Đề tài A blueprint for sustainable smallholder pig production in Central

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 13th 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 -80oC freezer at NIVR. Backup freeze dried cultures are also held at NIVR in case of a catastrophic freezer failure (if the -80oC freezer breaks down, the strains can be held at -20oC 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 37oC, but not at 18oC for both strains, confirming the production of adhesins (ie fimbriae) at 37oC (Table 1). Table 3: Haemagglutination results of two 5F- ETEC strains Cultures grown at: 37oC 18oC 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 1010 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 (37oC, overnight) PBS (1010 bacteria/ml) Add 2% (v/v) aluminum hydroxide to a final concentration of 20% Freeze-dried cultures 2 ml TSB (37oC, overnight) SBA (37oC, overnight) Appropriate culture media (37oC, 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 109 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