Vaccine 24 (2006) 9971008

Analysis of the immune response to FMDV structural and non-structural proteins in cattle in Argentina by the combined use of liquid phase and 3ABC-ELISA tests
Blanca Robiolo a , Cristina Seki a , Norberto Fondevilla b , Pablo Grigera a , Eduardo Scodeller c , Osvaldo Periolo a , Jose La Torre a , Nora Mattion a,
c a Centro de Virologa Animal (CEVAN-CONICET), Serrano 669, 1414 Buenos Aires, Argentina Centro de Investigaci n de Ciencias Veterinarias y Agron micas (CICVyA-INTA), CC77, 1708 Mor n, Argentina o o o Instituto de Medicina y Biologa Experimental de Cuyo (IMBECU-CONICET), Av. Ruiz Leal s/n, 5500 Mendoza, Argentina b

Received 17 March 2005; accepted 3 August 2005 Available online 7 September 2005

Abstract The successful sanitary campaign implemented to control the 20002002 outbreaks of foot-and-mouth disease virus (FMDV) in Argentina was greatly assisted by the combination of an ELISA test (3ABC-ELISA) that detects antibodies directed against FMDV viral non-structural proteins (NSPs) and a liquid phase blocking competitive ELISA (lpELISA) for the detection of antibodies against the viral structural proteins (SPs). The combined use of these two assays in large-scale analysis of eld samples allowed for a clear differentiation between infected and uninfected animals, with high specicity and sensitivity, regardless of the animals vaccination status. In order to validate the application in indirect vaccine potency assays and assessment of vaccination efciency, a preliminary correlation between serological response and protection from challenge with O1/Campos and A/Arg/01 FMD virus strains was established with data derived from commercial vaccine series challenge trials. Determination of antibodies to NSPs in vaccinated and revaccinated animals proved helpful in the analysis of vaccine purity. A review and discussion of the epidemiological status of cattle herds and real time monitoring of FMD in Argentina using these assays before, during and after the outbreaks is presented. 2005 Elsevier Ltd. All rights reserved.
Keywords: Foot-and-mouth disease virus; 3ABC-ELISA; Serology; Vaccination; Surveillance

1. Introduction Argentina has emerged from an exceptionally difcult sanitary situation caused by a large number of FMDV outbreaks. The rst outbreak took place in July 2000, followed by many others (2562 total outbreaks) that continued through to January 2002, when the last virus isolation was reported. FMDV serotypes O and A were responsible for all the cases, but the main sanitary problems were caused by serotype A strains belonging to two different lineages, designated as A/Argentina/2000 (A/Arg/00) and

Corresponding author. Tel.: +54 11 4773 0505; fax: +54 11 4773 0505. E-mail address: mattion@bertel.com.ar (N. Mattion).

A/Argentina/2001 (A/Arg/01). The kinetics of the epidemic and the control measures taken, as well as the molecular and antigenic characterization of the FMDV strains involved, have been previously described [1,2]. A major difculty confronted by sanitary authorities dealing with FMDV outbreaks in countries like Argentina where massive vaccination is applied, is how to differentiate infected animals from those that remained uninfected. Identication of the former group of animals is extremely important since they may become virus carriers posing a major risk as reservoirs of FMDV variants with potential to originate new outbreaks [3]. Infected animals, whether symptomatic or not, usually elicit antibodies (Abs) to both the FMDV structural cap-

0264-410X/$ see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.08.071

998

B. Robiolo et al. / Vaccine 24 (2006) 9971008

sid proteins (SPs) and non-structural proteins (NSPs). Since NSPs are exclusively synthesized during the FMDV replication cycle, the presence of anti NSP Abs in animal sera is a clear indication of either past or present viral activity. Non-infected vaccinated animals, on the other hand, should only elicit Abs to SPs, the major viral immunogens present in commercial vaccines. However, FMDV vaccines are generally made from partially puried inactivated virions grown in cell cultures and could still contain trace amounts of viral NSPs, particularly the viral polymerase 3D [4], which may be immunogenic in the presence of strong adjuvants. The use of anti-NSPs Ab titres as a criterion to detect past viral activity in vaccinated animals requires therefore a careful selection of the NSP antigens that are to be used in a particular immunological test. Several FMDV NSPs have been tested as antigens in the last decade for this purpose, and in general, there is consensus that polypeptide 3ABC [57] is the most appropriate antigen because of its high immunogenicity and its relative low concentration in infected cell lysates. NS FMDV polypeptides 2C [8], 3A, 3B, 3AB, and 3D [9] have been also used by different investigators with interesting results using either Western blot (EITB) [5,10] or ELISA formats [5,11,12]. However, there is still no international reference test (gold standard) for NSP detection. This paper describes a 3ABC-ELISA for detection of NSPs in sera from vaccinated or non vaccinated animals and its combined use in conjunction with an lpELISA in order to simultaneously monitor the levels of protection (herd immunity) and virus circulation (infection) in large populations of a susceptible animal. The lpELISA was originally standardised as an indirect potency test for FMDV strains A/Arg/79, A/Arg/87, O1/Caseros, and C3/Arg/85, which were the strains present in the Argentine vaccines before the year 1999 [2,13]. This test was successfully used for ofcial vaccine potency control from 1995 to 1999. Approximately 7000 serum samples from animals vaccinated and challenged were tested for Abs against each of the four viruses with this assay. This is equivalent to approximately 400 series of commercial tetravalent vaccine (450 million doses). After the year 2000 outbreaks, the emergency vaccines used in Argentina were at rst composed of FMDV strains A24/Cruzeiro, A/Arg/00 and O1/Campos. Later, A/Arg/01 virus strain was also incorporated into the vaccines [2]. The potency of the emergency vaccines was adjusted in virus challenge trials. For indirect potency control, a correlation between serological response and protection from virus challenge must be established for each new FMDV strain, as recommended by the OIE. In the case of A24/Cruzeiro and O1/Campos regional strains, the correlations established by Panaftosa (PAHO, WHO, Brazil) were originally used, although they are currently under validation. Correlation data between serology and protection from podal generalisation (PPG) after challenge is described in this work for A/Arg/01 and O1/Campos virus strains.

2. Materials and methods 2.1. Cloning and expression of FMDV 3ABC polypeptide The genomic fragment encoding the 3ABC polypeptide of FMDV strain O1/Campos was cloned into the IPTG inducible plasmid pET as previously described [14]. Briey, the fragment containing nucleotides 50816391 was amplied from total genomic RNA and cloned into the NcoI and BamHI sites of plasmid pET30 (Novagen) under the control of the phage T7 promoter (pET-3ABC plasmid). Self-processing of expressed p3ABC was largely prevented by site-directed mutagenesis of a single cysteine residue at position 383 (Cys387Gly substitution), which renders inactive the proteolytic activity of FMDV 3C [15]. Site-directed mutagenesis of p3ABC (p3ABCm) was engineered by overlap PCR extension [16]. BL21(DE3) competent cells were transformed with the pET-3ABCm plasmid and recombinant polypeptide (r3ABCm) expression was induced with 1 mM IPTG at 37 C [14]. For use in the ELISA tests, inclusion bodies were puried, and the r3ABCm polypeptide was solubilized in 7 M urea and titrated. 2.2. Monoclonal antibodies Monoclonal Abs (MAbs) against the FMDV r3ABCm polypeptide were generated using standard protocols [17,18]. MAb 3H7 (IgG1 isotype, directed to an epitope on the FMDV 3A polypeptide), was selected [14]. The preparation of MAbs against SPs of different FMDV serotypes was previously reported [1]. 2.3. Indirect ELISA for the detection of anti FMDV 3ABC antibodies Ninety-six at bottom well plates (Nunc Maxisorb) were rst coated with the MAb 3H7 diluted in carbonate/bicarbonate buffer, pH 9.6, in a 50 l volume and incubated overnight at 4 C. After four washes with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS-T), 50 l of 1:2000 partially puried r3ABCm in diluting buffer (PBS-T containing 10% equine serum, PBS-T/E) were added to alternate columns of wells, incubated for 2 h at 37 C, and washed four times with PBS-T. The assay was used as a single dilution test. Serum samples diluted 1:40 in PBS-T/E were added in duplicate to two different wells, one coated with the antigen (Ag+) and the other without antigen (Ag) and incubated for 30 min at 37 C. After four washings, 50 l of horseradish peroxidase-conjugated goat anti-bovine serum (Jackson ImmunoResearch Laboratories Inc., Pennsylvania) diluted in PBS-T/E was added to the wells and incubated for 30 min at 37 C. Finally, colour was developed using the chromogen/substrate mixture ABTS/H2 O2 (ABTS: 2,2 -azinobis [3-ethylbemziazoline-6-sulfonic acid]), and stopped after 30 min by the addition of 50 l of 0.2% NaF. The absorbance in each well was read at 415 nm.

B. Robiolo et al. / Vaccine 24 (2006) 9971008

999

The net OD for each serum tested was obtained by subtracting the corresponding OD values obtained in the wells without antigen from the values obtained in the wells with antigen (net ODsample = ODAg+ ODAg ). An antiserum from an FMD convalescent animal was used as a positive control (C+). The negative control (C) was a pool of sera obtained from animals belonging to the Argentine FMDV-free without vaccination area. The nal result for each sample was expressed as a percentage of the positive control (PP) using the formula: PP = net ODsample /net ODC+ 100. For the test system to be valid, the net OD of the C+ must be higher than 0.50 and at least three times higher than the net OD of the C, and the coefcient of variation (CV) intra-assay should be 10%. Three pools of sera with high, medium and low titres of anti NSP Abs, as well as the pool of negative sera were used as inter-assay standards. The reference sera were used in duplicates and no more than 2 standard deviations (S.D.) were admitted. To assess repeatability, two positive (weak and strong) and one negative sera were tested in 35 different assays performed at different days. Limits of variation were established, as well as the mean and the S.D. For specicity and sensitivity studies, panels of negative and positive sera were used. Negative sera (1626) were derived from FMD-free regions without vaccination (1057) and from vaccinated animals of similar origin used in vaccine potency assays (569) and bled at 60 days post vaccination. One hundred and forty-six (146) positive sera were obtained from animals experimentally infected by contact with two calves infected by the intradermolingual route with FMDV O1/Campos strain. Serial bleedings were performed from day 0 to day 570, but only bleedings from day 19 to 233 were considered to determine the assay sensitivity. The sera were analysed for the presence of anti SP and NSP Abs. The sensitivity of the 3ABC-ELISA was preliminary compared with four other NS tests, designated ELISA A, B, C and D. Serology was performed on 73 (A and B) or 60 (C and D) positive serum samples. The four tests were: ELISA A, Ceditest FMDV-NS, Cedi Diagnostics BV, Lelystad, The Netherlands; ELISA B, UBI FMDV NS EIA cattle, United Biomedical Inc., Hauppauge, USA; ELISA C, Chekit FMDV3ABC, Bommeli Diagnostics, Intervet, Liebefeld, Switzerland; ELISA D, Panaftosa (PAHO/WHO), Brazil. The manufacturers instructions were strictly followed. 2.4. Liquid-phase blocking competitive ELISA for detection of Abs against FMDV structural proteins The lpELISA, originally described by Hamblin et al. [19,20] and modied by Robiolo et al. [1], was carried out as previously described [1,2,13]. Ab titres were expressed as the reciprocal log10 of serum dilutions giving 50% of the absorbance recorded in the control wells (wells with virus but without serum). Six control sera of known titres were simul-

taneously titrated, as internal standards in each ELISA plate. The cut-off levels for each virus strain have been derived from the correlation data from challenge trials. 2.5. Virus strains The vaccine strains mentioned in this study are: O1/Campos/Brazil/58 (O1/Campos), O1/Caseros/Arg/68 (O1/Caseros), A24/Cruzeiro/Brazil/55 (A24/Cruzeiro), A/Argentina/79 (A/Arg/79) and A/Argentina/87 (A/Arg/87), C3/Argentina/85 (C3/Arg/85), C3/Indaial/Br/71 (C3/Indaial), A/Argentina/00 (A/Arg/00); and A/Argentina/01 (A/Arg/01). Inactivated suspensions of FMDV strains were provided by the National Reference Laboratory of Argentina (SENASA), after inactivation with binary ethyleneimine. The identity of each strain had been established by sequencing, mapping with a panel of MAbs, complement xation (CF) and serum neutralisation (SN). 2.6. Experimental studies and animal challenge procedures Protection data from animal challenge trials were provided by SENASA, while the serology was performed with the lpELISA described above. The animal challenge test in use in Argentina, PPG, has been previously described [2]. Briey, sixteen Hereford breed cattle, aged 1824 months and free from FMDV Abs, are used for each test. Challenge is performed at 90 days post vaccination (dpv) by inoculation of 10,000 suckling mice lethal dose 50% (SMLD50 ) by the intradermolingual route. Two unvaccinated cattle are included in each trial as controls. Seven days after challenge, the animals are examined for foot lesions of FMDV. Animals are considered unprotected when typical FMDV lesions develop on at least in one foot. At least 13 out of 16 animals must be protected in order to pass the test (PPG 81%). The unvaccinated control animals should show in all cases the characteristic foot lesions caused by the virus. Animal sera are obtained at 0 and 60 dpv. Additional bleedings at 30 and 90 dpv are also performed to follow up the kinetics of the immune response. To establish a correlation of serology with challenge, data from 156 and 138 cattle challenged by intradermolingual inoculation with A/Arg/01 and O1/Campos FMDV strains, respectively, were analysed. 2.7. Field studies Field studies have been carried out with more than 100,000 cattle sera. Abs to FMDV SPs and NSPs were examined between the years 2000 and 2004 in sera from infected and healthy cattle, whether vaccinated or not-vaccinated, and with or without clinical signs. Representative examples from well dened epidemiological eld situations that occurred before, during and after the clinical FMDV outbreaks were selected as follows:

1000

B. Robiolo et al. / Vaccine 24 (2006) 9971008

Case 1: Field situationfree without vaccination. Chascomus county, Province of Buenos Aires (PBA). No outbreaks of the disease had been reported since 1995, and last vaccination was in April 1999. Case 2: Field situationimmediately before the ofcial reports of FMD. Three premises in the Magdalena county, PBA, located approximately 90 km Northeast from Chascomus (Case 1) were studied during JuneJuly 2000. The animals showed neither clinical symptoms of FMD nor signs of past lesions, but a number of adult animals purchased in the Province of Formosa (Northeast of Argentina) had been introduced a few weeks before the bleedings. A few days later, the rst ofcial cases of clinical FMD were reported in Formosa, which marked the beginning of the 20002002 Argentine epidemic. A few weeks later, the county of Magdalena was also heavily affected by clinical FMD. Case 3: Field situationspreading of the disease and massive emergency vaccination. The samples were obtained in May 2001 from a farm located in Salto, Northwest region of PBA, where clinical FMD was reported in four male adult animals, which were immediately culled and eliminated according to federal regulations. No other clinical cases were reported in this farm afterwards. Case 4: Field situationemergency vaccination, farms that remained uninfected. Serum samples (n = 339) were obtained in April 2001 from adult animals (2 years of age) that had been vaccinated twice. The animals belonged to a farm located in the centre of PBA, at approximately 100 km of the nearest FMD outbreak Case 5: Field situationemergency vaccination, subclinical infections in vaccinated and re-vaccinated cattle. Serum samples (n = 104) were obtained in May 2001 from a farm located in the south of the Province of San Luis (centre of Argentina) located at approximately 25 km of a FMDV outbreak. Animals had been vaccinated twice and had no signs of clinical FMD at bleeding time. At this stage of the epidemic, only A/Arg/01 virus strain was circulating in the eld, but this strain had not been yet included in the current vaccines [2]. Case 6. Field situationimmediately post eradication to the present. A set of sera were obtained in August 2002 from animals (n = 80) belonging to a dairy farm in the county of Trenque Lauquen (West PBA). Sera was collected from: (i) animals (n = 20) that had presented clinical signs of FMD and remained isolated from others until they recovered from disease; (ii) animals (n = 20) from the same farm that had never presented clinical signs of FMD; (iii) calves (n = 20, females, 12 years of age) born soon after the outbreak in that farm; and (iv) females 612 months old born from mothers of the rst two groups (n = 20). Another set of sera were obtained in the year 2004 from animals randomly selected in the county of Lujan (55 km Northwest of Buenos Aires city): (i) Calves 612 months old (n = 366) vaccinated once or twice; (ii) young animals 1224 months old (n = 125) vaccinated two to three times; and (iii) vaccinated cows that had suffered FMDV infections (n = 84).

3. Results 3.1. Expression of FMDV r3ABCm polypeptide The r3ABCm polypeptide expressed in Escherichia coli/BL21(DE3) transformed with pET-3ABCm showed an apparent molecular weight of approximately 55 kDa in SDS polyacrylamide gels [14], and reacted with bovine convalescent serum and with MAb 3H7 in Western blots (data not shown). 3.2. Standardisation of the 3ABC-ELISA and determination of the cut-off value To assess assay repeatability, two positive (weak and strong) and one negative sera were tested in 35 different assays performed at different days. The test was highly repeatable as was demonstrated by the low dispersion of the values obtained for these three sera. The mean and S.D. for the strong, weak, and negative sera were 77 7.4, 53 6.8, and 6 3.1, respectively. Fig. 1 shows the frequency distributions of 3ABC-ELISA PP values obtained from the analysis of 1626 serum samples from negative bovines (naive or vaccinated) and 146 sera from experimentally infected animals. Two different cut-off values, 15 and 20%, are shown by dashes. Using a cut-off corresponding to a PP 15% the sensitivity of the assay was 98.6% and the specicities were 98.9 and 98.4% for naive or vaccinated animals, respectively. On the other hand, using a cut-off of 20%, the sensitivity was 96.6% and the specicity 99.6% for both groups of uninfected animals (Table 1). Based on this analysis, the cut-off was rst established as follows: positive, PP 20%; negative, PP 15%; suspicious, PP >15 and <19%. Serum samples classied as suspicious were conrmed by enzyme-linked immunoelectrotransfer blot assay (EITB) [5]. Specicity and sensitivity using a cut-off of PP 20% with the corresponding condence intervals (CI), are displayed in Table 1. Results from a preliminary comparison of sensitivity with four other commercial tests are shown in Table 2, with their corresponding 95% CIs. These results are not conclusive because only a limited number of serum samples were anal-

Table 1 Determination of specicity and sensitivity of the 3ABC-ELISA Naive Positives Negatives Total Specicitya (%) Sensitivitya (%) 95% CI 4 1053 1057 99.6 99.3100 Vaccinated 2 567 569 99.6 99.2100 Experimentally infected 141 5 146 96.6 93.699.5

CI: condence interval. a Cut-off PP 20%. Using a cut-off PP 15% the sensitivity was 98.6% and the specicities 98.9 and 98.4% for naive and vaccinated, respectively.

B. Robiolo et al. / Vaccine 24 (2006) 9971008

1001

Fig. 1. Frequency distribution of positive and negative values from 1626 sera of uninfected (naive or vaccinated) and 146 sera from experimentally infected cattle, tested with the 3ABC-ELISA test. The assay was run as described in Section 2. Two different cut-off values, 15 and 20% of the positive control (PP), are indicated by dashes. The percentages of samples included in a given PP value are shown below the gure. Samples that fell between both cut-off values were conrmed by EITB [5]. Infected ( ); uninfected ( ).

ysed with each kit and conrmation assays were not performed. Comparison of specicity was not done. 3.3. Duration of immunity to 3ABC The kinetics of Abs against 3ABC was followed up in 11 experimentally infected animals and presented up to 233 days post infection (dpi) in Fig. 2. The levels of Abs to FMDV 3ABC polypeptide increased with time reaching a maximum at approximately 20 dpi. After that, in most animals Ab levels progressively diminished, although some remained stable and a few animals showed an increase at some points. Some animals remained positive at 570 dpi, when the last samples were collected (not shown). 3.4. Experimental studies Fig. 3A shows a typical reactivity pattern obtained with the combined analysis performed in sera from 196 animals used in several vaccine trials. Abs to SP of every strain present in the vaccines were measured by lpELISA, but only titres
Table 2 Comparison of sensitivity of 3ABC-ELISA with other NS ELISAs ELISA A Positives Negatives Total Sensitivity (%) 95% CI 68 13 73 93.2 87.498.9 ELISA B 60 13 73 82.2 73.490.9

to A/Arg/01 strain are shown in the gure for practical reasons. A positive signal for 3ABC was detected in one of the animals, which was later conrmed as negative by EITB [5]. Sera from 11 animals bled 7 days after challenge (dpc) with A/Arg/01 were also analysed. In this case, it was found that 8 (8/11) animals developed Abs to 3ABC by 7 dpc, while a strong increase in FMDV SP Abs was found in all animals (data not shown). No reactivity to FMDV 3ABC was found in revaccinated animals. Fig. 3B shows the results obtained from 32 animals, which had been vaccinated and re-vaccinated either once or twice. The same results were found in a recent work when sera from multiply-vaccinated cattle were examined and all animals showed to be negative, irrespectively of the number of doses that they had received [21]. 3.5. Correlation between lpELISA titres and protection from challenge with A/Arg/01 and O1/Campos FMDV strains Correlation data between serology and challenge trials with A/Arg/01 and O1/Campos virus strains were collected.

ELISA Ca 59 1 60 98.3 95.0100.0

ELISA Da 59 1 60 98.3 95.0100.0

3ABC-ELISAb 71 2 73 97.3 93.6100.0

CI: condence interval. a Only 60 sera were analysed with this assay. b Cut-off value PP 20%.

1002

B. Robiolo et al. / Vaccine 24 (2006) 9971008

Fig. 2. Kinetics of Abs to FMDV r3ABCm polypeptide in animals experimentally infected with FMDV strain O1/Campos. Reactivity of sera from serial bleedings of 11 animals tested by 3ABC-ELISA and followed until 233 dpi is shown. The cut-off level (PP = 20%) is shown as a dashed line.

The results from 156 animals challenged with A/Arg/01 FMDV strain are plotted in Fig. 4A. The bars represent the percentage of animals that were protected from intradermolingual challenge at 90 dpv for a given lpELISA Ab titre measured at 60 dpv. In order to achieve the minimum 81%

protection required by Argentine authorities, the lpELISA titre might be over 2.2 log units. An Ab titre >2.7 protected all animals challenged with this strain. Similar results for 138 animals challenged with O1/Campos FMDV strain are shown in Fig. 4B. For this strain, a titre >2.1 may pos-

Fig. 3. FMDV antibodies found after experimental vaccination. Antibody levels to A/Arg/01 FMDV strain SPs as measured by lpELISA (right y-axis) ( ) and to FMDV NSPs (all strains) as measured by 3ABC-ELISA (left y-axis) ( ) are shown, for animals vaccinated with one or more doses of tetravalent vaccine (A24/Cruzeiro, O1/Campos, A/Arg/00, A/Arg/01). Panel A: group of 196 cattle vaccinated once and bled at 0, 30, 60 and 90 dpv. Panel B: group of 32 cattle vaccinated and revaccinated once and twice, bled at 30 dpv or revaccination. The 3ABC-ELISA cut-off (PP = 20%) is shown as a line. (C+) Positive control serum; (C) negative control serum. (*) Negative when analysed by EITB [5].

B. Robiolo et al. / Vaccine 24 (2006) 9971008

1003

but only titres to A/Arg/87 strain are shown for practical reasons. Similar results were obtained for the other strains. 3.6.2. Situation immediately before the ofcial reports of FMD Two of the three sampled farms presented serological proles similar to Section 3.6.1. The results obtained in the third farm are shown in Fig. 5B. As expected, unvaccinated calves, 612 months of age were negative when tested for Abs against both SPs (for all the strains mentioned in Section 3.6.1) and 3ABC. However, adult animals showed a high level of Abs against SPs and a surprisingly large number of individuals were found positive for NSP Abs. Moreover, in many cases, values of 3ABC reactivity were equal or even greater than the reference positive control sera. The analysis of these positive samples were repeated three times, and the positive reactivity was further conrmed by Western blot. 3.6.3. Spreading of the disease and massive emergency vaccination As in the two previous cases, we have analysed samples coming from 2 to 6 month-old-calves and from adult cattle (35 years old) carrying multiple previous vaccinations. Fig. 5C shows that both young calves and adult cattle tested positive for FMDV SPs and 3ABC Abs. Judging by their 3ABC reactivity, several animals were clearly infected (although not sick) probably by direct or indirect contact with the sick culled bulls (see Section 2). Abs to A/Arg/00 SPs are shown in Fig. 5C. 3.6.4. Farms that remained uninfected during emergency vaccination As can be seen in Fig. 6A, all animals elicited high levels of Abs against SPs (compatible with the two vaccinations with strains A24/Cruzeiro, O1/Campos and A/Arg/00) [2], but were clearly negative when tested for 3ABC Abs, showing that animals were well protected from disease and had not been in contact with live virus. SPs Ab levels shown in the gure correspond to A/Arg/00 FMDV strain. 3.6.5. Sub-clinical infections in vaccinated and re-vaccinated cattle High Ab levels to A/Arg/00 FMDV strain are shown in Fig. 6B. However, in this case most animals tested positive for NSP Abs, notwithstanding the lack of clinical symptoms during or after the bleedings. This case illustrates clearly a situation of a vaccinated-infected population with no detectable clinical disease. 3.6.6. Multiply vaccinated cattle population in the post eradication period The results from several sets of sera belonging to a dairy farm in the county of Trenque Lauquen are shown in Fig. 7A. In Group 1, the animals still presented Abs to NSPs derived from previous infection (16 months after the onset of clinical

Fig. 4. Correlation between protection from virus challenge (PPG) and levels of anti SPs Abs as measured by lpELISA for A/Arg/01 (n = 156) and O1/Campos (n = 138) FMDV strains. Cattle vaccinated with one dose of tetravalent vaccine were challenged with FMDV strains A/Arg/01 or O1/Campos. The bars show the percentage of animals that were protected from challenge at 90 dpv for a given lpELISA Ab titre obtained at 60 dpv, for each strain in trial.

sibly be needed for 81% protection. More data will be required in order to establish an accurate correlation for both strains. 3.6. Field studies 3.6.1. Status free without vaccination Fig. 5A shows that young calves ranging in age from 5 to 12 months were negative for both SP (titres <1.4 by lpELISA) and NSP (3ABC-ELISA) Abs, while adult animals with four or more vaccinations presented a high level of SPs Abs, but were negative for 3ABC Abs. Abs to SPs of A/Arg/79, A/Arg/87, A24/Cruzeiro, O1/Campos, and C/Arg/85 FMDV strains were measured by lpELISA,

1004

B. Robiolo et al. / Vaccine 24 (2006) 9971008

Fig. 5. Field studies in years 2000 and 2001. Ab levels to serotype A FMDV SPs as measured by lpELISA (right y-axis) ( ) and to FMDV NSPs as measured by 3ABC-ELISA (left y-axis) ( ) are shown. The 3ABC-ELISA cut-off (PP = 20%) is shown as a line. (C+) Positive control serum; (C) negative control serum. (A) Status free without vaccination, January 2000, Chascomus, Buenos Aires. Calves were uninfected and unvaccinated (n = 24). Cows are uninfected but had been multiply vaccinated in the previous campaign (n = 20). Abs levels to A/Arg/87 SPs are shown. (B) Immediately before the ofcial reports of FMD, Magdalena, Buenos Aires, July 2000. Calves were uninfected and unvaccinated (n = 33). Cows had several previous vaccinations (n = 63); subclinically infected adult animals were possibly introduced from infected areas. Ab levels to A/Arg/87 SPs are shown. (C) Spreading of the disease and massive emergency vaccination. Samples were obtained in May 2001 from infected animals in Salto, Buenos Aires. Ab levels to A/Arg/00 SPs are shown. Many unvaccinated calves (n = 30) and cows (n = 30) are subclinically infected.

symptoms). In Group 2, composed of cattle that had never presented clinical signs of FMD, only one of the sera tested positive for 3ABC Abs, probably derived from a sub-clinical infection (conrmed by analysis with three other available kits). The third panel (Group 3) shows the results of the analysis of sera from calves born soon after the outbreak in that farm. Surprisingly all of them except one tested negative for Abs to NSPs. Group 4 represents the results from females 6 to 12 months old born from mothers of Groups 1 or 2. They showed no reactivity to 3ABC and a good level of protective Abs, as expected for vaccinated animals. The SP Ab

levels shown in the gure correspond to A/Arg/01 FMDV strain. The results shown in Fig. 7B were obtained from samples coming from a massive survey carried out approximately 2 years after the last outbreak. Calves and young adults showed high levels of Abs to each of the four vaccine virus strains, compatible with 80% potential protection [1]. As expected, these animals tested negative for NSP Abs. On the other hand, older adult animals (2 years of age) also showed high levels of Abs against FMDV SPs of the vaccine strains in use. Some of them (5 out of 84) tested slightly positive for Abs against

B. Robiolo et al. / Vaccine 24 (2006) 9971008

1005

Fig. 6. Field studies during emergency vaccination. Ab levels to A/Arg/00 FMDV strain SPs as measured by lpELISA (right y-axis) ( ) and to FMDV NSPs as measured by 3ABC-ELISA (left y-axis) ( ) are shown. The 3ABC-ELISA cut-off (PP = 20%) is shown as a line. (C+) Positive control serum; (C) negative control serum. Circulating strain: A/Arg/01. (A) Farms that remained uninfected, April 2001, Buenos Aires Province, located 100 km from a clinical outbreak. Two-year-old animals (n = 339) that had been vaccinated twice (A/24/Cruzeiro, O1/Campos, A/Arg/00) are uninfected. (B) Subclinical infections in vaccinated and re-vaccinated cattle, May 2001, in the south of the Province of San Luis, located at 25 km from a clinical outbreak. Animals (n = 63) that had been vaccinated twice (A/24/Cruzeiro, O1/Campos, A/Arg/00) are sub-clinically infected.

NSPs in the 3ABC-ELISA, a sign that they might have had contact with live virus during the years of the FMDV epidemic. Ab levels shown in Fig. 7B correspond to A/Arg/01 FMDV strain.

4. Discussion The results of the combined application of immunological assays for FMDV SPs and NSPs have been critical for both the in-course validation of the 3ABC-ELISA assay and for a more precise understanding of the epidemiological situation and the follow up of the eradication campaigns. The continuous monitoring using an appropriate sampling schedule, which includes the younger population born after the outbreaks, has proved to be very useful. These assays have also been used with satisfactory results in related susceptible species (pigs and goats), which are not vaccinated in Argentina (data not shown). In addition, Abs against FMDV SPs were found excellent markers of vaccination efciency in NSPs negative animals. The lpELISA test described in this work had been ofcially used by SENASA from year 1995 to 1999 as an indirect potency test for vaccine control, and was standardised with correlation data between challenge protection and

Abs to FMDV SPs for the FMDV strains used in the former vaccine campaigns [1]. Preliminary data from ongoing serology/PPG correlation studies for A/Arg/01 and O1/Campos FMDV strains presented herein suggest that vaccines should elicit lpELISA titres over 2.2 log units in order to guarantee protection from disease. Further work done after submission of this work has conrmed these values. The 3ABC-ELISA used with different cut-off values can be exibly adapted to different needs. High specicity and sensitivity (over 98%) were found using a cut-off of PP 15%. Very high specicity (99.6%), although lower sensitivity (96.6%), was found when using a cut-off of PP 20%. Preliminary comparison with four other NS ELISAs using a cut-off of PP 20% gave acceptable results, although only a small number of serum samples were analysed. The peculiarity of Argentina resides in that FMD had been eradicated through a massive vaccination campaign that was later stopped for a period of 1 year and resumed again after the recurrence of outbreaks, but this second time using vaccines with different strain composition [2]. For this reason, there were at the same time adult animals, which had received several vaccinations, and two or three generations of naive young calves, that either had never been vaccinated or had received only one vaccine dose. The early detection of viral infection in the absence of clinical signs

1006

B. Robiolo et al. / Vaccine 24 (2006) 9971008

Fig. 7. Field studies in the post eradication period in a multiply vaccinated cattle population. Ab levels to A/Arg/01 FMDV strain SPs as measured by lpELISA (right y-axis) ( ) and to FMDV NSPs as measured by 3ABC-ELISA (left y-axis) ( ) are shown. The 3ABC-ELISA cut-off (PP = 20%) is shown as a line. (C+) Positive control serum; (C) negative control serum. (A) Cross-age study performed in Trenque Lauquen, Buenos Aires in August 2002. Group 1: cattle that had presented clinical signs of FMD in April 2001 and recovered from disease (n = 20). Group 2: idem Group 1 but without clinical signs of FMD (n = 20). Group 3: vaccinated young cattle 1224 months of age. Group 4: vaccinated calves 612 months old (n = 20) born after the outbreaks. (B) seroepidemiological survey in Lujan, Buenos Aires (year 2004). Group 1: calves 612 months old (n = 366) vaccinated once or twice. Group 2: young animals 1224 months old (n = 125) uninfected and vaccinated. Group 3: older animals (n = 84), vaccinated and that had suffered FMDV infections.

of disease, as well as the establishment of the appropriate protection levels of Abs elicited by vaccines against the emerging strains (vaccine matching), was therefore of critical importance. Among the several ELISA assays developed by different investigators to distinguish infected animals from uninfected naive or vaccinated animals [22,23], the detection of Abs to the NS polypeptide 3ABC was found to be the most reliable single marker of infection [7,24]. Moreover, Abs to 3ABC appeared early after infection and were detected for longer periods than Abs to any other of the NSPs examined [6,7]. In this work we have found anti 3ABC Abs in experimentally infected animals for at least 570 dpi, although a high variability in magnitude and duration of immunity at the individual animal level existed. As already shown [22], these assays should be judged only at the herd level, although the use in conjunction with lpELISA tests for the detection of SPs may eventually circumvent this problem. In the case by case analysis, we found evidence that some vaccinated animals can support viral replication without showing clinical signs, which is due to the fact that vaccines induce protection from clinical disease but not from infection. However, the data presented herein support the evaluation of anti-3ABC activity as a conclusive assay to

certify a previous infection with FMDV in vaccinated animals. Before the outbreaks (Case #1), the results obtained were typical of a post eradication period in a cattle population which had been previously intensively vaccinated. The calves, which were all born after vaccination had stopped, represented the fully susceptible population within cattle herds and had no FMDV Abs, while adult animals showed only SPs Abs, resulting from vaccination campaigns prior to the year 2000 (Fig. 5A). The monitoring assays were able to rapidly detect the introduction of several infected animals, purchased at the border with Paraguay by farms located in central Argentina (Case #2), through the detection of anti 3ABC Abs in adult vaccinated animals without clinical signs of the disease (Fig. 5B). Few weeks later (Case #3), the spreading of infection led to the generation of Abs to NSPs and SPs in calves and adult animals (Fig. 5C). In this case, Abs against SPs in adults could be a consequence of previous vaccinations (36 doses) whereas Abs in calves were only due to infection, although no clinical signs appeared except in four culled bulls. During the emergency vaccination period (Cases #4 and #5) different immunological patterns were found, in conjunction with the presence or absence of clinical disease. Numer-

B. Robiolo et al. / Vaccine 24 (2006) 9971008

1007

ous herds remained uninfected, as judged by the absence of both anti 3ABC Abs and clinical signs (Fig. 6A), while vaccinated-infected population with no detectable clinical disease showed high levels of Abs to 3ABC as well as to SPs (Fig. 6B). Vaccinated-infected populations with FMD clinical signs presented an immunological pattern indistinguishable from that of Fig. 6B. At the end of the Argentine epidemic (last virus isolation was in January 2002) more than 80% of the animals had been vaccinated, most of them several times. The Ab patterns that were observed t perfectly with the sanitary history of the cattle herds (Fig. 7). While cattle involved in the past clinical outbreaks still showed low levels of anti 3ABC Abs, calves born after the end of the epidemic were totally free from them, and anti-3ABC Abs were present in few young adult animals born subsequently or in animals that never showed FMD clinical signs. Similar results were found in epidemiological surveys conducted recently. Outbreaks of FMD in Taiwan (1997) and Europe (2001) have been contained by mass slaughter of infected and at-risk animals, a procedure that could not be applied in Argentina, and that might also be unacceptable in the future for political, economic, ethical and environmental reasons. The results shown in this work encourage vaccination accompanied by the regular use of combined serological surveys for both anti SP and NSP Abs as an alternative to the control of FMD by mass slaughtering of animals.

Acknowledgements We acknowledge the active collaboration of DILABSENASA with this work. We also thank Mrs. Carmen Devicenzo, Marcela Iglesias and Maria Rodriguez for their technical assistance. CEVAN-CONICET and CICVyA-INTA are members of the Inter-institutional FMD Research and Development Network (RIIDFA) of Argentina. This work was possible due to economical support from CONICET and FEVAN.

References
[1] Robiolo B, Grigera PR, Periolo OH, Seki C, Bianchi T, Maradei E, et al. Assessment of foot-and-mouth disease vaccine potency by liquid-phase blocking ELISA: a proposal for an alternative to the challenge procedure in Argentina. Vaccine 1995;13:134652. [2] Mattion NM, K nig G, Seki C, Smitsaart E, Maradei E, Robiolo o B, et al. Reintroduction of foot-and-mouth disease in Argentina: characterisation of the isolates and development of tools for the control and eradication of the disease. Vaccine 2004;22:414962. [3] Doel TR, Williams L, Barnett PV. Emergency vaccination against foot-and-mouth disease: rate of development of immunity and its implications for the carrier state. Vaccine 1994;12:592600. [4] Grigera PR, Tisminetzky SG, Lebendiker MB, Periolo OH, La Torre JL. Presence of a 43-kDa host-cell polypeptide in puried aphthovirions. Virology 1988;165:5848.

[5] Bergmann I, Malirat V, Neitzert E, Beck E, Panizzutti N, S nchez C, a et al. Improvement of a serodiagnostic strategy for Foot-and-Mouth Disease Virus surveillance in cattle under systematic vaccination: a combined system of an indirect ELISA-3ABC with an enzymelinked immunoelectrotransfer blot assay. Arch Virol 2000;145:473 89. [6] De Diego M, Brocchi E, Mackay D, De Simone F. The non-structural polyprotein 3ABC of Foot-and-Mouth Disease Virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle. Arch Virol 1997;142:202133. [7] Mackay D, Forstyth MA, Davies PR, Berlinzani A, Beilsham GJ, Flint M, et al. Differentiating infection from vaccination in FMD using a panel of recombinant non-structural proteins in ELISA. Vaccine 1998;5:44659. [8] Lubroth J, Brown F. Identication of native foot-and-mouth virus non-structural protein 2C as a serological indicator to differentiate infected from vaccinated livestock. Res Vet Sci 1995;59: 708. [9] Alonso Fern ndez A, G mes MPD, Martins MA, Sondahl MS. a o Detection of Foot-and-Mouth Disease Virus infection associated antigen antibodies: comparison of the enzyme-linked immunosorbent assay and agar gel immunodiffusion test. Prev Vet Med 1990;9:23340. [10] Bergmann IE, Aug de Mello P, Neitzert E, Beck E, Gomes I. Diage nosis of persistent aphtovirus infection and its differentiation from vaccination response by use of enzyme linked immunoelectrotransfer blot analysis with bioengineered non-structural viral antigens. Am J Vet Res 1989;54:82531. [11] Rodriguez A, Dopazo J, Saiz JC, Sobrino F. Immunogenicity of non-structural proteins of Foot-and-Mouth Disease Virus differences between infected and vaccinated swine. Arch Virol 1994;136: 12331. [12] Shen F, Chen PD, Waleld AM, Ye J, House J, Brown F, et al. Differentiation of convalescent animals from vaccinated against footand-mouth disease by a peptide ELISA. Vaccine 1999;17:3039 49. [13] Periolo OH, Seki C, Grigera PR, Robiolo B, Fernandez G, Maradei E, et al. Large-scale use of liquid-phase blocking sandwich ELISA for the evaluation of protective immunity against aphthovirus in cattle vaccinated with oil-adjuvanted vaccines in Argentina. Vaccine 1993;11:75476. [14] Capozzo AV, Burke DJ, Fox JW, Bergmann IE, La Torre JL, Grigera PR. Expression of foot and mouth disease virus non-structural polypeptide 3ABC induces histone H3 cleavage in BHK21 cells. Virus Res 2002;90:919. [15] Grubman MJ, Zellner M, Bablanian G, Mason PW, Piccone ME. Identication of the active-site residues of the 3C proteinase of Footand-Mouth Disease Virus. Virology 1995;10:5819. [16] Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Genes 1989;15:519. [17] Earley EM, Rener JC. Hybridoma technology I: murine monoclonal antibodies. J Tissue Cult Methods 1985;9:12987. [18] Harlow, David Lane, editors. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory; 1988 [chapters 6 and 7]. p. 139282. [19] Hamblin C, Barnett ITR, Crowther JR. A new enzyme linked immunosorbent assay (ELISA) for the detection of antibodies against FMD Development of a method for ELISA. J Immunol Methods 1986;93:11521. [20] Hamblin C, Kitching RP, Donaldson AI, Crowther JR, Barnett ITR. Enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against FMDV. III. Evaluation of antibodies after infection and vaccination. Epidemiol Infect 1987;99:733 44. [21] Espinoza AM, Maradei E, Mattion NM, Cadenazzi G, Maddonni G, Robiolo B, et al. Foot and mouth disease polyvalent oil vaccines inoculated repeatedly in cattle do not induce detectable antibodies

1008

B. Robiolo et al. / Vaccine 24 (2006) 9971008 to differentiate between cattle infected with or vaccinated against Foot-and-Mouth Disease Virus. Vet Microbiol 2004;99:93 101. [24] Clavijo A, Zhou E, Hole K, Galic B, Kitching P. Development and use of e biotinylated 3ABC recombinant protein in a solid-phase competitive ELISA for the detection of antibodies against Foot-andMouth Disease Virus. J Virol Methods 2004;120:21727.

to non structural proteins when evaluated by various assays. Vaccine 2004;23:6977. [22] Clavijo A, Wright P, Kitching P. Developments in diagnostic techniques for differentiating infection from vaccination in foot-andmouth disease. Vet J 2004;167:922. [23] Moonen P, Linde E, Ch nard G, Dekker A. Comparable sene sitivity and specicity in three commercially available ELISASs