Downloaded from http://veterinaryrecord.bmj.com/ on June 19, 2015 - Published by group.bmj.com
Research
Research EDITORIAL
Detecting mycobacteria in cattle blood Benjamin M. C. Swift, Catherine E. D. Rees The standard tests used to identify bovine TB in cattle rely on monitoring the immune response as an indicator of infection. While the skin test provides a simple and costeffective assay, it cannot differentiate between infected animals and those that have been vaccinated against infection. In countries where the disease is endemic, vaccination – of both cattle and potential wildlife reservoirs – is considered to be the best long-term strategy to reduce the threat of bovine TB. However, introducing routine vaccination negates the value of the current standard diagnostic tests. There is therefore a real need for new tests that can differentiate between naturally infected and vaccinated animals (termed DIVA). One approach would be to directly detect Mycobacterium bovis, the main causative agent of bovine TB, in samples from infected animals. While this approach can be used for many bacterial infections, it is problematic when working with mycobacteria. The slow growth of some pathogenic mycobacteria makes detection by traditional culture extremely difficult. For instance, culture results for M bovis can take up to eight weeks. Similarly, Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne’s disease, can take up to 16 weeks to culture. Even rapid, automated culture methods for bovine TB take up to 15 days. The lengthy incubation times and poor levels of sensitivity achieved when culturing mycobacteria from blood limits the diagnostic power of this method and it has not been used for many years.
Benjamin M. C. Swift, BSc (Hons), MRes, Catherine E. D. Rees, BA (Oxon), PhD University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK e-mail: [email protected]
522 | Veterinary Record | November 30, 2013
Molecular methods are often used to detect bacterial DNA as an alternative to culture. Blood assays based on PCR amplification of genomic signature sequences from Mycobacterium have been described, but these do not differentiate between living and dead cells. When trying to confirm infection, it is important that only viable cells are detected rather than residual DNA from cells that have been inactivated, either by the host immune system or by treatment. Unfortunately, PCR-based bovine TB detection methods have been found to be limited both by specificity and sensitivity (Parra and others 2008). These difficulties have led to the use of the intradermal skin test as the standard method of identifying bovine TB-infected cattle. The sensitivity of the skin test is known to be highly variable and results are affected by factors such as the stage and severity of disease and cross-reactions to other mycobacterial infections. Hence a positive skin test always requires further tests to confirm the diagnosis; this may be visible lesions in the carcase or culture of lymph node material. Interferon-γ tests can also be used to confirm infection; however, false-positives can occur when animals are infected with other pathogenic mycobacteria and, again, this test does not differentiate between infected and vaccinated animals. The licensed vaccine for both human and bovine TB is Bacillus Calmette-Guérin (BCG), an attenuated strain of M bovis, but the level of protection achieved is variable (Hope and Vordermeier 2005). More importantly, use of the BCG vaccine in cattle is incompatible with the current diagnostic tests for bovine TB. Research is being undertaken to identify recombinant vaccines based on specific antigens that
Downloaded from http://veterinaryrecord.bmj.com/ on June 19, 2015 - Published by group.bmj.com
Research prevent cross-reaction with the skin test, but this is in its early stages (Whelan and others 2010). PCR-based methods have been described that can differentiate between M bovis and BCG cells (Huard and others 2003), but their usefulness in the clinical setting is also limited by issues of sensitivity and an inability to differentiate between living and dead cells.
Bacteriophage-based detection Bacteriophages are viruses that infect bacterial cells. They have a specific host range and will only replicate within a viable cell. These features have been exploited for the development of many bacteriophagebased detection methods (Monk and others 2010). A commercial phage-based detection platform has been developed for the detection of M tuberculosis in sputum samples as a diagnostic test in people (Albert and others 2002). For the past six years we have been working on new applications of this assay, specifically focusing on methods to detect and enumerate viable MAP in milk and bovine blood samples (the RapidMAP assay). This has led to a range of assay formats that can report on the presence of viable mycobacterial cells in a sample within two days (Stanley and others 2007, Botsaris and others 2010, Swift and others 2013). Rather than waiting for the growth of the mycobacterial cells, the assay monitors the replication of the bacteriophage in a viable host cell. The identity of the cell detected is then confirmed by PCR (Fig 1). This can be done as a species-specific test (Swift and others 2013) or multiplex PCR assays have been developed that will simultaneously report on the presence of a range of different pathogenic mycobacteria (bovine TB and MAP) (Stanley and others 2007). These phage-based assays are low cost and do not need investment in specialist equipment or expensive reagents, and results are available within 48 hours. RapidMAP uses mycobacteriophage D29 that has a broad host range within the Mycobacterium genus, including the faster growing non-pathogens. The assay has been successfully used to detect viable MAP in the blood of cattle with Johne’s disease (Swift and others 2013). Results show that it is able to reproducibly detect low numbers of viable cells with better sensitivity than direct PCR and it even detected bacteria in the early stages of infection in blood ELISA-negative animals. Having developed a method to identify viable mycobacteria in blood, this opens up the possibility of creating a bovine TB DIVA test using the PCR step to distinguish between wildtype bovine TB and BCG, or other vaccine strains. There is a good prospect that the assay can be applied to sensitively detect low levels of bovine TB in blood samples and hence the development of a rapid bacteriophage-based DIVA test that differentiates between viable and non-viable
cells is a real possibility. In its current format, the RapidMAP test is labour intensive; the need to incubate plates determines the test time and is not readily automatable so the existing technology would be challenging if applied on a national herd level. Recently we have patented a tube format that is able to detect viable mycobacteria within eight hours which removes the need for agar plates and could be automated. The method retains the advantages of the RapidMAP technology in that it still detects only viable cells and incorporates a species-specific PCR to identify the cell detected. We are now starting to further develop this technology, not only as a potential DIVA test but as a method to rapidly detect and enumerate viable mycobacteria in a range of samples, from blood to milk (Rees and Botsaris 2012). This technology therefore has potential to be applied in a number of ways to facilitate future bovine TB research, including significantly shortening the period required for confirmatory culture of samples from positive skin test animals.
References
ALBERT, H., HEYDENRYCH, A., BROOKES, R., MOLE, R. J., HARLEY, B., SUBOTSKY, E., HENRY, R. & AZEVEDO, V. (2002) Performance of a rapid phagebased test, FASTPlaqueTB, to diagnose pulmonary tuberculosis from sputum specimens in South Africa. International Journal of Tuberculosis and Lung Disease 6, 529-37 BOTSARIS, G., SLANA, I., LIAPI, M., DODD, C., ECONOMIDES, C., REES, C. & PAVLIK, I. (2010) Rapid detection methods for viable Mycobacterium avium subspecies paratuberculosis in milk and cheese. International Journal of Food Microbiology 141, S87-S90 HOPE, J. C. & VORDERMEIER, H. M. (2005) Vaccines for bovine tuberculosis: current views and future prospects. Expert Review of Vaccines 4, 891-902 HUARD, R. C., LAZZARINI, L. C. D., BUTLER, W. R., VAN SOOLINGEN, D. & HO, J. L. (2003) PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions. Journal of Clinical Microbiology 41, 1637-1650 MONK, A. B., REES, C. D., BARROW, P., HAGENS, S. & HARPER, D. R. (2010) Bacteriophage applications: where are we now? Letters in Applied Microbiology 51, 363-9 PARRA, A., GARCIA, N., GARCIA, A., LACOMBE, A., MORENO, F., FREIRE, F., MORAN, J. & DE MENDOZA, J. H. (2008) Development of a molecular diagnostic test applied to experimental abattoir surveil-
FIG 1: Assay for detecting viable MAP in milk and bovine blood. Specimens that may contain mycobacteria (1) are mixed with phage D29 for one hour (2) to allow the virus to infect its target cells. Any extracellular phage that have not infected a host cell are chemically inactivated using a virucide (3) that does not affect the viability of mycobacterial cells in the sample. Only phage that have infected their host are protected from the virucide and continue to replicate (4). The new phage released from these infected target cells are then detected using a plaque assay. This uses a fast growing nonpathogenic mycobacteria (M smegmatis) that phage D29 also infects. Zones of lysis in the bacterial lawn (or plaques) (5, 6) indicate the position of one original phage-infected Mycobacterium in the test sample. The number of mycobacteria in the specimen is represented by the number of plaques formed. The time it takes from blood collection to plaques on a plate is 24 hours. To determine the identity of these cells, DNA is extracted from the centre of the plaque (6) and PCR is used to amplify diagnostic signature sequences present in the mycobacterial genome (7) lance on bovine tuberculosis. Veterinary Microbiology 127, 315-324 REES, C. R. & BOTSARIS, G. (2012) The Use of phage for detection, antibiotic sensitivity testing and enumeration. In Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis. Ed P. J. Cardona. Rijeka: InTech STANLEY, E. C., MOLE, R. J., SMITH, R. J., GLENN, S. M., BARER, M. R., MCGOWAN, M. & REES, C. E. D. (2007) Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours. Applied and Environmental Microbiology 73, 1851-1857 SWIFT, B. M., DENTON, E. J., MAHENDRAN, S. A., HUXLEY, J. N. & REES, C. E. (2013) Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp paratuberculosis in blood within 48 h. Journal of Microbiological Methods 94, 175-179 WHELAN, A. O., CLIFFORD, D., UPADHYAY, B., BREADON, E. L., MCNAIR, J., HEWINSON, G. R. & VORDERMEIER, M. H. (2010) Development of a skin test for bovine tuberculosis for differentiating infected from vaccinated animals. Journal of Clinical Microbiology 48, 3176-81
doi: 10.1136/vr.f7067 November 30, 2013 | Veterinary Record | 523
Downloaded from http://veterinaryrecord.bmj.com/ on June 19, 2015 - Published by group.bmj.com
Detecting mycobacteria in cattle blood Benjamin M. C. Swift and Catherine E. D. Rees Veterinary Record 2013 173: 522-523
doi: 10.1136/vr.f7067 Updated information and services can be found at: http://veterinaryrecord.bmj.com/content/173/21/522
These include:
References Email alerting service
This article cites 9 articles, 3 of which you can access for free at: http://veterinaryrecord.bmj.com/content/173/21/522#BIBL Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article.
Notes
To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://group.bmj.com/subscribe/
Research
Research EDITORIAL
Detecting mycobacteria in cattle blood Benjamin M. C. Swift, Catherine E. D. Rees The standard tests used to identify bovine TB in cattle rely on monitoring the immune response as an indicator of infection. While the skin test provides a simple and costeffective assay, it cannot differentiate between infected animals and those that have been vaccinated against infection. In countries where the disease is endemic, vaccination – of both cattle and potential wildlife reservoirs – is considered to be the best long-term strategy to reduce the threat of bovine TB. However, introducing routine vaccination negates the value of the current standard diagnostic tests. There is therefore a real need for new tests that can differentiate between naturally infected and vaccinated animals (termed DIVA). One approach would be to directly detect Mycobacterium bovis, the main causative agent of bovine TB, in samples from infected animals. While this approach can be used for many bacterial infections, it is problematic when working with mycobacteria. The slow growth of some pathogenic mycobacteria makes detection by traditional culture extremely difficult. For instance, culture results for M bovis can take up to eight weeks. Similarly, Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne’s disease, can take up to 16 weeks to culture. Even rapid, automated culture methods for bovine TB take up to 15 days. The lengthy incubation times and poor levels of sensitivity achieved when culturing mycobacteria from blood limits the diagnostic power of this method and it has not been used for many years.
Benjamin M. C. Swift, BSc (Hons), MRes, Catherine E. D. Rees, BA (Oxon), PhD University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK e-mail: [email protected]
522 | Veterinary Record | November 30, 2013
Molecular methods are often used to detect bacterial DNA as an alternative to culture. Blood assays based on PCR amplification of genomic signature sequences from Mycobacterium have been described, but these do not differentiate between living and dead cells. When trying to confirm infection, it is important that only viable cells are detected rather than residual DNA from cells that have been inactivated, either by the host immune system or by treatment. Unfortunately, PCR-based bovine TB detection methods have been found to be limited both by specificity and sensitivity (Parra and others 2008). These difficulties have led to the use of the intradermal skin test as the standard method of identifying bovine TB-infected cattle. The sensitivity of the skin test is known to be highly variable and results are affected by factors such as the stage and severity of disease and cross-reactions to other mycobacterial infections. Hence a positive skin test always requires further tests to confirm the diagnosis; this may be visible lesions in the carcase or culture of lymph node material. Interferon-γ tests can also be used to confirm infection; however, false-positives can occur when animals are infected with other pathogenic mycobacteria and, again, this test does not differentiate between infected and vaccinated animals. The licensed vaccine for both human and bovine TB is Bacillus Calmette-Guérin (BCG), an attenuated strain of M bovis, but the level of protection achieved is variable (Hope and Vordermeier 2005). More importantly, use of the BCG vaccine in cattle is incompatible with the current diagnostic tests for bovine TB. Research is being undertaken to identify recombinant vaccines based on specific antigens that
Downloaded from http://veterinaryrecord.bmj.com/ on June 19, 2015 - Published by group.bmj.com
Research prevent cross-reaction with the skin test, but this is in its early stages (Whelan and others 2010). PCR-based methods have been described that can differentiate between M bovis and BCG cells (Huard and others 2003), but their usefulness in the clinical setting is also limited by issues of sensitivity and an inability to differentiate between living and dead cells.
Bacteriophage-based detection Bacteriophages are viruses that infect bacterial cells. They have a specific host range and will only replicate within a viable cell. These features have been exploited for the development of many bacteriophagebased detection methods (Monk and others 2010). A commercial phage-based detection platform has been developed for the detection of M tuberculosis in sputum samples as a diagnostic test in people (Albert and others 2002). For the past six years we have been working on new applications of this assay, specifically focusing on methods to detect and enumerate viable MAP in milk and bovine blood samples (the RapidMAP assay). This has led to a range of assay formats that can report on the presence of viable mycobacterial cells in a sample within two days (Stanley and others 2007, Botsaris and others 2010, Swift and others 2013). Rather than waiting for the growth of the mycobacterial cells, the assay monitors the replication of the bacteriophage in a viable host cell. The identity of the cell detected is then confirmed by PCR (Fig 1). This can be done as a species-specific test (Swift and others 2013) or multiplex PCR assays have been developed that will simultaneously report on the presence of a range of different pathogenic mycobacteria (bovine TB and MAP) (Stanley and others 2007). These phage-based assays are low cost and do not need investment in specialist equipment or expensive reagents, and results are available within 48 hours. RapidMAP uses mycobacteriophage D29 that has a broad host range within the Mycobacterium genus, including the faster growing non-pathogens. The assay has been successfully used to detect viable MAP in the blood of cattle with Johne’s disease (Swift and others 2013). Results show that it is able to reproducibly detect low numbers of viable cells with better sensitivity than direct PCR and it even detected bacteria in the early stages of infection in blood ELISA-negative animals. Having developed a method to identify viable mycobacteria in blood, this opens up the possibility of creating a bovine TB DIVA test using the PCR step to distinguish between wildtype bovine TB and BCG, or other vaccine strains. There is a good prospect that the assay can be applied to sensitively detect low levels of bovine TB in blood samples and hence the development of a rapid bacteriophage-based DIVA test that differentiates between viable and non-viable
cells is a real possibility. In its current format, the RapidMAP test is labour intensive; the need to incubate plates determines the test time and is not readily automatable so the existing technology would be challenging if applied on a national herd level. Recently we have patented a tube format that is able to detect viable mycobacteria within eight hours which removes the need for agar plates and could be automated. The method retains the advantages of the RapidMAP technology in that it still detects only viable cells and incorporates a species-specific PCR to identify the cell detected. We are now starting to further develop this technology, not only as a potential DIVA test but as a method to rapidly detect and enumerate viable mycobacteria in a range of samples, from blood to milk (Rees and Botsaris 2012). This technology therefore has potential to be applied in a number of ways to facilitate future bovine TB research, including significantly shortening the period required for confirmatory culture of samples from positive skin test animals.
References
ALBERT, H., HEYDENRYCH, A., BROOKES, R., MOLE, R. J., HARLEY, B., SUBOTSKY, E., HENRY, R. & AZEVEDO, V. (2002) Performance of a rapid phagebased test, FASTPlaqueTB, to diagnose pulmonary tuberculosis from sputum specimens in South Africa. International Journal of Tuberculosis and Lung Disease 6, 529-37 BOTSARIS, G., SLANA, I., LIAPI, M., DODD, C., ECONOMIDES, C., REES, C. & PAVLIK, I. (2010) Rapid detection methods for viable Mycobacterium avium subspecies paratuberculosis in milk and cheese. International Journal of Food Microbiology 141, S87-S90 HOPE, J. C. & VORDERMEIER, H. M. (2005) Vaccines for bovine tuberculosis: current views and future prospects. Expert Review of Vaccines 4, 891-902 HUARD, R. C., LAZZARINI, L. C. D., BUTLER, W. R., VAN SOOLINGEN, D. & HO, J. L. (2003) PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions. Journal of Clinical Microbiology 41, 1637-1650 MONK, A. B., REES, C. D., BARROW, P., HAGENS, S. & HARPER, D. R. (2010) Bacteriophage applications: where are we now? Letters in Applied Microbiology 51, 363-9 PARRA, A., GARCIA, N., GARCIA, A., LACOMBE, A., MORENO, F., FREIRE, F., MORAN, J. & DE MENDOZA, J. H. (2008) Development of a molecular diagnostic test applied to experimental abattoir surveil-
FIG 1: Assay for detecting viable MAP in milk and bovine blood. Specimens that may contain mycobacteria (1) are mixed with phage D29 for one hour (2) to allow the virus to infect its target cells. Any extracellular phage that have not infected a host cell are chemically inactivated using a virucide (3) that does not affect the viability of mycobacterial cells in the sample. Only phage that have infected their host are protected from the virucide and continue to replicate (4). The new phage released from these infected target cells are then detected using a plaque assay. This uses a fast growing nonpathogenic mycobacteria (M smegmatis) that phage D29 also infects. Zones of lysis in the bacterial lawn (or plaques) (5, 6) indicate the position of one original phage-infected Mycobacterium in the test sample. The number of mycobacteria in the specimen is represented by the number of plaques formed. The time it takes from blood collection to plaques on a plate is 24 hours. To determine the identity of these cells, DNA is extracted from the centre of the plaque (6) and PCR is used to amplify diagnostic signature sequences present in the mycobacterial genome (7) lance on bovine tuberculosis. Veterinary Microbiology 127, 315-324 REES, C. R. & BOTSARIS, G. (2012) The Use of phage for detection, antibiotic sensitivity testing and enumeration. In Understanding Tuberculosis – Global Experiences and Innovative Approaches to the Diagnosis. Ed P. J. Cardona. Rijeka: InTech STANLEY, E. C., MOLE, R. J., SMITH, R. J., GLENN, S. M., BARER, M. R., MCGOWAN, M. & REES, C. E. D. (2007) Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours. Applied and Environmental Microbiology 73, 1851-1857 SWIFT, B. M., DENTON, E. J., MAHENDRAN, S. A., HUXLEY, J. N. & REES, C. E. (2013) Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp paratuberculosis in blood within 48 h. Journal of Microbiological Methods 94, 175-179 WHELAN, A. O., CLIFFORD, D., UPADHYAY, B., BREADON, E. L., MCNAIR, J., HEWINSON, G. R. & VORDERMEIER, M. H. (2010) Development of a skin test for bovine tuberculosis for differentiating infected from vaccinated animals. Journal of Clinical Microbiology 48, 3176-81
doi: 10.1136/vr.f7067 November 30, 2013 | Veterinary Record | 523
Downloaded from http://veterinaryrecord.bmj.com/ on June 19, 2015 - Published by group.bmj.com
Detecting mycobacteria in cattle blood Benjamin M. C. Swift and Catherine E. D. Rees Veterinary Record 2013 173: 522-523
doi: 10.1136/vr.f7067 Updated information and services can be found at: http://veterinaryrecord.bmj.com/content/173/21/522
These include:
References Email alerting service
This article cites 9 articles, 3 of which you can access for free at: http://veterinaryrecord.bmj.com/content/173/21/522#BIBL Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article.
Notes
To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://group.bmj.com/subscribe/
