Collaboration for Agriculture and Rural Development (CARD) Program 16 RESULTSONSTRENGTHENINGDIAGNOSTICCAPACITYANDCONTROLOFDIARRHOEAINSUCKLINGPIGS Project title: Diagnosis andcontrolofdiarrhoeainsucklingpigs Project c ode : CARD 001/04VIE Authors: Do Ngoc Thuy 1 , Cu Huu Phu 1 , Au Xuan Tuan 1 , Nguyen Xuan Huyen 1 , Truong Van Dung 1 , Tony Fahy 1 , Steve Driesen 2 , Karen Moore 2 , Aileen Vanderfeen 2 , Darren Trott 3 Collaborating organisations: 1 : National Institute of Veterinary Research 2 : Pig Research Unit, Vi ctorian Department of Primary Industry, Melbourne, Australia ; 3 : The University of Queensland , Australia SUMMARY During the implementation time (2005 - 2007), three objectives of the project, including: 1) Improved field and laboratory diagnosis of prewean ing 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 ofdiarrhoeainsucklingpigs 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 andin 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 diarrhoeainsuckling period. 1 . Introduction 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 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 ofsuckling diarrhoe a, 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 andcontrolofdiarrhoeainsuckling pigs) began with three objectives to solve th is problem: 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 This pr oject 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 appropria te 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. CARD 001/04 VIE – Controldiarrhoeainsucklingpigs 17 2 . Project contents and methods 2 .1 Project Contents The project focuses on: 1. Improved 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, training for field veterinarians 2 .2 Method ology - Experimental design based on groups and compare the significant difference (1 factor, multiple factors) - Isolati onand identification of bacteria ( E. coli , C. perfringens ) according to SOPs - Detection of virus (TGE virus, Rota virus) by ELISA (commercial kit, Institute Pourquier) - Detection of Cocci oocysts by standard faecal flotation , Cryptosporidium by modifi ed Ziehl - Neelsen staining of faecal smears - Detection of virulence factors of E. coli (fimbriae, toxins) by PCR - Production of vaccine according to SOP 3 . Research resultsand discussions 3 .1 Development of a procedure for quick diagnose some of th e most common infectious causes ofdiarrhoeainsucklingpigs A diagnostic procedure of six major causes ofdiarrhoeainsuckling pigs, including the infectious gastroenteritis virus (TGEV), Rota virus, E. coli, C. perfringens, Coccidiosis and Cryptospori dium 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 from commercial piggeries (participate d in the project) and small holder farmers in the nearby areas. Results are presented in table 1. Table 1: Prevalence of enteric pathogens in pre - weaning piglets with diarrhoea Agent(s) detected No. of positive samples (%) Commercial piggeries (n=117) Small holder farmers (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 infection 25 (21.2) RV ETEC 6 (5.1) RV TGEV 17 (14.4) 3 (6.7) Cocci RV 1 (0.8) 2 (4.4) Cocci Crypto 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) D. N Th u y, C . H Phú, A X. Tu a n, N. X.Huy e n, T.V. Dung, T. Fahy, S.Driesen, K. Moor e, A. Vanderfeen & D. Trott 18 Agent(s) detected No. of positive samples (%) Commercial piggeries (n=117) Small holder farmers (n=45) TGE V ETEC 7 (5.9) Cocci RV ETEC 2 (1.7) 3 (6.7) Cocci RV TGEV 4 (3.4) 3 (6.7) Cocci TGEV ETEC 2 (1.7) 2 (4.4) Cocci Crypto RV 1 (0.8) 1 (2.2) Crypto TGEV ETEC 5 (4.2) 4 (4.4) Crypto RV ETEC 1 (0.8) 3 (6.7) Cryp to TGEV C. per 1 (0.8) 1 (2.2) Crypto RV TGEV 3 (2.5) RV TGEV ETEC 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) TGEV ETEC C. per 2 (1.7) Crypto RV TGEV ETEC 2 (1.7) 1 (2.2) Crypto RV ETEC C. per 1 (0.8) 2 (4.4) Cocci Crypto RV TGEV 1 (0.8) 1 (2.2) Cocci Crypto TGEV C. per 1 (0.8) Cocci RV TGEV ETEC 4 (3.4) 1 (2.2) Cocci Crypto RV TGEV C. per 1 (0.8) 1 (2.2) Crypto RV TGEV ETEC C. per 1 (0.8) (3 6) (50) (97) (111) (76) (23) Total multiple infections 92 (78.8) 45 (100.0) 3.2 Production and testing of local produced E. coli vaccine A vaccine containing E. coli strains bearing full pathogenic factors (fimbriae, toxins) has been pro duced according to SOP. Specialised culture media were prepared in order to provide favourable growth conditions for the production of fimbriae. Each dose of vaccine (2 ml) contains of ~ 1.5 x 10 9 bacteria. Vaccine has been tested on sterility, safety and efficacy in laboratory andin the field (for small scale trial). CARD 001/04 VIE – Controldiarrhoeainsucklingpigs 19 Resultsof ELISA for efficacy testing of vaccine show that 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 2) Table 2 : Mean OD values of sera between tre atment andcontrol group Treatment group Mean Control 0.2260 a Litterguard 0.8129 b EcoVac 0.9406 b NIVR 0.8695 b * Means with different subscripts differ significantly p<0.005 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 diarrhoeain 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). 3. 3 Recommendations on herd health monitoring II.3.1. Sow management 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. 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. 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, appropriat e to the sow’s physiological needs (which vary during pregnancy) and to provide satiety without the sow becoming overweight. o Body score of breeding sows at farrowing should be 3.5 – 4. o Body score of breeding sows at weaning should be 3 or above. o Condition scoring is recommended as a way of assessing the adequacy of nutrition, along with monitoring of health and productivity. A guide to condition scoring ofpigs is given in the next part. o Drinkable water and/or other wholesome liquid must be easily availabl e to pigsin sufficient quantities to meet the 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 s hould be such that water requirements of different classes of pig are easily met. 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 envir onmental temperature , diet ingredients and live - weight. * Feed requirements for lactating sows The minimum feed recommended intakes for lactating sows as follows: D. N Th u y, C . H Phú, A X. Tu a n, N. X.Huy e n, T.V. Dung, T. Fahy, S.Driesen, K. Moor e, A. Vanderfeen & D. Trott 20 Table 3: Feed requirements for lactating sows Day of lactation Daily feed requirement ( kg) 1 2.5 2 3 3 3.5 4 4 5 4.5 6 5 7 5.5 8 6 9 7 10 8 11 - 15 9 16 - 20 10 21 - 28 10.5 * WATER REQUIREMENTS For planning purposes the following consumption estimates are provided for normal ambient temperatures: Table 4 : Average water consumption (litres per day)* Boar or dry sow 12 – 15 Sow and litter 25 – 45 Grower pig: 25 kg 45 kg 65 kg 3 – 5 5 – 7 7 – 9 90 kg 9 – 12 Flow rates will vary depending on the number of drinking points and care should be taken to ensure adequate pump capacityand supply to maintain flow. Recommended water flow rates and maximum water pressures Class Flow Rate (litres/minute) Maximum pressure (kPa) Weaners 0.5 85 - 105 Growers/Finishers 1.0 140 - 175 Dry sow 1.0 No limit* Lactating 2.0 No limit* * Care shou ld be made not to have excessive pressure as water wastage can occur. * TEMPERATURE REQUIR EMENTS Pigs, except the very young, are able to tolerate a wide range of temperatures without detriment to their well being, provided temperature changes do not o ccur 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 visite d was 37 - 38°C. CARD 001/04 VIE – Controldiarrhoeainsucklingpigs 21 The ranges of temperature that afford optimum comfort for different classes ofpigs 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 Weight (kg) Lower critical temp Evaporative critical temp Weaners 5 27 30 6 25 29 7 22 28 8 21 26 9 20 25 Growers 20 16 30 50 12 28 90 9 27 Dry sows & boars 150 15 27 Lactating sows 150 10 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) t he 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: D. N Th u y, C . H Phú, A X. Tu a n, N. X.Huy e n, T.V. Dung, T. Fahy, S.Driesen, K. Moor e, A. Vanderfeen & D. Trott 22 Condition scoring of sows Numerical Score Pelvic Bones, Tail Head Loin Vertebrae Ribs 1 Pelvic bones very prominent. Deep cavity around the tail head. Loin very narrow. Sharp edges on transverse spinal process. Flank very hollow. Prominent and sharp throughout the length of the backbone Individual ribs very prominent 2 Pelvic bones obvio us but some slight cover. Cavity around tail head. Loin narrow. Only very slight cover to edge of transverse spinal process. Flank rather hollow. Prominent Rib cage less apparent. Difficult to see individual ribs 3 Pelvic bones covered. Edge of transve rse spinal processes covered and rounded. Visible over the shoulder. Some cover further back. Covered but can be felt. 4 Pelvic bones only felt with firm pressure. No cavity around tail. Edge of transverse spinal processes felt only with firm pressure . Felt only with firm pressure Rib cage not visible. Very difficult to feel any ribs. 5 Pelvic bones impossible to feel. Root of tail set deep in surrounding fat. Impossible to feel bones. Flank full and rounded. Impossible to feel vertebrae. Not po ssible to feel ribs. 4 . Conclusions and recommendation s 4.1 Conclusions A diagnostic procedure of six major causes of diarrhea insuckling pigs, including the infectious gastroenteritis virus (TGEV), Rota virus, E. coli , C. perfringens , Coccidiosis and Cryptosporidium have been established. This procedue can be easily applied for diagnosis in laboratory conditions in Vietnam. A vaccine containing E. coli strains bearing full pathogenic factors (fimbriae, toxins) has been produced and proven safe and effective in CARD 001/04 VIE – Controldiarrhoeainsucklingpigs 23 laboratory andin 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 controldiarrhoeainsuckling pigs. An continuous improvement model, focused mainly on managemen t of sows during farrowing and lactation has been developed and utilized in order to improve pig production and minimize the losses due to diarrhoeainsuckling period. 4.2 Recommendations The E. coli vaccine should be commercialised as soon as possibl e so that it can be used for controlofdiarrhoeainsucklingpigsin Vietnam References 1. Cu Huu Phu, Nguyen Ngoc Nhien, Do Ngoc Thuy, Au Xuan Tuan, Nguyen Xuan Huyen, Van Thi Huong, Vu Ngoc Quy. Resultson investigation ofdiarrhoeainsucklingpigs i n 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. 2. Do Ngoc Thuy, Darre n 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 diarrhoeain North Viet Nam. Veterinary Sciences and Techniques. Vol. XII, No5. 2005.P 13 - 17. 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 Zealand societies for Microbiology. Auckland, New Zealand, 28 September - 2 October, 2003. p. 82. 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, A ustralia. 5. Do TN, Wilkie, I, Frost AJ and DJ Trott. 2004. Pathogenicity of Vietnamese ETEC strains in colostrum deprived, day - old piglets Proceedings of the 18 th IPVS Congress, Hamburg, Germany, June 28 to July 1, 2004. International Pig Veterinary Soc iety 2004. p. 285. 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 diarrhoeain North Vietnam. Proceedings of the 18 th IPVS Congress, Hamb urg, Germany, June 28 to July 1, 2004. International Pig Veterinary Society 2004. p. 318. 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 on e - day - old piglets. Veterinary Pathology 43 , 150 - 160. 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 Escherich ia coli isolated from pre - weaning pigsin North Vietnam. Journal of Medical Microbiology 55 , 93 - 99. 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 the six most common infectio us causes of pre - weaning diarrhoeain piglets in Vietnam. Proceedings of the 19 th IPVS Congress, Copenhagen, Denmark , July 16 - 19, 2006. International Pig Veterinary Society 2006. p. 318. 10. N. T. Do, H. P. Cu, N. N. Nguyen, X. H. Nguyen, X. T. Au, T. H. V an, N. Q. Vu and D. J. Trott. 2006. Antimicrobial resistance phenotypes of ETEC isolates from piglets with diarrhoeain North Vietnam. Annals of D. N Th u y, C . H Phú, A X. Tu a n, N. X.Huy e n, T.V. Dung, T. Fahy, S.Driesen, K. Moor e, A. Vanderfeen & D. Trott 24 the New York Academy of Sciences . 1081 : 543 - 545. 11. Trott DJ, Smith M, Do NT. 2006. Comparative resistance pr ofiles of Enterotoxigenic Escherichia coli isolated from Australia and Vietnam. Proceedings of the 19 th IPVS Congress, Copenhagen, Denmark , July 16 - 19, 2006. International Pig Veterinary Society 2006. p. 462 12. Do Ngoc Thuy. 2008. Virulence characterizati onof Vietnamese strains of E. coli causing diarrhoeain pigs. Proceedings of the 1 3 th Animal science congress of the Asian – Australasian association of animal production societies. September 22 - 26, 2008. Hanoi, Vietnam. p. 479. 13. D. N. Thuy, A. X. Tua n , 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 controlof pre - weaning Colibacillosis in Vietnam. Proceedings of the 14th Anima l Science Congress of the Asian – Australasian association of animal production societies. August 23 - 27, 2010. Pingtung, Taiwan, ROC. p. 459. 14. M. G. Smith, d. Jordan, t. A. Chapman, j. J - c chin, m. D. Barton, t. N. Do, v.a. fahy, j. M. Fairbrother, d . J. Trott. 2010 . Antimicrobial resistance and virulence gene profiles in multi - drug resistant enterotoxigenic escherichia coli isolated from pigs with post - weaning diarrhoea. Veterinary microbiology . [epub ahead of print ]. 15. Xi - Yang Wu, Toni Chapman, Darr en 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 commensal and Enterotoxigenic Escherichia coli isolates from weaned pigs. Applied an d Environmental Microbiology 73 , 83 - 91. . Collaboration for Agriculture and Rural Development (CARD) Program 16 RESULTS ON STRENGTHENING DIAGNOSTIC CAPACITY AND CONTROL OF DIARRHOEA IN SUCKLING PIGS Project title: Diagnosis and control of diarrhoea. quick diagnose some of th e most common infectious causes of diarrhoea in suckling pigs A diagnostic procedure of six major causes of diarrhoea in suckling pigs, including the infectious gastroenteritis. VIE – Control diarrhoea in suckling pigs 23 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