Báo cáo Nghiên cứu khoa học 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 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 A pr -0 5 M ay -0 5 Ju n- 05 Ju l-0 5 A ug -0 5 S ep -0 5 O ct -0 5 N ov -0 5 D ec -0 5 Ja n- 06 Fe b- 06 M ar -0 6 A pr -0 6 M ay -0 6 Ju n- 06 Month % P re W ea ni ng M or ta lit y 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. Objective 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 constituted 21.2% of total samples. By contrast, multiple agents were always detected in enteric disease samples from village-based pigs. In commercial piggeries, rotavirus and TGEV, or rotavirus, TGEV and enterotoxigenic E. coli (ETEC) were identified in 26.3% of samples, indicating that these diseases are most certainly endemic. These agents were also commonly detected as mixed infections in village piglets, though samples from these animals were more likely to contain a “mixed bag” of pathogens. Most of the samples were obtained from piglets older than 1 week, indicating that neonatal diarrhoea is largely controlled (by the use of expensive imported vaccines) and that haemolytic E. coli are most probably involved in scours in piglets greater than 14 days of age until weaning, with the two most common pathotypes being F4/Paa/STa/STb/LT/EAST1 and the new signature F19 pathotype, Paa/STa/STb/LT/EAST1. Coccidiosis was detected in 18% of commercial herd samples and 35% of village pig samples. This disease can be easily controlled by strategic preventive medication with toltrazuril (Baycox), administered at 3 days of age (adopted in the 004/05VIE project). Apart from providing an ideal creep environment that is dry and warm, some simple measures that may improve the diarrhoea due to rotavirus and TGEV include backfeeding a 1:20 dilution of the scour (in water) to dry and pregnant sows to provide some maternal colostral antibody, and as 47.9% of commercial pig and 60% of village pig samples contain ETEC, strategic medication with antimicrobials is also warranted. Use of drugs such as Lincospectin, trimethoprim/sulphonamide and amoxicillin are preferred to enrofloxacin (which is banned in food-producing animals in Australia). However, multiple drug resistance is likely to 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 at NIVR, but it is beyond the scope of this project. Table 4: Prevalence of enteric pathogens in pre-weaning piglets with diarrhoea (commercial vs village herds). Agent(s) detected # of positive specimens (%) Commercial (n=117) Village (n=45) Cocci 2 (1.7) Crypto 3 (2.5) RV 3 (2.5) TGEV 11 (9.3) ETEC 4 (3.4) C. per. 2 (1.7) Total single infections 25 (21.2) RV ETEC 6 (5.1) RV TGEV 17 (14.4) 3 (6.7) Cocci RV 1 (0.8) 2 (4.4) Cocci Crypto 1 (0.8) 2 (4.4) Crypto ETEC 3 (2.5) 1 (2.2) Crypto C. per. 1 (0.8) 1 (2.2) Crypto TGEV 6 (5.1) 2 (4.4) TGEV C. per. 1 (0.8) TGEV ETEC 7 (5.9) Cocci RV ETEC 2 (1.7) 3 (6.7) Cocci RV TGEV 4 (3.4) 3 (6.7) Cocci TGEV ETEC 2 (1.7) 2 (4.4) Cocci Crypto RV 1 (0.8) 1 (2.2) Crypto TGEV ETEC 5 (4.2) 4 (4.4) Crypto RV ETEC 1 (0.8) 3 (6.7) Crypto TGEV C. per. 1 (0.8) 1 (2.2) Crypto RV TGEV 3 (2.5) RV TGEV ETEC 14 (11.9) 7 (15.6) RV TGEV C. per. 1 (0.8) 1 (2.2) RV ETEC C. per. 2 (1.7) 3 (6.7) TGEV ETEC C. per. 2 (1.7) Crypto RV TGEV ETEC 2 (1.7) 1 (2.2) Crypto RV ETEC C. per. 1 (0.8) 2 (4.4) Cocci Crypto RV TGEV 1 (0.8) 1 (2.2) Cocci Crypto TGEV C. per. 1 (0.8) Cocci RV TGEV ETEC 4 (3.4) 1 (2.2) Cocci Crypto RV TGEV C. per. 1 (0.8) 1 (2.2) Crypto RV TGEV ETEC C. per. 1 (0.8) (36) (50) (97) (111) (76) (23) Total multiple infections 92 (78.8) 45 (100.0) Output 3.2: Characterization of ETEC virulence factors. Dr Thuy’s analysis of virulence factors in ETEC isolates obtained from both 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, the F19 strains were the second most common virulence profile identified after the typical F4 strains. These isolates were only identified in samples from commercial piggeries. In the case of the postweaning diarrhoea samples, a large number of pathotypes were identified, but 73.2% of the isolates possessed F18 fimbriae (and are likely to be serotype O141) whereas only 14.6% of the isolates carried the more common F4 (and are likely to belong to serotype O149). F4 strains are only associated with postweaning diarrhoea, whereas F18 is often associated with both postweaning diarrhoea and oedema disease. As an indication of this, stx2, the toxin mostly associated with oedema disease, was identified in 76.7% of the F18-positive isolates (63.4% of total isolates). In most pig-producing countries, oedema disease has become quite rare, but it is obviously still a 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) PWD (n=41) F4/STa/STb 1 F4/ Paa/STa/STb/LT/EAST1 5 F4/Paa/STb/LT/EAST1 5 F4/Paa/STb/LT/EAST1 2 F4/STa/STb/EAST1 1 F4/STa/STb/Aero 1 F5/Paa/STa 2 F18/STa/STb 2 F18/STa/EAST1 1 F18/AIDA-I/STa/STb 3 F18/Paa/AIDA-I/STa/Stx2 2 F18/AIDA-I/STb/Stx2 1 F18/LT/Stx2 1 F18/AIDA-I/STa/STb/Stx2 4 F18/Paa/AIDA-I/STa/STb/Stx2 3 F18/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 the design of NIVR Diagnostic Services Laboratory Submission and Recording Form and standard operating procedures (SOPs) from the Australian Pig Health and Research Unit (a NATA accredited laboratory) translated into Vietnamese to become SOPs for NIVR. Staff of the NIVR Bacteriology Laboratory (four full-time scientists) were competency assessed by Dr Do Ngoc Thuy and Dr Tony Fahy on their ability to identify and recognise the six major preweaning diarrhoea pathogens (enterotoxigenic E. coli, Clostridium perfringens type A [bacterial causes], rotavirus and transmissible gastroenteritis virus [viral causes], coccidiosis and Cryptosporidium [protozoan causes]) in faecal samples. Dr Thuy and members of her diagnostic transferred this technology to Hue University of Agriculture and Forestry as part of the 004/05VIE project in December 2009 and an ongoing project is examining causes of preweaning diarrhoea on smallholder farms in Thua Thien Hue. Dr Do Ngoc Thuy attended the IPVS Congress in Denmark in 2006 and the AAAP Congress in Hanoi in 2008. She had two papers accepted at both conferences, detailing preliminary and final results of characterization of diarrhoeal pathogens in preweaning pigs from commercial and village farms (Poster 1), and virulence gene pathotypes of enterotoxigenic E. coli isolates (Poster 2). These were both refereed conferences and the posters and abstracts have been submitted to CARD as part of previous milestone reports. In 2008 and 2010, Dr Thuy undertook further laboratory training at The E. coli OIE reference laboratory in Montreal under the direction of Prof John Fairbrother. Ten new gene primers were introduced, and Dr Thuy has identified new E. coli virulence gene profiles in Vietnam and shown why oedema disease is so prevalent in this country (discussed in 3.2 above). 5.3 Smallholder Benefits Smallholder farmers have been the direct recipients of the following major project interventions: 1) Characterization, efficacy and safety testing of the NIVR E. coli vaccine. Currently, the majority of commercial piggeries in Vietnam use Pfizer Littergard at a price of approximately $0.70 USD per dose. The majority of smallholder farmers currently do not practice vaccination and could not afford to routinely use the commercial vaccine, unless it was purchased 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 farms. The completed study by Dr Do Ngoc Thuy confirmed that in smallholder piggeries, single aetiological agents of preweaning diarrhoea were never identified and the most common, multifactorial diseases included TGEV, rotavirus and enterotoxigenic E. coli. Coccidiosis, a completely preventable disease, was also detected in over 30% of diarrhoea samples. This confirms that before any improvement in piglet health can be attained, we must start back at the basics and teach farmers about sow microclimate (cool, dry) and piglet microclimate (warm, dry). Stressed, wet piglets subjected to drafts and poor air quality succumb to enteric diseases. This goal was attained in project 004/05VIE encorporating knowledge and experience gained from 001/04VIE 3) Identification of ETEC pathotypes in smallholder farms. Characterization of the ETEC isolates obtained from smallholder farmers confirmed that the major ETEC pathotype in smallholder farm enterprises is F4:Paa:STa:STb:LT:EAST-1. This pathotype causes neonatal, 2-3 week old and postweaning scour and can be controlled by appropriate antimicrobial treatment or a combination of sow and piglet killed and live vaccines. Interestingly, the unusual F19 strains, so far, have not been identified in smallholder farms, which may be due to the overwhelming presence of F4:Paa:STa:STb:LT:EAST-1 pathotype. 5.4 Capacity Building Our research has shown that NIVR has produced a very successful vaccine that has been proven to be both safe and efficacious in small scale trials and elicits similar antibody levels to commercially available vaccines. Taking this to the next phase of commercialisation has proven to be difficult as our controlled field trial in smallholder farms in Central Vietnam (as part of the 004/05VIE project) proved difficult to manage with other more pressing project commitments (such as developing farmer clubs) taking precedence and was only completed in early 2010. Whilst this large field trial provided anaecdotal reports of excellent vaccine efficacy, it needs to be supported by field data from a much larger study. We therefore decided to conduct a field trial in the NIAH piggery that will conclude in November 2010. The transfer of technologies for laboratory testing for accurate diagnosis of pre-weaning diarrhoea has been another of the big success stories of this project. Technologies were transferred early in the project and diagnosticians at NIVR are now skilled in several techniques and confidently identified pathogens in samples. This confirmed that many piglets in Vietnam, both in commercial and village-based operations are infected with mixed pathogens, confirming that greater attention to pig husbandry (including appropriate preventative and treatment strategies including vaccination and prophylactic medication) must be initiated. The purchase of the new PCR machine and other laboratory equipment allowed the NIVR to accurately profile the pathotype of ETEC strains associated with preweaning and postweaning diarrhoea/oedema disease. This identified new virulence gene profiles and showed that the F19 strains, which were not identified in the early stages of the project are definitely still causing diarrhoea in Vietnam and must be encorporated into any locally produced vaccines. 5.5 Publicity Over the course of the project, the following opportunities for publicity were attained. 1) Attendance and presentation of research papers at the 2006 and 2010 IPVS Congresses. 2) Attendance and presentation of research papers at the 2008 and 2010 AAAP Congresses. Other opportunities for publicity are detailed in the 004/05VIE report. 5.6 Project Management Operational project management was shared between The University of Queensland, Victorian Department of Primary Industry (VicDPI), and the National Institute of Veterinary Research. The University of Queensland was responsible for overall management of the project, with VicDPI responsible for the delivery of training material, organisation of farm visits and data analysis. NIVR was responsible for the production and testing of E. coli vaccines for the project and providing advice and assistance on disease investigation and surveillance through laboratory diagnosis. 6. Report on Cross-Cutting Issues 6.1 Environment Antibiotic usage and antibiotic resistance: Further development of the NIVR E. coli vaccine and accurate identification of the causes of preweaning diarrhoea will lead to reduced reliance on antimicrobials for the treatment and prevention of disease. The majority of smallholder farmers use enrofloxacin, a top shelf antimicrobial for the treatment of diarrhoea, regardless of the cause. The widespread availability of the NIVR vaccine would lead to a dramatic reduction in antimicrobial usage with significant public health benefits. Other major issues of biosecurity and processing of piggery effluent were explored in the 004/05VIE project final report. 6.2 Gender and Social Issues These are more completely explored in the 004/05VIE project final report. 7. Implementation & Sustainability Issues 7.1 Issues and Constraints Issue 1: Commercialisation of the NIVR vaccine. Constraints: There is considerable investment in time and money involved in the path to commercialisation of 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 improvement model was not achieved to an extent that 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 production (eg NAVETCO), with a royalty stream flowing back to NIVR and the inventors which can be used to fund future research. The research conducted to date can be used to support the application, including the planned field trials in central Vietnam to coincide with 004/05VIE project aims. This model could be successfully used for other NIVR vaccines that are equally effective, but not yet registered and this could be an extremely effective marketing tool. The assistance of the CARD programme management team is sought to assist in this process by reiterating that the availability of a viable, cheap locally produced vaccine for smallholder farmers is the major outcome we are seeking to achieve and the assistance of the relative authorities is required so that this can be fast tracked to completion. Issue 2: 004/05VIE focused 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 engagement, improvement and profitability. 8. Next Critical Steps 1) An application for CARD project extension funds was granted to continue promoting project sustainability for the remainder of 2010. Furthermore, project award funds were used to send a number of scientists to the 2010 AAAP Congress in August 2010 to further promote the success of the project. 2) This will form the background for a large, multi-disciplinary application to Atlantic Philanthropies to be made in 2011 (budget ~$5.0 million) to continue the model in other provinces and possibly into Laos and Cambodia, employing project farmers as trainers. 3) The CARD conclusion Workshop in November represents a critical phase to galvanise stakeholders to take this project to the next level. 9. Conclusion This final report details the considerable research and effort by NIVR and Australian scientists to achieve project success 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 South East Asia. Apart from the final characterization of the F19 antigen, all objectives have been fulfilled. Strategies for commercialization of the vaccine will provide a technology platform for taking additional locally produced vaccines through to final licensing and will provide a continued funding stream for agencies such as NIVR to continue their research. Persistent application of the CIP model on the selected test commercial farms did show a difference in preweaning mortality compared to the control commercial farms and the lessons learnt in technology transfer have been applied to 004/05VIE to work more specifically with smallholder farmers.