Appendix 19

Figure 4 Protection rates (%) for cattle in (a) Sivas-Ulas; (b) Ankara-Bala; (c) Urfa-Ceylanpinar; (d) Aksaray and (e) Samsun-Gelemen.

(a) n=30 (b) n=30

100

80

60

40

20

0 86.66 90

70

28. day PV O

A

Asia-1 100 90 80 70 60 50 40 30 20 10 0 93

86.66 100

28. day PV

(c) n=100 (d) n=41

100 90 80 70 60 50 40 30 20 10 0 97

98 93

28. day PV O

A

Asia-1 100 90 80 70 60 50 40 30 20 10 0 97.56

90.24

28. day PV 100

(e) n=33

100 90 80 70 60 50 40 30 20 10 0 90

90 100

28. day PV O

A

Asia-1 O

A

Asia-1

O

A

Asia-1

Appendix 19

Discussion paper on guidelines for control of Foot-and-Mouth Disease (FMD) vaccine quality and performance in the field

Simon .J. Barteling1*, Hagai Yadin² & Paul Sutmoller3 1 Consultant Veterinary Vaccines, Amsterdam, The Netherlands, simbar@planet.nl 2 Kimron Veterinary Institute, Beit Dagan, Israel, hagaiy@moag.gov.il 3Animal Health Consultant, The Netherlands, paul.sutmoller@inter.nl.net

Abstract

In many parts of the world with endemic FMD or intrusions from neighboring countries, control of the disease is relying on vaccination of cattle and other susceptible species. In principle, vaccines should be produced and should perform according the directions of the European Pharmacopoeia (E.P.) with a guarantee of safety (correct inactivation plots) and sufficient potency. Also, ideally, vaccines should be purified and not induce antibodies against non-structural proteins. The last will enable the verification of freedom of FMD in vaccinated herds. In particular if the numbers of doses that are needed are difficult to predict, or if purified vaccine is required we recommend purchasing concentrated antigen. The formulation and bottling can be carried out locally. If the purchased antigen is stored frozen at ultra-low temperature (vaccine bank principle), the quantities needed can be produced at any time. Also, it will reduce the price of the vaccine (low transport costs), and guarantee a shelf life of at least 5 years. The veterinary authorities of the receiving country must, independently, control the vaccine efficacy. Therefore, it is important that veterinary services have clear guidelines on how to control and verify the quality of the vaccines, their performance in the field, and vaccine coverage. Israel has a long standing experience in this respect. Tests must be simple and relatively easy to perform and the use of live virus in the test should be avoided (no VNT!). The LPB-ELISA (with inactivated antigen) offers the internationally accepted test to verify these conditions. The World Reference Laboratory and the National Control Laboratories in South America consider a LPB titer of 1:100 (2 logs) as protective and – with some variation between countries - in South America 12 out of 16 cattle must reach that level shortly (6 weeks) after vaccination. Such titers are considered to be protective against (the heavy) challenge under laboratory conditions. The Russian FMD Institute FGI-ARRIAH considers 1:40 (1.6 logs) as sufficient protective. Cattle with these levels are probably protected under field conditions. This paper discusses the various aspects of FMD vaccine control and performance in the field with emphasis on vaccine control in Israel.

Introduction

In many countries there is a continuous threat from neighboring countries or from foci within the country where vaccination is not well possible (e.g. wild-life reservoirs). In particular for areas with a high cattle density FMD will spread rapidly. Even though mortality can be high in young animals, in adult cattle it usually is not more than 5%. However, recovered cattle may suffer serious sequels, such as unthrift ness, mastitis, abortion, infertility, and chronic heat intolerance syndrome (HIS, also known as “panters”). Also, in areas where oxen are used to plough and harvest of crops FMD outbreaks can have serious socio-economic consequences because recovered oxen often loose their ability for hard labor. Dependent on the local husbandry systems FMD can –like in South America and in Europe in the past – be controlled by the vaccination of cattle only. However, in other areas like the Middle East the roaming herds of sheep and goats and the intensive marketing system of these animals enables FMD virus to remain circulating in those herds. Under such circumstances vaccination of sheep and goats is necessary as well. When pig herds are endangered, the boars and sows could be vaccinated (with oil emulsion vaccines), however, around outbreaks all pigs must be vaccinated. Because oil emulsion vaccines induce, in general, a longer lasting immunity, and can be used for cattle, sheep, and pigs, this type of vaccine is recommended. For successful control it is essential that the vaccine is of good quality and contains the correct vaccine strains that cover the local field strains. Logistics of transport (cold chain) must guarantee that the quality of the vaccine is maintained until used in the field. To reach sufficient herd protection and to stop spreading of disease, it is essential that more then 80% of the animals be vaccinated. Because producers, whether private or governmental, want to market their products, independent control of the quality of the vaccines is required. Also, duration of immunity and herd coverage are important elements to make sure that FMD cannot spread and is effectively controlled. Here we will give guidelines to perform all steps in a proper way.

Guidelines for control of FMD by vaccination 1. General, zonal, or incidental vaccination

1.1. Situations where general vaccination is recommended:

• Frequent occurrence of outbreaks all over the country • Continuous and frequent threat from foci at the borders or within the country (e.g. wild-life reservoirs); • Sufficient financial means to enable general vaccination programs; • Low incidence but large (valuable) cattle population; • Good infrastructure, with consistent FMD surveillance ; • In the context of a regional eradication program e.g. like in South America.

1.2. Situations where incidental or zonal vaccination is recommended:

• Limited financial means; • Incidental threat from outside, buffer zone vaccination at the borders to prevent outbreaks inside the country; • Consistent surveillance systems all over the country, a pen-side test may contribute in rapid diagnosis (Kitching, 2004); • Good contingency plans (including vaccine bank) for ring-vaccination around outbreaks available.

2. Species that must be vaccinated

2.1. All susceptible species:

• In the face of an outbreak (s); • When small ruminants are regularly brought to markets or when they are not pastured at fixed places (like in the Middle East) and when pigs are kept in large holdings. In the latter case breeding stock (boars and sews) must be vaccinated; However, in the face of outbreaks vaccination of all pigs is required. Depending on the type of oil adjuvant, local reactions may occur and cause problems up to one month after vaccination. Suckling piglets can be vaccinated with a (double) oil emulsion vaccine intra-peritoneally to avoid local lesions at time of slaughter (Augé de Melho et al. 1987 and 1988). Double emulsions, in general, cause little local reaction.

2.2. Cattle only if:

• There is a small population of small ruminants; • Small ruminants housed or pastured (mainly) at fixed places. E.g. in Uruguay only cattle are vaccinated. They graze in open pastures mixed with sheep and sometimes pigs. In general they live there until slaughter.

3. Vaccine strains needed

Effective vaccination campaigns can be carried out with vaccines containing standard vaccine strains. Only occasionally has the incorporation of an emerging field strain been necessary. In general, such emerging strains will only be identified if other reasons for vaccination failure can be excluded. Breakthroughs in vaccinated livestock can often be controlled by re-vaccination with a potent vaccine containing standard antigens. The difficulties of adapting field isolates to the conditions of vaccine production and all the required testing must not be underestimated e.g. with regard to industrial growth of the virus in cell systems, proper inactivation and purification, antigen stability and shelf life of vaccines.

Vaccine strains incorporated in the vaccine can be selected on the basis of:

• Advice of local laboratory (if available and capable); • Advice of WRL; • Advice of (potential) producers;

• Strains selected in the context of regional co-operation (favored situation).

4. Purchasing vaccine or concentrated antigen?

Conditions and arguments for buying concentrated antigen:

• Ultra-low temperature (ULT) storage facilities (+ back-up power unit) • Facilities for formulation and bottling available (e.g. at local laboratory or pharmaceutical plant) • Flexibility, counts in particular when vaccines are incidentally required; • Possibility to increase the payload of the vaccines for rapid control of outbreaks; • Economical.

Note: Purchasing of commercial completely formulated vaccine has the advantage that the producer is responsible for failures. If vaccine is formulated from concentrated antigen that is bought elsewhere, the local laboratory that carries out the formulation and bottling of the vaccine becomes responsible for eventual failures or other problems.

5. Type of adjuvant:

• Aqueous (Al(OH)3 – saponin) for ruminants only (not for pigs); • Oil emulsion for all species.

6. Tender:

• No tender when there is a fixed relationship with a producer (e.g. a national laboratory); • Tender for commercial producers (at least 3), preferably working according to (internationally approved and certified) Good Manufacturing Practice (GMP). If a company or laboratory claims to work according to a Quality-based Operational System (Q-BOS) – not all requirements for GMP (e.g. for equipment) fulfilled, however, fixed procedures and controls and quality of product guaranteed (Barteling, 2004) – this must be verified by an audit (e.g. once every 3 year) if possible by an independent inspector/specialist in the field of (FMD) vaccine production; • Judgment of obtained data; • Selection of producer (price per dose).

7. Testing / verifying the quality of vaccine batches to be purchased:

“You get what you inspect not what you expect” is a slogan that certainly holds true in the field of veterinary vaccines. Also, it is essential to make sure that the vaccine can do the job. If this testing is not incorporated one is always behind the facts. This testing is necessary for successful control of FMD as the history from South America (and other places) has learnt us. If the vaccine is of insufficient quality, there might be incidental cases of FMD in the vaccinated population and the farmers will loose their believing in vaccination and will become less cooperative. If sufficient quality of the vaccine is verified only by monitoring the campaign it may become a frustrating business in case of poor quality vaccine. One can only hope for the best and that next time the producer will provide better quality, however, one has no guarantee. Also, negative results in the survey may be explained by the producer by failures in the campaign or cold-chain and breakthroughs by strain differences.

• Checking safety test results of the antigen batches that are incorporated in the vaccine batch (es), as presented by the producer: correct inactivation plots, and in vitro (cells) safety tests. • Testing of overall protein concentration of the vaccine or of the concentrated antigen (must be in accordance with producers purity estimations/guarantee). This can be carried out by using a Lowry test, or a commercial test kit (e.g. from Pierce). • Testing for absence of non-structural proteins (NSP) by ELISA (if required). In principle, the a-NSP ELISA can be used in a direct mode for testing concentrated (purified) antigen for the presence of traces of NSP. It is possible that this test can also be applied for testing vaccines directly.

• Anyhow animals that have repeatedly (up to 5 times) been vaccinated with vaccine from the same producer must remain negative (if absence of NSP is claimed by the producer). • Testing of potency: Vaccinate 16 “clean” calves (no significant maternal antibody levels). In 3 (aqueous) or 6 weeks (oil) time 13 out of 16 must reach antibody levels (LPB-ELISA,

Hamblin et al., 1987) of 2.0 logs (to guarantee 1.6 later on after vaccination).

8. Monitoring quality upon arrival: see 7

9. Storage and cold-chain facilities

Upon arrival the vaccine should be stored in a very reliable cool house with technical backup. Also, there must be cold-chain provisions for transportation of the vaccine to the local veterinary posts and for the distribution of the vaccine in the field.

10. Vaccination campaigns

Experience from the Middle East and from Europe in the past learned that FMD outbreaks occur (or start) in the winter time February/March. Therefore, it is recommended that the annual vaccination campaign (s) should be carried out at least 3 month in advance which means October / November and vaccination of new born young calves (3 month old) in January. By this schedule one may face the FMD period with completely vaccinated livestock.

11. Monitoring vaccination campaign(s)

Monitoring of vaccination campaigns is very dependent on local facilities: presence of sufficiently large farms (with at least 20 clean calves in one age group) located in different area's of the country (to make it unlikely that these farms become all involved in outbreaks) and laboratories (+staff) and logistics and other infrastructural requirements to do all the tests. The same counts for the sheep and/or goats. In general, it will be difficult to find such farms. In fact on such farms veterinary services / laboratory must take over the responsibility of all FMD vaccination on the farm. Farmers also want guarantees in case anything happens. The sera of the animals from the test can also be used to monitor protection against outbreak strains. For an overall picture one can take at random samples at slaughterhouses that are representative for the different districts. If monthly a total of 1.000 (?) sera of slaughtered animals are tested one may get a fair impression of overall herd immunity throughout the year. After all, every animal will end up in the slaughterhouse.

Vaccination schedule: The first 3 vaccinations (of calves) every 6 months, thereafter once a year.

Verifying endurance of immunity: Not less than 3 groups of 20 calves not previously vaccinated, on farms located in different areas of the country, will be followed during 6 months after vaccination. After that they will be revaccinated and followed for another 6 months. Serum samples will be taken every month.

Evaluation of herd immunity (can be carried out on the same farms as mentioned under 2):

• Method: Testing of serum samples by LPB-ELISA. Titer of >1.6 (1:40) can be considered protective against a first take. • When? Testing should preferably be carried out shortly before and 3 – 6 weeks after vaccination campaigns. • What?

Testing of serum samples from slaughterhouses; Testing of serum samples from “controlled farms (see 2) and, if possible, randomly selected (representative) dairy, beef, and sheep (if applicable) farms; Districts represented in the testing; • Requirement: at least 80% of the animals within herds must be protected throughout the year.

12. Surveillance for sub-clinical infections in vaccinated herds by a-NSP antibodies

• Routinely, e.g. on slaughterhouse sera; • Around outbreaks.

13. Annual evaluation and reporting

• Evaluation of monitoring and surveillance systems; • Evaluation of the results of vaccination campaigns; • Reporting to: o The ministry; o The farmers organization (s); o The press; o The regional FMD organization (if applicable); o The FAO / OIE and/or sponsoring organizations like the EU

Evaluation and annual planning

On the basis of the results of the past year and the current situation (outbreaks in surrounding countries (new field strains etc.) the FMD situation must be evaluated as well as current monitoring and surveillance systems. If necessary these systems must be improved. In accordance, the annual vaccination campaigns must be planned.

Discussion

The guidelines above are a step by step approach of what is – in our views – required in order to control FMD by vaccination in an endemic area. Israel is a good example of how a consistent vaccination policy can control FMD in an environment of continuous external disease pressure and their procedures stood model for the above guidelines.

Situations in many countries with a continuous threat of FMD may differ, however, with those in Israel:

- Not all countries have highly valuable livestock like in Israel, with an highly organized veterinary infrastructure. - In many countries farmers cannot afford to pay the world market prizes for FMD vaccine. - For above or other reasons veterinary services may be bound to the use of vaccines that are locally produced often by a national laboratory of the country itself or of a neighboring country. - Vaccine producers in developing countries often cannot afford or don’t have access to the high standards of equipment or of building facilities that are internationally required for

GMP accreditation. However, their procedures might be according to GMP with fixed production steps in a closed system described in standard operating procedures and adequate controls of each production step (e.g. sterility). Such a Quality-based operational system carried out by well-trained operators can guarantee the production of quality vaccines as well. If, the production unit is large enough the vaccine can probably be produced for competitive prizes. - An alternative can be to buy concentrated (purified) antigen from the big international producers against competitive prizes. The formulation and bottling being carried out locally.

Tenders can be made per virus strain. E.g. O1 Campos, a vaccine strain with a proven broad protection (e.g. against O1 Taiwan) can be bought from one of the South American producers working under a GMP regime while other strains are bought elsewhere.

Whatever the situation, history has learned that, in general, the quality of the vaccines must be tested by a laboratory or institution that is independent from the producer (or formulator) of the vaccine. In addition, reporting of results of tests (like in South America) and evaluation of the performance in the field (+ reporting) are other elements that will stimulate producers to make the quality of the vaccines to the highest standards.

References

Augé de Mello, P., Gomes, I., Alonso Fernandez, A. & Mascarenhas, J.C. 1978. Foot-and-mouth disease oil-adjuvanted vaccine for pigs. Intraperitoneal vaccination of young pigs with double emulsion vaccine. Bol. Centro Panamericano fiebre Aphtosa 31-32: 21-27.

Augé de Mello, P. 1979. Reflections on the prevention of foot-and-mouth disease in swine. Bol. Centro Panamericano fiebre Aphtosa 39-36: 59-61.

Barteling, S.J. 2004. Modern inactivated Foot-and-Mouth Disease (FMD) vaccines: Historical background and key elements in production and use. In: “Foot-and –Mouth Disease. Current perspectives”, Sobrino, F. and Domingo, E. editors, Horizon Bioscience, Wymondham, Norfolk, U.K. p.305-334

Hamblin, C., Kitching, R.P., Donaldson, A.I., Growther J.R. & Barnett, I.T. 1987. Enzymelinked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouthdisease virus. III. Evaluation of antibodies after infection and vaccination. Epidemiol. Infect. 99, 733-744

Kitching, R. P. 2004. Diagnosis and control of foot-and-mouth Disease In: : “Foot-and – Mouth Disease. Current perspectives”, Sobrino, F. and Domingo, E. editors, Horizon Bioscience, Wymondham, Norfolk, U.K. p.411-424.

Shimshoni, A. 2002. Foot-and-mouth disease: The Israeli approach. http://www.israel-embassy.org.uk/web/pages/fmd isr.htm

ADDENDUM

A model of technical specifications for FMD vaccines for a tender (as applied in Israel)

A. Requirements for Trivalent FMD vaccine for cattle

1. Antigen composition: trivalent vaccine should be composed of the following serotypes and isolates: 1.1 O1 Manisa, + O1 Isr. 2/85 (Geshur),+ O1 3039. 1.2 A22 Iran 87, +A turkey1/98 or equivalent. 1.3 Asia1 Isr. 3/89 (Shamir). 2. Antigen purification. It is required that the antigen will be purified for 90% and higher of (non-FMD) proteins. The purification process should use PEG or ultra-filtration (or both). 3. Adjuvant: the adjuvant should be double oil emulsion (w/o/w) Isa 206 or equivalent. 4. Safety. 4.1. The producer /Supplier should present the inactivation curves of The antigens presented in the vaccine. 4.2. The vaccine should be accompanied with results of safety control In cell culture and cattle. 4.3. The producer /Supplier should present evidence that the vaccine does not cause any allergic reactions in cattle and pregnant cows, even after repeated vaccinations. 5. Efficacy. 5.1. The vaccine documents should contain the results of efficacy test in cattle. The vaccine should contain not less then 6 PD50 per type per dose.

5.2. The antigen load expressed as 146S per type per dose should be mentioned in the vaccine documents. 5.3 The vaccine documents should contain the results of virus load before inactivation expressed as TCID50 per 1 ml. 6. Expiry: expiry date should be 18 month started at the last day of production. 7. Doses for cattle: 2 ml for trivalent cattle vaccine 8. Packaging: 100 doses per bottle. 9. Supply date will be fixed. 10. The label on each bottle should contain: producer name, place of production, batch number, quantity of dose, volume vaccine in ml per bottle, production date and expiry date.

11. Responsibility: the producer/supplier of the vaccine will be responsible for any harm caused by neglecting the indicated requirements.

B. Requirements for Monovalent O1 FMD vaccine for Sheep and goats.

1. Antigen composition. The vaccine (against sero-type O) should be composed of the following vaccine strains:

O1 Manisa, + O1 Isr. 2/85 (Geshur),+ O1 3039.

2. Antigen purification. It is required that the antigen will be purified for 90% and higher of non-FMD proteins. The purification should be carried out by PEG-precipitation and/or by ultrafiltration methods.

3. Adjuvant. The adjuvant should be double oil emulsion (w/o/w) Isa 206 or equivalent.

4. Safety. 4.1. The producer /Supplier should present the inactivation curves of The antigens presented in the vaccine. 4.2. The vaccine should be accompanied with results of safety control In cell culture and sheep. 4.3. The producer /Supplier should present evidence that the vaccine does not cause any allergic reactions in sheep and pregnant sheep, even after repeated vaccinations. 5. Efficacy. 5.1. The vaccine documents should contain the results of efficacy test in sheep, it should contain not less then 6 PD50 for sheep and goat per dose.

5.2. The results of antigen load expressed as 146S per dose should be added to the vaccine documents. 5.4 The vaccine documents should contain the results of virus load before inactivation expressed as TCID50 per 1 ml. 6. Expiry Expiry date should be 18 month started at the last day of production.

7. Dose Doses, for sheep/goat: 1ml for monovalent sheep/goat vaccine. 12. Packaging: 100 doses per bottle (100ml). 13. Supplying date will be fixed. 14. The label on each bottle should contain: producer name, place of production, quantity of dose, volume of vaccine in ml per bottle, batch number, production date and expiry date. 15. Responsibility; The producer/supplier of the vaccine will be responsible for any harm caused by neglecting the indicated requirements. The producer/supplier of the vaccine will be responsible for any harm caused by neglecting the indicated requirements.