Final Report Summary - CLEANHIVE (Detecting the pathogen that threatens European honey bees)
Honeybees have two very important roles with great economic impact on the beekeeping sector: as honey producers and as pollinators of crops and wild flowers. However, in recent years, the beekeeping sector is facing a serious crisis due to the worldwide honey bee colony loss phenomenon known as colony collapse disorder (CCD) or depopulation syndrome, which is causing an alarming decrease in the number of productive hives all over Europe and other continents.
Some of the most important consequences of this phenomenon for the beekeeping sector are:
1. reduction of honey production;
2. lower number of colonies to hire for pollination;
3. increase in production costs (hives restocking becomes very difficult and expensive);
4. reduction in overall competitiveness;
5. threat to the continuity of the beekeeping sector.
Several aetiology factors and pathogens have been identified as possible causes for CCD, including the infection by nosema apis and nosema ceranae. The latter, a pathogen initially restricted to Asiatic bees and now widespread due to the international trade of bees and hive equipment, has shown to be highly virulent and to induce high colony losses. It affects beehives during the entire year (while the first one has shown to affect beehives only in spring) and its infection is frequently asymptomatic until the point of colony collapse, impeding fast treatment and the control of the disease.
Thus, correct in-field diagnosis of both nosema species is fundamental to control the disease and also to stop its spread during bee and hive equipment trade, reducing its dramatic economic impact on the beekeeping sector. This project has the purpose of improving the competitiveness of beekeeping small and medium sized enterprises (SMEs) and SME associations (SME-AGs), guaranteeing the continuity of the sector by developing an efficient, portable, cost-effective, quick and accurate system to enable detection of nosema ceranae and nosema apis specifically, which can be used directly by beekeepers in their beehives.
The proposed system combines immunochromatography and optical scanner technologies. Immunochromatography assays, or lateral flow tests, are based on the recognition of a target analyte by antibodies that react specifically with the analyte and which are recognised by a second labelled antibody producing a detectable signal (e.g. colorimetric, fluorescent). Therefore, one of the key project objectives is the development and production of specific antibodies for nosema apis and nosema ceranae suitable for use in lateral flow assays.
As antibodies are developed against the nosema apis and nosema ceranae spores, the bees have to be processed before starting the immunochromatography assay for spore isolation. Hence, also a sample collection and processing protocol suitable to be performed in field to ensure maximum sensibility of the test and minimise cross-reactivity risks is required.
Although lateral flow assays are effective diagnostic tools, they can lead to interpretation errors as typically the read out of the signal is made using the naked eye. CLEANHIVE will overcome interpretation problems by means of a low-cost optical reader. This reader will accurately read the signal generated at the lateral flow assay, removing subjective factors that cause human error. The reader is based on the use of a solid state light source, such as light emitting diode (LED) or laser diode, to excite the fluorescence emission of the labelled antibody bound to the pathogen. Such a system will provide high sensitivity to measure fluorescence emission.
This reader will also have the ability to record, store and export data to a personal computer through the development of simple data-handling software that will allow communication of the reader with a personal computer (PC). This innovative feature in the beekeeping sector will be essential for professional beekeepers, veterinarians and inspectors that have to sample and analyse large numbers of apiaries and will also be very useful during the trade of large number of hives or queens.
Project context and objectives:
Work package one (WP1) objectives:
To gain a more detailed understanding of the market needs previously identified by the consortium SME-AGs in terms of pathogen detection in the beekeeping sector as well as to define the specifications for each of the equipment components.
WP2 objectives:
To develop and characterise a panel of monoclonal and polyclonal antibodies specific for nosema apis and nosema ceranae. Antibodies are produced by injecting immunogens to mice (monoclonal antibody) or to rabbits (polyclonal antibody). The spleen cells from the best animals are taken and fused with myeloma cells. The resulting hybrids are characterised and used to produce antibodies in large quantities, which later on will be purified and characterised.
Following problems with developing antibodies to nosema apis and nosema ceranae spores purified using Percoll in period one, the objective for period two was to develop a new spore purification method and using spores purified with this new method to develop and characterise a panel of monoclonal antibodies specific for nosema apis and nosema ceranae.
WP3 objectives:
To establish bee sampling conditions and design protocols for sample collection and preparation suitable to be performed in field conditions. For this purpose, the parasitic burden in different types of samples in different organs as well as the right moment will be checked. Different methods to sacrifice and macerate bees will be tested with infected colonies. Finally, the protocols developed will be evaluated.
WP4 objectives:
To develop a lateral flow device (LFD) for the detection, quantification and discrimination of nosema apis and nosema cerana. Following the transfer of the first panel of antibodies (developed using Percoll purified spores) to a LFD format, the objective for period two was to transfer antibodies developed using Percoll free spores for nosema apis and nosema ceranae. The LFDs are developed by conjugating the antibodies developed in WP2 to latex beads and colloidal gold and spraying them onto chromatographic membrane with a range of pore sizes. The most effective combinations of membrane and antibody coated latex / gold particles will be used for producing prototype LFDs which will be validated in the field and under laboratory conditions in WP6.
WP5 objectives:
To design and develop a low-cost, robust fluorescence analyser able to detect nosema apis and nosema ceranae using a lateral-flow assay ensuring maximum sensitivity while keeping the cost of the system below EUR 300. The unit will be a portable device with a user-friendly interface and will provide the required functionality to store the information in an internal memory as well as to transfer it into an external database. For this purpose, the most appropriate optical and electronic design will be carried out, the components will be assembled and the functionality of the prototype tested and optimised.
WP6 objectives:
To integrate the different components of the system into a prototype and define the validation protocol. To validate in the laboratory the LFDs being produced in WP4. To demonstrate that the CLEANHIVE system can compete with current detection methods in terms of speed, accuracy, sensitivity and specificity. To validate the optical reader and the software. To demonstrate that the system is capable of detecting nosema under field conditions.
WP7 objectives:
To design, prepare, test and deliver comprehensive training via carefully prepared training material, in order to ensure that SME and SME-AG participants will be able to assimilate the project results. To train SME-AG members on the use of the CLEANHIVE technology.
WP8 objectives:
To ensure that non-confidential information about the CLEANHIVE project and its results are disseminated to a wide audience in order to extend the impact of the project results. All consortium members will be actively involved in carrying out dissemination activities and will contribute with their networks of contacts.
WP9 objectives:
To facilitate the take-up of results by the SME-AGs, by promoting knowledge management activities and intellectual property rights (IPR) protection, as well as defining a plan for use and dissemination of foreground (PUDF) and a business plan. This will be done to support participating SME-AGs in protecting and using the research results to their best advantage, exploit the results and increase the competitiveness of the SME-AGs and SME participants.
WP10 objectives:
To review and assess the work being carried out by optimising the application of technical resources within the project, among partners and subcontracted research and technological development (RTD) performers and to establish exploitation mechanisms. To ensure that all aspects of the European Community (EC) requirements for communication and reporting are met.
Project results:
Initially, a market study was carried out to better understand the market needs previously identified by the consortium SME-AGs in terms of bee pathogen detection. Based on this information, system specifications were outlined to allow a better adaptation of the CLEANHIVE system to the sector needs, ensuring that the developed system fully meets them (WP1).
In WP2, a panel of monoclonal antibodies specific for nosema apis and nosema ceranae was developed and characterised. After the matrix interferents (basically Percoll) found to affect the performance of the initially developed antibodies, a new spore purification method was developed and used before new immunisations were carried out for the production of a new panel of species specific monoclonal antibodies.
In WP3, the ideal bee sampling conditions for detecting nosema infections were established by determining the parasitic burden in different types of samples, times of the day and months of the year. Also sample processing protocols suitable to be performed in field were designed by testing and evaluating different methods for simplicity in terms of easiness of use and time required for beekeepers, reliability and cost.
In WP4, the nosema ceranae antibodies developed in WP2 were transferred to LFDs by binding them to membranes as well as to latex and colloidal gold. These were tested using selected protein extracts produced from NC-infected, NA-infected and nosema-free bees. The first prototypes produced were optimised and the resulting prototype LFDs were subjected to laboratory validation. In this WP, also the final format for the LFD was evaluated and methods to enable efficient testing of bee material were developed and optimised.
In WP5, the portable, easy to use, low-cost CLEANHIVE fluorescent reader to accurately read the signal generated at the lateral flow assay as well as the simple data-handling software that allows to record, store and export data from an internal memory to a personal computer were fully developed and optimised.
All efforts on WP6 have been devoted to the system integration and validation protocol, the laboratory validation and the validation of the optical reader and software. During the laboratory validation, no correlation was observed between the presence of nosema in the samples and the positive results by LFD. This led to the conclusion that the specificity of the monoclonal antibodies was altered by binding them in a solid phase and transferring them into the LFD format. Significant efforts were dedicated to improve the performance of those antibodies on the LFDs, but unfortunately these were not suitable enough to proceed with the in field / industrial validation.
Training (WP7) to consortium SME and SME-AGs as well as to the SME-AG members was successfully achieved according to a training plan using different types of training materials created during the project life.
As part of the dissemination activities (WP8), a project website was created to serve as vehicle of dissemination of the project and its results. Other dissemination material has been prepared (several posters, a banner, a project leaflet, a PC multimedia guide, etc.) and presented in all the dissemination events to which different partners have assisted to.
Regarding preparation for exploitation activities (WP9), a knowledge management seminar was given, technology and market watch activities were performed regularly. Several exploitation meetings were held in which the strategy and actions required to protect and exploit the foreground derived from the project was discussed. The outcome of these discussions and the initial agreed exploitation strategy are reflected in the final PUDF.
Regarding the consortium management activities, monitoring of project progress and achievements has been done regularly by the project coordinator and through the general project meetings. CRIC has ensured that all milestones are met and all reports are prepared in accordance with contractual requirements.
Potential impact:
The CLEANHIVE system consists of two parts:
1. a LFD in which the target analyte (both nosema spores involved in the beehive depopulation syndrome) will be recognised by antibodies that react specifically with them;
2. a portable, cost-effective, easy to use optical reader, which will help beekeepers to accurately interpret the results generated by the LFD.
The main features of this tool are speed, specificity, sensitivity, accurateness, portability, inexpensive, easy to use by non-technical staff.
The proposed solution is highly relevant not only for consortium members but also for the beekeeping sector as a whole, as with the development of this technology:
1. the beekeeping sector will increase its productivity and profitability by reducing losses caused by direct disease impact;
2. competitiveness will improve by reducing the time and costs required for the treatment of bee colonies and by producing a high-quality and healthy product;
3. continuity of the beekeeping sector will be guaranteed which will have an impact on both job preservation and creation as well as on the development of European rural areas.
For this reason, dissemination actions were strategically planned to ensure market penetration. Besides, the European Union (EU) wide dissemination and utilisation of the proposed product would allow advancing in the control of this epidemic disease, which depends on the systematic and collective implementation of hive management measures.
Even though not all project objectives were fully met during the project life, significant technical progress was made and there is a real prospect of having tests available for nosema ceranea and nosema apis. For that reason and considering the commercial potential of CLEANHIVE, some of the consortium partners plan to continue working in the development / validation of such tests and collectively share the costs of bringing those tests to the market.
On the other hand, the developed portable, cost-effective, easy to use optical reader is considered to have a significant potential market, not only within the beekeeping sector for the detection of both nosema species involved in the depopulation syndrome, but for a wide range of applications.
Project website:
http://www.cleanhive.eu
Contact:
irene.gonzalez@cric.cat
Some of the most important consequences of this phenomenon for the beekeeping sector are:
1. reduction of honey production;
2. lower number of colonies to hire for pollination;
3. increase in production costs (hives restocking becomes very difficult and expensive);
4. reduction in overall competitiveness;
5. threat to the continuity of the beekeeping sector.
Several aetiology factors and pathogens have been identified as possible causes for CCD, including the infection by nosema apis and nosema ceranae. The latter, a pathogen initially restricted to Asiatic bees and now widespread due to the international trade of bees and hive equipment, has shown to be highly virulent and to induce high colony losses. It affects beehives during the entire year (while the first one has shown to affect beehives only in spring) and its infection is frequently asymptomatic until the point of colony collapse, impeding fast treatment and the control of the disease.
Thus, correct in-field diagnosis of both nosema species is fundamental to control the disease and also to stop its spread during bee and hive equipment trade, reducing its dramatic economic impact on the beekeeping sector. This project has the purpose of improving the competitiveness of beekeeping small and medium sized enterprises (SMEs) and SME associations (SME-AGs), guaranteeing the continuity of the sector by developing an efficient, portable, cost-effective, quick and accurate system to enable detection of nosema ceranae and nosema apis specifically, which can be used directly by beekeepers in their beehives.
The proposed system combines immunochromatography and optical scanner technologies. Immunochromatography assays, or lateral flow tests, are based on the recognition of a target analyte by antibodies that react specifically with the analyte and which are recognised by a second labelled antibody producing a detectable signal (e.g. colorimetric, fluorescent). Therefore, one of the key project objectives is the development and production of specific antibodies for nosema apis and nosema ceranae suitable for use in lateral flow assays.
As antibodies are developed against the nosema apis and nosema ceranae spores, the bees have to be processed before starting the immunochromatography assay for spore isolation. Hence, also a sample collection and processing protocol suitable to be performed in field to ensure maximum sensibility of the test and minimise cross-reactivity risks is required.
Although lateral flow assays are effective diagnostic tools, they can lead to interpretation errors as typically the read out of the signal is made using the naked eye. CLEANHIVE will overcome interpretation problems by means of a low-cost optical reader. This reader will accurately read the signal generated at the lateral flow assay, removing subjective factors that cause human error. The reader is based on the use of a solid state light source, such as light emitting diode (LED) or laser diode, to excite the fluorescence emission of the labelled antibody bound to the pathogen. Such a system will provide high sensitivity to measure fluorescence emission.
This reader will also have the ability to record, store and export data to a personal computer through the development of simple data-handling software that will allow communication of the reader with a personal computer (PC). This innovative feature in the beekeeping sector will be essential for professional beekeepers, veterinarians and inspectors that have to sample and analyse large numbers of apiaries and will also be very useful during the trade of large number of hives or queens.
Project context and objectives:
Work package one (WP1) objectives:
To gain a more detailed understanding of the market needs previously identified by the consortium SME-AGs in terms of pathogen detection in the beekeeping sector as well as to define the specifications for each of the equipment components.
WP2 objectives:
To develop and characterise a panel of monoclonal and polyclonal antibodies specific for nosema apis and nosema ceranae. Antibodies are produced by injecting immunogens to mice (monoclonal antibody) or to rabbits (polyclonal antibody). The spleen cells from the best animals are taken and fused with myeloma cells. The resulting hybrids are characterised and used to produce antibodies in large quantities, which later on will be purified and characterised.
Following problems with developing antibodies to nosema apis and nosema ceranae spores purified using Percoll in period one, the objective for period two was to develop a new spore purification method and using spores purified with this new method to develop and characterise a panel of monoclonal antibodies specific for nosema apis and nosema ceranae.
WP3 objectives:
To establish bee sampling conditions and design protocols for sample collection and preparation suitable to be performed in field conditions. For this purpose, the parasitic burden in different types of samples in different organs as well as the right moment will be checked. Different methods to sacrifice and macerate bees will be tested with infected colonies. Finally, the protocols developed will be evaluated.
WP4 objectives:
To develop a lateral flow device (LFD) for the detection, quantification and discrimination of nosema apis and nosema cerana. Following the transfer of the first panel of antibodies (developed using Percoll purified spores) to a LFD format, the objective for period two was to transfer antibodies developed using Percoll free spores for nosema apis and nosema ceranae. The LFDs are developed by conjugating the antibodies developed in WP2 to latex beads and colloidal gold and spraying them onto chromatographic membrane with a range of pore sizes. The most effective combinations of membrane and antibody coated latex / gold particles will be used for producing prototype LFDs which will be validated in the field and under laboratory conditions in WP6.
WP5 objectives:
To design and develop a low-cost, robust fluorescence analyser able to detect nosema apis and nosema ceranae using a lateral-flow assay ensuring maximum sensitivity while keeping the cost of the system below EUR 300. The unit will be a portable device with a user-friendly interface and will provide the required functionality to store the information in an internal memory as well as to transfer it into an external database. For this purpose, the most appropriate optical and electronic design will be carried out, the components will be assembled and the functionality of the prototype tested and optimised.
WP6 objectives:
To integrate the different components of the system into a prototype and define the validation protocol. To validate in the laboratory the LFDs being produced in WP4. To demonstrate that the CLEANHIVE system can compete with current detection methods in terms of speed, accuracy, sensitivity and specificity. To validate the optical reader and the software. To demonstrate that the system is capable of detecting nosema under field conditions.
WP7 objectives:
To design, prepare, test and deliver comprehensive training via carefully prepared training material, in order to ensure that SME and SME-AG participants will be able to assimilate the project results. To train SME-AG members on the use of the CLEANHIVE technology.
WP8 objectives:
To ensure that non-confidential information about the CLEANHIVE project and its results are disseminated to a wide audience in order to extend the impact of the project results. All consortium members will be actively involved in carrying out dissemination activities and will contribute with their networks of contacts.
WP9 objectives:
To facilitate the take-up of results by the SME-AGs, by promoting knowledge management activities and intellectual property rights (IPR) protection, as well as defining a plan for use and dissemination of foreground (PUDF) and a business plan. This will be done to support participating SME-AGs in protecting and using the research results to their best advantage, exploit the results and increase the competitiveness of the SME-AGs and SME participants.
WP10 objectives:
To review and assess the work being carried out by optimising the application of technical resources within the project, among partners and subcontracted research and technological development (RTD) performers and to establish exploitation mechanisms. To ensure that all aspects of the European Community (EC) requirements for communication and reporting are met.
Project results:
Initially, a market study was carried out to better understand the market needs previously identified by the consortium SME-AGs in terms of bee pathogen detection. Based on this information, system specifications were outlined to allow a better adaptation of the CLEANHIVE system to the sector needs, ensuring that the developed system fully meets them (WP1).
In WP2, a panel of monoclonal antibodies specific for nosema apis and nosema ceranae was developed and characterised. After the matrix interferents (basically Percoll) found to affect the performance of the initially developed antibodies, a new spore purification method was developed and used before new immunisations were carried out for the production of a new panel of species specific monoclonal antibodies.
In WP3, the ideal bee sampling conditions for detecting nosema infections were established by determining the parasitic burden in different types of samples, times of the day and months of the year. Also sample processing protocols suitable to be performed in field were designed by testing and evaluating different methods for simplicity in terms of easiness of use and time required for beekeepers, reliability and cost.
In WP4, the nosema ceranae antibodies developed in WP2 were transferred to LFDs by binding them to membranes as well as to latex and colloidal gold. These were tested using selected protein extracts produced from NC-infected, NA-infected and nosema-free bees. The first prototypes produced were optimised and the resulting prototype LFDs were subjected to laboratory validation. In this WP, also the final format for the LFD was evaluated and methods to enable efficient testing of bee material were developed and optimised.
In WP5, the portable, easy to use, low-cost CLEANHIVE fluorescent reader to accurately read the signal generated at the lateral flow assay as well as the simple data-handling software that allows to record, store and export data from an internal memory to a personal computer were fully developed and optimised.
All efforts on WP6 have been devoted to the system integration and validation protocol, the laboratory validation and the validation of the optical reader and software. During the laboratory validation, no correlation was observed between the presence of nosema in the samples and the positive results by LFD. This led to the conclusion that the specificity of the monoclonal antibodies was altered by binding them in a solid phase and transferring them into the LFD format. Significant efforts were dedicated to improve the performance of those antibodies on the LFDs, but unfortunately these were not suitable enough to proceed with the in field / industrial validation.
Training (WP7) to consortium SME and SME-AGs as well as to the SME-AG members was successfully achieved according to a training plan using different types of training materials created during the project life.
As part of the dissemination activities (WP8), a project website was created to serve as vehicle of dissemination of the project and its results. Other dissemination material has been prepared (several posters, a banner, a project leaflet, a PC multimedia guide, etc.) and presented in all the dissemination events to which different partners have assisted to.
Regarding preparation for exploitation activities (WP9), a knowledge management seminar was given, technology and market watch activities were performed regularly. Several exploitation meetings were held in which the strategy and actions required to protect and exploit the foreground derived from the project was discussed. The outcome of these discussions and the initial agreed exploitation strategy are reflected in the final PUDF.
Regarding the consortium management activities, monitoring of project progress and achievements has been done regularly by the project coordinator and through the general project meetings. CRIC has ensured that all milestones are met and all reports are prepared in accordance with contractual requirements.
Potential impact:
The CLEANHIVE system consists of two parts:
1. a LFD in which the target analyte (both nosema spores involved in the beehive depopulation syndrome) will be recognised by antibodies that react specifically with them;
2. a portable, cost-effective, easy to use optical reader, which will help beekeepers to accurately interpret the results generated by the LFD.
The main features of this tool are speed, specificity, sensitivity, accurateness, portability, inexpensive, easy to use by non-technical staff.
The proposed solution is highly relevant not only for consortium members but also for the beekeeping sector as a whole, as with the development of this technology:
1. the beekeeping sector will increase its productivity and profitability by reducing losses caused by direct disease impact;
2. competitiveness will improve by reducing the time and costs required for the treatment of bee colonies and by producing a high-quality and healthy product;
3. continuity of the beekeeping sector will be guaranteed which will have an impact on both job preservation and creation as well as on the development of European rural areas.
For this reason, dissemination actions were strategically planned to ensure market penetration. Besides, the European Union (EU) wide dissemination and utilisation of the proposed product would allow advancing in the control of this epidemic disease, which depends on the systematic and collective implementation of hive management measures.
Even though not all project objectives were fully met during the project life, significant technical progress was made and there is a real prospect of having tests available for nosema ceranea and nosema apis. For that reason and considering the commercial potential of CLEANHIVE, some of the consortium partners plan to continue working in the development / validation of such tests and collectively share the costs of bringing those tests to the market.
On the other hand, the developed portable, cost-effective, easy to use optical reader is considered to have a significant potential market, not only within the beekeeping sector for the detection of both nosema species involved in the depopulation syndrome, but for a wide range of applications.
Project website:
http://www.cleanhive.eu
Contact:
irene.gonzalez@cric.cat