1 Ministry of Agriculture & Rural Development Project Progress Report Diagnosis and control of diarrhoea in suckling pigs CARD Project 001/04VIE MILESTONES 3 and 6 2 nd and 3 rd SIX MONTHLY REPORTS (COMBINED) 2 Table of Contents 1. INSTITUTE INFORMATION 3 2. PROJECT ABSTRACT 4 3. EXECUTIVE SUMMARY 4 4. INTRODUCTION & BACKGROUND 6 5. PROGRESS TO DATE 6 5.1 RESPONSE TO APPRAISAL 6 5.2 IMPLEMENTATION HIGHLIGHTS 7 5.3 SMALLHOLDER BENEFITS 14 5.4 CAPACITY BUILDING 14 5.5 PUBLICITY 15 5.6 PROJECT MANAGEMENT 15 6. REPORT ON CROSS-CUTTING ISSUES 15 6.1 ENVIRONMENT 15 6.2 GENDER AND SOCIAL ISSUES 15 7. IMPLEMENTATION & SUSTAINABILITY ISSUES 15 7.1 ISSUES AND CONSTRAINTS 15 7.2 OPTIONS 15 7.3 SUSTAINABILITY 16 8. NEXT CRITICAL STEPS 16 9. CONCLUSION 17 10. STATUTORY DECLARATION 18 PROJECT PROGRESS AGAINST PROPOSED OBJECTIVES, OUTPUTS, ACTIVITIES AND INPUTS 21 APPENDIX ONE 27 APPENDIX TWO 34 APPENDIX THREE 37 3 1. Institute Information Project Name Diagnosis and control of diarrhoea in suckling pigs Vietnamese Institution National Institute of Veterinary Research (NIVR) Vietnamese Project Team Leader Dr. Truong Van Dung Australian Organisation The University of Queensland/Victorian Department of Primary Industry Australian Personnel Dr Darren Trott, Dr Ian Wilkie, Dr Tony Fahy Date commenced April 13 th 2005 Completion date (original) January 2007 Completion date (revised) April 2007 Reporting period March 2006-March 2008 Contact Officer(s) In Australia: Team Leader Name: Dr Darren Trott Telephone: 617 336 52985 Position: Associate Professor of Veterinary Microbiology Fax: 617 336 51355 Organisation School of Veterinary Science The University of Qld Email: d.trott@uq.edu.au In Australia: Administrative contact Name: Melissa Anderson Telephone: 61 7 33652651 Position: Manager Research Projects Office Fax: 61 7 33651188 Organisation School of Land and Food The University of Qld Email: In Vietnam Name: Dr Cu Huu Phu Telephone: 84 4 8693923 Position: Head of Bacteriology Department Fax: 84 4 8694082 Organisation NIVR Email: cuhuuphu@netnam.org.vn 4 2. Project Abstract This project is designed to improve productivity of smallholder pig farmers in Vietnam through improved health management, particularly of piglets during the pre-weaning period. Through consultation and dialogue with farmers and field veterinarians, an appropriate disease management plan will be developed. This will concentrate on the pre-weaning period where greatest losses occur, but will include principles of herd health management in general. Dissemination of the plan will be through training programmes for field staff and selected farmers. Additional to the health management plan the project will develop and implement appropriate rapid diagnostic tests for the principal strains responsible for enterotoxigenic colibacillosis, to improve speed and accuracy of laboratory diagnosis. The third part of the project is to improve the production and efficacy of locally-manufactured E. coli vaccines. In particular, this will involves including a unique local strain shown by previous research to be an important vector of p re-weaning disease in some, and possibly all, areas of Vietnam. 3. Executive Summary This report, which is a combination of two six-monthly reports (2 nd report and 3 rd report), documents progress on the following deliverables (linked to the project logframe objectives and milestone descriptions): 1. Vaccine efficacy and safety data (Production and testing of locally-produced E. coli vaccine- small scale and field trials Logframe Reference 1). 2. Enteric management plan and production parameter records at 10 selected farms (5 test and 5 control farms for a 12 month period) (Develop a management plan for preweaning diarrhoea using a continuous improvement model-Logframe reference 2a and 2b). 3. Development of polyclonal sera and/or PCR incl. rapid detection of novel fimbrial antigens (Improve diagnostics for preweaning diarrhoea-Logframe reference 3). Progress has been achieved against all three objectives according to the project logframe, although some significant problems were experienced in trying to identify the novel fimbrial antigen present in Vietnamese O8 strains (christened F19), even with collaboration from the world’s leading expert on enterotoxigenic E. coli in pigs for a period of 18 months (Professor John Fairbrother, E. coli Reference Laboratory University of Montreal, St Hyacinthe, Quebec, Canada). The E. coli reference laboratory is very close to identifying the protein, but has run out of resources for the project. However, the positive benefits of interaction and collaboration with Prof Fairbrother will lead to the development of a joint international funding application to continue research and extension in Vietnam beyond the life of the current AUSAID CARD programme at the end of 2009. Dr Fairbrother is planning to visit Vietnam in December 2009 and begin the process of applying for a Gates Foundation grant to continue collaborative research (focusing on a combination of holistic improvements to smallholder agriculture, public health and novel analysis of E. coli evolution and recombination within animal and human hosts). A/Prof 5 Trott’s move to the University of Adelaide has also provided an opportunity, with the availability of startup funds to attempt another large scale fimbrial purification in Prof Fairbrother’s laboratory to identify the novel antigen by two dimensional protein gel electrophoresis. The startup funds will provide Dr Do Ngoc Thuy with an airfare and living allowance to travel to Canada in the summer of 2010 and complete the work. In small scale trials conducted at NIVR, the ETEC vaccine (still encorporating F4, F5 and the new F19 antigens) was proven to be safe and efficacious when administered to pregnant sows (2 doses at 5 and 2 weeks before farrowing). It is now being supplied to selected piggeries in North Vietnam on a research only basis, with anecdotal reports of good efficacy against neonatal E. coli infection and no reports of side-effects. The vaccine has also been produced for 004/05VIE and used in the selected smallholder farms in central Vietnam in this related AUSAID project as part of a Continuous Improvement Model to integrate best management practices into a holistic pig production improvement plan. Production data for the five test and five control farms over a 12-month period were analysed and a statistically significant improvement in preweaning mortality was noted in the test farms (8.6% ± 3.6) over the trial period compared to the controls (15.6 ± 4.3; p<0.05). A bigger improvement may have been confounded by the small sample size, but problems in the adoption of the Continuous Improvement Model may also have had an impact (ie the benefits of using the vaccine were not being realised due to the many endemic disease and production problems that were beyond the scope of this initial project to improve). The major problem encountered from the farm visits was inadequate uptake of skills, knowledge and recommendations by piggery managers most probably caused by breakdown in communications between Vietnamese scientists and piggery workers in the intervening periods between site visits by Australian scientists. The National Institute for Veterinary Research scientists are, for the most part, laboratory based researchers and we identified a training requirement in veterinary extension. We therefore adopted a top down Train the Trainers approach in CARD004/05VIE which, for the large part, has been successful in creating a subset of successful smallholder farmers in Central Vietnam. Farm reports for September 2006 revealed that with a few notable exceptions, many of the farms had not maintained the changes and recommendations suggested during the visit in 2005. The major problems identified but still not addressed included poor ventilation, inadequate cooling mechanisms and unacceptable heat index recordings, poor environment for suckers and weaner pigs, restricted feeding of sows and poor breeding records (low number of growers for the total number of sows). These factors may be contributing to the high incidence of enteric disease in suckers and weaners as well as respiratory diseases in growers. Clearly addressing these multiple problems is beyond the scope of the current project and has been addressed in 04/005VIE. The PCR machine and rapid diagnostic assay kits purchased by the project continue to be used for NIVR research on preweaning enteric diseases. A complete analysis of diagnostic results on pre and post weaning diarrhoea, together with the results of safety and efficacy testing of the vaccine were presented as posters by Dr Do Ngoc Thuy at the Australasian Association of Animal Production Biennial Conference in Hanoi in September, 2008. A survey of 117 samples of preweaning diarrhoea from commercial farms and 45 samples from village-based smallholder farms confirmed the presence of multiple agents in both forms of agriculture, however, only the commercial farms recorded cases of diarrhoea due to a single agent. By far the most common agents identified were rotavirus and transmissible gastroenteritis virus, often as a mixed infection with enterotoxigenic E. coli (ETEC was only ever isolated from older pigs as neonatal diarrhoea seems to be controlled by vaccination with the imported vaccine Pfizer Littergard). These results confirm that care of the sow and piglets during the preweaning period on both village and 6 commercial piggeries in Vietnam is suboptimal, which has been the major focus of initiatives developed in 004/05VIE. Characterization of virulence factors from ETEC isolates obtained from cases of pre- and postweaning diarrhoea identified some interesting findings. Following the successful visit to Dr John Fairbrother’s laboratory by Dr Thuy, 10 additional virulence genes were included that have been linked with certain E. coli pathotypes in other studies. These included the genes for Paa, AIDA-1, EAST-1, stx2 (normally associated with oedema disease) and Aero (normally a marker for extraintestinal pathogenic E. coli) which were identified in the Vietnamese ETEC collection. Firstly in pre-weaning diarrhoea, F4:Paa:STa:STb:LT:EAST-1 was still the most common pathotype (it was also the most common pathotype observed during Dr Do Ngoc Thuy’s PhD studies). During studies in Dr John Fairbrother’s laboratory, he identified that the virulence factor pathotype Paa:STa:STb:LT:EAST-1 was a consistent marker for the O8 F19 isolates that possess the new fimbrial type (thus they can be rapidly identified by their virulence gene profile even though diagnostic antisera that was entirely specific for the O8 strains could not be produced in Vietnam). This pathotype was the second most prevalent in the pre-weaning diarrhoea isolates, indicating that it was still a significant pathogen in preweaning diarrhoea in Vietnam. In post- weaning diarrhoea, the major pathotypes were associated with F18 rather than F4 and the majority of F18 strains also possessed stx2 toxin, confirming that the isolates had the capability of causing both post-weaning diarrhoea and oedema disease. 4. Introduction & Background Diarrhoea during the suckling period has been recognised as the principle health problem affecting both smallholder and commercial pig production in Vietnam. Previous research has confirmed the presence of a new fimbrial type in E. coli strains causing colibacillosis in Vietnam that would not be controlled by existing vaccines. Existing vaccines are currently imported into Vietnam at considerable cost. In addition, there are many other causes of suckling diarrhoea, the significance of which is currently unknown in Vietnam, which are all affected by husbandry and management during farrowing and lactation. Project 001/04VIE (Diagnosis and control of diarrhoea in suckling pigs) began with three objectives to solve this problem: 1. Production and testing of locally-produced E. coli vaccines 2. Development of a management plan for preweaning diarrhoea using a continuous improvement (CIP) model 3. Improved field and laboratory diagnosis of preweaning diarrhoea 5. Progress to Date 5.1 Response to Appraisal In general the previous report was well received and there were only two major issues that required clarification: The proponents state that this project is still in the data gathering stage. This second six-monthly Milestone Report is about 10 months later than expected and given that the project is due for completion in March 2007 (according to the Contract) there is some concern that about delivery of 5 further milestones by this date. Can the project proponents please clarify this position? 7 All the data is presented and analysed in the current report. Unfortunately the requirements of the 004/05VIE project were overwhelming and prevented timely submission of reports, even though the work was completed within the specified timeframe (and the findings presented at two major international conferences). The presence of the new unique fimbrial type remains an issue. The proponents state that there is a need for a survey to determine its incidence. Given that a key component of this project is production of a vaccine for this (and other types) who is going to undertake such a survey and how will the economic necessity for incorporation of the new unique type into existing or Vietnamese produced vaccines be established? The research work conducted in the laboratory of Prof John Fairbrother confirmed the typical virulence gene pathotype of the unusual O8 (F19) strains, and work conducted by Dr Do Ngoc Thuy and presented in this report confirmed that these strains are still prevalent in commercial piggeries in Vietnam. The strains are likely to be present in smallholder farms, but have not been detected as yet in the small number of samples processed by NIVR. The cost of inclusion of this strain in the vaccine is minimal and far outweighs the risk of exclusion (ie widespread vaccination with F4 and F5 strains would only increase the prevalence of the F19 strains). 5.2 Implementation Highlights 1) Vaccine strain characterization and further identification of the novel (F19) fimbrial antigen present in O8 5F- strains (Note: A detailed report of activities is included in Appendix One: Characterization of Vaccine Strains). The NIVR E. coli vaccine strains were extensively and independently characterized. Attempts to produce diagnostic antisera that was 100% specific against the novel O8 strains (as previously reported) were unsuccessful and it was concluded that the laboratories in both Australia and Vietnam lacked the expertise and equipment to perform a large scale fimbrial extract required to purify the antigen. An opportunity for Dr Do Ngoc Thuy to visit the laboratory of Prof John Fairbrother (the world’s leading porcine E. coli expert) immediately following the IPVS Congress in 2006 was eagerly taken up. Dr Thuy performed initial experiments confirming to Prof Fairbrother that the novel Vietnamese ETEC did indeed produce a mannose-resistant adhesin at 37 o C (highly indicative of production of fimbriae capable of attachment to enterocytes), which was confirmed by transmission electron microscopy. An initial attempt at purification of the fimbrial extract revealed that there were still some contaminating proteins. Dr Thuy then returned to Vietnam and Prof Fairbrother continued the process of characterization. Initial attempts at identifying proteins in the purified fimbrial extract were unsuccessful. However, following a more rigorous extraction method, a 20KDa sized band (the right size!!) was identified in SDS-PAGE gels as the putative adhesin as it reacted in a Western Blot with hyperimmune serum obtained following immunization of rabbits with an E. coli O8 5F- whole cell extract grown at 37 o C to produce fimbriae and absorbed with sera obtained for the same strain grown at 18 o C, when it does not produce fimbriae. An N-terminal amino acid sequence obtained from the band cut from a one dimensional SDS-PAGE gel identified a protein closely related to Enterobacter ompX, but Prof Fairbrother is not convinced that this is the adhesin, even though it has a putative attachment role in other bacteria. A problem is that the adhesin does not seem to be expressed as much or as consistently as other fimbrial antigens and contaminating proteins may be being identified instead of the true fimbriae. However, Prof Fairbrother has produced diagnostic antisera that is now quite specific for the F19 antigen and screened a large number of isolates from his collection (n=140) including all of the Vietnamese strains. This identified the correlation between F19 and the virulence gene profile Paa/STa/ST/LT/EAST-1 8 which can be used as a marker for the strains. Prof Fairbrother is also assisting Dr Thuy in the characterization of post-weaning diarrhoea and oedema disease isolates for a major paper. 2) Safety and efficacy testing of NIVR E. coli vaccine. The NIVR prepared the vaccine for small scale trials according to the methodology detailed in Appendix Two: Vaccine production protocol. Protection, safety and efficacy studies are shown in Appendix Three: Results of Safety and Efficacy Studies of E. coli vaccine. In summary, the vaccine offered piglets significant protection from lethal homologous challenge infection and 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. Small amounts of the vaccine are now being supplied to selected herds in the North of Vietnam to obtain field data on its safety and anecdotal reports on efficacy. A true field trial is planned to coincide with major farmer to farmer training initiatives in 004/05VIE. 3) Analysis of all production records from the five test farms and five 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: Average preweaning mortalities observed in five test (T) and five control (C) piggeries during the 14-month observation period. 9 4) Farm visits to Northern Herds September 2006. A summary of the final farm visits to the Northern herds is detailed in Appendix Four: Farm reports all September. For comparative purposes, the farm audits from 2005 are included below each 2006 audit. 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. 5) Results of diagnostic investigations on pre-weaning diarrhoea. 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. 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 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. For scour occurring at the age of weaning or postweaning scour, the following regime was suggested by Dr Tony Fahy. This involves treatment with antibiotics, or if unsuccessful feeding a tame strain to sucker pigs 1 week before weaning:  Farm to ensure that new weaners are in a draught free pen. This means enclosing the pen with walls (eg used feed bags) and putting covering on about one third of the floor areas, in the zone heated area, where the pigs will lie (eg used feed bags).  Place zone heaters over the floor area that is covered by bags. 1 0  The temperature needs to be 30-32 ‘C in the first week after weaning. This should be lowered by 2 degrees each week by lifting the heaters up.  Piglets should be offered glucose and electrolyte solution in drinkers. A water soluble antibiotic (that Thuy will identify) must be added to this drinking solution. This solution is used for fourteen days post weaning.  Any pig that scours is to be treated (injectable) with the antibiotic recommended by Thuy.  Any piglets that scour are to have a rectal swab taken at the time of treatment (NOT AFTER treatment).  If this strategy is not successful you will have to orally vaccinate the sucker piglets one week before weaning. Oral Vaccine procedure This can only be done if the E. coli isolate does not have STa. To be cautious test it on two litters in the first week, four litters in the next week and then if there are no problems all litters can be treated the next week. If the vaccine causes a scour treat the scouring piglets with Baytril.  Thuy will provide a UHT skim milk inoculum for the production of the vaccine.  The vaccine will be made by adding 100 ml of inoculum to 900 ml of UHT skim milk and incubating overnight at around 37’C.  This vaccine is fed to the sucker piglets by adding 1 ml of it to 250ml of glucose electrolyte solution per pig.  This is done for four consecutive days for each litter.  Three days before the inoculum is added pigs are offered the glucose electrolyte solution so that they are used to drinking it.  This will provide an opportunity to determine how much fluid is drunk in one day.(ie if a whole litter of 9 pigs drinks 1500ml you will add 9 mls of inoculum to the 1500mls so the piglets get 1 ml each) Table 1: 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) [...]... rapidly and specifically identify the E coli O8 5F- strains (ie strains with NVP6 13 genotype): A) Crude whole cell method Step 1: Preparation of antigen and vaccination of rabbits: Method: - Grow isolate (NVP6 13 5F-) at 37 oC for 6- 8 hours on shaker - Inactivate with formalin (0 .3% v/v) overnight - Plate onto bacteriological agar to confirm inactivation - Wash in PBS and centrifuge at 30 00 rpm for 5-1 0 mins... in Table 2 for agglutination: Table 2: Agglutination of E coli strains in polyvalent rabbit sera prepared from a 5F- strain grown at 37 oC cross-absorbed with cells grown at 18oC Strains Growth temperature of cultures O-serotype Virulence factors Agglutination NVP612 37 oC O8 5F-/STa/STb/LT + NVP625 37 oC O8 5F-/STa/STb/LT +++ NVP 137 2* 37 oC O64 F5/STa - NVP 139 2 37 oC O149:K91 F4/STa/STb/LT - NVP1402* 37 oC... University of Queensland 3) Management and production workshop x 1 4) Farm audits, data recording system and ongoing data analysis, health and production knowledge, post mortem analysis 26 Ministry of Agriculture & Rural Development APPENDIX ONE Collaboration for Agriculture and Rural Development (CARD) Program Diagnosis and control of diarrhoea in suckling pigs Characterization of vaccine candidate strains... similar in of components, including novel the mechanisms of the novel produced vaccine pathogenicity to existing strains strain pathogen and will respond in a similar 1.b Formulation of vaccine Preparation of antigen for manner Preliminary work 1.c Efficacy testing of vaccine vaccination and diagnostic suggests this is true 1.d Field testing of vaccine reagents 1.e Commercial realisation of Production of. .. 30 00 rpm for 5-1 0 mins (x3) - Protein determination using the Lowry protein assay (an optic density of 1.0, determined at 280 nm contained an estimated protein concentration of 1 mg/ml) - Vaccination schedule: 2 rabbits were injected intravenously at 4-5 day intervals with 0.2; 0.4; 0 .6 and 1 mg of antigen, respectively - The rabbits were bled 6- 7 days after last injection and the serum harvested by... training of field commensurate with productivity health management techniques and laboratory staff continuing gains Information specific to local conditions 3 Improved diagnostics for preweaning diarrhoea Stakeholder beneficiaries and recipients of training / capacity building: Reagents to improve speed and accuracy of field and laboratory diagnosis Compilation and delivery of training materials and. .. from 26/ 6/20 06 to 14/7/20 06 A Characteristics of 5F- ETEC strains used in the experiments: Characteristics Designation O-serogroup Enterotoxins O8 5F-, F? STa, STb, LT O8 NVP612 Fimbirae 5F-, F? STa, STb, LT (CARD-VN1) EC-VN8 B Results of mannose-resistant haemagglutination: The two ETEC strains were examined for mannose-resistant haemagglutinating activity using Sheep Red Blood Cells Both strains were... presence of cells expressing the new fimbrial type However, it must be remembered that this is an inexpensive but crude technique and all cross-reactive antibodies may not be removed during cross-absorption 28 Method: - Grow isolate (NVP6 13 5F-) at 1 8-2 0oC (to prevent production of fimbriae) for 6- 8 hours - Inactivate with formalin (0 .3% v/v) overnight - Wash and centrifuge 3 times in PBS/saline Absorption:... Organise initial training workshops in Australia and Vietnam (2a) Initial Australian site visit by NIVR and 1st Vietnamese workshop completed 0 -3 months Sourcing co-operating farms (1, 2a, 2b) 0-2 months Test and control farms (5 each) selected in all provinces Each farm audited three times 24 Implementation of pig health recording services (2a, 2b) Training extension staff in data recording and risk... Outputs, Activities And Inputs Project Title: Diagnosis and control of diarrhoea in suckling pigs Vietnamese Implementing Institution: National Institute of Veterinary Research PROPOSAL Narrative Information Required Performance Measures PROGRESS REPORT Assumptions Information Required OBJECTIVES 1.a Identification and confirmation Confirmation and identification of 1.Production and testing of locallyNovel . 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. 1 Ministry of Agriculture & Rural Development Project Progress Report Diagnosis and control of diarrhoea in suckling pigs CARD Project 001/04VIE MILESTONES 3 and 6 2 nd and 3 rd . 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