Collaboration for Agriculture and Rural Development (CARD) Program

RESULTS ON STRENGTHENING DIAGNOSTIC CAPACITY AND CONTROL OF DIARRHOEA IN SUCKLING PIGS

Project title: Diagnosis and control of diarrhoea in suckling pigs

Project code: CARD 001/04VIE

Authors: Do Ngoc Thuy1, Cu Huu Phu1, Au Xuan Tuan1, Nguyen Xuan Huyen1, Truong Van Dung1,

1: National Institute of Veterinary Research 2: Pig Research Unit, Victorian Department of Primary Industry, Melbourne, Australia; 3: The University of Queensland, Australia

Tony Fahy1, Steve Driesen2, Karen Moore2, Aileen Vanderfeen2, Darren Trott3 Collaborating organisations:

SUMMARY

During the implementation time (2005-2007), three objectives of the project, including: 1) Improved field and laboratory diagnosis of preweaning diarrhoea; 2) Production and testing of locally- produced E. coli vaccines; 3) Development of a management plan for preweaning diarrhoea using a continuous improvement (CIP) model had been conducted and completed. Some achievements are: 1) A diagnostic procedure of six major causes of diarrhoea in suckling pigs has been applied successfully at Department of Bacteriology, NIVR. 2) A vaccine containing E. coli strains bearing full pathogenic factors (fimbriae, toxins) has been produced and proven safe and effective in laboratory and in the field. 3) An continuous improvement model, focused mainly on management of sows during farrowing and lactation has been developed and utilized in order to improve pig production and minimize the losses due to diarrhoea in suckling period.

2. Production and testing of locally-

1.

Introduction

produced E. coli vaccines

3. Development of a management plan for preweaning diarrhoea using a continuous improvement (CIP) model

is to project designed

type in E. coli through improved

farrowing during

through be field staff plan will for Diarrhoea during the suckling period has been recognised as the principle health problem affecting both smallholder and commercial pig production in Vietnam. Previous research (4, 7) has confirmed the presence of a new fimbrial 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 and management lactation. Project 001/04VIE (Diagnosis and control of diarrhoea in suckling pigs) began with three objectives to solve this problem: This improve productivity of smallholder pig farmers in health Vietnam 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 include principles of herd occur, but will health management in general. Dissemination of training the programmes and selected farmers. 1. Improved field and laboratory diagnosis of preweaning diarrhoea

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CARD 001/04 VIE – Control diarrhoea in suckling pigs

2.

Project contents and methods

modified Ziehl-Neelsen staining of faecal smears

2.1 Project Contents - Detection of virulence factors of E. coli (fimbriae, toxins) by PCR The project focuses on:

- Production of vaccine according to SOP 1. laboratory diagnosis of Improved preweaning diarrhoea

2. and testing of locally-

3.

Research results and discussions

Production produced E. coli vaccines

using

3.1 Development of a procedure for quick diagnose some of the most common causes of diarrhoea in infectious suckling pigs 3. Development of a management plan for preweaning a diarrhoea continuous improvement (CIP) model, training for field veterinarians

2.2 Methodology

- Experimental design based on groups and compare the significant difference (1 factor, multiple factors)

- Isolation and identification of bacteria (E. coli, C. perfringens) according to SOPs

commercial from - Detection of virus (TGE virus, Rota virus) Institute kit, by ELISA (commercial Pourquier)

A diagnostic procedure of six major causes of including the diarrhoea in suckling pigs, infectious gastroenteritis virus (TGEV), Rota virus, E. coli, C. perfringens, Coccidiosis and Cryptosporidium have been established, in accordance with the laboratory conditions at Department of Bacteriology, National Institute of Veterinary Research. This procedure has been used for diagnosis of faecal samples collected piggeries (participated in the project) and small holder farmers in the nearby areas. Results are presented in table 1. - Detection of Cocci oocysts by standard flotation, Cryptosporidium by faecal

Table 1: Prevalence of enteric pathogens in pre-weaning piglets with diarrhoea

No. of positive samples (%) Agent(s) detected

Small holder farmers (n=45)

Cocci

Crypto

RV

TGEV

ETEC

C. per

Total single infection

ETEC

TGEV

RV RV RV

Cocci Cocci

ETEC

C. per

Crypto Crypto Crypto Crypto 3 (6.7) 2 (4.4) 2 (4.4) 1 (2.2) 1 (2.2) 2 (4.4)

TGEV TGEV C. per Commercial piggeries (n=117) 2 (1.7) 3 (2.5) 3 (2.5) 11 (9.3) 4 (3.4) 2 (1.7) 25 (21.2) 6 (5.1) 17 (14.4) 1 (0.8) 1 (0.8) 3 (2.5) 1 (0.8) 6 (5.1) 1 (0.8)

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D.N Thuy, C.H Phú, AX. Tuan, N. X.Huyen, T.V. Dung, T. Fahy, S.Driesen, K. Moore, A. Vanderfeen & D. Trott

No. of positive samples (%) Agent(s) detected

Small holder farmers (n=45)

TGEV

ETEC ETEC

RV RV

TGEV TGEV ETEC

Cocci Cocci Cocci Cocci RV

TGEV

RV ETEC ETEC

C. per 3 (6.7) 3 (6.7) 2 (4.4) 1 (2.2) 4 (4.4) 3 (6.7) 1 (2.2)

Crypto Crypto Crypto Crypto Crypto

ETEC

TGEV TGEV TGEV TGEV

RV RV RV RV 7 (15.6) 1 (2.2) 3 (6.7)

C. per C. per C. per

TGEV TGEV

ETEC ETEC ETEC ETEC C. per

RV RV RV 1 (2.2) 2 (4.4) 1 (2.2)

Crypto Crypto Crypto Crypto C. per

ETEC

Cocci Cocci Cocci Cocci 1 (2.2) 1 (2.2)

Commercial piggeries (n=117) 7 (5.9) 2 (1.7) 4 (3.4) 2 (1.7) 1 (0.8) 5 (4.2) 1 (0.8) 1 (0.8) 3 (2.5) 14 (11.9) 1 (0.8) 2 (1.7) 2 (1.7) 2 (1.7) 1 (0.8) 1 (0.8) 1 (0.8) 4 (3.4) 1 (0.8) 1 (0.8)

RV RV RV (97) TGEV TGEV TGEV TGEV TGEV (111) ETEC (76) C. per C. per (23)

Crypto Crypto (36) (50) Total multiple infections 92 (78.8) 45 (100.0)

3.2 of local Production and testing produced E. coli vaccine

provide favourable growth conditions for the production of fimbriae. Each dose of vaccine (2 ml) contains of ~ 1.5 x 109 bacteria. Vaccine has been tested on sterility, safety and efficacy in laboratory and in the field (for small scale trial). A vaccine containing E. coli strains bearing full pathogenic factors (fimbriae, toxins) has been produced according to SOP. Specialised to culture media were prepared in order

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CARD 001/04 VIE – Control diarrhoea in suckling pigs

o When the body condition score of a pig falls below 2.5 (on the scale of 1-5, appendix 1), remedial action must be taken.

o All pigs

(excluding piglets) must be maintained at a body condition score of 2 or more.

o Feed must be provided in such a way as to prevent undue competition or injury.

Results of ELISA for efficacy testing of there was a significant vaccine show that 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 three vaccines were vaccines (p>0.1). All significantly different from the control group (p< 0.005) (Table 2)

Table 2: Mean OD values of sera between

treatment and control group

o Pregnant sows should be given enough bulky or high fibre feed to satisfy hunger. The feed intake for dry sows needs to be nutritionally balanced, appropriate to the sow’s physiological needs (which vary during pregnancy) and to provide satiety without the sow becoming overweight. Treatment group

o Body score of breeding sows at farrowing should be 3.5 – 4.

o Body score of breeding sows at weaning Control Litterguard EcoVac NIVR Mean 0.2260a 0.8129b 0.9406 b 0.8695 b should be 3 or above.

* Means with different subscripts differ significantly p<0.005

o Condition scoring is recommended as a way of adequacy of assessing the nutrition, along with monitoring of health and productivity. A guide to condition scoring of pigs is given in the next part.

to meet quantities o Drinkable water and/or other wholesome liquid must be easily available to pigs in the sufficient physiological needs of the pigs at all times

o Water provided should be fresh, palatable, and at a temperature that does not inhibit drinking.

o Drinker allocation per pen group and drinker design and flow rates should be such that water requirements of different classes of pig are easily met. Vaccine was tested in some piggeries in the North. Pregnant sows twice at 9 and 12 weeks of gestation each received 2 ml of vaccine (approximately 1.5 x 109 bacteria). Results show that under experimental conditions, the vaccine is both safe (100%) and efficacious (>85%). 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).

on herd health 3.3 Recommendations monitoring

II.3.1. Sow management o Pigs should have constant access to water or other suitable wholesome liquid. The daily consumption of water (or other wholesome liquid) by a pig will vary according to environmental temperature, diet ingredients and live-weight.

* Feed requirements for lactating sows

o Pigs must have access to feed at least daily and the diet must be nutritionally adequate to maintain health and meet the requirements for maintenance, growth, pregnancy and lactation. The minimum feed recommended intakes for lactating sows as follows:

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D.N Thuy, C.H Phú, AX. Tuan, N. X.Huyen, T.V. Dung, T. Fahy, S.Driesen, K. Moore, A. Vanderfeen & D. Trott

* WATER REQUIREMENTS Table 3: Feed requirements for lactating sows planning the

For following purposes consumption estimates are provided for normal ambient temperatures: Day of lactation Daily feed

Table 4: Average water consumption (litres requirement (kg)

per day)* 1 2.5

Boar or dry sow 12–15 2 3

Sow and litter 25–45 3 3.5

4 4 Grower pig:

5 4.5 25 kg 3–5

6 5 45 kg 5–7

7 5.5 65 kg 7–9

8 6 90 kg 9 – 12

9 7

10 8

11-15 9 Flow rates will vary depending on the number of drinking points and care should be taken to ensure adequate pump capacity and supply to maintain flow. 16-20 10

21-28 10.5

Recommended water flow rates and maximum water pressures

Class Maximum pressure Flow Rate

(litres/minute) (kPa)

Weaners 0.5 85-105

Growers/Finishers 1.0 140-175

Dry sow 1.0 No limit*

Lactating 2.0 No limit*

* Care should be made not to have excessive pressure as water wastage can occur.

* TEMPERATURE REQUIREMENTS

the very young, are able to Pigs, except tolerate a wide range of temperatures without detriment to their well being, provided temperature changes do not occur abruptly. During very hot weather (35°C or more) adult pigs are very susceptible to heat stress, and steps should be taken to alleviate distress and avoid deaths. Pigs may die if transported in very hot weather. The heat index in most sheds we visited was 37-38°C.

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CARD 001/04 VIE – Control diarrhoea in suckling pigs

The ranges of temperature that afford optimum comfort for different classes of pigs are:

Piglets — newborn 30–35°C

Piglets — 3 weeks of age 24–30°C (reducing to 26°C at 5 weeks of age)

Farrowing house 16-22°C

Weaners 28–30°C first week (start at 30C, reduce by 2°C per week to

22°C)

Growers Aim for 20-24°C, aim to keep daily fluctuations below 6°C

Finishers Aim for 20-24°C, aim to keep daily fluctuations below 6°C

Sows and boars 18 – 24°C, aim to keep daily fluctuations below 6°C

Table 5: Thermal comfort zone (assuming concrete floors, no drafts and 15% skin wetness)

Stock

Weaners

Growers

Dry sows & boars Lactating sows Weight (kg) 5 6 7 8 9 20 50 90 150 150 Lower critical temp 27 25 22 21 20 16 12 9 15 10 Evaporative critical temp 30 29 28 26 25 30 28 27 27 23

 At the lower critical temperature pigs will feel cold, huddle and change position to conserve heat. They will also need more feed to generate heat.

 At the upper limit (evaporative critical temperature) the pigs will begin to pant in an effort to cool down.

3.3.1. Condition scoring of sows

The following criteria can be used as reference for scoring sows:

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D.N Thuy, C.H Phú, AX. Tuan, N. X.Huyen, T.V. Dung, T. Fahy, S.Driesen, K. Moore, A. Vanderfeen & D. Trott

Condition scoring of sows

Loin Vertebrae Ribs Numerical Score

Individual ribs very prominent 1

Pelvic Bones, Tail Head Pelvic bones very prominent. Deep cavity around the tail head. Prominent and sharp throughout the length of the backbone

Prominent

2 Rib cage less apparent. Difficult to see individual ribs Pelvic bones obvious but some slight cover. Cavity around tail head. Loin very narrow. Sharp edges on transverse spinal process. Flank very hollow. Loin narrow. Only very slight cover to edge of transverse spinal process. Flank rather hollow.

Pelvic bones covered. Covered but can be felt.

3 Visible over the shoulder. Some cover further back. Edge of transverse spinal processes covered and rounded.

Felt only with firm pressure

4 Rib cage not visible. Very difficult to feel any ribs. Pelvic bones only felt with firm pressure. No cavity around tail. Edge of transverse spinal processes felt only with firm pressure.

Impossible to feel vertebrae. Not possible to feel ribs.

Impossible to feel bones. Flank full and rounded. 5

Pelvic bones impossible to feel. Root of tail set deep in surrounding fat.

have

4.

Conclusions and recommendations

applied 4.1 Conclusions Cryptosporidium been established. This procedue can be easily in diagnosis for laboratory conditions in Vietnam.

pathogenic full

 A vaccine containing E. coli strains bearing factors (fimbriae, toxins) has been produced and proven safe and effective in  A diagnostic procedure of six major causes of diarrhea in suckling pigs, including the infectious gastroenteritis virus (TGEV), Rota virus, E. coli, C. and perfringens, Coccidiosis

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CARD 001/04 VIE – Control diarrhoea in suckling pigs

laboratory and in the field. Vaccine can be used to vaccinate pregnant sows twice at 9 and 12 weeks of gestation, each received 2 ml of vaccine to control diarrhoea in suckling pigs. 4. Ngoc Thuy Do. 2004. The significance of enterotoxigenic E. coli as a cause of pre- weaning diarrhoea piglet in North Vietnam. Ph.D Thesis. The University of Queensland. Brisbane, Queensland, Australia.

the 18th

 An continuous improvement model, focused mainly on management of sows during farrowing and lactation has been developed and utilized in order to improve pig production and minimize the losses due to diarrhoea in suckling period. 5. Do TN, Wilkie, I, Frost AJ and DJ Trott. 2004. Pathogenicity of Vietnamese ETEC in colostrum deprived, day-old strains IPVS piglets Proceedings of Congress, Hamburg, Germany, June 28 to July 1, 2004. International Pig Veterinary Society 2004. p. 285.

4.2 Recommendations

The E. coli vaccine should be commercialised as soon as possible so that it can be used for control of diarrhoea in suckling pigs in Vietnam

6. TN Do, HP Cu, XH Nguyen, XT Au, NQ Vu, KM Townsend, B McCormick, AJ Frost and DJ Trott. 2004. Pathotypes and serotypes of ETEC isolates obtained from piglets with diarrhoea in North Vietnam. the 18th IPVS Congress, Proceedings of Hamburg, Germany, June 28 to July 1, 2004. International Pig Veterinary Society 2004. p. 318.

References

7. N.T. Do, I. Wilkie, S. J. Driesen, V. A. Fahy, and D. J. Trott. 2006. Pathogenicity of Vietnamese Enterotoxigenic Escherichia coli strains in colostrum-deprived one-day- old piglets. Veterinary Pathology 43, 150- 160. pigs

1. Cu Huu Phu, Nguyen Ngoc Nhien, Do Ngoc Thuy, Au Xuan Tuan, Nguyen Xuan Huyen, Van Thi Huong, Vu Ngoc Quy. Results on investigation of diarrhoea in suckling in North Vietnam, prevalence of antimicrobial resistance and virulence factors of obtained E. coli isolates. Proceedings of NIVR – 35 years of establishment and development, 1969- 2004. Agriculture Publishing House. 2004. Pp 126-136.

8. Thuy, N. Do, Phu H. Cu, Huyen X. Nguyen, Tuan X. Au, Quy N. Vu, Steve J. Driesen, Kirsty M. Townsend, James J-C. Chin and Darren J. Trott. 2006. Pathotypes and serogroups of enterotoxigenic Escherichia isolated from pre-weaning pigs in coli North Vietnam. of Medical Journal Microbiology 55, 93-99.

six most the

2. Do Ngoc Thuy, Darren Trot, Cu Huu Phu, Nguyen Xuan Huyen, Au Xuan Tuan, Van Thi Huong, Vu Ngoc Quy. Application of PCR on examination of virulence factors of E. coli strains obtained from piglets with diarrhoea in North Viet Nam. Veterinary Sciences and Techniques. Vol. XII, No5. 2005.P 13-17.

IPVS

9. Do NT, Cu HP, Van TH, Nguyen XH, Au XT, Vu NQ, Fahy VA, Driesen SJ, Moore K, Vanderfeen A, and Trott DJ. 2006. Prevalence of common infectious causes of pre-weaning diarrhoea in piglets in Vietnam. Proceedings of the 19th Copenhagen, Congress, Denmark, July 16-19, 2006. International Pig Veterinary Society 2006. p. 318.

societies

3. T-N Do, KM Townsend, AJ Frost and DJ. Trott. 2003. Pathogenicity of Vietnamese ETEC in day-old piglets. Proceedings of a combined annual scientific meeting and exhibition of the Australian and New for Microbiology. Zealand Auckland, New Zealand, 28 September-2 October, 2003. p. 82. 10. N. T. Do, H. P. Cu, N. N. Nguyen, X. H. Nguyen, X. T. Au, T. H. Van, N. Q. Vu and D. J. Trott. 2006. Antimicrobial resistance phenotypes of ETEC isolates from piglets with diarrhoea in North Vietnam. Annals of

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the New York Academy of Sciences. 1081: 543-545.

Congress of the Asian – Australasian association of animal production societies. August 23- 27, 2010. Pingtung, Taiwan, ROC. p. 459. profiles

J. Trott. Fairbrother,

11. Trott DJ, Smith M, Do NT. 2006. of resistance Comparative isolated Enterotoxigenic Escherichia coli from Australia and Vietnam. Proceedings the 19th IPVS Congress, Copenhagen, of Denmark, July 16-19, 2006. International Pig Veterinary Society 2006. p. 462 multi-drug in

12. Do Ngoc Thuy.

14. M. G. Smith, d. Jordan, t. A. Chapman, j. J-c chin, m. D. Barton, t. N. Do, v.a. fahy, j. M. 2010. d. Antimicrobial resistance and virulence gene resistant profiles enterotoxigenic escherichia coli isolated from pigs with post-weaning diarrhoea. Veterinary microbiology. [epub ahead of print].

2008. Virulence characterization of Vietnamese strains of E. coli causing diarrhoea in pigs. Proceedings of the 13th Animal science congress of the Asian – Australasian association of animal production societies. September 22- 26, 2008. Hanoi, Vietnam. p. 479.

and

15. Xi-Yang Wu, Toni Chapman, Darren J. Trott, karl Bettelheim, Thuy N. Do, Steve Driesen, Mark J. Walker, and James Chin. 2007. Comparative analysis of virulence genes, genetic diversity, and phylogeny of Enterotoxigenic commensal Escherichia coli isolates from weaned pigs. Applied and Environmental Microbiology 73, 83-91.

13. D. N. Thuy, A. X. Tuan, T. V. D. Kien, L. T. M. Hang, H. N. Duyet, N. V. Chao, C. H. Phu, T. T. B. Duyen, N. Q. Linh, C.R. Parke, D. J. Trott. Field Efficacy of a locally produced vaccine for the control of in Vietnam. pre-weaning Colibacillosis the 14th Animal Science Proceedings of

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