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

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

Ministry of Agriculture & Rural Development

Project Progress Report

A blueprint for sustainable smallholder pig production

in Central Vietnam CARD Project 001/04VIE

Milestone 8: FINAL REPORT

APRIL 2010

Table of contents

TABLE OF CONTENTS 2 

1 INSTITUTE INFORMATION 3 

2 PROJECT ABSTRACT 4 

3 EXECUTIVE SUMMARY 4 

4 INTRODUCTION & BACKGROUND 6 

5 PROGRESS TO DATE 6 

5.1 I MPLEMENTATION H IGHLIGHTS 6 

5.3 S MALLHOLDER B ENEFITS 15 

5.4 C APACITY B UILDING 15 

5.5 P UBLICITY 16 

5.6 P ROJECT M ANAGEMENT 16 

6 REPORT ON CROSS-CUTTING ISSUES 16 

6.1 E NVIRONMENT 16 

6.2 G ENDER AND S OCIAL I SSUES 16 

7 IMPLEMENTATION & SUSTAINABILITY ISSUES 16 

7.1 I SSUES AND C ONSTRAINTS 16 

7.2 O PTIONS 17 

7.3 S USTAINABILITY 17 

8 NEXT CRITICAL STEPS 17 

9 CONCLUSION 17 

1 Institute Information

Project Name Diagnosis and control of diarrhoea in suckling pigs

Vietnamese Institution National Institute of Veterinary Research (NIVR)

Vietnamese Project Team Leader Dr Truong Van Dung (Dr Cu Huu Phu)

Australian Organisation The University of Queensland/Victorian Department of

Primary Industry

Australian Personnel Dr Darren Trott, Dr Ian Wilkie, Dr Tony Fahy

Completion date (original) January 2007

Completion date (revised) April 2007

Reporting period March 2006-March 2008 and including data from

2009/2010

Contact Officer(s)

In Australia: Team Leader

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

Position: Manager Research Projects Office Fax: 61 7 33651188

Organisation School of Land and Food The

University of Qld

Email:

In Vietnam

Position: Head of Bacteriology Department Fax: 84 4 8694082

2 Project Abstract

This project is designed to improve productivity of smallholder pig farmers in Vietnam through improved health management, particularly of piglets during the pre-weaning period Through consultation and dialogue with farmers and field veterinarians, an appropriate disease

management plan will be developed This will concentrate on the pre-weaning period where

greatest losses occur, but will include principles of herd health management in general

Dissemination of the plan will be through training programmes for field staff and selected

farmers

Additional to the health management plan the project will develop and implement appropriate rapid diagnostic tests for the principal strains responsible for enterotoxigenic colibacillosis, to

improve speed and accuracy of laboratory diagnosis The third part of the project is designed to

improve the production and efficacy of locally-manufactured E coli vaccines In particular, this

will involve including a unique local strain shown by previous research to be an important

vector of pre-weaning disease in some, and possibly all, areas of Vietnam

3 Executive Summary

This final report documents progress on the following project deliverables (linked to the project logframe objectives and milestone descriptions):

1 Vaccine efficacy and safety data (Production and testing of locally-produced E coli

vaccine-small scale and field trials Logframe Reference 1)

2 Enteric management plan and production parameter records at 10 selected farms (5 test and 5

control farms for a 12 month period) (Develop a management plan for preweaning diarrhoea using

a continuous improvement model-Logframe reference 2a and 2b)

3 Development of polyclonal sera and/or PCR incl rapid detection of novel fimbrial antigens

(Improve diagnostics for preweaning diarrhoea-Logframe reference 3)

Whilst this project achieved outputs for all three objectives according to the project logframe, some significant problems were experienced in trying to identify the novel fimbrial antigen present in Vietnamese O8 strains (christened F19) and in developing an enteric management plan within a holistic continuous improvement framework A final attempt to purify the novel fimbrial antigen was undertaken with great success in mid-2010 using funds from the University of Adelaide and we are now awaiting identification of the amino acid and gene sequences for this unusual antigen

In small scale trials conducted at NIVR, the ETEC vaccine (still encorporating F4, F5 and the new F19 antigens) was proven to be safe and efficacious when administered to pregnant sows (2 doses at

5 and 2 weeks before farrowing) It is now being supplied to selected piggeries in North Vietnam on

a research only basis, with reports of good efficacy against neonatal E coli infection and no side-effects The vaccine has also been produced for the CARD 004/05VIE project and used in the

selected smallholder farms in central Vietnam in this related AUSAID project as part of a Continuous Improvement Model to integrate best management practices into a holistic pig production improvement plan A small scale field trial showed that the vaccine significantly reduced the occurrence of diarrhoea in general and in investigations of vaccinated herds that

reported diarrhoea, no enterotoxigenic E coli was isolated from faecal samples confirming that the

cause of the diarrhoea was not neonatal colibacillosis

Production data for the five test and five control farms over a 12-month period were analysed and a statistically significant improvement in preweaning mortality was noted in the test farms (8.6% ± 3.6) over the trial period compared to the controls (15.6 ± 4.3; p<0.05) A bigger improvement may have been confounded by the small sample size, but problems in the adoption of the Continuous Improvement Model may also have had an impact The major problem encountered from the farm visits was inadequate uptake of skills, knowledge and recommendations by piggery managers We therefore adopted different training approach in CARD 004/05VIE which has been extremely successful in creating successful, profitable smallholder farmers in Central Vietnam

The PCR machine and rapid diagnostic assay kits purchased by the project continue to be used for NIVR research on preweaning enteric diseases A complete analysis of diagnostic results on pre and post weaning diarrhoea, together with the results of safety and efficacy testing of the vaccine were presented as posters by Dr Do Ngoc Thuy at the Australasian Association of Animal Production Biennial Conference in Hanoi in September, 2008 A survey of 117 samples of preweaning diarrhoea from commercial farms and 45 samples from village-based smallholder farms confirmed the presence of multiple agents in both forms of agriculture, however, only the commercial farms recorded cases of diarrhoea due to a single agent By far the most common agents identified were

rotavirus and transmissible gastroenteritis virus, often as a mixed infection with enterotoxigenic E

coli in older pigs These results confirm that care of the sow and piglets during the preweaning

period on both village and commercial piggeries in Vietnam is suboptimal, which has been the major focus of initiatives developed in 004/05VIE

Characterization of virulence factors from ETEC isolates obtained from cases of pre- and postweaning diarrhoea identified some interesting findings Ten additional virulence genes were

included that have been linked with certain E coli pathotypes in other studies These included the

genes for Paa, AIDA-1, EAST-1, stx2 (normally associated with oedema disease) and Aero

(normally a marker for extraintestinal pathogenic E coli), which were identified in the Vietnamese

ETEC collection In pre-weaning diarrhoea, F4:Paa:STa:STb:LT:EAST-1 was still the most common pathotype and the pathotype Paa:STa:STb:LT:EAST-1 was a consistent marker for the O8 F19 isolates that possess the new fimbrial type This pathotype was the second most prevalent in the pre-weaning diarrhoea isolates, indicating that it was still a significant pathogen in preweaning diarrhoea in Vietnam In post-weaning diarrhoea, the major pathotypes were associated with F18 rather than F4 fimbriae and the majority of F18 strains also possessed stx2 toxin, confirming that the isolates had the capability of causing both post-weaning diarrhoea and oedema disease

In summary, the NIVR vaccine has been shown to be safe, efficacious and now must be registered

as soon as possible and licensed throughout the country An ongoing field trial will conclude in November 2010 and on the basis of this data, partnerships should be sought with local vaccine companies such as NAVETCO for the mass production and distribution of the vaccine A large number of pathogens have been isolated from preweaning pigs with diarrhoea confirming that greater attention to disease prevention through better husbandry and management, introduction of the NIVR vaccine, key preventative medications and minimal antimicrobial use will contribute strongly towards maintaining the profitability of smallholder farmers

4 Introduction & Background

Diarrhoea during the suckling period has been recognised as the principle health problem affecting

both smallholder and commercial pig production in Vietnam Previous research has confirmed the

presence of a new fimbrial type in E coli strains causing colibacillosis in Vietnam that would not be

controlled by existing vaccines Existing vaccines are currently imported into Vietnam at

considerable cost In addition, there are many other causes of suckling diarrhoea, the significance of

which is currently unknown in Vietnam, which are all affected by husbandry and management

during farrowing and lactation Project 001/04VIE (Diagnosis and control of diarrhoea in suckling

pigs) began with three objectives to solve this problem:

1 Production and testing of locally-produced E coli vaccines

2 Development of a management plan for preweaning diarrhoea using a continuous improvement

(CIP) model

3 Improved field and laboratory diagnosis of preweaning diarrhoea

5. Progress to Date

Objective 1: Production and testing of local produced vaccine

Output 1.1: Identification and confirmation of components, including novel strain.

The vaccine Master Seed (50 x 1ml vials of each of the three vaccine strains in Brain Heart Infusion

broth plus 12% glycerol) is held in a -80oC freezer at NIVR Backup freeze dried cultures are also

held at NIVR in case of a catastrophic freezer failure (if the -80oC freezer breaks down, the strains

can be held at -20oC for a short duration) Each time the vaccine is prepared according to the

protocol outlined in 1.3 below, a new vial of the Master Seed is subcultured and checked for purity

This then becomes the Working Seed for vaccine preparation, with the number of subcultures kept

to an absolute minimum and culture conditions used for maximum fimbriae expression Backup

cultures are also held at The AQIS approved laboratory of The University of Queensland School of

Veterinary Science and the OIE E coli reference laboratory at The University of Montreal

(managed by Prof John Fairbrother) The virulence characteristics (OK-antigen serogroup, fimbriae

and enterotoxins) of the three strains selected for vaccine production were independently confirmed

by The Pig Health and Research Unit (PHRU), Victorian Department of Primary Industry (Table 1)

These strains have been stored as freeze dried specimens in three separate laboratories (NIVR, UQ

and PHRU)

Table 1: E coli strains used for the preparation of vaccine

Virulence Characteristics

Designation of E coli

vaccine strains

O-serogroup Fimbriae Enterotoxin(s)

NVP613

(CARD-VN1)

(CARD-VN2)

NVP1372

(CARD-VN3)

O64 F5 STa

* Negative for all five recognized fimbriae associated with porcine enterotoxigenic E coli (F4, F5,

F6, F18 and F41) May therefore possess a novel fimbrial antigen

Output 1.2: Characterization of the novel fimbrial antigen

The two 5F- ETEC strains were examined for mannose-resistant haemagglutinating activity using

Sheep Red Blood Cells Mannose-resistant haemagglutination was observed at 37oC, but not at

18oC for both strains, confirming the production of adhesins (ie fimbriae) at 37oC (Table 1)

Table 3: Haemagglutination results of two 5F- ETEC strains

Cultures grown at:

Strain

NaCl 0.85% 1.5% D-Mannose NaCl 0.85% 1.5% D-Mannose

CARD-VN1

Transmission electron microscopy photographs taken at low and high magnification showed the

presence of hair-like structures on the surface of the bacteria cells Research conducted between

2006-2008 in the OIE Reference Laboratory for E coli by Dr Do Ngoc Thuy, came extremely close

to purifying and characterizing the new fimbrial antigen, however contaminating proteins in the

preparation obscured the identification In a return visit by Dr Do Ngoc Thuy in July 2010

sponsored by the University of Adelaide, the new fimbrial type was successfully purified without

contaminating proteins and we are eagerly awaiting confirmation of the identity

Output 1.3 Formulation of vaccine

Specialised culture media were prepared in order to provide favourable growth conditions for the

production of fimbriae For efficient expression of F4, strain CARD-VN2 was grown on Buffered

Glucose Nutrient Agar whereas for the production of F5 fimbriae on strain CARD-VN3, Minca

agar was used For the strain with currently uncharacterized fimbriae (CARD-VN-1), buffered

Glucose Nutrient Agar was shown to enhance production of the new fimbrial type The procedure

used to prepare the vaccine is summarised in Figure 1

Figure 1: Preparation of E coli multivalent vaccine (1 ml of vaccine contains approximately 10

bacteria)

Output 1.4: Efficacy testing of vaccine

The NIVR prepared the vaccine for small scale protection, safety and efficacy trials In summary, the vaccine produced no unacceptable side effects in vaccinated gilts and their progeny When compared to Littergard and Ecovac, two commercially available vaccines from Pfizer and Intervet,

respectively, the NIVR vaccine produced statistically similar specific antibody titres to an E coli F4

fimbriae strain This confirms that under experimental conditions, the vaccine is both safe and efficacious in generating anti-F4 agglutinating antibodies Small amounts of the vaccine were supplied to selected herds in the North of Vietnam and to smallholder farmers in Central Vietnam as part of the 004/05VIE project No side effects or vaccine reactions were reported and anecdotal reports suggest the vaccine is highly efficacious, though in central Vietnam it was not possible to identify causes of preweaning diarrhoea Therefore some episodes of diarrhoea in piglets from vaccinated sows could have been caused by other agents such as coccidiosis, rotavirus or transmissible gastroenteritis virus, all of which have been demonstrated in Vietnamese smallholder farms

20 ml TSB (37oC, overnight)

PBS (1010 bacteria/ml)

Add 2% (v/v) aluminum hydroxide to a final concentration of 20%

Freeze-dried

cultures

2 ml TSB (37oC, overnight)

SBA (37oC, overnight)

Appropriate culture media (37oC, overnight)

Purity testing

10% (v/v) bufferred formaldehyde to a final concentration of 0.3%

Mix with equal colume of each bacterin

Dispense into sterile bottles and label

Sterility testing

Sterility testing

Output 1.5: Field testing of vaccine

Field trials were conducted at two communes in Thua Thien Hue and three communes in Quang Tri in 2009/2010 Pregnant sows each received 2 ml of vaccine (approximately 1.5 x

vaccinated No local or systemic reaction to the vaccine was observed and all sows gave birth at the correct stage of gestation to an average of 9.3 healthy piglets per sow The prevalence of pre-weaning diarrhoea in piglets born from vaccinated sows at 1, 2 or 3 weeks

of age were: 16.1; 22.7 and 26.5%, compared with those of 48.1; 33.8 and 37.5%, respectively from control group (P<0.005)

Random faecal samples (n=37) taken from piglets with diarrhoea were assayed for the

prevalences of transmissible gastroenteritis virus (TGEV), rotavirus (RV) and coccidiosis were not

significantly different between vaccinated or non-vaccinated groups Clostridium perfringens was only found in non-vaccinated group None of the E coli isolates obtained from the vaccinated group possessed toxin genes, whilst the E coli strains isolated from the non-vaccinated group all still

carried STa, STb and/or LT toxin genes This study demonstrated that the implementation of locally

produced E coli vaccine, not only reduced the prevalence of pre-weaning diarrhoea, but also may suppress the presence of toxigenic E coli strains in the gut of piglets

A second field trial is currently being undertaken at the National Institute of Animal Husbandry research piggery and will conclude in November 2010 This is the final experiment required to generate data for the licensing of the vaccine

Output 1.6: Commercial realisation of vaccine

NIVR continues to produce the vaccine for research purposes, as attested by the vaccine records (MS3 and 6 reports), but registration requires a detailed document to be submitted to the Department of Animal Health Most of the requirements for registration, including safety and efficacy have been met by the current project, with the current field trial providing necessary field efficacy data NIVR Bacteriology Laboratory is not experienced in the commercialization of its discoveries, therefore we suggest partnership between the two major local vaccine manufacturers that hold GMP/GLP licenses, NAVETCO (for the south of Vietnam) and the National Veterinary Factory (for the north) to complete the registration dossier Prior to this occurring however, we advise that a patent attorney is hired to assist Dr Do Ngoc Thuy, the inventor of the vaccine to lodge

a patent application with the Office of Intellectual Property of Vietnam within the Ministry of Science and Technology Once this is obtained, negotiations may be commenced whereby the level

of royalties returning to the sole inventor and the NIVR laboratory are clearly indicated The assistance of the CARD programme management team is also requested to foster negotiations with the Department of Animal Health and other major stakeholders This will ensure that delays are kept to a minimum and that the vaccine becomes readily available for use by smallholder farmers Such a strategy towards commercialization could also be used for other NIVR vaccines, such as the NIVR oedema disease vaccine that has excellent efficacy but is currently unregistered

Objective 2: Enteric management plan for pre-weaning diarrhoea through adoption of a continuous improvement plan

Output 2.1 Field data collected at test and control farms

An analysis of preweaning mortality reported over a 14-month observation period established that the test farms, which were subject to a number of recommendations during the life of the project, had a significantly lower average pre-weaning mortality compared to the control farms (8.6% ± 3.6

vs 15.6 ± 4.3; p<0.05) One of the control farms was removed from the trial due to an outbreak of hog cholera For the majority of test farms, consistently lower pre-weaning mortalities were sustained over the trial period, however for Dong May farm in Thai Binh, pre-mortalities of close to 20% were reduced to 10% towards the end of the observation period It is difficult to determine

whether this reduction in preweaning mortality was associated with uptake of any of the previous visit’s recommendations as the same problems were still observed on the second visit

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

Month

Anh De Thai Binh C Anh Thiet Hung Yen C Trang Due Hai Phong C Minh Duong Ha Tay C Dinh Dung Binh Dinh C Dong My Thai Binh T Anh Hiep Hung Yen T Anh Tinh Hai Phong T Thanh Bich Ha Tay T Nhon Hoa Binh Dinh T

Figure 2: Average preweaning mortalities observed in five test (T) and five control (C) piggeries

during the 14-month observation period The Anh Thiet farm was discontinued due to an outbreak

of hog cholera

Output 2.3: Continuous improvement model for smallholder farmers

Summaries of the results of field visits to test and control farms were submitted with MS3 and MS6 reports Overall, whilst some improvements were noted on individual farms, many of the recommendations made on previous visits were not being followed Drip coolers that had been installed were removed on some farms, the farms were not operating to full capacity in terms of the number of sows vs the number of growers and care of neonatal and weaner pigs was still not ideal Some of the disease problems were clearly linked to the unacceptably high heat index recorded in some of the sheds, restricted feed intake and the large number of sows with low condition scores and poor ventilation Anh Hiep Farm (Hung Yen Province) perhaps showed the greatest improvements over the life of the project, but this farm achieved consistently low rates of preweaning mortality throughout the year

The overall objective of the continuous improvement model was, through the farm visits, to provide Vietnamese scientists with training in herd health monitoring (focused on preweaning mortality) whilst creating demonstration farms that could be utilized for smallholder training workshops However, we soon realised that this model was unworkable and that the resources allocated were inadequate With advice and assistance from the CARD Programme Management Unit, Project number 004/05VIE (A blueprint for smallholder pig production in Central Vietnam) was developed

as a holistic plan for capacity building, focused on smallholder farmers in Quang Tri and Thua Thien Hue This project was extremely successful and details are provided in the final report However, it must be stressed that without the experience gained from 001/04VIE, we would not have achieved such a good outcome There was considerable crossover between the two projects,

particularly in that the E coli vaccine produced by NIVR was provided free to smallholder farmers

selected in the 004/05VIE project for further training and capital improvement