Appendix 16

Virus Inactivation Kinetics Appendix 15

Soren Alexandersen Danish Institute for Food and Veterinary Research, Department of Virology, Lindholm, DK-4771 Kalvehave, Denmark

At the session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease in Gerzensee, Berne, Switzerland in September 2003 a review of methods for describing the effect of temperature and time upon virus survival in products was presented (Have, 2003). The Research Group recommended that “specific studies on heat inactivation should be designed to support further risk assessments for those identified as “high” risk commodities. These studies should make use of existing experimental data on D and Z-values or involve further experiments to fill any gaps” and that “the available data on inactivation of FMDV in milk and milk products should be reviewed in the light of current international trade standards. If necessary, additional studies on inactivation by heat treatment or lowering pH should be carried out”. It was decided to develop study plans for assessing D-values and Z-values for heat treatment of milk and pork from FMD-infected animals and to consult the industry in regard to the planning and funding of such studies.

As mentioned by Have (2003) the scientific literature regarding inactivation kinetics of FMDV from relevant sources and under relevant conditions is fragmented and sporadic and the difficulties of making quantitative assessments from various data sources on virus inactivation e.g. in the environment has been reviewed by Bartley et al (2002). As explained (Have, 2003); the effect of heating is determined by a combination of time and temperature and the inactivation kinetics are often assumed to be first-order although this is not always the case. The decimal reduction time DT is the time needed to reduce the viable population by 90% at the temperature T. Semi-log plots of Dvalues against temperature often yield linear relationships, from which z-values can be calculated as the number of degrees temperature required to change D by one log unit. The z-value is often considered to be constant for a given strain of microorganism in a given product. Heat treatment includes a heating phase and a cooling phase and to account for the combined effect during heating and cooling, the temperature/time relationship data can be used to calculate lethal rates over the entire process and integrating into a cumulated lethal effect, expressed relative to a standard treatment at a chosen reference temperature and taking advantage of knowing the value of z (Peleg, 2003).

In regard to FMDV, clearly also other conditions, apart from the temperature, such as product type, pH, ionic strength and a number of other factors are also of importance for the kinetics of inactivation.

Conclusions

• Inactivation studies of FMDV need to be done in a systematic way employing kinetic data under relevant conditions.

Recommendations

• The recommendations of the 2003 meeting need to be taken immediately forward. It is suggested to make a small working group with the responsibility of urgently drawing up a study plan and to take this plan to the industry for consultation in regard to the practical planning and funding of such studies.

References

Bartley, L. M., Donnelly, C.A. & Anderson, R.M. 2002. Review of foot-and-mouth disease virus survival in animal excretions and on fomites. Vet. Rec. 151:667-669. Have, P. An assessment of guidelines for treatment of meat from a FMD vaccination zone. Session of the Research Group of the Standing Technical Committee, European Commission for the Control of Foot-and-Mouth Disease 2003: 149-152. Peleg, M. 2003. Calculation of the non-isothermal inactivation patterns of microbes having sigmoidal isothermal semi-logarithmic survival curves. Crit Rev. Food Sci. Nutr. 43:645-658

Appendix 16

Screening for FMD virus in vaccinated herds affected by field infection

Hagai Yadin1*, Dalia Chai1, Jacob Brener1, Zamir Oved2, Yuval Hadany2, Alexandra Kusak2 . 1Kimron Veterinary Institute, Bet Dagan 50250, Israel. 2 Regional Veterinary Services, Bet Dagan 50250, Israel.

Introduction

Israel is located in the Middle East in an FMD endemic area and since August 1999 had been free of FMD outbreaks. In January 2004 the country faced numerous FMD outbreaks, eight farms were affected; two dairy and fattening herds, four calf feedlots, one sheep flock and one farm keeping game animals. Although morbidity approached 30% in the feedlots, there were no clinically affected animals in the dairy operations. The objective of this study was to estimate the prevalence of subclinical infection in vaccinated herds by identifying NSP seropositive animals.

Material and methods:

Five FMD-affected farms were blood sampled between 30 and 80 days after the outbreak had occurred on each premises; two of these were dairy farms with associated feedlots, two were standalone feedlot operations and one was a sheep-rearing enterprise (Table 1). As controls, animals were also blood-sampled in three dairy farms, one feedlot and one sheep farm, all of which were unaffected. Commercial ELISA kits were used for evaluation of the presence of NSP antibodies; a direct ELISA for Bovine sera (Svanova) and an indirect ELISA for both ovine and bovine sera (Cedidiagnostics).

Table 1 Sampled animals in feedlots, dairy farms and sheep farms

Date of outbreak FARM Enterprise Type Morbidity Last FMD Vaccn

19/01/04 2. Givati I Feedlot 65/360 May 2003 Dairy (n = 331) 23/01/04 3. Givati II Feedlot 37/365 May 2003 26/01/04 5. Ein Hashofet Feedlot 80/650 April 2003 Dairy (n = 550) 11/02/04 7. Beit Zarzir Sheep farm 5/80 No vaccination 18/03/04 8. Arab Zbeidat Feedlot 4/80 June 2003

Controls: 1 x Feedlot, n =110 calves (7 groups on 3 farms) 3 x Dairy farms, n = 398 cows (Elifaz, n=319; K. Warburg I, n=43; K. Warburg II, n=36) 1 x sheep farm, n =131 dairy sheep (Vulcany center)

Summarised interpretation of the presence of NSP Antibody in serum

NSP + Disease - virus multiplication - (± vaccination). NSP + Subclinical Disease - virus multiplication (± vaccination) NSP + No disease - No virus multiplication - multiple vaccination with unpurified vaccine. NSP - No disease - No virus multiplication - efficient vaccination.

Results

Although the two dairy farms were free of clinical disease, calves in the adjacent feedlots developed typical signs of FMD. On those dairy farms, 27% of the animals were sampled (239 of 881 animals; 132 of 456 cows and 107 of 425 calves and heifers) and eight of the sampled animals were NSP positive; seven were cows aged more than three years which had been vaccinated between five and eight times and one was a heifer which had received three vaccinations. At the four feedlots, 194 calves were sampled (from a total population of 1400) and 137 of these were NSP seropositive. Ninety-two of the seropositive calves were between six and ten months of age and were between four and seven months after receiving a single vaccination. In the unvaccinated flock of 80 sheep, four clinical cases were seen and 60 of the 69 sheep that were sampled were NSP seropositive. From multiply-vaccinated but clinically-unaffected dairy herds, only one of the 398 cows that were tested was NSP seropositive whilst only a single seropositive sheep was detected in a control flock of 131 dairy sheep.

Table 2: Screening for NSP antibody in dairy herds

(i) FMD vaccinated dairy herds after outbreak Farm No. 2

Age Morbidity Days postoubreak NSP Ab- Svanova NSP AbCeditest

7m-5y 0/331 80 2/119 2/119 <6m 0/135 70 1/58 >6m 0/196 31 1/61

Farm No. 5

Age Morbidity Days postoubreak NSP Ab- Svanova NSP AbCeditest 2m-5y 0/550 80 6/120 6/120 <6m 0/290 70 0/49 >6m 0/260 31 6/71

(ii) FMD-vaccinated control dairy herds (No outbreak)

Farm No. Age NSP Ab- Svanova I 43 2y-5y 0/43 II 36 2y-5y 0/36 III 319 2y-5y 0/319

(iii) FMD vaccinated feedlots after outbreak Farm No. 2

Age Morbidity Days postoubreak NSP Ab- Svanova 2m-10m 65/360 31,56 38/128 <6m 1/120 56 4/61 >6m 64/240 31 34/67

Farm No. 5

Age Morbidity Days postoubreak NSP Ab- Svanova < 7m 4/33 56 9/29 > 8m 37/41 56 32/37

Farm FMD Enterprise Age Morbidity Days postoubreak NSP Ab- Svanova NSP AbCeditest

3 YES Feedlot 6 – 7m 35/365 30 10/44 9/44 8 YES Feedlot 7 - 8m 4/80 28 32/37

1 NO Feedlot (Control) 4.5m 0 0/110

(iv) Screening in sheep flocks after outbreak (FMD vaccination status known or not?) vaccinated

Farm FMD Enterprise Age Morbidity post-outbreak NSP Ab- Ceditest 5 YES sheep 6 – 7 m 5/80 31 days 61/64 IV NO sheep (control) 2 – 85 m 0/131 56 days 1/131

Discussion

Dairy farms were well protected by vaccination against clinical as well as subclinical infection and very few animals were NSP seropositive. It seems that even the young calves before first vaccination are well protected by colostral antibody. However, feedlots with imported calves from FMD free-countries were susceptible to infection as early as four month after a single dose of vaccine had been administered. Unvaccinated lambs may be considered as potential vectors of FMD virus as they may spread the infection without showing clinical signs of disease.