Ministry of Agriculture & Rural Development CARD Project Technical Report Development of an Improved Capability in support of National Bio-security for the Surveillance and Control of Foot & Mouth Disease in Cattle and Pigs Milestone Epidemiological and sero-surveillance programs operational By Debbie Eagles & Chris Morrissy Table of Contents Institute Information _ Project Abstract _ 3 Executive Summary Introduction & Background _ Epidemiological and Sero-surveillance programs 5.1 5.2 Capacity Building _ 36 5.3 Implementation Highlights Publicity _ 36 Implementation & Sustainability Issues _ 37 6.1 Issues and Constraints _ 37 6.2 Options 37 6.3 Sustainability _ 37 Next Critical Steps 38 Conclusion 39 1 Institute Information Project Name Vietnamese Institution Regional Animal Health Centre, Ho Chi Minh City (RAHO - ), South Vietnam Vietnamese Project Team Leader Dr Dong Manh Hoa Australian Organisation Australian Personnel Australian Animal Health Laboratory (AAHL), PMB 24, Geelong, 3213, Australia Mr Chris Morrissy Date commenced 01/06/2005 Completion date (original) 01/06/2008 Completion date (revised) Reporting period Contact Officer(s) In Australia: Team Leader Mr Chris Morrissy Diagnostic Virologist Supervisor Mammalian Virology Organisation Australian Animal Health Laboratory (AAHL), PMB 24, Geelong, 3213, Australia Name: Position: Telephone: Fax: +61 5227 5000 +61 5227 5555 Email: chris.morrissy@csiro.au In Australia: Administrative contact Mr Chris Morrissy Name: Patents Contracts Officer Position: Organisation Australian Animal Health Laboratory (AAHL), PMB 24, Geelong, 3213, Australia Telephone: Fax: Email: +61 5227 5000 +61 5227 5555 christopher.morrissy@csiro.au In Vietnam Dr Dong Manh Hoa Name: Director Position: Organisation Regional Animal Health Centre, Ho Chi Minh City (RAHO - ), South Vietnam Telephone: Fax: Email: + 84 8568220 + 84 8569050 rahchcmc@hcm.vnn.vn Project Abstract The project’s purpose was twofold - to develop capacity for FMD (and other disease) surveillance and diagnosis at both a laboratory and field level, and to investigate the serotypes of FMDV circulating in Vietnam and the reason for vaccine failures Regional laboratories were set up with the reagents and methods to allow a diagnostic capability for FMDV diagnosis and serology Control strategies for understanding of FMD epidemiology have been implemented through veterinary and laboratory training workshops The project has highlighted the importance of having a laboratory network to identify what is happening in the field and how to prevent and control disease outbreaks The pilot zones were established in provinces near the borders of Vietnam to study serotypes circulating in Vietnam and to determine their origin The number and quality of samples increased with each round of the project giving more data on the FMD situation in Vietnam Virus isolation and molecular studies can now be carried out on FMD samples from the field and molecular epidemiological studies of the FMDV isolates in these provinces has provided insights into the effectiveness of border control and origin of circulating FMDV Improved diagnostic capacity for FMD allows for the early detection and identification of disease enabling better control of disease and helps reduce loss of livestock and therefore increases productivity Executive Summary The CARD FMD project was ambitious in that it had very broad and diverse aims The first objective was capacity building – at the laboratory, epidemiological and field levels The second major aim was to investigate possible causes of vaccination failure by evaluating isolates of circulating strains and sero-surveillance data The project was very successful at achieving its objective in relation to capacity building As documented in the final report, the four collaborating Vietnamese laboratories improved their FMD diagnostic capacity and have been able to apply their new skills to disease investigations and surveillance projects In addition, there have been important and measurable improvements in both epidemiological and field areas When this project began there was no epidemiology department at any of the laboratories There is now a fully functional epidemiology department, with full-time staff, at RAHO – This group has been instrumental in supporting this and other international projects and has provided advice and training to field and provincial veterinarians They have also been crucial to investigation of disease outbreaks such as HPAI and PRRS, particularly in southern Vietnam Through implementation of this project one of the lessons learnt has been the importance of complete and accurate field information Whereas the laboratories were already accustomed to recording results, the recording of information such as vaccination and infection data in the field was not commonplace Data collection and management has improved dramatically throughout the project The only downside of this is that data quantity and quality for the initial rounds is difficult to analyze for the purpose of investigation of vaccine failure In addition to this the accuracy of some of the data is questionable, as is discussed throughout this report With the laboratory and epidemiological capacity now available in the collaborating laboratories, particularly HCMC, there is now the potential for a smaller, more focused study on vaccination failure This would be best limited to a smaller number of provinces in southern Vietnam, with a study protocol aimed specifically at investigating vaccination effectiveness Introduction & Background Serum samples and information were initially to be collected from 10 provinces – An Giang, Binh Phuoc, Dong Thap, Kien Giang, Kom Tum, Lang Son, Long An, Quang Nam, Quang Ninh and Tay Ninh No samples were ever collected from Lang Son Samples were also only collected intermittently from the other northern province in the project, Quang Ninh The central provinces of Kom Tum and Quang Nam provided samples for all but the 3rd round In the southern provinces samples were not available from Long An in the final round and An Giang did not provide pig samples in round 1, or The epidemiological support and interest at RAHO - is almost certainly a contributing factor to the better provision of samples from the southern provinces A combination of fewer specialized staff in the northern provinces and laboratories and the required allocation of resources to outbreak response is likely to have reduced their ability to collect samples NCVD did not have a epidemiology section In 2005, the field data collection and the use of forms was not well developed As a result, for most provinces the only information collected was species, sampling location (district/commune/village) and in some instances vaccination information, sampling date and animal age In 2006 the data collection form was further developed and standardised with the following fields – district, commune, village, species, age, sex, sampling date, vaccination date, vaccine name/manufacturer/serotypes, last date of infection, last serotype of infection and field sample number This form improved data collection dramatically although not all fields were completed in every round for each province Epidemiological and Sero-surveillance programs 5.1 Implementation Highlights Analysis by Province The analysis for the information in each province is divided into cattle and pigs For each species there are tabular results and a graph followed by a description of the results for each round The graph only displays information for rounds and serotypes for which animals have been vaccinated ≤ months prior to sampling An Giang Cattle Year (Round) Vaccination Type % 3ABC + %O ELISA + %A ELISA + % Asia 1+ Previous Infection date in province (species, serotype) August 2005 (pigs, O) 36 89 100 51 August 2005 (pigs, O) August 2005 (pigs, O) August 2005 (pigs, O) August 2005 (pigs, O) 2008 (6) O,A Vaccinationsampling interval 2mths 2008 (5) O,A 5mths 10 10 22 2007 (4) O,A 1mth 21 61 58 2007 (3) O,A 6-7mth 37 66 47 31 2006 (2) O,A 6mth 38 19 56 34 August 2005 (pigs, O) 2005 (1) O,A Unknown 76 63 70 56 August 2005 (pigs, O) Comment Higher proportion Asia +ves amongst 3ABC+ group Proportions across the 3ABC/3ABC+ similar Unknown history of infection Percent cattle O/A positive when vaccinated for that serotype 100 90 80 70 60 Percentage 50 O 40 A 30 20 10 Round 2005 (Round 1) Without vaccination or infection information available no judgment can be made on vaccination response 75% cattle were NSP ELISA positive, and more than half of these are positive for all serotypes This suggests that these cattle have been vaccinated with either a bivalent or trivalent vaccine, given that infection with more than one serotype simultaneously is rare Further testing of the sera is necessary to determine the serotypes present, ie titration of the sera to a endpoint against each sera type There was a reported outbreak of serotype O infection in pigs in 2005 2006 (Round 2) All cattle were vaccinated for serotypes O/A, 6months prior to sampling The greatest serological response was to serotype A at 50% It is likely that there was a problem with the sensitivity of the O ELISA for this batch of samples, as it would be unlikely for animals to have been vaccinated for serotype A and not serotype O It is almost certain that some animals were also vaccinated for Asia 1, given the serological response to this serotype, absence of outbreak history and the negative 3ABC result in the majority of those that were Asia positive 2007 (Round 3) Vaccination was months prior to sampling with O/A vaccine Despite lack of infection history, 37% were 3ABC ELISA positive In general the serological response to serotype O was better than in the previous round which may be more indicative of changes to the assay as opposed to differences in vaccine response Close to a third of cattle were also positive on the Asia ELISA and of these 2/3 were also 3ABC positive This is suggestive of both unrecorded vaccination and/or unreported infection (which may be related to animal movement) 2007 (Round 4) Vaccination occurred one month prior to sampling with an approximately 60% response rate to both serotype O and A (bivalent vaccine administered) A smaller % were positive on the 3ABC ELISA in this round than in round 2008 (Round 5) The serological response to the O/A vaccine is very poor, regardless of the month interval between vaccination and sampling Following the protocol listed below in Appendix (Investigating Vaccination Failure Checklist) may assist in determining the reasons for vaccine failure Records suggest that the same vaccine was used in each round 2008 (Round 6) There was an excellent serological response to vaccine in this round, in which the vaccination-sampling interval was month Over 50% of cattle also seroconverted to Asia despite no history of recent vaccination for this serotype Half of these were also 3ABC positive, despite no infection history in the sampled animals or the province (see below) Conclusions The % of cattle 3ABC was > 1/3 in of the rounds In the absence of outbreak history or isolates from this province since 2005 this suggests that: There has been movement (transboundary or between provinces) of infected (diseased or carrier) animals There have been unreported or undetected (due to mild clinical signs) infections in the surveyed communes There is a large number of animals previously exposed to FMD or carriers that remain – at least intermittently – NSP ELISA positive Some variation in the results between years may also be due to the inclusion of communes (An Phu, Tinh Bien, An Nong and Nhon Hung) which were variably sampled in the different rounds Vaccination response was only – in the final around - above the required herd protected level of 80% Pigs Year Vaccination Type % 3ABC + %O ELISA + %A ELISA + % Asia 1+ Previous Infection date in province (species, serotype) Comment Vaccinationsampling interval 2008 (6) No samples 2008 (5) No samples 2007 (4) O 1mth 0 August 2005 (pigs, O) 2007 (3) O 6mths 0 August 2005 (pigs, O) 2006 (2) O mths 0 0 August 2005 (pigs, O) 2005 (1) No samples Percent pigs O/A positive when vaccinated for that serotype 100 90 80 70 60 Percent 50 O 40 30 20 10 Round Samples were collected from vaccinated pigs in 2006 and both rounds of 2007 The highest serological response for serotype O was 5% despite the fact that on one occasion vaccination was administered just one month prior to sampling In rounds and all sampled piglets were >6 months In the remaining rounds some piglets were as young as 2.5 months, so could not have been vaccinated on the date recorded Binh Phuoc Cattle Year Vaccination Type % 3ABC + %O ELISA + %A ELISA + % Asia 1+ Previous Infection date in province (species, serotype) Comment 24 94 93 93 2006 (Cattle, pig; O) 2006 (Cattle, pig; O) Last infection Aug 05 ½ that have been infected are Asia + 2008 (6) O,A,Asia1 Vaccinationsampling interval 1mth 2008 (5) O,A,Asia1 mths 28 34 51 18 2007 (4) O, A mth 14 89 88 38 2007 (3) O,A 5-6mths 41 81 55 2006 (2) O,A,Asia1 mths 11 50 35 35 2005 (1) O, A mths 10 20 24 2006 (Cattle, pig; O) 2006 (Cattle, pig; O) 2006 (Cattle, pig; O) Previous infection (O/A?) April, Aug, Oct 2005 (Cattle, O/A; pigs, O) Percent of cattle O/A/Asia1 positive when vaccinated for that serotype 100 90 80 70 60 50 O 40 A 30 Percentage Asia1 20 10 Round 2005 (Round 1) There is little field information available for this year, as the forms for information collection had not been developed Cattle were vaccinated months prior to the sampling date with O/A vaccine, which is just at the extent of the expected vaccination protective period The vaccination response rate in this year was very poor (20% positive for serotype O and 5% positive for serotype A), however it is difficult to pass judgment on first year results from either field or laboratory perspective 2006 (Round 2) In this year the recorded information shows that vaccination was with a serotype A vaccine called Trivale However discussions at sub-DAH confirm that, as the vaccine name would suggest, this is more likely a trivalent vaccine The manufacturer is unknown The vaccination date was months prior to sampling date, so it is not surprising that the seropositives were relatively low at 50%, 35% and 35% for serotypes O, A and Asia respectively 2007 (Round 3) In this year records suggest that all cattle were vaccinated with serotypes O/A vaccine SubDAH staff again suggested that cattle may have been vaccinated for Asia1 This would fit the ELISA results, with 41%, 81% and 55% positive for O, A and Asia1 serotypes respectively The vaccine was administered months prior to sampling, which may contribute to the variation in % positive for each serotype 2005 (Round 1) As with other provinces there was little history provided with these samples and thus the results are relatively meaningless There is indication that a small proportion of the population has been previously infected 2006 (Round 2) There were no samples for this round 2007 (Round 3) No samples 2007 (Round 4) In this round cattle were vaccinated one month prior to sampling with a trivalent vaccine and the ELISA result suggests excellent response to the vaccine 2008 (Round 5) There is no history available for this round, and it appears that samples were only tested by 3ABC and O ELISA It is almost certain that the cattle have recently been vaccinated against at least serotype O, due to the 77% of positives on this assay 2008 (Round 6) No samples Conclusions There was only one round from Quang Ninh for which there were sufficient samples and information about which to make a judgment of vaccine response The vaccine response was excellent in that round However both sample and data collection need to improve so that Quang Ninh can actively participate in the National Control Program 28 Pigs Year Vaccination % 3ABC + %O ELISA + %A ELISA + % Asia + Comment June/July 2008 (Buffaloes; O) Type Previous Infection date in province (species, serotype) No samples Vaccinationsampling interval 2008 (6) 2008 (5) 2007 (4) No samples Unknown 1mth 0 0 2007 (3) February 2007 (unknown, O) No samples 2006 (2) March 2006 (Goats; A) No samples 2005 (1) January 2000 (unknown; O), May 2001 (goats;O) Samples were only collected from pigs in round of the project, and all pigs were negative on all ELISAs 29 Tay Ninh Cattle Year Vaccination % 3ABC + %O ELISA + %A ELISA + % Asia + Comment Type Vaccinationsampling interval 2008 (6) O,A mth 20 35 90 2008 (5) none none 42 27 2007 (4) O,A 1mth 69 69 2007 (3) O,A mth 15 2006 (2) O,A mth 48 96 54 12 2006 (pig, buffalo; O) No record of infection 2005 (1) Unknown Unknown 27 35 May 2005 (pig; O) No infection or vacc information No history of vacc or infection Pecent cattle Elisa positive when vaccinated for serotype 100 90 80 70 60 Percent 50 O 40 A 30 20 10 Round 2005 (Round 1) There are no vaccination records for cattle or pigs for the first round About ¼ of the cattle were 3ABC positive, suggesting previous infection Of these, the majority were also O 30 ELISA positive, with a small number A ELISA positive Around 15% of the cattle were O ELISA positive but not previously infected (3ABC negative) indicating vaccination 2006 (Round 2) In this year 50% of cattle showed signs of previous infection Outbreaks recorded for previous years were only in pigs and buffalo It is possible that there was either Movement of infected animals (from other provinces or countries) Unreported/undetected (mild clinical signs) infection in cattle Large number of carriers that remain (at least intermittently) NSP ELISA positive It is difficult to determine the serotype to which these cattle had been exposed, as the relative proportions of O ELISA and A ELISA positives between infected and non-infected groups is approximately the same Titration of positive samples would also be required There is some discrepancy between the O and A ELISA results, with almost 100% positive to the O ELISA and 50% positive on the A ELISA after vaccination one month previously with bivalent vaccine This could be due to either a vaccine-ELISA antigen mismatch, or vaccination of some cattle with monovalent (O) vaccine only 2007 (Round 3) Cattle were administered O/A vaccine months prior to sampling The vaccination response rate is extremely poor – so low that it is unlikely to be just waning antibodies postvaccination It is more likely that different animals were presented for vaccination and sampling or inaccurate recording of vaccination There was no indication of previous infection in cattle The village used in this round was also one of the villages sampled in round 2, in which there was a very high proportion of 3ABC positive cattle The dramatic decrease in positives on this ELISA suggests few were still carrying the virus, or that animals were moved out of the village 2007 (Round 4) In the second round of 2007 cattle were vaccinated with O/A vaccine and sampled month later The vaccination response was reasonable with 70% cattle positive on both the O and A ELISA About 10% were also positive for antibodies to Asia without any positive to the ABC ELISA, suggesting previous vaccination with Asia also 2008 (Round 5) Vaccination occurred later than normal in this year as vaccine was not available for the normal March vaccination period As such, the cattle were not vaccinated prior to sampling and the ELISA results are consistent with this 31 Again, there is a relatively high (40%) 3ABC positive ELISA response and most of the O ELISA positives were also 3ABC positive suggesting that this was the serotype of infection in these animals (however this would need to be confirmed by titration of the ELISA positive samples) These animals came from different communes Without recent outbreaks of serotype O in cattle in the province this is suggestive of: Movement of infected animals (from other provinces or countries) Unreported/undetected (mild clinical signs) infection in cattle Large number of carriers that remain (at least intermittently) NSP ELISA positive 2008 (Round 6) In this round cattle were vaccinated month prior to sampling with O/A vaccine There was excellent response to the A component of the vaccine (100% positive on the ELISA) but only 25% on the O ELISA This is most likely to be a problem with the sensitivity of the O ELISA, given the response to the A component of the vaccine and assurances that O/A vaccine was administered to all cattle Conclusions Vaccination responses in cattle were very variable in Tay Ninh In addition, 3ABC ELISA results varied and were not entirely consistent with outbreak history Combined these results suggest significant movement of cattle into or through this province 32 Tay Ninh - Pigs Year Vaccination % 3ABC + %O ELISA + %A ELISA + % Asia + Comment Type Vaccinationsampling interval 2008 (6) O,A 1-2mth 21 41 2008 (5) None NA 0 0 2007 (4) O 1mth 0 0 2007 (3) None NA 0 0 2006 (2) None NA 0 2006 (pig, buffalo; O) 2005 (1) None NA 0 0 May 2005 (pig; O) Almost all 12 months, and vaccination-sampling interval ranged from 20 – 122 days (12months and only ½ were also O ELISA positive, while most of those that were Asia 1+ were aged 3-4 months and predominantly O ELISA positive No pigs had either a history of infection or a 3ABC positive result The A ELISA positives are almost certainly due to previous vaccination for this serotype, particularly due to the age of the pigs It is possible some of the younger pigs also received different vaccines % Pigs Elisa positive after O vaccine 80 % 60 40 20 O A Asia1 Elisa type Forty cows ranging in age from 12months – years were vaccinated with O/A/Asia1 with a vaccination-sampling interval of 43-154 days (1.5-5 months) The seropositives by ELISA type were 70%, 80% and 100% for O, A and Asia1 respectively 38% of cattle were also 3ABC positive, despite no infection history These cattle ranged in age from 1.5-6years Those at the upper end of the age range may have had multiple vaccinations It is known that NSP ELISAs can be positive in animals that have received multiple vaccinations (or unpurified vaccines) The representation of O/A/Asia1 positives between the 3ABC ELISA positive and negative groups was similar Although 19 buffalo were also vaccinated as part of this trial, the vaccine dates and serotypes are unknown, thus limiting the evaluation of the data 34 Vaccine Trials In this trial, 20 cows were vaccinated with O/A vaccine and 76 with O/A/Asia Samples were taken weeks after the 2nd vaccination All were Merial vaccines The overall vaccine response was >88% (up to 100%) for each serotype where administered No cattle were 3ABC positive Ages are not available It would also have been useful to test these animals at 5-6 months post-vaccination, given that in the field there appears to be a reduction in herd immunity over the vaccination coverage period Trial Conclusions Vaccination and sampling in the vaccine trial group was obviously performed under ideal conditions In these conditions vaccine response was excellent, and would be sufficient for herd immunity In HCMC trials the vaccine and sampling were conducted under conditions similar to those seen in the field (for example, a range of animal ages and vaccination-sampling intervals) However, in this environment the vaccination response rate was significantly higher in both the pig group than seen in the provincial results This suggests that properly administered vaccination is effective in pigs and indicates that pigs in the field may not be correctly receiving vaccine or that there are different animals vaccinated and sampled, as appears to be the case on a number of occasions Vaccination response rates in cattle appeared to be better than in most rounds in the provinces Conclusions • In cattle the vaccines not appear to be lasting the full months, with antibody levels regularly good at 1-3 months post vaccination but poor 4-6 months after vaccination • In pigs the vaccination response is generally very poor The best response to vaccination with serotype O was just under 50% However in at least 13 rounds where serotype O vaccine was administered