Báo cáo Nghiên cứu khoa học Diagnosis and control of diarrhoea in suckling pigs: 2nd six monthly report

Ministry of Agriculture & Rural Development CARD Project Progress Report 001/04VIE: Diagnosis and control of diarrhoea in suckling pigs MS 3: 2nd SIX-MONTHLY REPORT October 2005 – March 2006 1 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 October 2005 – March 2006 Contact Officer(s) In Australia: Team Leader Name: Dr Darren Trott Telephone: 617 336 52985 Position: Senior Lecturer School of Veterinary Science Fax: 617 336 51355 Organisation School of Veterinary Science The University of Qld Email: d.trott@uq.edu.au In Australia: Administrative contact Name: May Montecino Telephone: 61 7 33652651 Position: Manager Research Projects Office Fax: 61 7 33651188 Organisation School of Land and Food The University of Qld Email: m.montecino@uq.edu.au 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 2. Project Abstract 4 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. Additional to the health management plan the project will develop and implement appropriate rapid diagnostic tests for the principal causes of preweaning diarrhoea to improve speed and accuracy of laboratory diagnosis. The third part of the project seeks to improve the production and efficacy of locally-manufactured E. coli vaccines. During the second six months of the project, we are still in a data gathering phase. Test farms were revisited and further recommendations made. An unexpected solution to problems in producing diagnostic antisera for the unusual fimbrial type that appears to be unique to Vietnam has emerged. Project findings will be presented at the 2006 International Pig Veterinary Society Congress. 3. Executive Summary This project began with three major objectives: 1. Production and testing of locally-produced vaccine 2. Develop a management plan for preweaning diarrhoea using a continuous improvement model 3. Improve diagnostics for preweaning diarrhoea During the second six months of the project, progress has been made towards all three objectives according to the project logframe, although some complications were encountered. Fimbriae could not be purified for the unusual Vietnamese serotype O8 strains that appear to carry a novel unrecognised attachment antigen at NIVR (or in Australia) but an opportunity to conduct this vital research at an internationally recognised laboratory has arisen. Test and control farms were visited again, but several farms are not implementing the recommended changes or supplying adequate production records for the pig health recording system put in place to monitor improvements. Diagnostic submissions continue to be sent to NIVR to determine the importance of different causes of preweaning diarrhoea and the findings of the first 8-months of collection (118 specimens in total) will be presented at the International Pig Veterinary Society Congress in Copenhagen by Dr Do Ngoc Thuy. The success of the project is reliant on adequate training of Vietnamese scientists in a holistic approach to understanding and identifying production constraints in a parallel project (004/05VIE) to conduct our first training sessions to smallholder farmers. Two high performing piggeries have been identified as excellent candidate premises where training can be undertaken. 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 5 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 of 1st six monthly report 1. Vietnam is a tropical country with hot-humid climate conditions, especially in Northern part of Vietnam. Diarrhoea in pre-weaning pigs is caused by complex and series of pathogen agents. Under such circumstances a number of questions such as identification of novel pathogen with changes in pathogenicity over time and in different environments should be considered. The aim of this project was to set up a number of test and control farms and monitor production parameters and enteric disease in suckling pigs over a period of time to include potential seasonal effects on disease incidence. This also coincided with laboratory training to accurately identify the causative agents. The data collected over a period of time will be ready for analysis (and will be reported) during the next six monthly report. 2. It is noted from farm reports that most farms surveyed were comparatively large. With small scale production (1-3 pig per householders), how could developed management systems be applied sustainably? Our initial investigations identified: 1) Large deficiencies in training, management and record keeping on intensive pig operations in North Vietnam 2) Deficiencies in disease diagnosis and farm auditing by veterinary service providers We therefore developed a top down, train the trainers approach that has been further extended into CARD project 004/05VIE. For CARD project 001/04VIE, it was important to initially set up the large scale trial piggeries where management changes could be easily implemented and audited. These piggeries would then serve as training institutions for technology transfer. Whilst the benefits of this scheme to the smallholder farmer may not be immediately apparent it was essential for the long term uptake of the continuous improvement model by smallholder farmers that this key infrastructure was put into place. What applies for 100 sows applies equally for one sow; good husbandry is good husbandry whichever way it goes. 3. There are two issues that seem to be related and need to be elaborated on: a. You have not been able to identify Vietnamese serotype 08 strains in any of the samples collected so far; and, b. The sera obtained from rabbits so far is not specific against 5F-08 strains Some important questions arise: (i) Is this an analytical technique issue or an absence of the strain issue? If it is the latter: - a. What are the implications of this on the delivery of Milestone 5 – Vaccine produced and efficacy tested? b. What is the evidence that the 08 strains have significant economic importance? c. If the lack of 08 strains is generic what is the point of local vaccine production? 6 The absence of the unusual 5F-O8 ETEC strains containing the proposed new fimbrial type is interesting, given that it was the second most prevalent pathotype in commercial piggeries in North Vietnam in the work conducted by Dr Do Ngoc Thuy for her PhD thesis (Do et al., J Med Microbiol. 2006 55:93-9). I can assure the M&E team that these strains are indeed pathogenic as they contain the full complement of enterotoxins and caused disease in baby pigs (Do et al., Vet Pathol. 2006 43:150-60). Its general absence could be explained by the fact that it may not be as virulent as the more cosmopolitan enterotoxigeic E. coli strains such as F4 and F5 (for example if the novel fimbrial antigen on the surface of the 08 strains has an inferior binding capacity to porcine intestinal epithelium). However if the more pathogenic F4 and F5 strains are controlled by vaccination, this will leave a vacuum for the 5F-O8 strains to fill. We have seen this phenomenon many times in our own herds. Analysis of submissions from our 10 test farms (see below) has shown that endemic transmissable gastroenteritis and rotavirus and to a lesser extent coccidiosis were important pathogens of the preweaning period. It is highly possible that on our test farms, the O8 strain is entirely absent as there have been very few episodes of diarrhoea in 1-4 day old pigs indicating that the current vaccination programme for ETEC (utilising an imported vaccine known as Littergard) is working well). We proposed that poor management is the number one reason for the high incidence of multiple enteric diseases endemic in suckling pigs in Vietnam and the farm reports and laboratory data attest to this. The failure to produce rabbit sera that was specific for the O8 serotype is not unusual given that the bacterial preparations used to immunize the rabbits were crude whole cell preparations. However it was worth trying this simpler method at NIVR in preference to a much more difficult whole fimbrial extract. It soon became apparent that NIVR lacked the equipment required to produce the purified fimbrial extract as well and we lacked the expertise in Australia to advise. We discussed this problem with our colleague Dr John Fairbrother from the E. coli laboratory at the Faculté de médecine vétérinaire Université de Montréal. Dr Fairbrother is the world’s leading expert on enterotoxigenic E. coli in pigs (his laboratory has been designated an official E. coli international reference laboratory by the OIE World Organization for Animal Health). He is extremely excited about the possibility of a unique fimbrial type being present in Vietnam and invited Dr Thuy to work in his laboratory which is fully equipped to produce the fimbrial extract in the middle of 2006 to coincide with attendance at the International Pig Veterinary Society Congress. Without a nationwide survey it is impossible to determine to what extent the new fimbrial type is present in Vietnam and whether it is of economic significance. Its identification in all age groups of suckling pigs its haemolysis pattern and the fact that it produces all three enterotoxins would suggest to me that it may be more common in Vietnam than we think. Until this is known it is best to incorporate this fimbrial type in the locally produced multivalent vaccine. It must be remembered that this vaccine is not only being produced against just the 08 strains but will also include the more prevalent F4 and F5 fimbriated strains. The major reason for using a locally made vaccine is cost. It can be produced in Vietnam at a fraction of the cost of the imported Littergard vaccine. 4. It is useful to note from the farm data that there have been a number of issues that appear to have a significant impact on pig productivity. The identification of mange and the widespread under-feeding of sows appear to be more significant than the incidence of diarrhoea – at least in the surveyed farms. This survey supports the development of a holistic approach to pig productivity improvement and the incorporation of financial returns and profitability. This issue has been addressed in the new CARD project 004/05VIE. A 1st six monthly report is due for this project at the end of September 2006 (Indeed one of the major reasons for the long delay in finalising this report is the large amount of time and effort that has gone into setting up 7 this large collaborative project involving 7 institutions and providing an extensive training programme to six visiting Vietnamese scientists). 5. It is noted that some equipment has been provided to NIVR. This includes provision of reagents and the PCR machine. Although the contract negotiated is a lumpsum contract we are required to assure AusAID that contracted inputs have been provided. The Statutory Declaration is designed for this purpose and should be completed detailing the inputs that have occurred during each 6-monthly reporting period. Can you please ensure that the CARD PMU Office receives a signed Statutory Declaration as soon as possible? 6. When we look at the contract (or the proposal budget) we do not see the PCR machine listed. Have we missed something? There is no problem in variation in the detail of equipment provided as long as the total sum is not increased. If you have jointly decided that equipment priorities have changed could you please advise the PMU office? We had to move quickly on this piece of equipment and the chance to send an additional $8000 worth of free laboratory equipment to NIVR with the offer presented by the company. A signed statutory declaration will accompany this report. A letter advising the change was sent to Hassell and Associates. 5.2 Implementation Highlights 1) Third trip to Vietnam by Australian scientists November 2005. Farm audits were again conducted by Australian scientific to determine the uptake of knowledge and skills suggested on previous visits. In summary: a) Concern was expressed as to the random nature of vaccination schedules followed by each operation. Dr Tony Fahy developed a more organised vaccination schedule for implementation on test farms. b) Several of the piggeries were not taking up the full recommendations made on previous visits. We believe that transfer of technology is only truly possible when Vietnamese scientists are trained to conduct the farm audits and recommend changes with authority in Vietnamese. This was the major impetus for CARD project 004/05VIE, as many of the problems were holistic in nature rather than just being indicative of enteric disease in suckling pigs (though this is certainly a big problem!). On a positive note, the test farm in Binh Dinh province showed excellent progress and will be an ideal demonstration farm for future training programmes 2) Acceptance of two conference poster abstracts detailing project outcomes (written by NIVR scientist Dr Do Ngoc Thuy) to be presented at the 19th International Pig Veterinary Society Congress in Copenhagen, Denmark, July 2006. Abstract 1 detailed the results of laboratory investigations of 117 faecal specimens submitted from the 10 test farms to NIVR for diagnosis of enteric disease in suckling pigs over an 8–month period. The results confirm the presence of multiple pathogens on all farms. In summary: a) Very few cases of ETEC were reported in 1-4 day old piglets indicating that on these particular farms, imported ETEC vaccines are probably working. b) By far the most common agents were endemic TGEV and rotavirus. A high incidence of these diseases may be reflective of poor management. c) Coccidiosis and Cryptosporidium are present in Vietnam. The former is completely preventable via strategic use of Baycox during the suckling period. 8 d) Clostridium perfringens has been identified as the aetiological agent in several cases of suckling pig diarrhoea in Vietnam. This is most probably Type A which is becoming an important cause of neonatal diarrhoea worldwide. Abstract 2 detailed a molecular investigation of virulence factors in E. coli isolated from Vietnamese pigs with oedema disease and post-weaning diarrhoea. This was the first time such a study has been conducted in Vietnam and it utilised the new PCR machine purchased as part of CARD project 001/04VIE. Determining the virulence factors of post-weaning strains has assisted the production of locally produced oedema disease vaccine. 3) Planning of ETEC vaccine trials at NIVR for March 2006. 4) Fourth trip to Vietnam by Australian scientists February 2006. This was planned to coincide with a planning meeting in Hanoi to discuss the formation of the large collaborative 004/05VIE project and presented an opportunity to expose experienced pig veterinarian and collaborator Dr Trish Holyoake to pig production in Vietnam. Dr Holyoake made the following observations with respect to future training programmes: • Visiting Vietnamese trainees should be taught during their time in Australia to conduct on- farm questioning to conduct problem solving. Basic skills such as “How long have the pigs been ill?”, “Have you treated them with anything?” “What was the response?” • It would be good to develop pictorial flow charts for the common diseases present on-farm and link them with the treatment of choice. These pictorial charts could be hung in the piggery office. Include a pictorial reference to drugs not suitable for treating pigs • Trainees need instruction on pig flow-facility utilization, setting mating targets, defining facility bottlenecks, the importance of AIAO vs continuous flow • Need for more production records 5) Official invite from Dr John Fairbrother for Dr Thuy to visit his laboratory in Montreal and continue work on the unusual O8 strains as well as other Vietnamese ETEC from suckling and post- weaning pigs. 5.3 Smallholder Benefits 1) Established and proven diagnostic capability for pre-and post-weaning enteric diseases at NIVR. 2) Clear identification of two model farms (one in Binh Dinh; one in Anh Tinh) that will be used for smallholder farmer training workshops during year 2 of the project and in 004/05VIE. 3) Continued emphasis on the need for a top down, train the trainers approach to training workshops advocating accurate record keeping and continuous improvement that will be fully implemented in 004/05VIE. 5.4 Capacity Building No changes from the previous report. Project is still in a data-gathering phase. The opportunity to conduct the fimbrial purification in Dr Fairbrother’s laboratory is timely in order to develop specific antisera for accurate diagnosis of the unusual fimbrial type. 5.5 Publicity There are two excellent opportunities for publicity: 1) Attendance and presentation of research material by Dr Thuy at the IPVS congress in Copenhagen. 9 2) Visit to the E. coli reference laboratory in Montreal to conduct further research. We would be happy to provide an article and pictures for the next CARD newsletter. 5.6 Project Management Operational project management continues to be shared between the National Institute of Veterinary Research Bacteriology Department, the Victorian Department of Primary Industry and The University of Queensland. Dr Steve Driesen has retired from the Victorian DPI and Dr Tony Fahy is now in charge of the day to day operations of the project. The Faculty Research Projects Office in the School of Land and Food at The University of Queensland, who have many years experience in managing international agricultural research projects, is responsible for administration of the project. 6. Report on Cross-Cutting Issues 6.1 Environment No changes from previous report 6.2 Gender and Social Issues No changes from previous report 7. Implementation & Sustainability Issues 7.1 Issues and Constraints As identified previously, the major constraint to the success of our project is the lack of appropriately trained veterinary extension workers who can conduct independent farm audits accurately and efficiently so that necessary management changes will be adopted to improve production and profitability. This will take time to be addressed properly. 7.2 Options We have developed a farm audit checklist that can be used to “train the trainers” as well as give an objective assessment of the major constraints to production at each farm visited. Use of this checklist will enable our scientists to evaluate the level of competency of Vietnamese personnel conducting the audit. However, to fully implement this methodology it will require extensive training of Vietnamese scientists in Australia, which is being undertaken in the new CARD projects (004/05VIE and 020/05VIE). The farm audit questionnaire and checklist will be included in the 1st six monthly report for 004/05VIE. 7.3 Sustainability Not applicable to comment on at this stage of the project. 8. Next Critical Steps Key steps in the next six months of the project: 1) Safety and efficacy testing of locally produced E. coli vaccine at NIVR as per the developed protocol 10 2) Importation of strains and fimbrial purification at the E. coli reference laboratory in Montreal 3) Finalise production records from test and control farms 4) Site visit and training programme for Vietnamese scientists (for 004/05VIE) so they can deliver the first CARD training programme for 001/04VIE in September 2006. 9. Conclusion We are essentially still in a data gathering phase at this stage of the project. Problems associated with methodology for antisera production and fimbrial purification of the novel O8 strains have been addressed. A strategy to circumvent the continual frustration experienced at discovering that the recommendations of Australian Scientists are not be taken on board at the level of the test and control piggeries during the farm visits has been identified for the new CARD project. This will allow for excellent synergy and resources to be shared between the two projects.