Báo cáo Nghiên cứu khoa học Diagnosis and control of diarrhoea in suckling pigs: 2nd and 3rd six monthly reports (combined)

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 2nd and 3rd 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 13th 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 pre-weaning disease in some, and possibly all, areas of Vietnam. 3. Executive Summary This report, which is a combination of two six-monthly reports (2nd report and 3rd 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 37oC (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 37oC to produce fimbriae and absorbed with sera obtained for the same strain grown at 18oC, 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 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: 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. 10  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) 11 RV TGEV 17 (14.4) 3 (6.7) Cocci RV 1 (0.8) 2 (4.4) CocciCrypto 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) TGEVETEC 7 (5.9) Cocci RV ETEC 2 (1.7) 3 (6.7) Cocci RV TGEV 4 (3.4) 3 (6.7) Cocci TGEVETEC 2 (1.7) 2 (4.4) CocciCrypto RV 1 (0.8) 1 (2.2) Crypto TGEVETEC 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 TGEVETEC 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) TGEVETEC C. per. 2 (1.7) Crypto RV TGEVETEC 2 (1.7) 1 (2.2) Crypto RV ETEC C. per. 1 (0.8) 2 (4.4) CocciCrypto RV TGEV 1 (0.8) 1 (2.2) CocciCrypto TGEV C. per. 1 (0.8) Cocci RV TGEVETEC 4 (3.4) 1 (2.2) CocciCrypto RV TGEV C. per. 1 (0.8) 1 (2.2) Crypto RV TGEVETEC C. per. 1 (0.8) (36) (50) (97)(111) (76) (23) Total multiple infections 92 (78.8) 45 (100.0) 12 6) Characterization of E. coli 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 F19 O8 strains, 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. 13 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 14 5.3 Smallholder Benefits Whilst this initial project has focused on monitoring herd health in commercial-sized piggeries, considerable downstream benefits will flow to smallholder farmers as follows: 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 could be produced at $0.15 USD per dose. Our intended purpose is to register this vaccine, but in order to do so we must conduct a comprehensive field trial. The farms developed in 004/05VIE present an ideal opportunity for this to occur as the farmers are now raising piglets with good hygiene in dry, warm surroundings. In addition, there is a ready supply of “control” farms located nearby where data could be gathered on the incidence of diarrhoeal diseases, including ETEC 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 diseases. 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. The unusual F19 strains, so far, have not been identified in smallholder farms. 5.4 Capacity Building NIVR has developed skills in the isolation and identification of major pig pathogens causing preweaning diarrhoea, using a combination of culture, ELISA test kits and faecal floatation/faecal microscopic examination. This capacity could easily be scaled up so that NIVR then teaches other laboratories, including diagnostic laboratories how to accurately identify pig pathogens, particularly those involved in pre-weaning diarrhoea. We have demonstrated that all the major causes of pre-weaning diarrhoea on a worldwide basis are present in Vietnam, often in mixed infections, with rotavirus, TGEV and ETEC predominating in commercial pigs and the same agents dominating in village based smallholder farmer pigs with the important addition of coccidiosis. Accurate identification of these agents is required, both on clinical suspicion (each agent causes different clinical signs and affects pigs at different ages, however in the case of mixed infections this may be more difficult to detect) as well as laboratory grounds. Two major disease causing agents (ETEC in the first week of life and coccidiosis at 6-14 days of age) are easily preventable by vaccination of the dam in the case of ETEC and preventative treatment with toltrazuril at 3 days of age in the case of coccidiosis. However, without extreme improvements in the care and husbandry of piglets during the crucial pre-weaning period, any perceived benefits will not be attained. 15 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 and we have been unwilling to attempt any controlled field trials until such we are confident that our smallholder farmers have improved their standards of husbandry. A large field trial is planned in central Vietnam as part of 004/05VIE in order to finalise both projects under our direction. All data will be obtained in such a way as to support eventual registration of the vaccine. 5.5 Publicity Unfortunately the negative publicity surrounding NIVR and the production and sale of unregistered vaccines in 2008 is not the sort of marketing tool that we like to brandish. However, this does present an opportunity to understand why some of these events occurred. There is a tremendous need in Vietnam for locally produced, cheap and safe vaccines that can be used by smallscale farmers and NIVR has produced some very successful vaccines. However, they have limited experience and resources in following the path to commercialization and registration. We will use the ETEC vaccine as the paradigm for effective interrelationships with MARD and the Department of Animal Health to conduct a field trial and successfully negotiate the path to commercialisation. We will also seek to patent Dr Thuy’s vaccine through the Office of Intellectual Property of Vietnam within the Ministry of Science and Technology. This will enhance the reputation of NIVR as a quality research establishment and provide much needed funds through a royalty stream to upgrade facilities and continue to conduct first rate research. Other vaccines, such as the oedema disease vaccine could be taken along a similar path once the ETEC vaccine is successfully launched into the market. 5.6 Project Management No major developments have taken place apart from the retirement of Dr Steve Driesen from the Victorian Department of Primary Industries. 6. Report on Cross-Cutting Issues 6.1 Environment Environmental impacts, gender and social issues are more appropriately discussed in the O4/005VIE project reports as these have significant downstream benefits to smallholder farmers, their families and the immediate environment. 6.2 Gender and Social Issues 7. Implementation & Sustainability Issues 7.1 Issues and Constraints 7.2 Options Issue 1: Commercialisation of the NIVR vaccine. 16 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. Options: 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 will seek the advice and assistance of a patent attorney and seek to 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. 7.3 Sustainability The issues of sustainability as we are coming to the end of this project can be discussed under three broad schemes: 1. Production and testing of locally-produced E. coli vaccines This has been discussed at length. The availability of leftover funds once the 004/05VIE project is completed will need to be used to further the path to commercialization and complete the analysis of field trials in Central Vietnam. 2. Development of a management plan for preweaning diarrhoea using a continuous improvement (CIP) model. This theme has been carried into 004/05VIE where we are seeking to develop a core of successful farmers and has been extended to a holistic approach to pig production. We are now at the stage in 004/05VIE where we can conduct a field trial on the NIVR vaccine AND a prevalence study for causes of preweaning diarrhoea in test farms (those that have received an upgrade to their facilities and knowledge and skills) and control farms (those that were initially surveyed but did not receive advice). Our legacy will be a nucleus herd in central Vietnam that becomes the model for successful smallholder production in other regions. 3. Improved field and laboratory diagnosis of preweaning diarrhoea. As part of the fulfilment for 2), NIVR will transfer their knowledge and technology obtained in the diagnosis of preweaning diseases to Hue University of Agriculture and Forestry which will then provide the diagnostic service for the region. 8. Next Critical Steps 1) Complete vaccine field trial and prevalence study of enteric diseases in central Vietnam (Dec 2009-Feb 2010). 2) Transfer technology for diagnosis of preweaning enteric diseases to Central Vietnam (Dec 2009-Feb 2010). 3) Continue negotiations and pathways towards registration of the NIVR E. coli vaccine (Oct 2009-April 2010). 17 9. Conclusion This combined milestone report details the considerable research and effort by NIVR and Australian scientists to achieve project success within the logframe. Apart from the complete characterization of the F19 antigen, all objectives have been fulfilled and remaining milestones will be relatively straightforward. Even the further characterization of the F19 antigen from non- project funds provides an opportunity for continued sustainability of the project and longterm international funding collaborations. Difficulties experienced in seeking the right pathway for commercialization of the vaccine will provide a technology platform for taking additional locally produced vaccines through to commercialization and will provide a continued funding stream for NIVR. 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. 18 10. Statutory Declaration COLLABORATION FOR AGRICULTURE AND RURAL DEVELOPMENT PROGRAM CARD Project Title: - Diagnosis and control of diarrhoea in suckling pigs CARD Project Number: - 001/04/VIE We the undersigned hereby declare that during the period 01/04/2006 to 31/09/2008 we have delivered the following inputs to assist in implementation of the above project. 1: PERSONNEL INPUTS Australian Personnel Provided (Name) Days in Vietnam Days in Australia Trips to Vietnam Dr Tony Fahy 20 12 2 Ms Karen Moore 10 12 1 Dr Trish Holyoake 0 0 0 Dr Darren Trott/Dr Ian Wilkie 10 12 1 Total 40 36 4 Vietnamese Personnel Provided Days in Vietnam Dr. Truong Van Dung 0 Dr. Cu Huu Phu 24 Dr. Do Ngoc Thuy 48 NIVR Diagnostic Laboratory Staff 24 19 2: EQUIPMENT AND OTHER SERVICES Equipment & Other Services Description Budget Limit Signed for the Australian Institution by a duly authorised officer in the presence of witness Signature of Witness Insert Name and Title Insert Name and Title 3; EQUIPMENT AND SERVICES HANDOVER This is to certify that the above personnel inputs have been delivered and the equipment and services identified above provided has been handed over to the Lead Vietnamese Institution Signed for the Vietnamese Institution by a duly authorised officer in the presence of witness Signature of Witness 20 Insert Name and Title Insert Name and Title 21 Project Progress Against Proposed Objectives, Outputs, Activities And Inputs Project Title: Diagnosis and control of diarrhoea in suckling pigs Vietnamese Implementing Institution: National Institute of Veterinary Research PROPOSAL PROGRESS REPORT Narrative Information Required Performance Measures Assumptions Information Required OBJECTIVES 1.Production and testing of locally- produced vaccine 2a. Enteric management plan for preweaning diarrhoea 1.a. Identification and confirmation of components, including novel strain 1.b. Formulation of vaccine 1.c. Efficacy testing of vaccine 1.d. Field testing of vaccine 1.e. Commercial realisation of vaccine Quantitative data on piglet health, survival and growth rates Incidence and nature of preweaning diseases Confirmation and identification of the mechanisms of the novel pathogen Preparation of antigen for vaccination and diagnostic reagents Production of vaccine Safety and efficacy tests completed in NIVR Field trials completed Vaccine batch records scrutinized Workshop with stakeholders produces acceptable management plan. Implementation of plan on demonstration farms Training of facilitators to disseminate to other provinces and regions Supply of training material Decrease in preweaning mortality Workshop feedback surveys, certified training records Novel pathogen is similar in pathogenicity to existing strains and will respond in a similar manner. Preliminary work suggests this is true. Workshops with stakeholder group will result in a coherent approach and workable management plan. Characterization of the vaccine strain, in particular the isolation of novel fimbriae and identification of the genes encoding the fimbriae was undertaken by Dr Do Ngoc Thuy in the laboratory of Dr John Fairbrother, Montreal Canada. The purified fimbriae can be used to produce diagnostic antisera. Characterization is complete (apart from the gene identification of the F19 positive strains) Efficacy and safety testing is completed After our first workshop and farm visits, we immediately identified that there would be a breakdown in information and technology transfer from the Australian Scientists to the producers because of a general lack of training of veterinarians with herd health management skills. We therefore 22 2.b Continuous improvement plan (CIP) 3. Improved diagnostics for preweaning diarrhoea Stakeholder beneficiaries and recipients of training / capacity building: -smallholder farmers -large commercial piggeries -NIVR laboratory staff -NIVR vaccine production Subinstitute Analysis of data acquired during and after project Extension workers trained in herd health management techniques Information specific to local conditions. Reagents to improve speed and accuracy of field and laboratory diagnosis Disease monitoring and surveillance, implementation of pig management recording services Protocols developed, instructional materials prepared, training of field and laboratory staff continuing Compilation and delivery of training materials and successful completion of training modules for laboratory staff (Aust. and Vietnam) Availability of validated reagents Training of field staff in gross diagnostic techniques Ability to assign mortality to a category That stakeholder demand will maintain interest and input commensurate with productivity gains Adequate laboratory capability Timely development of reagents developed the philosophy of “train the trainers” and applied this to 004/05VIE Selected demonstration farms collected data on preweaning mortality-test farms were significantly different from controls. The CIP model is being reinforced with each visit to Vietnam. As above, we have identified another layer of training with highly trained Vietnamese scientists giving the workshops and facilitating hands on training. This will be more fully explored in CARD project 004/05VIE Techniques have successfully been transferred, accurate data on causes of enteric diseases in suckling pigs has been compiled for both commercial and smallholder farmers OUTPUTS 1. Local ETEC strains confirmed and characterised. Technologies: Large scale production of locally produced vaccine that is efficacious against all strains of ETEC Data compiled, strains isolated and preserved Vaccine strains cultured and combined As above Whole cell immunization of rabbits for production of polyclonal antisera to identify novel O8 F- strains was successfully completed in Canada 23 2a. Enteric management plan for preweaning diarrhoea 2.b Continuous improvement plan (CIP) Institutional capacity: Improved diagnostic reagents produced at NIVR for accurate investigation of disease on farm/vaccine field trials with particular respect to new fimbrial type. Financial benefits: NIVR Subinstitute increased vaccine production for distribution throughout Vietnam. Improved production at reduced cost through use of cheap local vaccine, less reliance on antimicrobials, reduced build up of antimicrobial resistance (both commercial and smallholder producers) Export products: Pork products- reduced public health concern re antimicrobial resistance, improved production at reduced cost. Vaccine could be exported to neighbouring countries such as Laos and Cambodia Knowledge: Farm reports and farm audits; herd health monitoring Institutional capacity: Scientists trained to conduct audits/provide advice; Financial benefits: creation of model farms for demonstrating how changes in management can improve production and financial profitability; records and farms can Data from field trials collected and compiled Data collected from farms Reduced preweaning mortality Field data collection and analysis Income estimations of farmers Written and verbal communication records/evidence of ongoing fieldwork Record of training certificates issued to animal health workers/producers and feedback survey results As above and identified a unique virulence gene profile for the O8 strains (now christened F19 strains). An unexpected opportunity arose for NIVR staff to receive further training to identify the novel antigen in another laboratory working with world experts in the field who are very interested in the novel strains. The work will lead to future international funding applications. With full and ongoing support from NIVR, test farms have been audited twice more to continually reinforce the CIP model. Some farms have adopted the recommended changes whilst others have not. However without further appropriate training of Vietnamese veterinarians to be able to independently conduct farm audits and recommend changes, full adoption of management changes and continuous improvement will be limited and 24 3. Improved diagnostics for preweaning diarrhoea then be used in future workshops for training smallholder farmers Social outcomes: Meaningful training for rural women in herd management Technologies and Knowledge: Supply of rapid diagnostic reagents and appropriate training to NIVR for rapid and accurate diagnosis of the six major causes of pre- weaning diarrhoea Institutional capacity: Capacity for accurate and timely complete diagnostic service at NIVR for specimens received from trial farms (faecal specimens and faecal swabs). Financial benefits: improved knowledge of major diseases limiting production and best methods for control Social outcomes: Majority of NIVR lab staff are women Improved ability to diagnose disease accurately and rapidly More accurate analysis of causes and age at mortality As above unsustainable. This is being addressed in CARD project 004/05VIE. NIVR laboratory is accurately and rapidly determining the causes of preweaning diarrhoea on the test and control farms. The data has been compiled and presented at two major international conferences. ACTIVITIES Organise initial training workshops in Australia and Vietnam (2a) Sourcing co-operating farms (1, 2a, 2b) 0-2 months 0-3 months Initial Australian site visit by NIVR and 1st Vietnamese workshop completed Test and control farms (5 each) selected in all provinces. Each farm audited three times. 25 Implementation of pig health recording services (2a, 2b) Training extension staff in data recording and risk analysis identification (2a, 2b) Characterise novel ETEC strain (1) 0-6 months 0-12 months (revised to 0-24 months) 0-6 months (revised to 0-24 months) Pig health recording services initiated for each farm. Data being collected and farm audited by Australian Scientists three time during the reporting period (November 2005; February 2006; September 2006) After the first workshop, it was clear that training of extension staff will require additional time and resources not allocated in the present project. “Train the trainers” in a “holistic approach to improving all stages of production” has become the theme for CARD project 004/05VIE focusing on smallholder production. The farms established in the current project together with the production records obtained will be important training resources. Analysis of herd records for the test and control farms confirmed that preweaning mortality was significantly reduced on the test farms Lack of specificity in the prepared diagnostic antisera, necessitated technical changes to antigen production and partly because of new opportunities arising to conduct further characterisation in another laboratory. Specific antisera has been developed in Cnada, but the gene for the novel 26 Develop and/or acquire and implement suitable diagnostic reagents Develop vaccine strains. Transfer of vaccine technology and production of vaccine 0-8 months 0-12 months (revised to 0-18 months) fimbriae remains elusive. All diagnostic reagents have been acquired from Australia, laboratory staff have been trained in using them and all diagnostic techniques have now been implemented at NIVR apart from the diagnostic O8 F- antisera. PCR Machine purchased and implemented for pathotyping ETEC strains. Data collected to be presented at two international conferences Vaccine strains have been selected and their pathotypes independently verified. Vaccines have been produced and efficacy and safety tested INPUTS University of Queensland Pig Health and Research Unit NIVR 1) Diagnostic reagents and laboratory training (Australia and Vietnam) 2) Laboratory equipment 3) Management and production workshop x 1 4) Farm audits, data recording system and ongoing data analysis, health and production knowledge, post mortem analysis 27 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 for E. coli vaccine 28 1. Serogroup, fimbriae and enterotoxins possessed by the candidate strains 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 (CARD-VN2) O149: K91 F4 STa/STb/LT 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. 2. Attempts to produce diagnostic antisera at NIVR to rapidly and specifically identify the E. coli O8 5F- strains (ie strains with NVP613 genotype): A) Crude whole cell method Step 1: Preparation of antigen and vaccination of rabbits: Method: - Grow isolate (NVP613 5F-) at 37oC 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 3000 rpm for 5-10 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 centrifugation. Step 2: Preparation of antigen for cross-absorption: Note: E. coli grown at 18-20oC will not produce fimbriae. Therefore, to prepare sera that is specific for the possible new fimbrial type, the antisera from rabbits is mixed with inactivated NVP613 cells harvested from an 18-20oC culture. Non-specific antigen:antibody complexes are then removed by centrifugation and the remaining antisera should only agglutinate in the 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. 29 Method: - Grow isolate (NVP613 5F-) at 18-20oC (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: - Make a 20% cell suspension with the inactivated cells grown at 18-20oC in the rabbit antisera - Incubate 1 hour at 37oC, then refrigerate overnight - High spin to pellet cells - Remove antisera - Coagglutinate antisera - Test against cells grown at 37C + Isolate NVP613: + + F4: - + F5: - + Field strain (NVP625 5F-): + Results: Prepared antisera was tested against the enterotoxigenic E. coli strains listed in Table 2 for agglutination: Table 2: Agglutination of E. coli strains in polyvalent rabbit sera prepared from a 5F- strain grown at 37oC cross-absorbed with cells grown at 18oC. Strains Growth temperature of cultures O-serotype Virulence factors Agglutination NVP612 37oC O8 5F-/STa/STb/LT + NVP625 37oC O8 5F-/STa/STb/LT +++ NVP1372* 37oC O64 F5/STa - NVP1392 37oC O149:K91 F4/STa/STb/LT - NVP1402* 37oC O149:91 F4/STa/STb/LT - NVP613* 37oC O8 5F-/STa/STb/LT +++ 20oC +++ NVP1272 37oC O8:G7 F4/STa/STb - - no agglutination observed + Weak agglutination (25%) ++ Moderate agglutination (50%) +++ Strong agglutination (100%) * vaccine strains Conclusion: The agglutination reaction with the vaccine strain prepared at both 37oC and 20oC suggests that the antisera prepared from a crude culture extract may not be specific against the unknown F-antigen only. It might contain other antigenic components present in the O8 strain cell wall. Therefore, we conclude that in order to prepare diagnostic antisera that is 100% specific, we will need to purify and identify the unknown 5F- antigen before immunizing rabbits again. Our 30 respective laboratories at NIVR and in Australia neither have the expertise nor the equipment to prepare purified fimbrial extracts. Following presentation of her work at the 2006 International Pig Veterinary Society Congress, Prof John Fairbrother (OIE E. coli laboratory at the Faculté de médecine vétérinaire Université de Montréal) invited Dr Do Ngoc Thuy to his laboratory to begin work on the further characterization of the new fimbrial type. 3. Summary results of experiments in OIE Escherichia coli Reference Laboratory, Montreal for the period from 26/6/2006 to 14/7/2006 A. Characteristics of 5F- ETEC strains used in the experiments: Characteristics Designation O-serogroup Fimbirae Enterotoxins NVP612 (CARD-VN1) O8 5F-, F? STa, STb, LT EC-VN8 O8 5F-, F? STa, STb, LT 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 tested using overnight cultures grown at 18oC and 37oC. The density of bacteria in each culture was adjusted to OD=1 (=660 m) in NaCl 0.85% using a PYE Unicam PU-8600 UV/VIS spectrophotometer (Philips), prior to mixing with red blood cells. The results of haemagglutination of cultures of each strain are presented in Table 1. Mannose-resistant haemagglutination was observed at 37oC, but not at 18oC for both strains, confirming the production of adhesins (ie fimbriae) at 37oC. 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 C. Observation of E. coli 5F- cell morphology by Transmission Electron Microscopy: Specimen preparation for transmission electron microscopy was carried out as follows: One drop from the surface of a culture of strain CARD-VN1 grown in nutrient broth at 37oC overnight was placed on a glow discharged (carbon and formvar) coated copper 200 mesh grid, and allowed to absorb for 2 min. Excess liquid was then removed with filter paper and one drop of 1% ammonium molybdate (pH=6.5) was added. After 30 sec, the specimen was blotted dry with filter paper and observed under a Transmission Electron Microscope operated at 80 kV. Transmission electron microscopy photographs taken at low and high magnification showed the presence of hair-like structures on the surface of the bacteria cells (an example is shown in Figure 1). 31 5F- ETEC strain (grown overnight in BHI broth) under transmission electron microscopy Scale bar = 2 m Scale bar = 1 m Scale bar = 200 m 32 D. Preliminary results on purification of 5F- fimbriae: The purification of the 5F- fimbriae was performed using the OIE E. coli Laboratory protocol EcL1000 (Production of Fimbriae) with some modifications. All steps were exactly the same as described in the protocol, except that: - In the Precipitation step, the concentration of sulfate ammonium was increased to 30% and 40% - In the Dialysis step: citric acid 2.5% or sodium desoxycholate were used to precipitate the non- fimbriae proteins. After sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified fimbrial extract, the results confirmed that a protein band was present in the gel with a molecular weight of ~26,000-27,000 kDa. This is the same size as most recognized E. coli fimbrial proteins. However, the protein precipitation and wash steps require further development since the final fimbrial extract also appeared to still be contaminated with several other proteins. These were promising results to be followed up by the OIE E. coli Reference Laboratory following Dr Thuy’s return to NIVR. 4. Further characterization of the unknown fimbriae type by the OIE Escherichia coli Reference Laboratory, (Report dated September 2008). Here is a quick update on F19 (Thuy’s new fimbrial type) to let you know what we are doing. As I already mentioned, we did a new fermentation early in the year and ran the heat-treated extract on SDS-PAGE. We sent off for N-terminal aa sequencing the 20K band which we had identified by western using an adsorbed antiserum as the putative fimbrial band (present on the strain grown at 37°C but not at 18°C.). This came back as the following sequence: ATSTVWGGYY. On Blast, this did not show close similarity with anything, but was not a long enough sequence. We proceeded to run another gel and send off the band for Mass Spectrometry as this seems to be the approach of choice now. It came back identified with high confidence factor as OmpX (see attached), with predicted molecular mass of 17kDa. I was not too convinced that this is our adhesin as it is an Omp and not a fimbriae, and our band showed up at 20kDa on western. I have read some articles on OmpX- it could be involved in adherence to and invasion of cells in culture and is related to Ail in Yersinia, and pagC in Salmonella. As I am not fully convinced, we will run another SDS-PAGE and this time, we will cut out any bands we observe around 18 to 22 kDa that seem to be less expressed at 18°C, and send them off for MS. It costs around $60 per reaction. We are doing this at the moment. As the DNA sequence of the gene in Enterobacter is known, we could also test by PCR or probe (see pdf attached) phenotypically positive and negative isolates to see if there is any correlation. I have tested all of the 140 isolates in our collection from Thuy by immunofluorescence using the adsorbed anti-F19 serum. There is a good correlation with the virotype Paa,STa,STb,LT,East1. All of the isolates have been virotyped for 20 virulence genes. Also, the postweaning isolates (about 40) have been tested by MIC, for the paper with Thuy. We did not do the preweaning isolates for MIC, as you had said that these isolates had already been tested and will be in another paper. Several of the PWD isolates are enroflox resistant, hence my interest to test for the qnr genes. I am compiling all these results at the moment. I know that the F19 characterisation has been taking a lot of time to get results. It is a question of man power. When my technician has had spare time, she has worked on it. However, she went off on pregnancy leave! Now that the summer holidays are finished, I have put another technician on the project. We will run more gels this week or next and send off bands for MS. Our problem has always been that the adhesin band is not produced in great quantities as we observed with F4, F165 etc, so it is not easy to identify the correct band. 33 Do you think that at this point you will have enough for AUSAID? I think that if we want to go further, it would be a great project for a student. Another approach if we still have problems could be DIGE, the proteomic approach. We could send the 18C and 37C preps off for 2-D gel electrophoresis and MS. Let me know what you think. John PS How is Matthew progressing? PPS Is Thuy still in Vietnam? We have to advance the PWD paper once I compile the data. John M. Fairbrother, BVSc, PhD Director / Directeur Reference Laboratory for Escherichia coli john.morris.fairbrother@umontreal.ca T 450.773.8521 or 514.345.8521, ext. 18234 F 450.778.8108 Cell 450.230.2720 GREMIP, Faculté de médecine vétérinaire Université de Montréal 3200 Sicotte, C.P. 5000 Saint-Hyacinthe (Quebec) Canada J2S 7C6 34 APPENDIX TWO Vaccine production protocol 1. An inactivated whole cell multivalent vaccine was prepared according to the protocol of the Bacteriology Laboratory, NIVR. The vaccine contained three E. coli strains with the O-antigen and virulence characteristics of each listed in Table 1. Table 1: E. coli strains used for the preparation of vaccine Characteristics Designation of E. coli vaccine strains O-serogroup Fimbriae Enterotoxin(s) CARD-VN1 O8 F? STa/STb/LT CARD-VN2 O149: K91 F4 STa/STb/LT CARD-VN3 O64 F5 STa 2. 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 as described by Jones & Rutter (1972), whereas for the production of F5 fimbriae on strain CARD-VN3, Minca agar as described by Guinee et al. (1977a) was used. For the strain with currently uncharacterized fimbriae (CARD-VN-1), it was shown in Appendix One that Buffered Glucose Nutrient Agar was suitable for the production of a possible new fimbrial type. The procedure used to prepare the vaccine is summarised in Figure 1. In brief, cultures of each strain were revived from freeze-dried tubes, inoculated into TSB and incubated at 37oC overnight with agitation (150 rpm). The broth cultures were flood seeded onto Buffered Glucose Nutrient Agar or Minca Agar, incubated at 37oC for 18 h and harvested in PBS to an optical density equivalent to approximately 1010 cells/ml by viable count of diluted samples on SBA plates. The purity and identity of each bacterial suspension were assessed by streaking a loopful onto SBA incubated aerobically and anaerobically, as well as Sabouraud Dextrose Agar containing chloramphenicol. The suspensions were mixed with buffered formaldehyde (10%) (v/v) to a final concentration of 0.3% (v/v) and incubated at 37oC for 24 h with shaking. Sterility testing was performed by placing 0.5 ml of the killed vaccine into 25 ml of enrichment broth, incubated at 37oC for 24 h and subcultured onto SBA (aerobic and anaerobic incubation), MCA and SC. All plates were kept at 37oC for up to 10 days. The vaccine preparation was considered sterile when there was no evidence of bacterial or fungal growth. Once the results of sterility testing were known, equal volumes of each of the five cultures were blended together with sterile 2% (v/v) aluminum hydroxide (Alhydrogel) (Brenntag, Denmark) to a final concentration of 20%. The vaccine was dispensed into sterile bottles in 10 ml aliquots. A final sterility check was performed on the leftover vaccine (Figure 2). 35 Figure 1: Preparation of E. coli multivalent vaccine (1 ml of vaccine contains approximately 1010 bacteria) 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 36 Figure 2: NIVR E. coli vaccine References: Jones, G. W. & Rutter, J. M. (1972). Role of the K88 antigen in the pathogenesis of neonatal diarrhoea caused by Escherichia coli in piglets. Infection and Immunity 6, 918-927. Guinee, P. A. M., Veltkamp, J. & Jansen, W. H. (1977a). Improved Minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli. Infection and Immunity 15, 676-678. 37 APPENDIX THREE Results of safety and efficacy studies conducted on NIVR’s E. coli vaccine in comparison to commercially available vaccines (Pfizer Litterguard and Intervet EcoVac) 1. Protection studies During Dr Do Ngoc Thuy’s PhD studies, the NIVR vaccine, which initially encorporated five strains was tested for the ability to protect newborn piglets from challenge infection. Table 1: ETEC strains used for vaccine preparation and for challenge Characteristics Designation of ETEC strains O-serogroup Fimbriae Enterotoxin(s) NVP613 O8 5F- STa/STb/LT NVP1402 O149:K91 F4 STa/STb/LT NVP1271* O8:G7 F4 STa/STb NVP2081* O101 F4 STa/STb NVP1372 O64 F5 STa * These strains were not included in the final NIVR vaccine as protection against these strains is provided by the F4 CARD-VN2 strain. Twelve pregnant gilts (crossbred Landrace x Mong Cai x Yorkshire) were obtained from a local commercial piggery and maintained on an experimental farm belonging to the National Institute of Veterinary Research (NIVR), Hanoi, Vietnam. During pregnancy, ten gilts were vaccinated subcutaneously (SC) in the neck twice with 5 ml of the inactivated multivalent whole cell vaccine at 6 weeks and then again at 2 weeks before parturition. The two remaining gilts, which acted as non-immunised controls, received subcutaneous injections of PBS at the same time. During late pregnancy, the gilts were separated into isolated farrowing rooms (two vaccinated gilts or one unvaccinated gilt per room). Each inoculum used for challenge administration was prepared from cultures grown overnight on SBA plates. After incubation at 37oC, the growth was harvested into sterile PBS and the suspension was adjusted to a density of approximately 1010 bacteria/ml. Following birth, piglets were allowed access to colostrum and nursed a further 24 h, after which they were intragastrically inoculated with 1 ml of bacterial suspension (1010 bacteria/ml) via a stomach tube. Piglets born to both vaccinated gilts and non-vaccinated gilts were challenged with a homologous bacterial suspension prepared from one of the five vaccine strains (one strain per two litters). In each litter, five piglets were challenged with a single E. coli strain and the remaining piglets were left as non-inoculated in-contact control piglets. Only two strains (NVP613 and NVP1402) were used to challenge the piglets from the control gilts. None of the vaccinated or control gilts showed abnormal clinical signs in the 24 h period immediately following vaccination or administration of the placebo. From each gilt, 10-12 piglets were born alive. All piglets were healthy and had an average weight of 0.9-1.2 kg/piglet. 38 Table 2: Designation of groups of piglets used for challenge No. of piglets Gilt Group (litter number) Challenge In-contact Strain challenged 1 (I, II) 10 11 NVP613 2 (III, IV) 10 12 NVP1402 3 (V, VI) 10 12 NVP1271 4 (VII, VIII) 10 11 NVP2081 Vaccinated gilts 5 (IX, X) 10 13 NVP1372 6 (XI) 5 6 NVP613 Unvaccinated gilts 7 (XII) 5 7 NVP1402 Results of the protection studies are shown in Tables 3-5. Vaccinated pigs had less diarrhoea, showed reduced faecal shedding of the challenge strain and fewer pigs became moribund and required euthanasia. These results confirm that the vaccine does protect from significant homologous challenge infection. 39 Table 3: Frequency of diarrhoea and excretion of challenge strain in piglets born from vaccinated gilts and unvaccinated controls No. of piglets with diarrhoea (%) No. of piglets excreting the challenge strain (%) Gilt Group (litter number) Strain challenged Challenge In-contact Challenge In-contact 1 (I, II) NVP613 3/10 a (30.0) 1/11b (9.1) 4/10a (40.0) 3/11b (27.3) 2 (III, IV) NVP1402 3/10 a (30.0) 1/12b (8.3) 6/10 (60.0) 3/12b (25.0) 3 (V, VI) NVP1271 3/10 a (30.0) 1/12b (8.3) 4/10a (40.0) 2/12c (16.7) 4 (VII, VIII) NVP2081 2/10 b (20.0) 0/11b (0.0) 3/10a (30.0) 2/11b (18.2) Vaccinated gilts 5 (IX, X) NVP1372 3/10 a (30.0) 1/13b (7.7) 5/10 (50.0) 4/13b (30.8) 6 (XI) NVP613 5/5 (100.0) 5/6 (83.3) 5/5 (100.0) 6/6 (100.0) Unvaccinated gilts 7 (XII) NVP1402 5/5 (100.0) 5/7 (71.4) 5/5 (100.0) 7/7 (100.0) a: P<0.05 (vaccinated groups vs. control group) b: P<0.01 (vaccinated groups vs. control group) c: P< 0.001 (vaccinated groups vs. control group) (two-tailed Fisher’s exact test) 40 Table 4: Duration of diarrhoea and excretion of challenge strains in piglets born from vaccinated gilts and unvaccinated controls Diarrhoea (average days) Excretion of challenge strain (average days) Gilt Group (litter number) Strain challenged Challenge In-contact Challenge In-contact 1 (I, II) NVP613 2.0a 1 2.5a 1.3 2 (III, IV) NVP1402 2.0a 1 2.3a 1.7 3 (V, VI) NVP1271 1.7 1 1.8 2.0 4 (VII, VIII) NVP2081 1.5 - 2.0 1.5 Vaccinated gilts 5 (IX, X) NVP1372 1.7 1 2.0 1.8 6 (XI) NVP613 3.5b 1.8 5.5b 2.5 Unvaccinated gilts 7 (XII) NVP1402 4c 1.4 6c 2.2 a: Average number excluded the two piglets submitted for necropsy at 24 h p.i. b: Average number excluded the three piglets euthanased at 16-24 h p.i. and submitted for necropsy c: Average number excluded the four piglets euthanased at 16-25 h p.i. and submitted for necropsy -: None piglet in the group had diarrhoea Table 5: Morbidity requiring euthanasia in piglets born from vaccinated gilts and unvaccinated controls Gilts Group (Litter number Strain challenged No. of piglets euthanased/inoculated (%) No. of piglets euthanased /in-contact (%) 1 (I, II) NVP613 0/10 (0)a 0/11 (0) 2 (III, IV) NVP1402 0/10 (0)a 0/12 (0) 3 (V, VI) NVP1271 0/10 (0)a 0/12 (0) 4 (VII, VIII) NVP2081 0/10 (0)a 0/11 (0) Vaccinated gilts 5 (IX, X) NVP1372 0/10 (0)a 0/13 (0) 6 (XI) NVP613 3/5 (60.0) 2/6 (33.3) Unvaccinated gilts 7 (XII) NVP1402 4/5 (80.0) 1/7 (14.3) a: P<0.05 (vaccinated groups vs. control group) (two-tailed Fisher’s exact test) 41 2. Vaccine safety study: Sixteen healthy pregnant sows were allocated into two groups: * Group 1: 8 sows, vaccinated with Pfizer Litterguard vaccine * Group 2: 8 sows, vaccinated with NIVR's vaccine Each sow were vaccinated subcutaneously twice with 2 ml of vaccine at 9 weeks and again at 12 weeks of gestation. The following criteria were recorded for each farrowing sow: + Piglets born alive + Stillbirths + Mummies or (died before parturition) + Deformities eg splay legs + Abortion Differences in each of the above criteria for each group of sows were assessed by using the Student's t-test (two-tailed). All statistical significance was set at a P (two-sided) value of < 0.05, using the statistical program GraphPad Instat version 3.01. A summary of the results is shown in Table 2. Table 2: Recorded criteria on safety study Recorded criteria Group 1 (8 sows) Group 2 (8 sows) Piglets born alive 80 82 Stillbirths 3 4 Mummies or (died before parturition) 2 2 Deformities eg splay legs 4 5 Abortion 0 0 There were no significant differences between two groups of sows (all P values >0.05). 42 3. Efficacy study: For the efficacy study, 4 groups of pigs (12 weeks of age) were divided as follows: Control group n = 5 pigs Litterguard group n = 10 pigs Intervet vaccine n = 10 pigs NIVR vaccine n = 10 pigs The procedure for bleeding and vaccination as follows: Age of pigs (weeks) 12 Bleeding and 1st vaccination 16 2nd vaccination 18 Bleeding All pigs showed excellent health during and after the experiment, except that 1 pig from the control group developed shock and died after the first bleeding. A total of 34 sera samples were examined for antibody level against F4 antigens by ELISA test (according to the protocol from E. coli Lab, DPI. Bendigo, Victoria, Australia). 43 Table 4: Statistical analysis raw data Group Pre vaccination antibody levels Post vaccination antibody levels Post-pre diff litterguard 1.337 1.772 0.435 litterguard 1.136 2.013 0.877 litterguard 1.214 1.944 0.73 litterguard 1.215 2.005 0.79 litterguard 1.004 1.965 0.961 litterguard 0.648 1.538 0.89 litterguard 0.981 1.842 0.861 litterguard 1.176 1.843 0.667 litterguard 1.376 2.182 0.806 litterguard 0.914 2.026 1.112 ecovac 1.11 2.162 1.052 ecovac 1.164 2.173 1.009 ecovac 1.954 2.252 0.298 ecovac 0.906 2.088 1.182 ecovac 0.76 2.299 1.539 ecovac 0.432 1.961 1.529 ecovac 0.83 1.701 0.871 ecovac 1.062 1.826 0.764 ecovac 1.236 1.699 0.463 ecovac 0.944 1.643 0.699 NIVR 0.562 1.871 1.309 NIVR 1.192 1.715 0.523 NIVR 1.071 2.122 1.051 NIVR 1.124 1.943 0.819 NIVR 0.34 1.599 1.259 NIVR 1.028 1.893 0.865 NIVR 1.135 1.798 0.663 NIVR 1.41 2.023 0.613 NIVR 1.014 2.048 1.034 NIVR 1.121 1.68 0.559 control 1.447 1.664 0.217 control 0.8 0.91 0.11 control 0.593 0.729 0.136 control 0.611 1.052 0.441 44 Table 5: Summary statistics Pre- vaccination mean OD values Group Mean Variance Control 0.863 0.1605 Litterguard 1.100 0.0475 EcoVac 1.040 0.1571 NIVR 1.000 0.0985 Post- vaccination mean OD values Group Mean Variance Control 1.089 0.1654 Litterguard 1.913 0.0309 EcoVac 1.980 0.0614 NIVR 1.869 0.0292 Pre-Post vaccination mean OD values Group Mean Variance Control 0.2260 0.0226 Litterguard 0.8129 0.0328 EcoVac 0.9406 0.1683 NIVR 0.8695 0.0814 Statistical analysis An analysis of the difference between the Pre and post vaccination OD values was done using linear regression with pairwise differences. Statistical Analysis was preformed by Genstat 8th Edt, Laws Agricultural Trust, Rothamsted Experimental Station. Results There was a significant difference between the OD levels for the treatment groups (p<0.003). There was no significant difference between the antibody response elicited (as demonstrated by OD values) by Litterguard, EcoVac or NIVR vaccines (p>0.1). All three vaccines were significantly different from the control group (p< 0.005) (Table 1) Table 1 Treatment group Mean Control 0.2260a Litterguard 0.8129b EcoVac 0.9406 b NIVR 0.8695 b * Means with different subscripts differ significantly p<0.005