14-ATC2 - Vaccination based control of fasciolosis in farmed ruminants (TSB App # 45264-298219)
Lead Research Organisation:
University of Nottingham
Department Name: School of Veterinary Medicine and Sci
Abstract
F. hepatica is the cause of fasciolosis, an economically important parasitic disease of cattle and sheep which is the cause of chronic liver infection in an estimated 700 million animals worldwide. Infection results in an estimated annual global loss of US$2000 million through livestock mortality, reductions in productivity and condemnation of animal products that should go into the human food chain. Costs of this infection to UK industry is estimated to be around £14M/annum in beef cows, £13.5M/annum in dairy cattle and £3.1M/annum in sheep, although these are most certainly conservative and relate to
direct losses only. The proposed project is a pre-industrial research project that will enable us to validate a collection of important components of F. hepatica that could be combined into sub-unit vaccine formulations for protecting ruminants against infection with this pathogen. Infection by F. hepatica is acquired by the ingestion of water or vegetation that is contaminated by parasitic metacercariae which excyst to produce newly excysted juveniles (NEJs) , these migrate across the intestine and peritoneal cavity to the liver and develop into sexually mature hermaphrodite adults. The adults release eggs into the bile ducts, which are transported to the intestine and are released into the environment via faeces. These parasites cause reduced milk yields, poor fertility and high perinatal loses, and chronic weight loss in cattle. Acute disease will cause sudden death from haemorrhage and liver damage in sheep. Control of fluke infection is heavily reliant on a limited number of anthelmintic compounds, which due to their widespread and often indiscriminate use has led to problems associated with drug resistance. There are also emerging concerns regarding the use of these drugs in food production animals, with recent legislation banning the use of four major classes of these drugs in dairy animals where milk is destined for the food chain. The most viable alternative to drug treatment would be vaccination against Fasciolosis. Currently no vaccine preparation is commercially available to control F. hepatica. The delay in bringing a commercial vaccine to market is a result of the complex lifecycle of the parasite, its large genome and complex biology, and the strategies it employs to evade host immunity. F. hepatica is well known to alter the host immune responses thereby deflecting attacks aimed at it which could damage and eventually kill the parasite. These strategies employ a number of molecules termed immunomodulators. Furthermore, the naturally occurring immune responses against this parasite often provide limited protection to the host. We now know that full protection will be offered by the two distinct arms of the immune system the antibody and the cellular arms. This project aims to characterise i) both the antibody and cellular immune response to this organism in animals that show variation in levels of fluke burdens and ii) a novel set of recently identified Immunomodulator proteins, produced by F. hepatica that are likely to suppress the host immune response. Aim i) will be conducted by a selection method exploiting the ability of antibodies against F. hepatica to bind to small proteins (peptides) - we will then test the reaction of infected animals to these peptides to determine their usefulness. Aim ii) will use a novel set of immunomodulators generated in the applicants lab that will be tested for their ability to subvert normal host immune functions. Upon completion this 18 month study will evaluate potential vaccine candidates that have the greatest potential for formulation into subunit vaccine preparations that will show efficacy in either preventing infection or reducing worm burden. Our project will benefit from incorporating a range of proteins/peptides into the final vaccine formulation, presenting antigens that stimulate an optimal Th1/Th2 response as well as overcoming parasite immunosuppression.
direct losses only. The proposed project is a pre-industrial research project that will enable us to validate a collection of important components of F. hepatica that could be combined into sub-unit vaccine formulations for protecting ruminants against infection with this pathogen. Infection by F. hepatica is acquired by the ingestion of water or vegetation that is contaminated by parasitic metacercariae which excyst to produce newly excysted juveniles (NEJs) , these migrate across the intestine and peritoneal cavity to the liver and develop into sexually mature hermaphrodite adults. The adults release eggs into the bile ducts, which are transported to the intestine and are released into the environment via faeces. These parasites cause reduced milk yields, poor fertility and high perinatal loses, and chronic weight loss in cattle. Acute disease will cause sudden death from haemorrhage and liver damage in sheep. Control of fluke infection is heavily reliant on a limited number of anthelmintic compounds, which due to their widespread and often indiscriminate use has led to problems associated with drug resistance. There are also emerging concerns regarding the use of these drugs in food production animals, with recent legislation banning the use of four major classes of these drugs in dairy animals where milk is destined for the food chain. The most viable alternative to drug treatment would be vaccination against Fasciolosis. Currently no vaccine preparation is commercially available to control F. hepatica. The delay in bringing a commercial vaccine to market is a result of the complex lifecycle of the parasite, its large genome and complex biology, and the strategies it employs to evade host immunity. F. hepatica is well known to alter the host immune responses thereby deflecting attacks aimed at it which could damage and eventually kill the parasite. These strategies employ a number of molecules termed immunomodulators. Furthermore, the naturally occurring immune responses against this parasite often provide limited protection to the host. We now know that full protection will be offered by the two distinct arms of the immune system the antibody and the cellular arms. This project aims to characterise i) both the antibody and cellular immune response to this organism in animals that show variation in levels of fluke burdens and ii) a novel set of recently identified Immunomodulator proteins, produced by F. hepatica that are likely to suppress the host immune response. Aim i) will be conducted by a selection method exploiting the ability of antibodies against F. hepatica to bind to small proteins (peptides) - we will then test the reaction of infected animals to these peptides to determine their usefulness. Aim ii) will use a novel set of immunomodulators generated in the applicants lab that will be tested for their ability to subvert normal host immune functions. Upon completion this 18 month study will evaluate potential vaccine candidates that have the greatest potential for formulation into subunit vaccine preparations that will show efficacy in either preventing infection or reducing worm burden. Our project will benefit from incorporating a range of proteins/peptides into the final vaccine formulation, presenting antigens that stimulate an optimal Th1/Th2 response as well as overcoming parasite immunosuppression.
Technical Summary
F. hepatica is the cause of fasciolosis, an economically important parasitic disease of cattle and sheep which is the cause of chronic liver infection in an estimated 700 million animals worldwide. Infection results in an estimated annual global loss of US$2000 million through livestock mortality, reductions in productivity and condemnation of animal products that should go into the human food chain. Costs of this infection to UK industry is estimated to be around £14M/annum in beef cows, £13.5M/annum in dairy cattle and £3.1M/annum in sheep, although these are most certainly conservative and relate to
direct losses only. The proposed project is a pre-industrial research project that will enable us to validate a collection of important components of F. hepatica that could be combined into sub-unit vaccine formulations for protecting ruminants against infection with this pathogen. These parasites cause reduced milk yields, poor fertility and high perinatal loses, and chronic weight loss in cattle. Control of fluke infection is heavily reliant on a limited number of anthelmintic compounds, which due to their widespread and often indiscriminate use has led to problems associated with drug resistance. The most viable alternative to drug treatment would be vaccination against Fasciolosis. Currently no vaccine preparation is commercially available to control F. hepatica. Upon completion this 18 month study will evaluate potential vaccine candidates that have the greatest potential for formulation into subunit vaccine preparations that will show efficacy in either preventing infection or reducing worm burden. Our project will benefit from incorporating a range of proteins/peptides into the final vaccine formulation, presenting antigens that stimulate an optimal Th1/Th2 response as well as overcoming parasite immunosuppression.
direct losses only. The proposed project is a pre-industrial research project that will enable us to validate a collection of important components of F. hepatica that could be combined into sub-unit vaccine formulations for protecting ruminants against infection with this pathogen. These parasites cause reduced milk yields, poor fertility and high perinatal loses, and chronic weight loss in cattle. Control of fluke infection is heavily reliant on a limited number of anthelmintic compounds, which due to their widespread and often indiscriminate use has led to problems associated with drug resistance. The most viable alternative to drug treatment would be vaccination against Fasciolosis. Currently no vaccine preparation is commercially available to control F. hepatica. Upon completion this 18 month study will evaluate potential vaccine candidates that have the greatest potential for formulation into subunit vaccine preparations that will show efficacy in either preventing infection or reducing worm burden. Our project will benefit from incorporating a range of proteins/peptides into the final vaccine formulation, presenting antigens that stimulate an optimal Th1/Th2 response as well as overcoming parasite immunosuppression.
Planned Impact
Endemic and chronic bacterial diseases are a major health and welfare issue for animals and undermine the continued
production of a global food supply in a sustainable fashion. In our project, we aim to address one of the most insidious
parasitic diseases within the UK and temperate climates of the globe - Fasciolosis. Our research programme has
immediate and long term beneficial impacts in the areas of livestock production, the veterinary pharmaceutical industry,
consumer perception, scientific community, and the environment. The 'Pathways to Impact' of this proposal describes how
end-users and other stakeholders will be engaged. Livestock producers face increasing pressure to provide a more
sustainable product at a fixed price for growing markets. The consumer, environment, and pharmaceutical industry are also
all affected by this. To realistically meet this demand, stakeholders must improve disease control with an alternative to
chemotherapy, i.e. vaccination. Key is securing successful disease control is a novel approach to vaccine candidate
selection which our project will deliver. Our novel approach to this problem will have immediate impacts on the
pharmaceutical industry and a longer term impact on livestock stakeholders.
Development of effective vaccines and a consequent reduction in use of anthelmintics concerns both livestock
stakeholders and the veterinary pharmaceutical industry. The current ineffective control of fasciolosis leads to wasted
resources, animal welfare issues and disease outbreaks and spread. We will develop both a novel approach to vaccine
candidate selection but also a novel portfolio of vaccine candidates for a sub-unit vaccine against one of the most
devastating parasitic infections thereby improving producers' chances of using vaccination strategies to reduce the
incidence of disease. The pharmaceutical industry will be able to develop solutions based on our results, leading to novel
vaccine formulations that are applicable both in the UK and Globally. Reductions in rates of clinical disease will mean a decrease in the use of anthelmintics, positively impacting upon the consumer and environment. Furthermore, the
generation of data and outputs from this project will make a meaningful contribution of the knowledge-based economy of
the UK in coming years through the generation of revenue via IP.
Impacting upon environmental wellbeing will parallel the consumer perception of the farming industry. Our research
programme will result in a more sustainable and environmentally friendly farm incorporating less waste through reduced
disease burden and increased production. These will cause subsequent reductions in the total carbon footprint of farming
and UK economy, key priorities going forward for UK PLC. Publicising this will positively influence consumer perception as
improvements in animal welfare and environmental awareness with regard to origin of food products are foremost in the
consumer mind. Improvements and benefits at this level will improve consumer confidence, growing the home and
international UK beef market but also increasing demand for translation of UK practice to foreign markets.
Our research programme offers a plethora of benefits to the scientific community. It can act as a training ground for
collaborating scientists at it embodies a diverse yet fully integrated range of disciplines. Our data sets to be made public
include large transcriptomics data, via NCBI, immunological data sets via publication, and a collection of valuable
resources including RNA/cDNA libraries and recombinant proteins. Once published these, resources permitting, can be
made available upon request. Our published data will represent novel integrated data sets validated in target species with
significant translatable outcomes. Thereby, allowing veterinary and human scientists to build upon our observations in
fields including immunology; infection biology and vaccine development
production of a global food supply in a sustainable fashion. In our project, we aim to address one of the most insidious
parasitic diseases within the UK and temperate climates of the globe - Fasciolosis. Our research programme has
immediate and long term beneficial impacts in the areas of livestock production, the veterinary pharmaceutical industry,
consumer perception, scientific community, and the environment. The 'Pathways to Impact' of this proposal describes how
end-users and other stakeholders will be engaged. Livestock producers face increasing pressure to provide a more
sustainable product at a fixed price for growing markets. The consumer, environment, and pharmaceutical industry are also
all affected by this. To realistically meet this demand, stakeholders must improve disease control with an alternative to
chemotherapy, i.e. vaccination. Key is securing successful disease control is a novel approach to vaccine candidate
selection which our project will deliver. Our novel approach to this problem will have immediate impacts on the
pharmaceutical industry and a longer term impact on livestock stakeholders.
Development of effective vaccines and a consequent reduction in use of anthelmintics concerns both livestock
stakeholders and the veterinary pharmaceutical industry. The current ineffective control of fasciolosis leads to wasted
resources, animal welfare issues and disease outbreaks and spread. We will develop both a novel approach to vaccine
candidate selection but also a novel portfolio of vaccine candidates for a sub-unit vaccine against one of the most
devastating parasitic infections thereby improving producers' chances of using vaccination strategies to reduce the
incidence of disease. The pharmaceutical industry will be able to develop solutions based on our results, leading to novel
vaccine formulations that are applicable both in the UK and Globally. Reductions in rates of clinical disease will mean a decrease in the use of anthelmintics, positively impacting upon the consumer and environment. Furthermore, the
generation of data and outputs from this project will make a meaningful contribution of the knowledge-based economy of
the UK in coming years through the generation of revenue via IP.
Impacting upon environmental wellbeing will parallel the consumer perception of the farming industry. Our research
programme will result in a more sustainable and environmentally friendly farm incorporating less waste through reduced
disease burden and increased production. These will cause subsequent reductions in the total carbon footprint of farming
and UK economy, key priorities going forward for UK PLC. Publicising this will positively influence consumer perception as
improvements in animal welfare and environmental awareness with regard to origin of food products are foremost in the
consumer mind. Improvements and benefits at this level will improve consumer confidence, growing the home and
international UK beef market but also increasing demand for translation of UK practice to foreign markets.
Our research programme offers a plethora of benefits to the scientific community. It can act as a training ground for
collaborating scientists at it embodies a diverse yet fully integrated range of disciplines. Our data sets to be made public
include large transcriptomics data, via NCBI, immunological data sets via publication, and a collection of valuable
resources including RNA/cDNA libraries and recombinant proteins. Once published these, resources permitting, can be
made available upon request. Our published data will represent novel integrated data sets validated in target species with
significant translatable outcomes. Thereby, allowing veterinary and human scientists to build upon our observations in
fields including immunology; infection biology and vaccine development
Publications
Sachdev D
(2017)
The Chronic Stages of Bovine Fasciola hepatica Are Dominated by CD4 T-Cell Exhaustion.
in Frontiers in immunology
Japa O
(2015)
TGF-ß superfamily members from the helminth Fasciola hepatica show intrinsic effects on viability and development.
in Veterinary research
Neill DR
(2018)
Origins and evolution of innate lymphoid cells: Wardens of barrier immunity.
in Parasite immunology
Flynn RJ
(2016)
Myeloid-derived suppressor cell, arginase-1, IL-17 and cl-CD95L: an explosive cocktail in lupus?
in Annals of translational medicine
Chukwuanukwu R
(2017)
Modulation of the immune response to Mycobacterium tuberculosis during malaria/ M. tuberculosis co-infection
in Clinical and Experimental Immunology
Musah-Eroje M
(2018)
Fasciola hepatica, TGF-ß and host mimicry: the enemy within.
in Current opinion in microbiology
Garza-Cuartero L
(2016)
Fasciola hepatica infection reduces Mycobacterium bovis burden and mycobacterial uptake and suppresses the pro-inflammatory response.
in Parasite immunology
Flynn R
(2020)
Fasciola hepatica - Methods and Protocols
Beesley N
(2018)
Fasciola and fasciolosis in ruminants in Europe: Identifying research needs
in Transboundary and Emerging Diseases
Poissonnier A
(2016)
CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice.
in Immunity
Description | We have characterized the expression and function of a number of molecules from the parasite Fasciola hepatica - liver fluke. These molecules appear to be similar to a number of growth factors that appear in mammals, including the main mammalian hosts of Liver Fluke. We believe that these molecules initially carried out functions, motility [muscle development] or reproductive, within the parasite before developing subsequent roles in parasitism. Their roles in interacting with the host immune system seem to be primarily aimed at subverting the immune system to enable passage through the gut. In addition we have also characterized a second molecule that is involved in nutrient acquisition in a non-mechanical manner. We have also identified a number of proteins/peptides that are novel immunogens of liver fluke. |
Exploitation Route | The novel immunogens may be useful in helping to design novel control strategies. Elsewhere the novel immunomodulators might be used by others, we are actively pursuing these collaborations, as methods of therapy in other diseases either infectious or not. |
Sectors | Agriculture, Food and Drink |
Description | Investigators from this project have delivered a number of talks to farming/production animal groups. These talks have not focused on the specifics of results within this application but instead on the processes behind vaccine design and testing. These talks were given in the context of emerging problems with current drug usage and aimed at educating and highlighting the hurdles and time required in vaccine development. These talks have resulted in ongoing discussions with stakeholder groups with a view to developing management strategies that incorporate ongoing vaccine work, existing drugs and husbandry measures. In addition there have been a number of talks with public outreach panels in 2017 discussing animal disease, its control and the roles of government. Also R Flynn has contributed to an expert review in the area of Fasciola hepatica for EU DisContools. |
First Year Of Impact | 2016 |
Sector | Agriculture, Food and Drink,Government, Democracy and Justice |
Impact Types | Societal,Policy & public services |
Description | I contributed to an updated expert review of Fasciola as an entire disease area for the EU Discontools Project. |
Geographic Reach | Europe |
Policy Influence Type | Implementation circular/rapid advice/letter to e.g. Ministry of Health |
URL | http://www.discontools.eu/database.html |
Description | BBSRC TDRF |
Amount | £148,800 (GBP) |
Funding ID | BB/M018520/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 07/2015 |
End | 11/2016 |
Description | Follow On Pathfinder |
Amount | £15,681 (GBP) |
Funding ID | BB/R005451/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2017 |
End | 03/2018 |
Description | NA - PhD Distribution and function of Bone Morphogenic Proteins (BMPs) in Fasciola hepatica. |
Amount | £103,500 (GBP) |
Organisation | Saudi Arabian Cultural Bureau |
Sector | Public |
Country | Canada |
Start | 01/2019 |
End | 12/2022 |
Description | OJ - personal visiting grant to perform RNAi experimental at UoL. |
Amount | £15,000 (GBP) |
Organisation | Newton Fund |
Sector | Public |
Country | United Kingdom |
Start | 05/2018 |
End | 09/2018 |
Description | Exchange of IMs with Biomedical Researchers |
Organisation | University of Liverpool |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | A collaboration has a risen whereby we have exchanged parasite immunomodulators with a group of biomedical researchers interested in testing the potential therapeutic activity of these IMs in various airway disease models. This has included visits, by this team, to the collaborators groups to see how their models of disease are established and measured. |
Collaborator Contribution | Our collaborators have begun to test a parasite IM in their system for its effects on alleviating the course of airway disease |
Impact | None as of yet |
Start Year | 2016 |
Description | Phenotyping of Fasciola hepatica knockdowns |
Organisation | University of Phayao |
Country | Thailand |
Sector | Academic/University |
PI Contribution | Under the auspicious of a Newton Fund/British Council award we are hosting a research from the UNiversity of Phayao to develop knock-down phenotypes for Fasciola hepatica. We are providing molecular biology and genomic experience alongside parasitology support. |
Collaborator Contribution | Molecular biology and RNAi |
Impact | Knowledge exchange to date. |
Start Year | 2018 |
Description | Vaccine Formulation and Delivery Partnership |
Organisation | Test location |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Making use of novel antigens we have begun a collaboration with SiSaf to best determine the methods of delivery for prolonged immunity. |
Collaborator Contribution | The partners have contributed reagents/intellectual input |
Impact | Currently there is a further funding application being written jointly. |
Start Year | 2017 |
Description | Association of Veterinary Teaching and Research Workers Meeting 2017 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | PG presentation to a veterinary focused research meeting. |
Year(s) Of Engagement Activity | 2017 |
Description | Edinburgh Invited Talk |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | Invited seminar at the University of Edinburgh Immunology Group to speak on the role of anergy in developing protective immunity/vaccination against Fasciola hepatica. |
Year(s) Of Engagement Activity | 2019 |
Description | Immunity to Veterinary Pathogens (Vaccinology) - Keystone Symposium |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | PhD student AZ presented work related to the above project at an internationally attended conference. The audience including fellow researchers along with key stakeholders from relevant funding bodies and industry. There was discussion as a result of the presentation and some subsequent requests for material exchange afterwards. |
Year(s) Of Engagement Activity | 2015 |
Description | Invited Seminar University of Liverpool regional Immunology group |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Postgraduate students |
Results and Impact | Invited seminar for the British Society of Immunology Liverpool/Merseyside regional group. The topics of liver fluke disease and host immunity were discussed. As a result of the talk there has already been a collaboration established. |
Year(s) Of Engagement Activity | 2016 |
Description | Keystone Symposium mmunity to Veterinary Pathogens: Informing Vaccine Development (A3) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Third sector organisations |
Results and Impact | A Postgraduate student attended and presented data related to the discovery of diagnostic/control targets at this meeting. There was approximately 200-250 people in attendance. The presentation was well received and encouraged discussion afterwards with fellow researchers, since then there has been interactions with other groups (both nationally and internationally). |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.keystonesymposia.org/index.cfm?e=Web.Meeting.Program&Meetingid=1340 |
Description | Molecular and Cellular Biology of Helminths XI |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | PG presentation at International Parasitology meeting |
Year(s) Of Engagement Activity | 2017 |
Description | Moredun Research Institute Invited Seminar - RF |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | RF was invited to give a talk at the Moredun Research Institute (Edin.) in 2017 to present findings on current vaccine based control approaches to Fasciola hepatica. There was discussion afterwards which in turn has led to a new research collaboration being initiated. |
Year(s) Of Engagement Activity | 2017 |
Description | Parasitology/Immunology CPD |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Was asked to speak at the British Cattle Veterinary Association annual meeting as a sponsored session by Boehringer Ingelheim. Presented on mucosal immunology in the context of infection. Main audience was practising vets and some industry members. |
Year(s) Of Engagement Activity | 2018 |
Description | Presentation at British Society for Parasitology 2017 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | PG student presentation |
Year(s) Of Engagement Activity | 2017 |
Description | Public Involvement Panel - Speaking Engagement at University of Liverpool |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | A 20 min overview of research in the fasciola area was presented and potential problems and future directions were explained. There was discussion afterwards specific to the topic and more generally around public perception of disease control, government involvement and animal experimentation. |
Year(s) Of Engagement Activity | 2017 |
Description | european multicolloquium of parasitology 2016 - Finland Conference |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | MME, a Phd student associated with this project has made an oral presentation at EMOP 2016, a parasitology conference. Presenting some of her data related to the outcomes of the above project. The talk was well received and there was much discussion afterwards - including subsequent requests for collaboration and materials exchange. |
Year(s) Of Engagement Activity | 2016 |