HAL (Le Centre pour la Communication Scientifique Directe), Jun 22, 2014
International audienceBackground: Unlike other MAC members, Mycobacterium avium subsp. paratuberc... more International audienceBackground: Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis does not produce the GPL on the surface of the cell wall but a lipopentapeptide called L5P or LP01. The molecular and genetic characterization of this antigen demonstrated that L5P is specific to Mycobacterium avium subsp. paratuberculosis. L5P produced by chemical synthesis was used to show that this molecule is suitable to detect Mycobacterium avium subsp. paratuberculosis infected animals. Currently available diagnostic tests are based on the use of whole cell antigen that are mainly not Mycobacterium avium subsp. paratuberculosis specific and that require a pre-absorption step with antigen of Mycobacterium phlei. These diagnostics detect animals with a late infection but are not sensitive enough to detect early infection. We hypothesize that the pre-absorption step prevents detection of informative populations of immunoglobulins, especially in animals with “asymptomatic shedding” not detected by the current serology-based diagnostics. Objective: Assess the potential of L5P and soluble variants for the serological diagnosis of Mycobacterium avium subsp. paratuberculosis infection using collections of sera from different contexts. Method: In order to find the best compound for use in serology we chemically synthesized L5P and derivatives of L5P including water-soluble forms. These pure compounds were evaluated on three collections of serum, each well characterized from infected and non-infected cattle, goats and sheep. Results: ROC analysis showed that L5P and also water-soluble derivatives are suitable for the development of a serological diagnostic test. Advantageously, these pure synthetic Mycobacterium avium subsp. paratuberculosis specific antigens can be produced at low cost
HAL (Le Centre pour la Communication Scientifique Directe), Jun 20, 2016
National audienceBackground: Unlike other MAC members, Mycobacterium avium subsp. paratuberculosi... more National audienceBackground: Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce GPL on the surface of the cell wall but a lipopentapeptide called L5P or ParaLP01. The molecular and genetic characterization of this antigen demonstrated that L5P is specific to MAP. Currently available diagnostic tests are based on the use of whole cell antigens that are mainly not MAP specific and that require a pre-absorption step with antigens of M. phlei. This diagnosis detect animals with a late stage infection but are not sensitive enough to detect early stage infection. We hypothesize that the pre-absorption step prevents detection of informative populations of immunoglobulins, especially in animals with subclinical faecal shedding of MAP that are not detected by the current serology-based diagnostics. The L5P antigen has now been used in various studies to evaluate a MAP specific sero-diagnostic. This synthetic molecule was used as a pure product, not requiring pre-absorption step. However a number of parameters must be assessed including the formulation of L5P and with reference to a panel of well defined serum samples.Objective: To assess the potential of L5P and its hydrosoluble variants for the serological diagnosis of MAP infection using collections of sera from different contexts.Method: In order to find the best compound for use in serology we chemically synthesized L5P and derivatives of L5P including water-soluble variants. These pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle, goats and sheep. In addition, the panels of sera included animals infected with M. bovis and M. avium subsp. hominissuis.Results: ROC analysis showed that L5P and also its water-soluble derivatives are suitable for the development of serological diagnosis. Advantageously, these pure synthetic MAP specific antigens can be produced at low cost. The use of L5P has not been validated in the contexts of ovine paratuberculosis. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests
Abstract This study was designed to develop a model of analysis of space-time dynamics in bovine ... more Abstract This study was designed to develop a model of analysis of space-time dynamics in bovine tuberculosis (bTB) possibly leading to a re-orientation of surveillance measures. A database compiling Mycobacterium bovis strains isolated between 1995 and 2006 was elaborated based on three molecular typing techniques. The classification of M. bovis strains allowed the identification of one predominant strain in Belgium. Several parameters or potential risk factors (n= 49) were tested using a multiple stepwise logistic regression. Two ...
Abstract The aims of the present study were to elaborate a model of bTB space-time dynamics analy... more Abstract The aims of the present study were to elaborate a model of bTB space-time dynamics analysis and to identify potential risk factors for the disease in order to suggest a re-orientation of surveillance measures. A database including Mycobacterium bovis strains isolated during the 1995-2006 period was compiled using three molecular typing techniques. Mycobacterium bovis strains were classified into twelve lineages (I to XII): lineage VII appeared as being predominant in the country. Several potential risk factors (n ...
Annales de la Société belge de médecine tropicale, 1986
L'influence du stress thermique a ete etudiee chez des souris consanguines C 3 H/H 2 inoculee... more L'influence du stress thermique a ete etudiee chez des souris consanguines C 3 H/H 2 inoculees par voie intraperitoneale avec 500 formes metacycliques de T.b.b. Un groupe a ete soumis a une ambiance thermique chaude (35°C), un autre groupe a ete maintenu a la temperature ambiante de laboratoire. Les souris stressees comme les non stressees montrent une forte augmentation du nombre des formes longues de T. au cours de la periode precedant la mort. Aucune difference significative n'a ete observee entre les 2 groupes en ce qui concerne la parasitemie le temps de survie moyen et le pleomorphisme parasitaire
In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) ... more In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) was screened for specific coding sequences that could be very valuable in the design of a sensitive and specific Map detection serological assay. Eighty-seven Map-specific sequences were retained. Among these, three candidate antigens have been analysed for their serodiagnostic potential. These antigens were selected on the basis of their putative immunogenicity as predicted by in silico analysis. The antigens were cloned in Escherichia coli, expressed, and purified before testing in an antibody detection ELISA test, using a well characterized panel of 18 and 48 sera from Map infected and uninfected cattle, respectively. Two of these antigens, antigen 6 and MAP1637c, yielded in our conditions a sensitivity of 72% and 82%, respectively, for a specificity of 98%. It is particularly noticeable that, when probed with the same serum panel, the most widely used European paratuberculosis commer...
This assessment aimed to elaborate a statistical nationwide model for analyzing the space-time dy... more This assessment aimed to elaborate a statistical nationwide model for analyzing the space-time dynamics of bovine tuberculosis in search of potential risk factors that could be used to better target surveillance measures. A database comprising Mycobacterium bovis molecular profiles from all isolates obtained from Belgian outbreaks during the 1995-to-2006 period ( n = 415) allowed the identification of a predominant spoligotype (SB0162). Various databases compiling 49 parameters to be tested were queried using a multiple stepwise logistic regression to assess bovine tuberculosis risk factors. Two isolate datasets were analyzed: the first included all Mycobacterium bovis isolates, while the second included only data related to the SB0162 type strain. When all Mycobacterium bovis isolates were included in the model, several risk factors were identified: history of bovine tuberculosis in the herd ( P < 0.001), proximity of an outbreak ( P < 0.001), cattle density ( P < 0.001), ...
Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivati... more Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivatives from Mycobacterium bovis and M. avium is the standard for diagnosing bovine tuberculosis. However, improved tests based on defined, specific antigens are urgently needed. In the present study, a combination of bioinformatics, molecular biology, and bovine models of infection were used to screen mycobacterial proteins for their potential as diagnostic reagents which could be used in a whole-blood assay for diagnosis of tuberculosis. Initial screening of 28 proteins selected in silico and expressed as recombinants in Escherichia coli indicated that CFP-10, ESAT-6, TB27.4, TB16.2, TB15.8, and TB10.4 induced strong gamma interferon responses in experimentally infected cattle. A more thorough investigation over time in two groups of animals infected with a high (10 6 CFU) and a low (10 4 CFU) dose of M. bovis revealed that, for both groups, the strength of the in vitro response to individ...
Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools ... more Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis, respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations. The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups, indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diag...
The characterization of protective antigens is essential for the development of an effective, sub... more The characterization of protective antigens is essential for the development of an effective, subunit-based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well-known protective antigens of Mycobacterium tuberculosis and Mycobacterium bovis. Culture filtrate, prepared from Mycobacterium avium subsp. paratuberculosis ATCC 19698 grown as a surface pellicle on synthetic Sauton medium, was strongly and early recognized in experimentally infected B6 bg/bg beige mice and cattle, as indicated by elevated spleen cell gamma interferon (IFN-γ) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-γ responses against antigen 85 (Ag85) complex (a major protein component from M. bovis BCG culture filtrate) could be detected in cattle as early as 10 weeks after oral M. avium subsp. paratuberculosis infection. Synthetic pe...
HAL (Le Centre pour la Communication Scientifique Directe), Jun 22, 2014
International audienceBackground: Unlike other MAC members, Mycobacterium avium subsp. paratuberc... more International audienceBackground: Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis does not produce the GPL on the surface of the cell wall but a lipopentapeptide called L5P or LP01. The molecular and genetic characterization of this antigen demonstrated that L5P is specific to Mycobacterium avium subsp. paratuberculosis. L5P produced by chemical synthesis was used to show that this molecule is suitable to detect Mycobacterium avium subsp. paratuberculosis infected animals. Currently available diagnostic tests are based on the use of whole cell antigen that are mainly not Mycobacterium avium subsp. paratuberculosis specific and that require a pre-absorption step with antigen of Mycobacterium phlei. These diagnostics detect animals with a late infection but are not sensitive enough to detect early infection. We hypothesize that the pre-absorption step prevents detection of informative populations of immunoglobulins, especially in animals with “asymptomatic shedding” not detected by the current serology-based diagnostics. Objective: Assess the potential of L5P and soluble variants for the serological diagnosis of Mycobacterium avium subsp. paratuberculosis infection using collections of sera from different contexts. Method: In order to find the best compound for use in serology we chemically synthesized L5P and derivatives of L5P including water-soluble forms. These pure compounds were evaluated on three collections of serum, each well characterized from infected and non-infected cattle, goats and sheep. Results: ROC analysis showed that L5P and also water-soluble derivatives are suitable for the development of a serological diagnostic test. Advantageously, these pure synthetic Mycobacterium avium subsp. paratuberculosis specific antigens can be produced at low cost
HAL (Le Centre pour la Communication Scientifique Directe), Jun 20, 2016
National audienceBackground: Unlike other MAC members, Mycobacterium avium subsp. paratuberculosi... more National audienceBackground: Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce GPL on the surface of the cell wall but a lipopentapeptide called L5P or ParaLP01. The molecular and genetic characterization of this antigen demonstrated that L5P is specific to MAP. Currently available diagnostic tests are based on the use of whole cell antigens that are mainly not MAP specific and that require a pre-absorption step with antigens of M. phlei. This diagnosis detect animals with a late stage infection but are not sensitive enough to detect early stage infection. We hypothesize that the pre-absorption step prevents detection of informative populations of immunoglobulins, especially in animals with subclinical faecal shedding of MAP that are not detected by the current serology-based diagnostics. The L5P antigen has now been used in various studies to evaluate a MAP specific sero-diagnostic. This synthetic molecule was used as a pure product, not requiring pre-absorption step. However a number of parameters must be assessed including the formulation of L5P and with reference to a panel of well defined serum samples.Objective: To assess the potential of L5P and its hydrosoluble variants for the serological diagnosis of MAP infection using collections of sera from different contexts.Method: In order to find the best compound for use in serology we chemically synthesized L5P and derivatives of L5P including water-soluble variants. These pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle, goats and sheep. In addition, the panels of sera included animals infected with M. bovis and M. avium subsp. hominissuis.Results: ROC analysis showed that L5P and also its water-soluble derivatives are suitable for the development of serological diagnosis. Advantageously, these pure synthetic MAP specific antigens can be produced at low cost. The use of L5P has not been validated in the contexts of ovine paratuberculosis. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests
Abstract This study was designed to develop a model of analysis of space-time dynamics in bovine ... more Abstract This study was designed to develop a model of analysis of space-time dynamics in bovine tuberculosis (bTB) possibly leading to a re-orientation of surveillance measures. A database compiling Mycobacterium bovis strains isolated between 1995 and 2006 was elaborated based on three molecular typing techniques. The classification of M. bovis strains allowed the identification of one predominant strain in Belgium. Several parameters or potential risk factors (n= 49) were tested using a multiple stepwise logistic regression. Two ...
Abstract The aims of the present study were to elaborate a model of bTB space-time dynamics analy... more Abstract The aims of the present study were to elaborate a model of bTB space-time dynamics analysis and to identify potential risk factors for the disease in order to suggest a re-orientation of surveillance measures. A database including Mycobacterium bovis strains isolated during the 1995-2006 period was compiled using three molecular typing techniques. Mycobacterium bovis strains were classified into twelve lineages (I to XII): lineage VII appeared as being predominant in the country. Several potential risk factors (n ...
Annales de la Société belge de médecine tropicale, 1986
L'influence du stress thermique a ete etudiee chez des souris consanguines C 3 H/H 2 inoculee... more L'influence du stress thermique a ete etudiee chez des souris consanguines C 3 H/H 2 inoculees par voie intraperitoneale avec 500 formes metacycliques de T.b.b. Un groupe a ete soumis a une ambiance thermique chaude (35°C), un autre groupe a ete maintenu a la temperature ambiante de laboratoire. Les souris stressees comme les non stressees montrent une forte augmentation du nombre des formes longues de T. au cours de la periode precedant la mort. Aucune difference significative n'a ete observee entre les 2 groupes en ce qui concerne la parasitemie le temps de survie moyen et le pleomorphisme parasitaire
In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) ... more In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) was screened for specific coding sequences that could be very valuable in the design of a sensitive and specific Map detection serological assay. Eighty-seven Map-specific sequences were retained. Among these, three candidate antigens have been analysed for their serodiagnostic potential. These antigens were selected on the basis of their putative immunogenicity as predicted by in silico analysis. The antigens were cloned in Escherichia coli, expressed, and purified before testing in an antibody detection ELISA test, using a well characterized panel of 18 and 48 sera from Map infected and uninfected cattle, respectively. Two of these antigens, antigen 6 and MAP1637c, yielded in our conditions a sensitivity of 72% and 82%, respectively, for a specificity of 98%. It is particularly noticeable that, when probed with the same serum panel, the most widely used European paratuberculosis commer...
This assessment aimed to elaborate a statistical nationwide model for analyzing the space-time dy... more This assessment aimed to elaborate a statistical nationwide model for analyzing the space-time dynamics of bovine tuberculosis in search of potential risk factors that could be used to better target surveillance measures. A database comprising Mycobacterium bovis molecular profiles from all isolates obtained from Belgian outbreaks during the 1995-to-2006 period ( n = 415) allowed the identification of a predominant spoligotype (SB0162). Various databases compiling 49 parameters to be tested were queried using a multiple stepwise logistic regression to assess bovine tuberculosis risk factors. Two isolate datasets were analyzed: the first included all Mycobacterium bovis isolates, while the second included only data related to the SB0162 type strain. When all Mycobacterium bovis isolates were included in the model, several risk factors were identified: history of bovine tuberculosis in the herd ( P < 0.001), proximity of an outbreak ( P < 0.001), cattle density ( P < 0.001), ...
Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivati... more Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivatives from Mycobacterium bovis and M. avium is the standard for diagnosing bovine tuberculosis. However, improved tests based on defined, specific antigens are urgently needed. In the present study, a combination of bioinformatics, molecular biology, and bovine models of infection were used to screen mycobacterial proteins for their potential as diagnostic reagents which could be used in a whole-blood assay for diagnosis of tuberculosis. Initial screening of 28 proteins selected in silico and expressed as recombinants in Escherichia coli indicated that CFP-10, ESAT-6, TB27.4, TB16.2, TB15.8, and TB10.4 induced strong gamma interferon responses in experimentally infected cattle. A more thorough investigation over time in two groups of animals infected with a high (10 6 CFU) and a low (10 4 CFU) dose of M. bovis revealed that, for both groups, the strength of the in vitro response to individ...
Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools ... more Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis, respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations. The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups, indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diag...
The characterization of protective antigens is essential for the development of an effective, sub... more The characterization of protective antigens is essential for the development of an effective, subunit-based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well-known protective antigens of Mycobacterium tuberculosis and Mycobacterium bovis. Culture filtrate, prepared from Mycobacterium avium subsp. paratuberculosis ATCC 19698 grown as a surface pellicle on synthetic Sauton medium, was strongly and early recognized in experimentally infected B6 bg/bg beige mice and cattle, as indicated by elevated spleen cell gamma interferon (IFN-γ) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-γ responses against antigen 85 (Ag85) complex (a major protein component from M. bovis BCG culture filtrate) could be detected in cattle as early as 10 weeks after oral M. avium subsp. paratuberculosis infection. Synthetic pe...
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