1

Ministry of Agriculture & Rural Development

Project Progress Report
Diagnosis and control of diarrhoea in suckling pigs
CARD Project 001/04VIE

MILESTONES 3 and 6
2
nd
and 3
rd
SIX MONTHLY REPORTS (COMBINED) 2
Table of Contents
1. INSTITUTE INFORMATION 3
2. PROJECT ABSTRACT 4
3. EXECUTIVE SUMMARY 4
4. INTRODUCTION & BACKGROUND 6
5. PROGRESS TO DATE 6
5.1 RESPONSE TO APPRAISAL 6
5.2 IMPLEMENTATION HIGHLIGHTS 7
5.3 SMALLHOLDER BENEFITS 14
5.4 CAPACITY BUILDING 14
5.5 PUBLICITY 15
5.6 PROJECT MANAGEMENT 15
6. REPORT ON CROSS-CUTTING ISSUES 15

th
2005
Completion date (original)
January 2007
Completion date (revised)
April 2007
Reporting period
March 2006-March 2008
Contact Officer(s)
In Australia: Team Leader
Name:
Dr Darren Trott
Telephone:
617 336 52985
Position:
Associate Professor of Veterinary
Microbiology
Fax:
617 336 51355
Organisation
School of Veterinary Science The
University of Qld
Email:

In Australia: Administrative contact
Name:
Melissa Anderson
Telephone:
61 7 33652651
Position:

greatest losses occur, but will include principles of herd health management in general.
Dissemination of the plan will be through training programmes for field staff and selected
farmers.

Additional to the health management plan the project will develop and implement appropriate
rapid diagnostic tests for the principal strains responsible for enterotoxigenic colibacillosis, to
improve speed and accuracy of laboratory diagnosis. The third part of the project is to improve
the production and efficacy of locally-manufactured E. coli vaccines. In particular, this will
involves including a unique local strain shown by previous research to be an important vector of
p
re-weaning disease in some, and possibly all, areas of Vietnam.

3. Executive Summary

This report, which is a combination of two six-monthly reports (2
nd
report and 3
rd
report),
documents progress on the following deliverables (linked to the project logframe objectives and
milestone descriptions):

1. Vaccine efficacy and safety data (Production and testing of locally-produced E. coli vaccine-
small scale and field trials Logframe Reference 1).

2. Enteric management plan and production parameter records at 10 selected farms (5 test and 5
control farms for a 12 month period) (Develop a management plan for preweaning diarrhoea
using a continuous improvement model-Logframe reference 2a and 2b).

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

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


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).

Thuy then returned to Vietnam and Prof Fairbrother continued the process of characterization.
Initial attempts at identifying proteins in the purified fimbrial extract were unsuccessful.
However, following a more rigorous extraction method, a 20KDa sized band (the right size!!)
was identified in SDS-PAGE gels as the putative adhesin as it reacted in a Western Blot with
hyperimmune serum obtained following immunization of rabbits with an E. coli O8 5F- whole
cell extract grown at 37
o
C to produce fimbriae and absorbed with sera obtained for the same
strain grown at 18
o
C, when it does not produce fimbriae. An N-terminal amino acid sequence
obtained from the band cut from a one dimensional SDS-PAGE gel identified a protein closely
related to Enterobacter ompX, but Prof Fairbrother is not convinced that this is the adhesin, even
though it has a putative attachment role in other bacteria. A problem is that the adhesin does not
seem to be expressed as much or as consistently as other fimbrial antigens and contaminating
proteins may be being identified instead of the true fimbriae. However, Prof Fairbrother has
produced diagnostic antisera that is now quite specific for the F19 antigen and screened a large
number of isolates from his collection (n=140) including all of the Vietnamese strains. This
identified the correlation between F19 and the virulence gene profile Paa/STa/ST/LT/EAST-1

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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,

Jun-05
Jul-05
Aug-05
Sep-05
Oct-05
Nov-05
Dec-05
Jan-06
Feb-06
Mar-06
Apr-06
May-06
Jun-06
Month
% Pre Weaning Mortality
Anh De Thai Binh C
Anh Thiet Hung Yen C
Trang Due Hai Phong C
Minh Duong Ha Tay C
Dinh Dung Binh Dinh C
Dong My Thai Binh T
Anh Hiep Hung Yen T
Anh Tinh Hai Phong T
Thanh Bich Ha Tay T
Nhon Hoa Binh Dinh T
Figure: Average preweaning mortalities observed in five test (T) and five control (C) piggeries
during the 14-month observation period.

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4) Farm visits to Northern Herds September 2006. A summary of the final farm visits to the

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.

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0
 The temperature needs to be 30-32 ‘C in the first week after weaning. This should be lowered
by 2 degrees each week by lifting the heaters up.
 Piglets should be offered glucose and electrolyte solution in drinkers. A water soluble
antibiotic (that Thuy will identify) must be added to this drinking solution. This solution is
used for fourteen days post weaning.
 Any pig that scours is to be treated (injectable) with the antibiotic recommended by Thuy.
 Any piglets that scour are to have a rectal swab taken at the time of treatment (NOT AFTER
treatment).
 If this strategy is not successful you will have to orally vaccinate the sucker piglets one week
before weaning.
Oral Vaccine procedure

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) 1
1

RV TGEV

17 (14.4) 3 (6.7)
Cocci


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


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)

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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

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


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

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.

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6
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


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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.

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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

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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


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

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 o
f

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


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

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

2a. Enteric management plan for
preweaning diarrhoea
2.b Continuous improvement plan
(CIP)

Institutional capacity:
Improved
diagnostic reagents produced at
N
IVR 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)


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

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

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Social outcomes:
Majority of
N
IVR 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.


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)

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