Báo cáo nghiên cứu khoa học "A blueprint for sustainable smallholder pig production in Central Vietnam - Milestone 8 " potx

Ministry of Agriculture & Rural Development Project Progress Report A blueprint for sustainable smallholder pig production in Central Vietnam CARD Project 001/04VIE Milestone 8: FINAL REPORT APRIL 2010 Table of contents TABLE OF CONTENTS 2 1. INSTITUTE INFORMATION 3 2. PROJECT ABSTRACT 4 3. EXECUTIVE SUMMARY 4 4. INTRODUCTION & BACKGROUND 6 5. PROGRESS TO DATE 6 5.1 IMPLEMENTATION HIGHLIGHTS 6 5.3 SMALLHOLDER BENEFITS 15 5.4 CAPACITY BUILDING 15 5.5 PUBLICITY 16 5.6 PROJECT MANAGEMENT 16 6. REPORT ON CROSS-CUTTING ISSUES 16 6.1 ENVIRONMENT 16 6.2 GENDER AND SOCIAL ISSUES 16 7. IMPLEMENTATION & SUSTAINABILITY ISSUES 16 7.1 ISSUES AND CONSTRAINTS 16 7.2 OPTIONS 17 7.3 SUSTAINABILITY 17 8. NEXT CRITICAL STEPS 17 9. CONCLUSION 17 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 (Dr Cu Huu Phu) 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 13 th 2005 Completion date (original) January 2007 Completion date (revised) April 2007 Reporting period March 2006-March 2008 and including data from 2009/2010 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 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 designed to improve the production and efficacy of locally-manufactured E. coli vaccines. In particular, this will involve 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 final report documents progress on the following project 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). Whilst this project achieved outputs for all three objectives according to the project logframe, some significant problems were experienced in trying to identify the novel fimbrial antigen present in Vietnamese O8 strains (christened F19) and in developing an enteric management plan within a holistic continuous improvement framework. A final attempt to purify the novel fimbrial antigen was undertaken with great success in mid-2010 using funds from the University of Adelaide and we are now awaiting identification of the amino acid and gene sequences for this unusual antigen. 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 reports of good efficacy against neonatal E. coli infection and no side- effects. The vaccine has also been produced for the CARD 004/05VIE project 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. A small scale field trial showed that the vaccine significantly reduced the occurrence of diarrhoea in general and in investigations of vaccinated herds that reported diarrhoea, no enterotoxigenic E. coli was isolated from faecal samples confirming that the cause of the diarrhoea was not neonatal colibacillosis. 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. The major problem encountered from the farm visits was inadequate uptake of skills, knowledge and recommendations by piggery managers. We therefore adopted different training approach in CARD 004/05VIE which has been extremely successful in creating successful, profitable smallholder farmers in Central Vietnam. 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 in older pigs. These results confirm that care of the sow and piglets during the preweaning period on both village and 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. Ten 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. In pre-weaning diarrhoea, F4:Paa:STa:STb:LT:EAST-1 was still the most common pathotype and the pathotype Paa:STa:STb:LT:EAST-1 was a consistent marker for the O8 F19 isolates that possess the new fimbrial type. 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 fimbriae 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. In summary, the NIVR vaccine has been shown to be safe, efficacious and now must be registered as soon as possible and licensed throughout the country. An ongoing field trial will conclude in November 2010 and on the basis of this data, partnerships should be sought with local vaccine companies such as NAVETCO for the mass production and distribution of the vaccine. A large number of pathogens have been isolated from preweaning pigs with diarrhoea confirming that greater attention to disease prevention through better husbandry and management, introduction of the NIVR vaccine, key preventative medications and minimal antimicrobial use will contribute strongly towards maintaining the profitability of smallholder farmers. 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 Implementation Highlights Objective 1: Production and testing of local produced vaccine Output 1.1: Identification and confirmation of components, including novel strain. The vaccine Master Seed (50 x 1ml vials of each of the three vaccine strains in Brain Heart Infusion broth plus 12% glycerol) is held in a -80 o C freezer at NIVR. Backup freeze dried cultures are also held at NIVR in case of a catastrophic freezer failure (if the -80 o C freezer breaks down, the strains can be held at -20 o C for a short duration). Each time the vaccine is prepared according to the protocol outlined in 1.3 below, a new vial of the Master Seed is subcultured and checked for purity. This then becomes the Working Seed for vaccine preparation, with the number of subcultures kept to an absolute minimum and culture conditions used for maximum fimbriae expression. Backup cultures are also held at The AQIS approved laboratory of The University of Queensland School of Veterinary Science and the OIE E. coli reference laboratory at The University of Montreal (managed by Prof John Fairbrother). The virulence characteristics (OK-antigen serogroup, fimbriae and enterotoxins) of the three strains selected for vaccine production were independently confirmed by The Pig Health and Research Unit (PHRU), Victorian Department of Primary Industry (Table 1). These strains have been stored as freeze dried specimens in three separate laboratories (NIVR, UQ and PHRU). Table 1 : E. coli strains used for the preparation of vaccine Virulence Characteristics Designation of E. coli vaccine strains O-serogroup Fimbriae Enterotoxin(s) NVP613 (CARD-VN1) O8 5F-* STa/STb/LT NVP1402 O149: K91 F4 STa/STb/LT (CARD-VN2) NVP1372 (CARD-VN3) O64 F5 STa * Negative for all five recognized fimbriae associated with porcine enterotoxigenic E. coli (F4, F5, F6, F18 and F41). May therefore possess a novel fimbrial antigen. Output 1.2: Characterization of the novel fimbrial antigen The two 5F- ETEC strains were examined for mannose-resistant haemagglutinating activity using Sheep Red Blood Cells. Mannose-resistant haemagglutination was observed at 37 o C, but not at 18 o C for both strains, confirming the production of adhesins (ie fimbriae) at 37 o C (Table 1). Table 3 : Haemagglutination results of two 5F- ETEC strains Cultures grown at: 37 o C 18 o C Strain NaCl 0.85% 1.5% D-Mannose NaCl 0.85% 1.5% D-Mannose CARD- VN1 H (1/1024) H (1/1024) Negative Negative EC-VN8 H (1/1024) H (1/1024) Negative Negative Transmission electron microscopy photographs taken at low and high magnification showed the presence of hair-like structures on the surface of the bacteria cells. Research conducted between 2006-2008 in the OIE Reference Laboratory for E. coli by Dr Do Ngoc Thuy, came extremely close to purifying and characterizing the new fimbrial antigen, however contaminating proteins in the preparation obscured the identification. In a return visit by Dr Do Ngoc Thuy in July 2010 sponsored by the University of Adelaide, the new fimbrial type was successfully purified without contaminating proteins and we are eagerly awaiting confirmation of the identity. Output 1.3 Formulation of vaccine Specialised culture media were prepared in order to provide favourable growth conditions for the production of fimbriae. For efficient expression of F4, strain CARD-VN2 was grown on Buffered Glucose Nutrient Agar whereas for the production of F5 fimbriae on strain CARD-VN3, Minca agar was used. For the strain with currently uncharacterized fimbriae (CARD-VN-1), buffered Glucose Nutrient Agar was shown to enhance production of the new fimbrial type. The procedure used to prepare the vaccine is summarised in Figure 1. Figure 1: Preparation of E. coli multivalent vaccine (1 ml of vaccine contains approximately 10 10 bacteria) Output 1.4: Efficacy testing of vaccine The NIVR prepared the vaccine for small scale protection, safety and efficacy trials. In summary, the vaccine produced no unacceptable side effects in vaccinated gilts and their progeny. When compared to Littergard and Ecovac, two commercially available vaccines from Pfizer and Intervet, respectively, the NIVR vaccine produced statistically similar specific antibody titres to an E. coli F4 fimbriae strain. This confirms that under experimental conditions, the vaccine is both safe and efficacious in generating anti-F4 agglutinating antibodies. Small amounts of the vaccine were supplied to selected herds in the North of Vietnam and to smallholder farmers in Central Vietnam as part of the 004/05VIE project. No side effects or vaccine reactions were reported and anecdotal reports suggest the vaccine is highly efficacious, though in central Vietnam it was not possible to identify causes of preweaning diarrhoea. Therefore some episodes of diarrhoea in piglets from vaccinated sows could have been caused by other agents such as coccidiosis, rotavirus or transmissible gastroenteritis virus, all of which have been demonstrated in Vietnamese smallholder farms. 20 ml TSB (37 o C, overni g ht ) PBS (10 10 bacteria/ml) Add 2% (v/v) aluminum hydroxide to a final concentration of 20% Freeze-dried cultures 2 ml TSB (37 o C, overnight) SBA (37 o C, overnight) Appropriate culture media (37 o C, overnight) Purity testing 10% (v/v) bufferred formaldehyde to a final concentration of 0.3% Mix with equal colume of each bacterin Dispense into sterile bottles and label Sterility testing Sterility testing Output 1.5: Field testing of vaccine Field trials were conducted at two communes in Thua Thien Hue and three communes in Quang Tri in 2009/2010. Pregnant sows each received 2 ml of vaccine (approximately 1.5 x 10 9 bacteria) at 9 and 12 weeks of gestation compared to the control group which were not vaccinated. No local or systemic reaction to the vaccine was observed and all sows gave birth at the correct stage of gestation to an average of 9.3 healthy piglets per sow. The prevalence of pre-weaning diarrhoea in piglets born from vaccinated sows at 1, 2 or 3 weeks of age were: 16.1; 22.7 and 26.5%, compared with those of 48.1; 33.8 and 37.5%, respectively from control group (P<0.005). Random faecal samples (n=37) taken from piglets with diarrhoea were assayed for the presence of the six most common enteric pathogens, causing pre-weaning diarrhoea. The prevalences of transmissible gastroenteritis virus (TGEV), rotavirus (RV) and coccidiosis were not significantly different between vaccinated or non-vaccinated groups. Clostridium perfringens was only found in non-vaccinated group. None of the E. coli isolates obtained from the vaccinated group possessed toxin genes, whilst the E. coli strains isolated from the non-vaccinated group all still carried STa, STb and/or LT toxin genes. This study demonstrated that the implementation of locally produced E. coli vaccine, not only reduced the prevalence of pre-weaning diarrhoea, but also may suppress the presence of toxigenic E. coli strains in the gut of piglets. A second field trial is currently being undertaken at the National Institute of Animal Husbandry research piggery and will conclude in November 2010. This is the final experiment required to generate data for the licensing of the vaccine. Output 1.6: Commercial realisation of vaccine NIVR continues to produce the vaccine for research purposes, as attested by the vaccine records (MS3 and 6 reports), but registration requires a detailed document to be submitted to the Department of Animal Health. Most of the requirements for registration, including safety and efficacy have been met by the current project, with the current field trial providing necessary field efficacy data. NIVR Bacteriology Laboratory is not experienced in the commercialization of its discoveries, therefore we suggest partnership between the two major local vaccine manufacturers that hold GMP/GLP licenses, NAVETCO (for the south of Vietnam) and the National Veterinary Factory (for the north) to complete the registration dossier. Prior to this occurring however, we advise that a patent attorney is hired to assist Dr Do Ngoc Thuy, the inventor of the vaccine to lodge a patent application with the Office of Intellectual Property of Vietnam within the Ministry of Science and Technology. Once this is obtained, negotiations may be commenced whereby the level of royalties returning to the sole inventor and the NIVR laboratory are clearly indicated. The assistance of the CARD programme management team is also requested to foster negotiations with the Department of Animal Health and other major stakeholders. This will ensure that delays are kept to a minimum and that the vaccine becomes readily available for use by smallholder farmers. Such a strategy towards commercialization could also be used for other NIVR vaccines, such as the NIVR oedema disease vaccine that has excellent efficacy but is currently unregistered. Objective 2: Enteric management plan for pre-weaning diarrhoea through adoption of a continuous improvement plan. Output 2.1 Field data collected at test and control farms. An analysis of preweaning mortality reported over a 14-month observation period established that the test farms, which were subject to a number of recommendations during the life of the project, had a significantly lower average pre-weaning mortality compared to the control farms (8.6% ± 3.6 vs 15.6 ± 4.3; p<0.05). One of the control farms was removed from the trial due to an outbreak of hog cholera. For the majority of test farms, consistently lower pre-weaning mortalities were sustained over the trial period, however for Dong May farm in Thai Binh, pre-mortalities of close to 20% were reduced to 10% towards the end of the observation period. It is difficult to determine whether this reduction in preweaning mortality was associated with uptake of any of the previous visit’s recommendations as the same problems were still observed on the second visit. 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Apr-05 May-05 Jun-05 Jul-05 Aug-05 Sep-05 Oct-05 Nov-05 Dec-05 Jan-06 Feb-06 Mar-06 Apr-06 May-06 Jun-06 Month % Pre Weaning Mortality Anh De Thai Binh C Anh Thiet Hung Yen C Trang Due Hai Phong C Minh Duong Ha Tay C Dinh Dung Binh Dinh C Dong My Thai Binh T Anh Hiep Hung Yen T Anh Tinh Hai Phong T Thanh Bich Ha Tay T Nhon Hoa Binh Dinh T Figure 2: Average preweaning mortalities observed in five test (T) and five control (C) piggeries during the 14-month observation period. The Anh Thiet farm was discontinued due to an outbreak of hog cholera. Output 2.3: Continuous improvement model for smallholder farmers Summaries of the results of field visits to test and control farms were submitted with MS3 and MS6 reports. Overall, whilst some improvements were noted on individual farms, many of the recommendations made on previous visits were not being followed. Drip coolers that had been installed were removed on some farms, the farms were not operating to full capacity in terms of the number of sows vs the number of growers and care of neonatal and weaner pigs was still not ideal. Some of the disease problems were clearly linked to the unacceptably high heat index recorded in some of the sheds, restricted feed intake and the large number of sows with low condition scores and poor ventilation. Anh Hiep Farm (Hung Yen Province) perhaps showed the greatest improvements over the life of the project, but this farm achieved consistently low rates of preweaning mortality throughout the year. The overall objective of the continuous improvement model was, through the farm visits, to provide Vietnamese scientists with training in herd health monitoring (focused on preweaning mortality) whilst creating demonstration farms that could be utilized for smallholder training workshops. However, we soon realised that this model was unworkable and that the resources allocated were inadequate. With advice and assistance from the CARD Programme Management Unit, Project number 004/05VIE (A blueprint for smallholder pig production in Central Vietnam) was developed as a holistic plan for capacity building, focused on smallholder farmers in Quang Tri and Thua Thien Hue. This project was extremely successful and details are provided in the final report. However, it must be stressed that without the experience gained from 001/04VIE, we would not have achieved such a good outcome. There was considerable crossover between the two projects, particularly in that the E. coli vaccine produced by NIVR was provided free to smallholder farmers selected in the 004/05VIE project for further training and capital improvement. [...]... F5/Paa/STa 2 F 18/ STa/STb 2 F 18/ STa/EAST1 1 F 18/ AIDA-I/STa/STb 3 F 18/ Paa/AIDA-I/STa/Stx2 2 F 18/ AIDA-I/STb/Stx2 1 F 18/ LT/Stx2 1 F 18/ AIDA-I/STa/STb/Stx2 4 F 18/ Paa/AIDA-I/STa/STb/Stx2 3 F 18/ Paa/STa/LT/Stx2 13 Paa/STa/LT/Stx2 2 Paa/STa/STb/LT/EAST1 4 AIDA-I/STb/EAST1 1 AIDA-I/STb/LT/EAST1 1 STa/STb 1 STb/EAST1 1 LT/Stx2 2 Output 3.3: Transfer of laboratory skills Diagnostic training manuals and procedures included... within the logframe The considerable time between project commencement and this final report should take into account that the two projects (001/04VIE and 004/05VIE) actually blended into a single entity that achieved great success for smallholder farmers in Central Vietnam and developed a model for pig production and further expansion into other provinces of Vietnam as well as neighbouring countries in. .. preweaning and postweaning piglets also provided some interesting findings and comparisons between commercial and village pigs Firstly, given the information from Prof John Fairbrother’s laboratory on the typical virulence gene profile possessed by the O8 strains expressing the new fimbrial antigen, Dr Thuy was able to demonstrate that in the case of preweaning diarrhoea samples from commercial pigs,... exclusively on creating a subset of successful smallholder farmers who were able to increase their production from 10 pigs/sow/year to 20 pigs/sow/year This would not have been achieved without the benefit of hindsight 7.3 Sustainability Summary: In combination, projects 001/04VIE and 004/05VIE are now ripe for large scale NGO funding to expand production of the NIVR vaccine and the model for smallholder farmer... serious disease in Vietnam (and has been observed in smallholder pig farms in Central Vietnam during our 04/005VIE project) NIVR does produce an effective oedema disease vaccine which requires further development and commercialization Table 2: Pathotype of E coli isolates from cases of pre-weaning and postweaning diarrhoea in commercial and village pigs Source of isolates Pathotype PrWD (n= 18) F4/STa/STb... 3: Improved diagnostics for preweaning diarrhoea Outputs 3.1: Prevalence of major causes of pre-weaning diarrhoea on large piggeries and smallholder farms Dr Thuy’s investigation of the causes of preweaning mortality in samples from commercial vs village based piggeries provided some interesting results Firstly, single disease agents were only ever identified in commercial piggeries, but these only... the vaccine This could result in many delays until the eventually availability of the vaccine for smallholder farmers Issue 2: Preweaning enteric management plan and creation of demonstration piggeries Constraints: The focus on large piggeries in 001/04VIE was designed to identify herds with a significant number of animals for further training programmes, however progress according to the continuous... be encountered (identified in Dr Thuy’s PhD thesis) and drugs that would probably be successful as second choice drugs include ceftiofur and apramycin In Australia, 2-3 week scour due to ETEC is controlled by feeding a milk vaccine to pregnant sows containing live “tame” E coli strains (ie they contain F4 antigen but no toxins) It should be possible to identify these strains in the E coli collection... large piggeries could become training venues 7.2 Options Issue 1: We have been advised that the best way forward is for Dr Thuy and NIVR to patent their vaccine through the Office of Intellectual Property of Vietnam In this endeavour, we recommend NIVR to seek the advice and assistance of a patent attorney and make partnerships with vaccine manufacturers in Vietnam who hold a GMP/GLP licenses for vaccine... by a co-operative of farmers The Vietnamese vaccine can be produced at $0.15 USD per dose Several thousand doses of this vaccine were provided free of charge to the smallholder farmers selected in 004/05VIE with anecdotal reports confirming that it was safe and efficacious in preventing neonatal diarrhoea in the first week of life 2) Identification of the causes of pre-weaning diarrhoea in smallholder . 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Apr-05 May-05 Jun-05 Jul-05 Aug-05 Sep-05 Oct-05 Nov-05 Dec-05 Jan-06 Feb-06 Mar-06 Apr-06 May-06 Jun-06 Month % Pre Weaning Mortality Anh De Thai Binh C Anh Thiet Hung Yen. Ministry of Agriculture & Rural Development Project Progress Report A blueprint for sustainable smallholder pig production in Central Vietnam CARD Project 001/04VIE Milestone 8: . number 004/05VIE (A blueprint for smallholder pig production in Central Vietnam) was developed as a holistic plan for capacity building, focused on smallholder farmers in Quang Tri and Thua