Đề tài Diagnosis and control of diarrhoea in suckling pigs - Milestones 3 and 6

1 Ministry of Agriculture & Rural Development Project Progress Report Diagnosis and control of diarrhoea in suckling pigs CARD Project 001/04VIE MILESTONES 3 and 6 2nd and 3rd SIX MONTHLY REPORTS (COMBINED) 2 Table of Contents 1. INSTITUTE INFORMATION ........................................................................................................................3 2. PROJECT ABSTRACT...................................................................................................................................4 3. EXECUTIVE SUMMARY..............................................................................................................................4 4. INTRODUCTION & BACKGROUND ..........................................................................................................6 5. PROGRESS TO DATE....................................................................................................................................6 5.1 RESPONSE TO APPRAISAL ....................................................................................................................................6 5.2 IMPLEMENTATION HIGHLIGHTS ...........................................................................................................................7 5.3 SMALLHOLDER BENEFITS...........................................................................................................................14 5.4 CAPACITY BUILDING..................................................................................................................................14 5.5 PUBLICITY..................................................................................................................................................15 5.6 PROJECT MANAGEMENT ............................................................................................................................15 6. REPORT ON CROSS-CUTTING ISSUES...................................................................................................15 6.1 ENVIRONMENT ...........................................................................................................................................15 6.2 GENDER AND SOCIAL ISSUES .....................................................................................................................15 7. IMPLEMENTATION & SUSTAINABILITY ISSUES ................................................................................15 7.1 ISSUES AND CONSTRAINTS .........................................................................................................................15 7.2 OPTIONS.....................................................................................................................................................15 7.3 SUSTAINABILITY ........................................................................................................................................16 8. NEXT CRITICAL STEPS .............................................................................................................................16 9. CONCLUSION..............................................................................................................................................17 10. STATUTORY DECLARATION...................................................................................................................18 PROJECT PROGRESS AGAINST PROPOSED OBJECTIVES, OUTPUTS, ACTIVITIES AND INPUTS ........21 APPENDIX ONE .....................................................................................................................................................27 APPENDIX TWO.....................................................................................................................................................34 APPENDIX THREE.................................................................................................................................................37 3 1. Institute Information Project Name Diagnosis and control of diarrhoea in suckling pigs Vietnamese Institution National Institute of Veterinary Research (NIVR) Vietnamese Project Team Leader Dr. Truong Van Dung Australian Organisation The University of Queensland/Victorian Department of Primary Industry Australian Personnel Dr Darren Trott, Dr Ian Wilkie, Dr Tony Fahy Date commenced April 13th 2005 Completion date (original) January 2007 Completion date (revised) April 2007 Reporting period March 2006-March 2008 Contact Officer(s) In Australia: Team Leader Name: Dr Darren Trott Telephone: 617 336 52985 Position: Associate Professor of Veterinary Microbiology Fax: 617 336 51355 Organisation School of Veterinary Science The University of Qld Email: d.trott@uq.edu.au In Australia: Administrative contact Name: Melissa Anderson Telephone: 61 7 33652651 Position: Manager Research Projects Office Fax: 61 7 33651188 Organisation School of Land and Food The University of Qld Email: In Vietnam Name: Dr Cu Huu Phu Telephone: 84 4 8693923 Position: Head of Bacteriology Department Fax: 84 4 8694082 Organisation NIVR Email: cuhuuphu@netnam.org.vn 4 2. Project Abstract This project is designed to improve productivity of smallholder pig farmers in Vietnam through improved health management, particularly of piglets during the pre-weaning period. Through consultation and dialogue with farmers and field veterinarians, an appropriate disease management plan will be developed. This will concentrate on the pre-weaning period where greatest losses occur, but will include principles of herd health management in general. Dissemination of the plan will be through training programmes for field staff and selected farmers. Additional to the health management plan the project will develop and implement appropriate rapid diagnostic tests for the principal strains responsible for enterotoxigenic colibacillosis, to improve speed and accuracy of laboratory diagnosis. The third part of the project is to improve the production and efficacy of locally-manufactured E. coli vaccines. In particular, this will involves including a unique local strain shown by previous research to be an important vector of pre-weaning disease in some, and possibly all, areas of Vietnam. 3. Executive Summary This report, which is a combination of two six-monthly reports (2nd report and 3rd report), documents progress on the following deliverables (linked to the project logframe objectives and milestone descriptions): 1. Vaccine efficacy and safety data (Production and testing of locally-produced E. coli vaccine- small scale and field trials Logframe Reference 1). 2. Enteric management plan and production parameter records at 10 selected farms (5 test and 5 control farms for a 12 month period) (Develop a management plan for preweaning diarrhoea using a continuous improvement model-Logframe reference 2a and 2b). 3. Development of polyclonal sera and/or PCR incl. rapid detection of novel fimbrial antigens (Improve diagnostics for preweaning diarrhoea-Logframe reference 3). Progress has been achieved against all three objectives according to the project logframe, although some significant problems were experienced in trying to identify the novel fimbrial antigen present in Vietnamese O8 strains (christened F19), even with collaboration from the world’s leading expert on enterotoxigenic E. coli in pigs for a period of 18 months (Professor John Fairbrother, E. coli Reference Laboratory University of Montreal, St Hyacinthe, Quebec, Canada). The E. coli reference laboratory is very close to identifying the protein, but has run out of resources for the project. However, the positive benefits of interaction and collaboration with Prof Fairbrother will lead to the development of a joint international funding application to continue research and extension in Vietnam beyond the life of the current AUSAID CARD programme at the end of 2009. Dr Fairbrother is planning to visit Vietnam in December 2009 and begin the process of applying for a Gates Foundation grant to continue collaborative research (focusing on a combination of holistic improvements to smallholder agriculture, public health and novel analysis of E. coli evolution and recombination within animal and human hosts). A/Prof 5 Trott’s move to the University of Adelaide has also provided an opportunity, with the availability of startup funds to attempt another large scale fimbrial purification in Prof Fairbrother’s laboratory to identify the novel antigen by two dimensional protein gel electrophoresis. The startup funds will provide Dr Do Ngoc Thuy with an airfare and living allowance to travel to Canada in the summer of 2010 and complete the work. In small scale trials conducted at NIVR, the ETEC vaccine (still encorporating F4, F5 and the new F19 antigens) was proven to be safe and efficacious when administered to pregnant sows (2 doses at 5 and 2 weeks before farrowing). It is now being supplied to selected piggeries in North Vietnam on a research only basis, with anecdotal reports of good efficacy against neonatal E. coli infection and no reports of side-effects. The vaccine has also been produced for 004/05VIE and used in the selected smallholder farms in central Vietnam in this related AUSAID project as part of a Continuous Improvement Model to integrate best management practices into a holistic pig production improvement plan. Production data for the five test and five control farms over a 12-month period were analysed and a statistically significant improvement in preweaning mortality was noted in the test farms (8.6% ± 3.6) over the trial period compared to the controls (15.6 ± 4.3; p<0.05). A bigger improvement may have been confounded by the small sample size, but problems in the adoption of the Continuous Improvement Model may also have had an impact (ie the benefits of using the vaccine were not being realised due to the many endemic disease and production problems that were beyond the scope of this initial project to improve). The major problem encountered from the farm visits was inadequate uptake of skills, knowledge and recommendations by piggery managers most probably caused by breakdown in communications between Vietnamese scientists and piggery workers in the intervening periods between site visits by Australian scientists. The National Institute for Veterinary Research scientists are, for the most part, laboratory based researchers and we identified a training requirement in veterinary extension. We therefore adopted a top down Train the Trainers approach in CARD004/05VIE which, for the large part, has been successful in creating a subset of successful smallholder farmers in Central Vietnam. Farm reports for September 2006 revealed that with a few notable exceptions, many of the farms had not maintained the changes and recommendations suggested during the visit in 2005. The major problems identified but still not addressed included poor ventilation, inadequate cooling mechanisms and unacceptable heat index recordings, poor environment for suckers and weaner pigs, restricted feeding of sows and poor breeding records (low number of growers for the total number of sows). These factors may be contributing to the high incidence of enteric disease in suckers and weaners as well as respiratory diseases in growers. Clearly addressing these multiple problems is beyond the scope of the current project and has been addressed in 04/005VIE. The PCR machine and rapid diagnostic assay kits purchased by the project continue to be used for NIVR research on preweaning enteric diseases. A complete analysis of diagnostic results on pre and post weaning diarrhoea, together with the results of safety and efficacy testing of the vaccine were presented as posters by Dr Do Ngoc Thuy at the Australasian Association of Animal Production Biennial Conference in Hanoi in September, 2008. A survey of 117 samples of preweaning diarrhoea from commercial farms and 45 samples from village-based smallholder farms confirmed the presence of multiple agents in both forms of agriculture, however, only the commercial farms recorded cases of diarrhoea due to a single agent. By far the most common agents identified were rotavirus and transmissible gastroenteritis virus, often as a mixed infection with enterotoxigenic E. coli (ETEC was only ever isolated from older pigs as neonatal diarrhoea seems to be controlled by vaccination with the imported vaccine Pfizer Littergard). These results confirm that care of the sow and piglets during the preweaning period on both village and 6 commercial piggeries in Vietnam is suboptimal, which has been the major focus of initiatives developed in 004/05VIE. Characterization of virulence factors from ETEC isolates obtained from cases of pre- and postweaning diarrhoea identified some interesting findings. Following the successful visit to Dr John Fairbrother’s laboratory by Dr Thuy, 10 additional virulence genes were included that have been linked with certain E. coli pathotypes in other studies. These included the genes for Paa, AIDA-1, EAST-1, stx2 (normally associated with oedema disease) and Aero (normally a marker for extraintestinal pathogenic E. coli) which were identified in the Vietnamese ETEC collection. Firstly in pre-weaning diarrhoea, F4:Paa:STa:STb:LT:EAST-1 was still the most common pathotype (it was also the most common pathotype observed during Dr Do Ngoc Thuy’s PhD studies). During studies in Dr John Fairbrother’s laboratory, he identified that the virulence factor pathotype Paa:STa:STb:LT:EAST-1 was a consistent marker for the O8 F19 isolates that possess the new fimbrial type (thus they can be rapidly identified by their virulence gene profile even though diagnostic antisera that was entirely specific for the O8 strains could not be produced in Vietnam). This pathotype was the second most prevalent in the pre-weaning diarrhoea isolates, indicating that it was still a significant pathogen in preweaning diarrhoea in Vietnam. In post- weaning diarrhoea, the major pathotypes were associated with F18 rather than F4 and the majority of F18 strains also possessed stx2 toxin, confirming that the isolates had the capability of causing both post-weaning diarrhoea and oedema disease. 4. Introduction & Background Diarrhoea during the suckling period has been recognised as the principle health problem affecting both smallholder and commercial pig production in Vietnam. Previous research has confirmed the presence of a new fimbrial type in E. coli strains causing colibacillosis in Vietnam that would not be controlled by existing vaccines. Existing vaccines are currently imported into Vietnam at considerable cost. In addition, there are many other causes of suckling diarrhoea, the significance of which is currently unknown in Vietnam, which are all affected by husbandry and management during farrowing and lactation. Project 001/04VIE (Diagnosis and control of diarrhoea in suckling pigs) began with three objectives to solve this problem: 1. Production and testing of locally-produced E. coli vaccines 2. Development of a management plan for preweaning diarrhoea using a continuous improvement (CIP) model 3. Improved field and laboratory diagnosis of preweaning diarrhoea 5. Progress to Date 5.1 Response to Appraisal In general the previous report was well received and there were only two major issues that required clarification: The proponents state that this project is still in the data gathering stage. This second six-monthly Milestone Report is about 10 months later than expected and given that the project is due for completion in March 2007 (according to the Contract) there is some concern that about delivery of 5 further milestones by this date. Can the project proponents please clarify this position? 7 All the data is presented and analysed in the current report. Unfortunately the requirements of the 004/05VIE project were overwhelming and prevented timely submission of reports, even though the work was completed within the specified timeframe (and the findings presented at two major international conferences). The presence of the new unique fimbrial type remains an issue. The proponents state that there is a need for a survey to determine its incidence. Given that a key component of this project is production of a vaccine for this (and other types) who is going to undertake such a survey and how will the economic necessity for incorporation of the new unique type into existing or Vietnamese produced vaccines be established? The research work conducted in the laboratory of Prof John Fairbrother confirmed the typical virulence gene pathotype of the unusual O8 (F19) strains, and work conducted by Dr Do Ngoc Thuy and presented in this report confirmed that these strains are still prevalent in commercial piggeries in Vietnam. The strains are likely to be present in smallholder farms, but have not been detected as yet in the small number of samples processed by NIVR. The cost of inclusion of this strain in the vaccine is minimal and far outweighs the risk of exclusion (ie widespread vaccination with F4 and F5 strains would only increase the prevalence of the F19 strains). 5.2 Implementation Highlights 1) Vaccine strain characterization and further identification of the novel (F19) fimbrial antigen present in O8 5F- strains (Note: A detailed report of activities is included in Appendix One: Characterization of Vaccine Strains). The NIVR E. coli vaccine strains were extensively and independently characterized. Attempts to produce diagnostic antisera that was 100% specific against the novel O8 strains (as previously reported) were unsuccessful and it was concluded that the laboratories in both Australia and Vietnam lacked the expertise and equipment to perform a large scale fimbrial extract required to purify the antigen. An opportunity for Dr Do Ngoc Thuy to visit the laboratory of Prof John Fairbrother (the world’s leading porcine E. coli expert) immediately following the IPVS Congress in 2006 was eagerly taken up. Dr Thuy performed initial experiments confirming to Prof Fairbrother that the novel Vietnamese ETEC did indeed produce a mannose-resistant adhesin at 37oC (highly indicative of production of fimbriae capable of attachment to enterocytes), which was confirmed by transmission electron microscopy. An initial attempt at purification of the fimbrial extract revealed that there were still some contaminating proteins. Dr Thuy then returned to Vietnam and Prof Fairbrother continued the process of characterization. Initial attempts at identifying proteins in the purified fimbrial extract were unsuccessful. However, following a more rigorous extraction method, a 20KDa sized band (the right size!!) was identified in SDS-PAGE gels as the putative adhesin as it reacted in a Western Blot with hyperimmune serum obtained following immunization of rabbits with an E. coli O8 5F- whole cell extract grown at 37oC to produce fimbriae and absorbed with sera obtained for the same strain grown at 18oC, when it does not produce fimbriae. An N-terminal amino acid sequence obtained from the band cut from a one dimensional SDS-PAGE gel identified a protein closely related to Enterobacter ompX, but Prof Fairbrother is not convinced that this is the adhesin, even though it has a putative attachment role in other bacteria. A problem is that the adhesin does not seem to be expressed as much or as consistently as other fimbrial antigens and contaminating proteins may be being ident