0.355 Overall Minimum 0.001 0.043 0.251 OD ~ean 0.587 0.551 0.821 ~iml~m 1.378 1.007 1.634 (n) (167) (35) (35) Horses which present with undiagnosed gastro-enteric signs cannot be differentiated from early stage (i.e. <28 days duration) cases of chronic Grass Sickness. However in the absence of confirmed diagnosis it cannot be assumed that inconclusive C novyi responses are non-specific. The data for horse 6 (colitis X) demonstrate that the test procedure can indicate the primary involvement o~
clostridia in non EGS disease processes; conversely serodiagnostic potential is limited by ~ailure to differentiate possible cases of EGS (horses 1, 2 and 3) from normal 'immune' animals in endemic area populations (d) Group IV Clinical non GE
A total of 25 specimens were submitted ~rom horses wit~ various diagnosed 'non abdominal' disorders or presenting signs. In this category 15/25 i e 60~ of specimens were seronegative to the C novyi antigen at a W 096/25669 PCT/~k5G,'~-323 serum dilution of 1:50. The following table lists the data for the three positive horses (OD ~ 0.8) and for the seven inconclusive animals OD 0.4 - 0.8.
TABI.E 6 Group IV horses : summary o~ diag~oses (or clinical signs) expressed in descending order of ELISA N50 OD for C.novyi antigen No. N50 OD Ref Duration Diagnosis Seropositive days (signs i.e. OD > 0.8 1 0.925 IN 3498 * lame 2 0.853 3611 * ND
3 0.831 4861 >728 lame Inconclusive 4 0.754 Cl >28 weight loss OD 0.4 - 0.8 5 0.698 IN 2995 * ND
6 0.639 ~6/27 >28 laminitis 7 0.592 3479 . ~ lame 8 0.516 A864 >28 'neurologic' 9 0.475 IN 3297 >28 l~m;n;tis 0.474 ~ 2302 ~ ND
*Missing data Four of the inclusive horses in the Group IV category were tested against the 'non specific' antigens and the results are presented in Table 7.
Pattern of responses to three indicator antigens in miscellaneous clinical cases 'inconclusive' to the C.novyi antigen :~orse Diagnosis N50 P50 S50 (signs ND 0.698 0.539 0.785 :
6 laminitis 9.639 0.912 1.49S
9 laminitis 0.475 0.780 0.950 ND 0.474 0.966 0.745 ~_ Overall Minimum 0.001 0-043 0 251 O~ Mean 0.587 0.551 0 821 ~laximum 1 378 1.007 1.634 n) (167) (35) ~35) CA 022l27l8 l997-08-08 - 21 - _ The two laminitis cases horses 6 and 9 both show evidence of non-speci~ic seroconversion which is consistent with responses to concomitant enterotoxaemia.
EXAMPLE 3 - Diagnoses per~ormed in normal horses.
Table 8 indicates the source and numbers o~ horses tested against the C.botulinum Group C indicator organism i.e.
C.novyi. All routine tests were carried out at a 1:50 serum dilution.
Categories of 'normal' horses tested*
ENDEMIC AREAS NON-ENDEMIC AREAS
EAST OF SCOTLAND WEST OF SCOTLAND
Group V VI VII VIII
Type Mainly older Horses High Mixed horses in plane grazing transit nutrition Populations 3 1 3 5 (n) * All groups contained one or more animals which were not clinically normal - mainly sporadic colic/diarrhoea or chronic 'weight loss'.
Summarised data demonstrates the obvious between-group di~erentiation o~ the selected populations.
ELISA OD values ~or 1:50 serum dilutions vs C.novvi antigen Group V VI VII VIII :~
n 35 16 11 36 Mean 0.781 0 720 a.586 0.478 Range 0.313- 0 462- 0 261- 0.179-1 287 1.378 1 026 1.08 variance 0 060 0 071 0 056 0.06 The normal horses tested according to this Example are grouped as follows:
GrouD V
Horses in this category were 'lifetime' residents in endemic EGS areas or NE Scotland and included 20 animals grazing within two miles of the original Barry camp outbreak (1909); 33/35 Group V horses were seroconverted, irrespective of age.
GrouD VI
The majority of horses in Group IV were imported to Scotland from Ireland and were therefore likely to have been seronegative on arrival. At the time of sampling they had been 'in residence' ~or periods of one week to six months; the statistical pattern of seroconversion was indistinguishable from that of the Group V horses.
These limited data suggest that premises which are subject to 'regular traffic' are likely to become mini-endemic areas as a result of recurrent 'contamination', therefore highly susceptible newcomers may be 'at risk' especially when subjected to intercurrent nutritlonal or climatic stresses during the incubation period.
GrouDs VII and VIII
Eventers or race horses in 'full work' or trainins on a high plane of nutrition (Group VII) were included as a control catesory for the miscellaneous 'non-endemic' population (Group VIII).
Table 10 shows the pattern o~ seroconversion to the C.novvi antigen ~or the four non-clinical categories .
Groups N50 v VI VII VIII
OD
Seronegative <0.4 2 0 l 13 Inconclusive 0.4-0.8 16 4 3 10 Seropositive >0.8 17 12 7 13 There~ore as a principle ~eature of the diagnostic procedure according to the invention, specimens ~rom clinical cases which demonstrate inconclusive or positive serological responses to the indicator cell wall antigens are re-examined ~or the presence o~ "antitoxins" viz.
antibodies to speci~ic clostridial toxins such as the Clostridium botulinum Type C1 neurotoxin. The results are presented in Tables 11 below:
TABT,E 11 GROUP AI Acut~ EGS (n = 7) I II III IV V
No./ Age E~ISA OD's Case in Cl novyi Cl botulinum re~. yrs 1:50 (C1 an~itoxin) 2 x 10 4 x 10 3 47.0 0.278 0.409 0.181 124.0 0.221 0.286 0.122 182.0 0.28~ 0.237 0.122 2012.0 0.636 0.552 0.279 221.0 0.283 0.252 0.130 272.0 0.377 0.274 0.127 314.0 0.380 0.271 0.132 Group AII Chronic EGS (n = 8) 29.0 0.335 0.172 0.087 55.0 0.378 0.250 0.123 244.0 0.698 0.425 0.208 2512 0 0.890 0.432 0.195 908 0 0.391 0.214 0.009 919.0 0.465 0.2~8 0.129 283.0 0.639 0.153 0.097 344.0 0.954 0.206 0.~96 W 096/25669 PCT/GB~ 323 TABLE 11 (Contd.) Group BI
Clinical (Antitoxin ELISA OD ~ O 10) No./ Age ELISA OD's Case in Cl novyiCl botulinum ref. yrs Diagnosis (Cl an~itoxin) (signs) 2 x 10- 4 x 10-3 19.0 Carcinoma 0.338 0.913 0.493 A 10.0 (Diarrhoea/ 1.221 0.312 0.182 Colic) 13 * (Pharyngeal 0.806 0.274 0.142 paralysis) 64 10.0 Epiploic 0.350 0.196 0.091 entrapment B 7.0 (lame) 0.592 0.185 0.083 7.0 (trauma) 0.212 0.183 0.080 C 8.0 (lame) 0.925 0.140 0.065 77 12.0 Ovarian 0.289 0.119 0.044 tumour TABLE 11 (Contd.) Group 3II
Clinical ( ~ titoxin ELISA OD < O.lO) No./ Age ELISA OD's Case in * Cl novyi Cl botulinum ref. yrs Diagnosis (C13antltox n) D 9.0 (respir- 0.256 0.042 0.020 atory) E 12.0 Pyelone- 0.371 0.041 0.020 phritis F 11.0 Colitis X 0.718 0.034 0.010 G 14.0 * 0.698 0.033 0.016 H 15 . O T.~m; n; tis 0.475 0.030 0.001 J * (lame) 0.087 0.022 0.027 K 0.5 (diarrhoea) 0.650 0.013 0.006 W 096/25669 PCT/~9G/~C323 TABLE 11 (Contd ) GROUP C
Seroco~v~rsion vs duratio~ of clinical signs (days) CT 0.5 - 3.0 days No. Ref Duration ELISA OD
(days) ( Clostridium novyi ) 1:50 1 E4 0.5 0.278 2 E22 0.5 0.283 3 E31 0.5 0.380 4 E27 1.5 0.377 E18 2.0 0.286 6 E12 3.0 0.221 CII lO.O - 15.0 days 1 N2 11.0 0.649 2 N8 11.0 0.437 3 E2 12.0 0.335 4 ~ 26/91 13.0 0.465 N6 14.0 0.358 6 N10 14.0 0.938 7 E24 15.0 0.698 CA 022l27l8 l997-08-08 TABLE 11 (Contd.) CIII > 15 days 1 r.28 16.0 0.639 2 N3 16.0 0.893 3 E25 18.0 0.890 4 N4 21.0 0.770 Nl3 21.0 0.727 6 N12 27.0 1.092 7 E34 33.0 0.954 W O 96/25669 PCT/GB~6/0~3 Demonstration of Clostridium botulinum Type Cl neurotoxin in the intestinal content of horses affected with EGS.
Faecal specimens were collected at autopsy in a series of horses affected with EGS; in all cases the diagnosis was confirmed prior to euth~n~ia by the presence o~ characteristic neuronal lesions in ileal biopsy specimens.
Approximately 20 gram specimens of faeces were transferred to 20 ml volumes of phosphate buffered saline pH 7.2 containing 0.2~ gelatin (PBSG) to preserve the toxin; the PBSG samples were mixed to disperse the specimen and then allowed to infuse at 4~C ~or 24 hours prior to long-term storage at -30~C.
The presence and potency o~ botulinum Cl neurotoxin was determined and assayed in the supernatant ~luid o~ PBSG
in~usions by a modi~ication of the ELISA protocol which involves precoating the reaction plates with a standardised C. botulinum Type Cl antitoxin.
Method The use o~ sandwich ELISA for the detection of botulinum Type Cl neurotoxin.
1. An ELISA plate was coated (100 ~l well) with guinea pig antibody speci~ic ~or Clostridium botulinum type C
~ 25 neurotoxin at 5-10 ~g/ml using phosphate buffered saline (PBS) as a diluent. The antibody solution was added and the plate was shaken for five minutes before incuba~ion -W 096/25669 PCTIGB~5'~~~23 - 30 - -~
overnight at 4~~.
2. The plate was washed once using PBS containing 0.1 Tween 20 (PBS-T).
3. The blanking solution o~ PBS-T containing 5% ~oetal bovine serum (FBS) was added (100 ~l/well) and the plate was incubated for 1 hour at 37~C with continuous shaking.
Unbound materials were removed by means o~ washing as above.
4. The antigen solution prepared in PBS-T containing 5%
FBS (10 ~g/ml; 1:5 dilutions) was added (100 ~l/well).
Incubation was carried out in plate ~or 60-90 minutes at 37~C with continuous shaking.
5. The plate was washed three times using PBS-T.
6. The second antibody-enzyme conjugate (100 ~l/well o~
guinea pig antibody labelled with horse radish peroxidase) prepared in PBS-T cont~ining 5% FBS was added. Incubation oi~ the plate was carried out ~or 60-90 minutes at 37~ C with continuous shaking.
7. The plate was washed three times using PBS-T.
8. The substrate* solution (TMB) was added and the enzyme was allowed to react.
9 The reaction was stopped with 50 ~l/well o~ H2S04, 2M
be~ore measuring the absorbance at 450mm.
* 10 mg TMB in 1 ml DMSO was dissolved in a dark glass tube. 100 ~l o~ the TMB solution was added to 10 ml ~hosphate/citrate bu~er, pH 5 0 containing 44 ~l 1% H2O2.
The results are shown below in Table 12.
W 096~2~669 PCT/~
T~3LE 12 Detection and assay of botulinum Cl neurotoxin in the intestinal content of horses affected with EGS.
CaseELISA OD VALUES Botulinum C
Ref: toxin*
Serum C.novyi PBSG extract (ng/ml) antibody botulinum C1 toxin 733 . 0.798 1~017 8.8 851 NS 0.729 6.3 869 0.897 NS
889 0.685 1.305 11.4 NS not sampled * 1.0 ng botulinum Cl toxin represents approximately 100 mouse MLD'S.
The demonstration of lethal concentrations of botulinum C1 neurotoxin in faecal infusions derived from 3/4 horses affected with EGS, combined with concomitant serological evidence of exposure to a phenotype of C. novyi ~urther attests to the toxicoinfectious nature o~ the disease process. The evidence also suggests that the contagious source of the Cl neurotoxin is located in the tissues, or lumen, of the bowel.
Confirmation of the location of a botulinum Cl toxin-producing organism in the tissues o~ horses a~fected with EGS.
The neuronal lesions of EGS invariably a~ect the ileum which is there~ore likely to be a predilection site ~or the establishment o~ toxicoin~ectious contagion.
W 096/25669 PCT/GD5GI~C323 Approximately 20 gram samples o~ ileum and/or spleen tissues were taken at autopsy o~ ~ive con~irmed cases o~
EGS and transferred immediately to 50 ml volumes o~
prereduced enrichment culture media. The selected medium J
5 (designated AIM/CMB/SSG) was a modi~ied version o~ cooked meat broth ~orti~ied with glucose (0.2~) and soluble starch (0.3~) to optimise conditions ~or primary isolation o~
~astidious anaerobes and also incorporated gentamycin (10 ml/l) to inhibit the overgrowth o~ dominant or cont~m;n~nt 10 species. The sample bottles were incubated anaerobically ~or 5 days at 30~C; supernatant ~luids were then harvested and tested ~or the presence of toxin by the ELISA technique as used in the preceding example.
The results are shown in the ~ollowing table:
W096/25669 CA 022l27l8 l997-08-08 PCT/GB~6/0~3?3 - 33 - = -TA~3LE 13 Isolation of botulinum C1 neurotoxin-producing C. botulinum from the tissues of horses affected with EGS.
Case Sample C. botulinum Type C1 neurotoxin 5 Ref type ELISA OD* ng/ml 733 Faeces~Ve Ileum - isolate pending identification Spleen - no toxin detected 10 830 Spleen 1.424 12.4 851 FaecestVe Ileum 1. 312 57.0**
869 Faeces~Ve Ileum 1.449 12. 6 Spleen - no toxin detected 889 Faeces~Ve Ileum - no toxin detected Spleen - no toxin detected Sample dilution * 1/5 ** 1/25 The demonstration of botulinum Cl neurotoxin in 3/5 AIM/CMB/SSG broth fluid supernates confirms the presence therein of a viable serotype of C. botulinum type C or D.
A combination of data presented in Examples S and 6 above indicates that the immunoassays successfully demonstrate the presence of lethal concentrations of botulinum C
neurotoxin in faecal fluids and/or in tissue culture supernates derived from five successive confirmed cases of EGS. These positive associations of a potent toxin and the parent toxinogenic anaerobe with clinical EGS confirms the v diagnostic validity of the differential serological test 5 here presented as the inventive procedure.
The sample containing the Group III antigen or analogue thereof may be a biological ~luid or tissue sample, and is pre~erably serum.
The diagnostic method according to the invention provides a rapid, non-invasive and conclusive method ~or confirming the diagnosis of equine dysautonomia, o~herwise known as grass sickness. The method allows for the differential diagnosis of clostridial involvement in non-speci~ic gastro intestinal and/or neurologic equine W 096t25669 PCT/GB96100323 disorders.
As a ~urther feature, the method of the invention may be used as a facility for monitoring responses to treatment o~ clinical toxicoinfectious clostridiosis, including Grass Sickness, in individual horses. Host responses to individual clostridial toxins involved in the toxinology o~
equine toxicoinfectious clostridiosis may also be identified and quantified by performing the method o~ the invention and interpreting the results obtained.
The diagnostic method of the invention may also be used as the basis of a technique for the parallel assessment of bacterial virulence ~actors or toxins which combine to determine the pathogenesis of field strains o~
toxinogenic clostridia.
Identification and quantification of host responses to individual clostridial toxins together with the parallel assessment of bacterial virulence ~actors or toxins responsible for the pathogenesis of specific toxinogenic clostridia are prerequisites ~or the epidemiological study of equine toxicoinfectious disease.
The invention is further illustrated by the ~ollowing hxamples. In the Examples, four categories of horses showlng clinical signs were identified. The groups are as follows:
Group I - Acute Grass Sickness (AGS) Group II - Chronic Grass Sickness (CGS) Group III - Clinical gastroenteric (GE) including acute W 096/25669 PCT/GB~Gi'~-~23 abdominal crises.
Group IV - Clinical miscellaneous including specimens submitted ~rom a broad spectrum o~ diagnosed cases or with - key presenting signs Example 1 - Determination o~ indicator antiaens A group o~ 34 clinical specimens was tested by the (IgG) ELISA technique at two serum dilutions i.e. 1/25 and 1/50. Each specimen was tested in duplicate and ELISA OD
values were presented as a mean o~ the two results.
Table 1 shows the mean OD values (with ranges and variance) ~or the four clinical groups, against the three indicator antigens.
I II III IV
AGS CGS GE nonGE
Antigen n 9 23 12 25 C novvimean 0 302 0 634 0.677 0 399 range0.001-0.6350.195-1.100 0 064-1 238 0.087-0 925 variance 0.029 0 066 0 121 0 057 C ~er~rinqens n 8 8 6 12 mean 0 480 0_622 0 502 0.596 range-0 043-0 9S4 0 173-0.866 0 184-1 007 0 337-0.966 variance 0 088 0 057 0.088 0.048 C s~oroqenes ~ ~ 3 ~ 12 ~ean 0 7710.996 Q 696 0 819 range 0 38S-1 165 0 479-1.634 0 251-1 067 0 511-1.499 variance 0.066 0.145 O.lOS 0 064 The representative data presented in Table 1 indicate that the overall range o~ ELISA OD values was broadly - 14 -=
similar for each of the three antigens. However, significant between-group variation was only evident for the C.novyi antigen.
The summarised clinical data provided in Table 1 support the assumptions that all Group I (AGS) and the majority of Group IV (miscellaneous clinical) horses have low levels of antibody to C. novyi and are therefore susceptible to EGS. Conversely horses in Group II (CGS) are expected to be variably seroconverted according to stage of 'incubation' at the time of sampling; inconclusive or positive results from the individual horses in the non-specific gastro-intestinal (Group III) ~nim~l S may indicate involvement of a toxicoinfectious clostridial component in the disease process. Group I (AGS) and Group IV
(miscellaneous clinical, non GE) may therefore be amalgamated to form a combined 'seronegative/inconclusive' category; similarly Group II (CGS) and III (clinical, GE) were statistically indistinguishable and may be combined to ~orm an 'inconclusive/positive' category.
Comparison o~ these two combined groups I + IV and II
III provides a basis for meaningful analyses of variance.
l~ABLE 2 Analyses of variance o~ ELISA OD values for the three antigens based on amalgamated clinical grouping CA 022l27l8 l997-08-08 W 096125669 PCT/~h,''~~323 Seronegative/ InConclusive/ VR Sig.
inconclusive Seroposi~ive 1:34 Groups I + IV Groups II ~ III
n 20 15 C. nov~ri 1:50 Q.358 0.554 8.03 p<0.01 1:25 0.514 0.616 2.03 NS
C . Derf rinqens 1:50 0.550 0.553 0.00 NS
1:25 0.492 0.535 0.21 NS
C sPoroqeneS
1:50 0.800 0.850 0.23 NS
1:25 0.852 0.816 0.09 NS
The individuality of the serological response to the three indicator antigens was further substantiated by the absence of significant correlation between the data sets.
TABLE 2A Correlation matrix ~or ELISA OD at 1:50 serum dilution C.novyi 1.000 C.Per~rinqes 0.476 * 1.000 20 C.s~oroqenes 0.429* 0.530** 1.000 C.novyi C.per~rinqens C.s~oroqenes EXAMPLE 3 ELISA of Clinical qrouDs (a) Group I Acute Grass Sickness (AGS) Nine specimens were submitted ~rom con~irmed cases of AGS and with ~he exception of one specimen all were seronegative to the C.novyi antigen (i.e. OD <0.4). The single inconclusive result (OD 0.635) was recorded ~or a 12 year old horse in Group I which was also characterised by above group average seroconversion to the two non-specific antigens (Table 3).
ELISA OD Values Horse 5 (D20.91) 0.635 0.539 1.165 Group I min 0.001 0.043 0.385 mean 0.302 0.480 0.771 max 0.635 0.954 1.165 variance 0.029 0.088 0.066 (b) Group II Chronic Grass Sickness (CGS) By definition horses susceptible to CGS are likely to be in the seronegative range (i.e. OD<0.4) at the outset o~
disease; clinical progression will be accompanied by a variable rate o~ seroconversion modi~ied according to individual animal responses to the toxicoin~ectious challenge. The majority of specimens included in Group II
(con~irmed CGS) were sampled at a relatively early stage in the disease process Mean duration at sampling 19 days range 11-35 days n = 23 (c) Group III Clinical GE
Table 4 provides a summary o~ the diagnoses or clinical signs recorded ~or the 12 horses included in this category Clinical GE - summary o~ diagnoses (or clinical signs) expressed in descenaing order oi~ ELISA OD ~or C novvi antigen at 1:50 serum dilution for Group III horses No. N50 OD RefDuration Diagnosis Seropositive days (signs) i.e. OD > 0.8 1 1.238 F456 LS ND
2 1.076 E50 ~S ND
3 1.052 F462 LS ND
4 0.806 86/13 ~ Pharyngeal paralysis Inconclusive i.e. OD 0.4 - 5 0.744 E55 ~S ND
0.8 6 0.718 926 * Colitis X
7 0.706 86/17 6.0 'colic' 8 0.660 967 ~ 'diarrhoea' 9 0.426 86.10 14.0 stenosis Sero-negative i.e. OD <0.4 10 0.350 92/64 1 0 Epiploic entrapment 11 0.292 Nl ~ Intussuscep-tion 12 0.064 939 * 'diarrhoea' Key Duration - stage of disease at time o~ sampling LS - long standing i.e. > 28 days ND - not diagnosed ~ - missing data Nine horses were either positive (OD ~0 8) or inconclusive (OD 0 4-0 8) to the ELISA N50 diagnostic test and were therefore serologically indistinguishable from the majority of chronic Grass Sickness cases (Group II) The remaining three listed horses were seronegative (OD < 0 4) and there~ore indistinguishable ~rom con~irmed cases o~
acute Grass Sickness (Group I); it is noteworthy that cases 10 and ll were acute abdominal crises and that the lowest OD value (0.064) was recorded from an ~undiagnosed~ case o~
diarrhoea in a foal.
Horses 1, 2, 3 and 5 were all from endemic Grass Sickness areas and presented signs consistent with a 'field diagnosis of chronic Grass Sickness i.e. sporadic inappetance, mild colic and weight loss. However they could not be di~ferentiated serologically from clinically normal animals in the same populations (Group V, mean OD
0.781).
Horse 6 (Colitis X) presented serological results which were indicative of C.perfrinqens involvement (OD
1.007) possibly associated with nonspecific seroconversion to the other two antigens (Table 5): conversely horse 4 (Pharyngeal paralysis) and horses 7 and 8 which were not diagnosed, did not show any evidence of non-speci~ic seroconversion. The pyloric stenosis case (horse 9) had been a~fected 14 days prior to samplingi the N50 reaction was borderline (OD 0.426) but specific.
TABLE 5 Group III Pattern of responses to three indicator antigens in four clinical cases with gastro-enteric diagnoses/signs ~ Hors~ Diagnosis/ N53 P50 s~O
'sisr.s~
4 Pharyngeal 0.806 0.560 0.751 paralysis 6 colitis X 0.718 1.007 0.875 7 'colic' 0.706 0.562 0.882 9 Pyloric stenosis 0.426 0.226 0.355 Overall Minimum 0.001 0.043 0.251 OD ~ean 0.587 0.551 0.821 ~iml~m 1.378 1.007 1.634 (n) (167) (35) (35) Horses which present with undiagnosed gastro-enteric signs cannot be differentiated from early stage (i.e. <28 days duration) cases of chronic Grass Sickness. However in the absence of confirmed diagnosis it cannot be assumed that inconclusive C novyi responses are non-specific. The data for horse 6 (colitis X) demonstrate that the test procedure can indicate the primary involvement o~
clostridia in non EGS disease processes; conversely serodiagnostic potential is limited by ~ailure to differentiate possible cases of EGS (horses 1, 2 and 3) from normal 'immune' animals in endemic area populations (d) Group IV Clinical non GE
A total of 25 specimens were submitted ~rom horses wit~ various diagnosed 'non abdominal' disorders or presenting signs. In this category 15/25 i e 60~ of specimens were seronegative to the C novyi antigen at a W 096/25669 PCT/~k5G,'~-323 serum dilution of 1:50. The following table lists the data for the three positive horses (OD ~ 0.8) and for the seven inconclusive animals OD 0.4 - 0.8.
TABI.E 6 Group IV horses : summary o~ diag~oses (or clinical signs) expressed in descending order of ELISA N50 OD for C.novyi antigen No. N50 OD Ref Duration Diagnosis Seropositive days (signs i.e. OD > 0.8 1 0.925 IN 3498 * lame 2 0.853 3611 * ND
3 0.831 4861 >728 lame Inconclusive 4 0.754 Cl >28 weight loss OD 0.4 - 0.8 5 0.698 IN 2995 * ND
6 0.639 ~6/27 >28 laminitis 7 0.592 3479 . ~ lame 8 0.516 A864 >28 'neurologic' 9 0.475 IN 3297 >28 l~m;n;tis 0.474 ~ 2302 ~ ND
*Missing data Four of the inclusive horses in the Group IV category were tested against the 'non specific' antigens and the results are presented in Table 7.
Pattern of responses to three indicator antigens in miscellaneous clinical cases 'inconclusive' to the C.novyi antigen :~orse Diagnosis N50 P50 S50 (signs ND 0.698 0.539 0.785 :
6 laminitis 9.639 0.912 1.49S
9 laminitis 0.475 0.780 0.950 ND 0.474 0.966 0.745 ~_ Overall Minimum 0.001 0-043 0 251 O~ Mean 0.587 0.551 0 821 ~laximum 1 378 1.007 1.634 n) (167) (35) ~35) CA 022l27l8 l997-08-08 - 21 - _ The two laminitis cases horses 6 and 9 both show evidence of non-speci~ic seroconversion which is consistent with responses to concomitant enterotoxaemia.
EXAMPLE 3 - Diagnoses per~ormed in normal horses.
Table 8 indicates the source and numbers o~ horses tested against the C.botulinum Group C indicator organism i.e.
C.novyi. All routine tests were carried out at a 1:50 serum dilution.
Categories of 'normal' horses tested*
ENDEMIC AREAS NON-ENDEMIC AREAS
EAST OF SCOTLAND WEST OF SCOTLAND
Group V VI VII VIII
Type Mainly older Horses High Mixed horses in plane grazing transit nutrition Populations 3 1 3 5 (n) * All groups contained one or more animals which were not clinically normal - mainly sporadic colic/diarrhoea or chronic 'weight loss'.
Summarised data demonstrates the obvious between-group di~erentiation o~ the selected populations.
ELISA OD values ~or 1:50 serum dilutions vs C.novvi antigen Group V VI VII VIII :~
n 35 16 11 36 Mean 0.781 0 720 a.586 0.478 Range 0.313- 0 462- 0 261- 0.179-1 287 1.378 1 026 1.08 variance 0 060 0 071 0 056 0.06 The normal horses tested according to this Example are grouped as follows:
GrouD V
Horses in this category were 'lifetime' residents in endemic EGS areas or NE Scotland and included 20 animals grazing within two miles of the original Barry camp outbreak (1909); 33/35 Group V horses were seroconverted, irrespective of age.
GrouD VI
The majority of horses in Group IV were imported to Scotland from Ireland and were therefore likely to have been seronegative on arrival. At the time of sampling they had been 'in residence' ~or periods of one week to six months; the statistical pattern of seroconversion was indistinguishable from that of the Group V horses.
These limited data suggest that premises which are subject to 'regular traffic' are likely to become mini-endemic areas as a result of recurrent 'contamination', therefore highly susceptible newcomers may be 'at risk' especially when subjected to intercurrent nutritlonal or climatic stresses during the incubation period.
GrouDs VII and VIII
Eventers or race horses in 'full work' or trainins on a high plane of nutrition (Group VII) were included as a control catesory for the miscellaneous 'non-endemic' population (Group VIII).
Table 10 shows the pattern o~ seroconversion to the C.novvi antigen ~or the four non-clinical categories .
Groups N50 v VI VII VIII
OD
Seronegative <0.4 2 0 l 13 Inconclusive 0.4-0.8 16 4 3 10 Seropositive >0.8 17 12 7 13 There~ore as a principle ~eature of the diagnostic procedure according to the invention, specimens ~rom clinical cases which demonstrate inconclusive or positive serological responses to the indicator cell wall antigens are re-examined ~or the presence o~ "antitoxins" viz.
antibodies to speci~ic clostridial toxins such as the Clostridium botulinum Type C1 neurotoxin. The results are presented in Tables 11 below:
TABT,E 11 GROUP AI Acut~ EGS (n = 7) I II III IV V
No./ Age E~ISA OD's Case in Cl novyi Cl botulinum re~. yrs 1:50 (C1 an~itoxin) 2 x 10 4 x 10 3 47.0 0.278 0.409 0.181 124.0 0.221 0.286 0.122 182.0 0.28~ 0.237 0.122 2012.0 0.636 0.552 0.279 221.0 0.283 0.252 0.130 272.0 0.377 0.274 0.127 314.0 0.380 0.271 0.132 Group AII Chronic EGS (n = 8) 29.0 0.335 0.172 0.087 55.0 0.378 0.250 0.123 244.0 0.698 0.425 0.208 2512 0 0.890 0.432 0.195 908 0 0.391 0.214 0.009 919.0 0.465 0.2~8 0.129 283.0 0.639 0.153 0.097 344.0 0.954 0.206 0.~96 W 096/25669 PCT/GB~ 323 TABLE 11 (Contd.) Group BI
Clinical (Antitoxin ELISA OD ~ O 10) No./ Age ELISA OD's Case in Cl novyiCl botulinum ref. yrs Diagnosis (Cl an~itoxin) (signs) 2 x 10- 4 x 10-3 19.0 Carcinoma 0.338 0.913 0.493 A 10.0 (Diarrhoea/ 1.221 0.312 0.182 Colic) 13 * (Pharyngeal 0.806 0.274 0.142 paralysis) 64 10.0 Epiploic 0.350 0.196 0.091 entrapment B 7.0 (lame) 0.592 0.185 0.083 7.0 (trauma) 0.212 0.183 0.080 C 8.0 (lame) 0.925 0.140 0.065 77 12.0 Ovarian 0.289 0.119 0.044 tumour TABLE 11 (Contd.) Group 3II
Clinical ( ~ titoxin ELISA OD < O.lO) No./ Age ELISA OD's Case in * Cl novyi Cl botulinum ref. yrs Diagnosis (C13antltox n) D 9.0 (respir- 0.256 0.042 0.020 atory) E 12.0 Pyelone- 0.371 0.041 0.020 phritis F 11.0 Colitis X 0.718 0.034 0.010 G 14.0 * 0.698 0.033 0.016 H 15 . O T.~m; n; tis 0.475 0.030 0.001 J * (lame) 0.087 0.022 0.027 K 0.5 (diarrhoea) 0.650 0.013 0.006 W 096/25669 PCT/~9G/~C323 TABLE 11 (Contd ) GROUP C
Seroco~v~rsion vs duratio~ of clinical signs (days) CT 0.5 - 3.0 days No. Ref Duration ELISA OD
(days) ( Clostridium novyi ) 1:50 1 E4 0.5 0.278 2 E22 0.5 0.283 3 E31 0.5 0.380 4 E27 1.5 0.377 E18 2.0 0.286 6 E12 3.0 0.221 CII lO.O - 15.0 days 1 N2 11.0 0.649 2 N8 11.0 0.437 3 E2 12.0 0.335 4 ~ 26/91 13.0 0.465 N6 14.0 0.358 6 N10 14.0 0.938 7 E24 15.0 0.698 CA 022l27l8 l997-08-08 TABLE 11 (Contd.) CIII > 15 days 1 r.28 16.0 0.639 2 N3 16.0 0.893 3 E25 18.0 0.890 4 N4 21.0 0.770 Nl3 21.0 0.727 6 N12 27.0 1.092 7 E34 33.0 0.954 W O 96/25669 PCT/GB~6/0~3 Demonstration of Clostridium botulinum Type Cl neurotoxin in the intestinal content of horses affected with EGS.
Faecal specimens were collected at autopsy in a series of horses affected with EGS; in all cases the diagnosis was confirmed prior to euth~n~ia by the presence o~ characteristic neuronal lesions in ileal biopsy specimens.
Approximately 20 gram specimens of faeces were transferred to 20 ml volumes of phosphate buffered saline pH 7.2 containing 0.2~ gelatin (PBSG) to preserve the toxin; the PBSG samples were mixed to disperse the specimen and then allowed to infuse at 4~C ~or 24 hours prior to long-term storage at -30~C.
The presence and potency o~ botulinum Cl neurotoxin was determined and assayed in the supernatant ~luid o~ PBSG
in~usions by a modi~ication of the ELISA protocol which involves precoating the reaction plates with a standardised C. botulinum Type Cl antitoxin.
Method The use o~ sandwich ELISA for the detection of botulinum Type Cl neurotoxin.
1. An ELISA plate was coated (100 ~l well) with guinea pig antibody speci~ic ~or Clostridium botulinum type C
~ 25 neurotoxin at 5-10 ~g/ml using phosphate buffered saline (PBS) as a diluent. The antibody solution was added and the plate was shaken for five minutes before incuba~ion -W 096/25669 PCTIGB~5'~~~23 - 30 - -~
overnight at 4~~.
2. The plate was washed once using PBS containing 0.1 Tween 20 (PBS-T).
3. The blanking solution o~ PBS-T containing 5% ~oetal bovine serum (FBS) was added (100 ~l/well) and the plate was incubated for 1 hour at 37~C with continuous shaking.
Unbound materials were removed by means o~ washing as above.
4. The antigen solution prepared in PBS-T containing 5%
FBS (10 ~g/ml; 1:5 dilutions) was added (100 ~l/well).
Incubation was carried out in plate ~or 60-90 minutes at 37~C with continuous shaking.
5. The plate was washed three times using PBS-T.
6. The second antibody-enzyme conjugate (100 ~l/well o~
guinea pig antibody labelled with horse radish peroxidase) prepared in PBS-T cont~ining 5% FBS was added. Incubation oi~ the plate was carried out ~or 60-90 minutes at 37~ C with continuous shaking.
7. The plate was washed three times using PBS-T.
8. The substrate* solution (TMB) was added and the enzyme was allowed to react.
9 The reaction was stopped with 50 ~l/well o~ H2S04, 2M
be~ore measuring the absorbance at 450mm.
* 10 mg TMB in 1 ml DMSO was dissolved in a dark glass tube. 100 ~l o~ the TMB solution was added to 10 ml ~hosphate/citrate bu~er, pH 5 0 containing 44 ~l 1% H2O2.
The results are shown below in Table 12.
W 096~2~669 PCT/~
T~3LE 12 Detection and assay of botulinum Cl neurotoxin in the intestinal content of horses affected with EGS.
CaseELISA OD VALUES Botulinum C
Ref: toxin*
Serum C.novyi PBSG extract (ng/ml) antibody botulinum C1 toxin 733 . 0.798 1~017 8.8 851 NS 0.729 6.3 869 0.897 NS
889 0.685 1.305 11.4 NS not sampled * 1.0 ng botulinum Cl toxin represents approximately 100 mouse MLD'S.
The demonstration of lethal concentrations of botulinum C1 neurotoxin in faecal infusions derived from 3/4 horses affected with EGS, combined with concomitant serological evidence of exposure to a phenotype of C. novyi ~urther attests to the toxicoinfectious nature o~ the disease process. The evidence also suggests that the contagious source of the Cl neurotoxin is located in the tissues, or lumen, of the bowel.
Confirmation of the location of a botulinum Cl toxin-producing organism in the tissues o~ horses a~fected with EGS.
The neuronal lesions of EGS invariably a~ect the ileum which is there~ore likely to be a predilection site ~or the establishment o~ toxicoin~ectious contagion.
W 096/25669 PCT/GD5GI~C323 Approximately 20 gram samples o~ ileum and/or spleen tissues were taken at autopsy o~ ~ive con~irmed cases o~
EGS and transferred immediately to 50 ml volumes o~
prereduced enrichment culture media. The selected medium J
5 (designated AIM/CMB/SSG) was a modi~ied version o~ cooked meat broth ~orti~ied with glucose (0.2~) and soluble starch (0.3~) to optimise conditions ~or primary isolation o~
~astidious anaerobes and also incorporated gentamycin (10 ml/l) to inhibit the overgrowth o~ dominant or cont~m;n~nt 10 species. The sample bottles were incubated anaerobically ~or 5 days at 30~C; supernatant ~luids were then harvested and tested ~or the presence of toxin by the ELISA technique as used in the preceding example.
The results are shown in the ~ollowing table:
W096/25669 CA 022l27l8 l997-08-08 PCT/GB~6/0~3?3 - 33 - = -TA~3LE 13 Isolation of botulinum C1 neurotoxin-producing C. botulinum from the tissues of horses affected with EGS.
Case Sample C. botulinum Type C1 neurotoxin 5 Ref type ELISA OD* ng/ml 733 Faeces~Ve Ileum - isolate pending identification Spleen - no toxin detected 10 830 Spleen 1.424 12.4 851 FaecestVe Ileum 1. 312 57.0**
869 Faeces~Ve Ileum 1.449 12. 6 Spleen - no toxin detected 889 Faeces~Ve Ileum - no toxin detected Spleen - no toxin detected Sample dilution * 1/5 ** 1/25 The demonstration of botulinum Cl neurotoxin in 3/5 AIM/CMB/SSG broth fluid supernates confirms the presence therein of a viable serotype of C. botulinum type C or D.
A combination of data presented in Examples S and 6 above indicates that the immunoassays successfully demonstrate the presence of lethal concentrations of botulinum C
neurotoxin in faecal fluids and/or in tissue culture supernates derived from five successive confirmed cases of EGS. These positive associations of a potent toxin and the parent toxinogenic anaerobe with clinical EGS confirms the v diagnostic validity of the differential serological test 5 here presented as the inventive procedure.
Claims (6)
1. A method for the diagnosis of equine toxicoinfectious clostridiosis which comprises detecting the presence of an antibody to a C. botulinum antigen or a phenotype thereof and/or an antibody to a clostridial toxin in a biological sample.
2. A method according to claim 1, for the diagnosis of equine grass sickness which comprises detecting the presence of an antibody to C. botulinum Group III antigen or a phenotype thereof and an antibody to a botulinum type C toxin.
3. A method according to any of claims 1 or 2, wherein the phenotype is derived from C. novyi or C.
perfringens.
perfringens.
4. A method according to claim 3 for the detection of anterior enteritis or colitis X, wherein the phenotype is derived from C. perfrinqens.
5. A kit for diagnosis of equine toxicoinfectious clostridiosis which comprises means for detecting an antibody to a C. botulinum antigen or a phenotype thereof, and/or means for detecting an antibody to a clostridial toxin.
6. A kit according to claim 5 for diagnosis of equine grass sickness, which comprises means for detecting the presence of an antibody to C. botulinum Group III
antigen or a phenotype thereof and means for detecting an antibody to a botulinum type C toxin.
antigen or a phenotype thereof and means for detecting an antibody to a botulinum type C toxin.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9502739.7A GB9502739D0 (en) | 1995-02-13 | 1995-02-13 | Diagnosis of toxicoinfections clostridiosis |
| GB9502739.7 | 1995-02-13 | ||
| GBGB9523473.8A GB9523473D0 (en) | 1995-11-16 | 1995-11-16 | Diagnosis of toxicoinfectious clostridiosis |
| GB9523473.8 | 1995-11-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2212718A1 true CA2212718A1 (en) | 1996-08-22 |
Family
ID=26306491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2212718 Abandoned CA2212718A1 (en) | 1995-02-13 | 1996-02-13 | Diagnosis of toxicoinfectious clostridiosis |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0811163A1 (en) |
| JP (1) | JPH11500221A (en) |
| AU (1) | AU4670696A (en) |
| CA (1) | CA2212718A1 (en) |
| WO (1) | WO1996025669A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7563874B2 (en) | 1998-08-31 | 2009-07-21 | The Regents Of The University Of California | Therapeutic monoclonal antibodies that neutralize botulinum neurotoxins |
| EP1850866A4 (en) | 2005-01-27 | 2009-07-08 | Univ California | Therapeutic monoclonal antibodies that neutralize botulinum neurotoxins |
| EP2134749B1 (en) | 2007-03-22 | 2013-11-06 | The Regents of the University of California | Therapeutic monoclonal antibodies that neutralize botulinum neurotoxins |
| CA2732003A1 (en) | 2008-07-31 | 2010-02-04 | James D. Marks | Antibodies that neutralize botulinum neurotoxins |
| WO2012047427A2 (en) | 2010-08-31 | 2012-04-12 | The Regents Of The University Of California | Antibodies for botulinum neurotoxins |
-
1996
- 1996-02-13 WO PCT/GB1996/000323 patent/WO1996025669A1/en not_active Application Discontinuation
- 1996-02-13 EP EP96902363A patent/EP0811163A1/en not_active Withdrawn
- 1996-02-13 CA CA 2212718 patent/CA2212718A1/en not_active Abandoned
- 1996-02-13 AU AU46706/96A patent/AU4670696A/en not_active Abandoned
- 1996-02-13 JP JP8524753A patent/JPH11500221A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| AU4670696A (en) | 1996-09-04 |
| EP0811163A1 (en) | 1997-12-10 |
| JPH11500221A (en) | 1999-01-06 |
| WO1996025669A1 (en) | 1996-08-22 |
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