Appendix 11

Appendix 10

A serosurvey to trace non-structural proteins to FMDV conducted with the sera from Thrace Region of Turkey

Bulut, A.N., Çokçalışkan, C., Alpay, B. FMD Institute (Sap Enstitutusu), PO Box: 714, 06044 Ankara, Turkey

Abstract

This paper summarises the results of 3ABC ELISA measuring antibodies to non-structural proteins (NSP’s) of foot-and-mouth disease (FMD) to differentiate infection from vaccination. For this purpose, in total 1310 sera, which were collected from cattle and small ruminants at day 28 and 60 post vaccination were tested by FMD-3ABC ready to use kit, which has been developed by Bommeli Diagnostics. 16 sera were detected as positive in total of those sera, 11 of which are from cattle and 5 from small ruminants. The ratio of positive samples in total was 1.22% (1.6% bovine and 0,7% ovine sera). After this work, a field active surveillance was conducted in epidemiological units where had been collected those positive sera, it was not defined any clinical infection. It was concluded that those animals detected positive relating to NSP antibody might be infected or sub-clinically infected in previous years and that antibody has been still remained as positive.

Keywords: 3ABC ELISA, non-structural proteins, FMD

1. Introduction

Foot-and-mouth disease (FMD) is highly contagious viral disease, which affects all clovenhoofed animals. It has an economically devastating impact affected countries, since trade barriers, which are imposed where the disease occurs. The detection of antibody to the polyprotein 3ABC of FMDV is the single most reliable indicator of infection (Diago, M de. et. al. 1997) all naive animals seroconvert to this protein following infection. It is known that vaccinated animals, which are exposed to infection, can become persistently infected with FMD without ever showing clinical signs (Mackay, D.K.J., et. al., 1998). Countries, where use vaccination to control FMD outbreaks, it is important to differentiate to antibody against structural protein from non-structural proteins (NSP’s) in order to discriminate virus which is circulated in field either clinical infection or persistence. In the study presented here, sera, which were collected from cattle and small ruminants in Thrace Region of Turkey, were examined by 3 ABC ELISA.

2. Material and Methods 2.1. Test sera

Two different group samples were used as test sera: 1st group: in total 1046 sera in 35 villages and 30 large and small ruminants from each village were selected and sera were collected at day 28 post vaccination. 2nd group: 260 sera in 9 villages but different from group first were selected and the blood sera collected at day 60 post vaccination.

2.2. 3ABC ELISA

Sera were tested by commercial ready to use ELISA kit (Bommeli Diagnostic/Intervet), which was donated EU for this serosurveillance. Test is direct ELISA and very simple to perform. The assays were made as described in the kits instructions.

3. Results

A total of 1310 sera collected from 651 large ruminants and 659 small ruminants (of 1046 sera from day 28. and 264 sera from day 60) were tested by 3ABC-ELISA against 3ABC proteins in Thrace Region including Edirne, Tekirdag, Kirklareli, Istanbul and Canakkale Provinces. 16 sera were detected as positive in total of 1310 sera, which were collected from day 28. (14/1046) and day 60 (2/ 264). The ratio of positive samples in total was 1.22 % (1.6% bovine and 0,7% ovine sera) (Tables 1 and 2 and Fig.1). Sera, which were detected positive, collected from animals that their ages were more than 2 except two cattle one of them from Istanbul and the other from Tekirdağ province, which were between 1 and 2 old.

Taking account each OD value of test serum, positive and negative control, it was estimated mean, maximum and minimum OD, standard deviation and coefficient of variation. Mean OD of positive control sera was 1.107. Detailed data is shown in table 3.

4. Discussion

In this study reported here, it was reflected a statistical percentage in terms of NSP antibody in selected animals from whole animal population of Thrace Region. It was detected very low percentage which was1.22% in total 1310 animals as positive for antibody to FMD NSP. This percentage is almost correlated with previous NSP surveillance. Sera, which were determined as positive, were collected from animals in different provinces and we don’t have any detail about number of applied vaccine. But we have certainly known that those animals have not had any FMD infection during the last one year. Lubroth et. al. (1996) reported that when sera from animals repeatedly vaccinated in the field were examined, occasional positive or doubtful antibody levels to NSP were detected. Likewise Bergman et. al. (1993) found that older, repeatedly vaccinated animals gave occasional positive reaction to one or more bands in the EITB. These and other studies (Mackay, D., et. al.) indicate that the detection of antibodies to FMD NSP is not clearly an indication of previous infection. Therefore age and previous history of seropositive animals must always be taken into account when designing a planning of srosurveillance to trace viral activity. In the light of all these findings, it is thought that those animals detected as positive for antibody to FMD NSP might be: • The rate of positive animals was too low to indicate active virus circulation in the region. • Animals previously infected with FMDV in Anatolian and introduced to Thrace region. • Animals previously infected with FMDV in Thrace region and antibody remained until test time. • False positive.

In this issue, since 14 animals of in total 16 detected as positive were more than two years old, it was supported this possibilities.

Furthermore this possibility was proven by a field active surveillance, which was conducted in epidemiological units where had been collected those positive sera, in conclusion it was not identified any clinical infection.

Perhaps since vaccinated animals have been under the risk to become carriers, this is another complication for interpretation in our results. In any case, the last FMD outbreak was reported in Malkara district, Tekirdag Province in the Thrace Region on 29 June 2001, it has not been luckily occurred any outbreak since this time.

3 ABC ELISA used in this study is very easy to perform, reproducible and specific. Although test instruction was recommended that OD value of positive control should be less than 0.900, we obtained 1.107 as a mean OD value, which was slightly higher absorbance than recommendation. Also not only positive control, at the same time we generally had got high absorbance in whole plate wells. However, we have had an opportunity retest those positive sera and some negative sera, which were tested as negative our study, during the FAO workshop in Sofia, Bulgaria in March 2002, then those 16 sera were scored as positive and rest of 16 sera as negative with low absorbance like recommendation. So we have been sure that those sera have been proven as positive with repeated test. It was concluded that test conditions such as high room temperature and incubator condition might be caused such high absorbance. Data generated from analysis of results are indicated that in generally reproducibility of test seems very plentiful because of the obtained low standard deviation and coefficient of variation which they are assigned less pipetting error and test stability.

In conclusion, this study was important to reveal the rate of animals previously contacted with FMDV in Thrace Region. But a further study is needed to clarify the real and implicit situation of the virus circulation in the region such as probang sampling and virus isolation.

Acknowledgements

ELISA kits, which were used in this study, were donated by the EU. The authors wish to thank members of the Sap Institute particularly Dr. Sinan Aktas and Lab. Technicians, who are Oktay Tezal and Yusuf Demir.

References

Bergman, I.E., et.al. (1993). Diagnosis of persistent aphtovirus infection and its differentiation from vaccination response in cattle by use of enzyme-linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens. Am. J. Vet.Res. 1993; 54(6): 825-31 Diago M. De., et.al. (1997).The non-structural polyprotein 3ABC of FMDV as a diagnostic antigen in ELISA to differentiated infected from vaccinated cattle. Arch. Virol.; 142:2021-33 Lubroth, J., et.al. (1996). Cattle response to FMDV nonstructural proteins as antigens within vaccines produced using different concentrations.Res. Vet.Sci. 59:70-8 Mackay, D.J.K., et. al. (1998). Antibody to the nonstructural proteins of FMDV in vaccinated animals exposed to infection. Vaccine16(5) 446-59

Number of sera Positive % SERA FROM DAY 28. 1046 14 %1.3 SERA FROM DAY 60. 264 2 %0. 7 TOTAL 1310 16 %1.22 Table.1. The results of the 3ABC-ELISA

Provinces Species Positive

Large Rum. Small Rum. Large Rum. Small Rum. No. No. No. % No. %

Edirne 161 169 0 0 1 1.29 Tekirdag 155 155 2 1.29 2 1.29 Kirklareli 165 165 2 1.22 1 0.60

Istanbul 125 125 5 4.0 0 0 Canakkale 45 45 2 4.44 1 2.22

TOTAL 651 659 11 1.67 5 0.76 1310 16 (1.22%)

Table 2: The results of the 3ABC-ELISA: Distribution of results by provinces and animal species

mean Standard deviation Coefficient of variation (%) <%2 %2-5 %5-10 >%10 OD of negative control 0,189 1.2 80 15 3 2 OD of positive control 1,107 1.6 76 20 3 1

OD of test sera - - 51 29 15 5 Table.3: Data generated from analysis of each test plate wells OD value. Standard deviation value was given as mean of cumulative. Coefficients of variation values were shown in different category of as percentage.

690 640 590 540 490 440 390 340 290 240 190 140 90 40 -10

Edirne(320 (310) Tekirdag Kirklareli(330)

Figure 1: The results of the 3ABC-ELISA

Istanbul(250) Canakkale(90) TOTAL(1310)

species large ruminants species small ruminants positive L. ruminants positive small ruminants

Appendix 11

Genetic characterisation of type O and A viruses isolated from Turkey between 2000-2002

Parlak Ü, Aktaş, S., Özyörük, F. FMD Institute, P.K. 714, Ulus, Ankara, Turkey

Introduction

Foot and mouth disease is one of the most important diseases of livestock due to direct and indirect losses. FMD has been endemic in Turkey causing several outbreaks every year. The disease has been controlled by mass vaccination of susceptible animals, restriction of animal and animal product movements, quarantine and other zoosanitary measures. Introductions of new virus strains from neighbouring countries have caused several epidemics in the past. Therefore it is very important to characterise virus strains and to determine the relationship between field viruses and vaccine strains. The objective of this study was to characterise FMD type O and A viruses isolated from Turkey between 2000 and 2002. For this purpose, sequences of approximately 170 nucleotides at the 3’ end of the gene coding for capsid protein 1D were determined.

Material and Methods

Viruses 9 FMD type A virus isolates and 4 type O virus isolates from Turkey between 2000 and 2002 were used in this study (Tables 1 and 2). All viruses were adapted to BHK-21 cells and the clarified infected cell culture supernatants were used for extraction of RNA. Additional sequences used in the study obtained from Genbank.

RNA Extraction, RT-PCR Total RNA was extracted using RNeasy kit (Qiagen) according to the manufacturer’s instructions. RT-PCR reaction was performed as described by Knowles and Samuel (1998). The primers used for RT-PCR were NK61, ARS4 and 1C562 (Table 3). The PCR products were purified using Promega Wizard Preps Kit.

Cycle Sequencing The fmolTM DNA sequencing kit (Promega, U.K.) which utilises the method described by Murray (1989) was used and the manufacturer’s sequencing protocol using an end-labelled primer (NK72) was followed.

Sequence analysis Sequence of 165 nucleotides at the 3’ end of the 1D gene of all samples subjected to phylogenetics analysis. For comparison type A and type O sequences from different regions reported earlier were also included in the study.

Nucleotide sequences were aligned with CLUSTALX (Thompson et al., 1997), Phylogenetic analysis was carried out with the SEQBOOT, DNADIST and FITCH algoritms in the PHYLIP ver 3.5c package. The phylogenetic tree was constructed as described by Felsenstein (1993).