Detection of Foot-and-Mouth Disease Virus-Infected Cattle by Assessment of Antibody Response in Oropharyngeal Fluids

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1995, p Vol. 33, No /95/$ Copyright 1995, American Society for Microbiology Detection of Foot-and-Mouth Disease Virus-Infected Cattle by Assessment of Antibody Response in Oropharyngeal Fluids IVONNE L. ARCHETTI, 1 MASSIMO AMADORI, 1 * ANNA DONN, 2 JEREMY SALT, 2 AND EZIO LODETTI 1 Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia, Brescia, Italy, 1 and Institute for Health, Pirbright Laboratory, Pirbright, United Kingdom 2 Received 29 June 1994/Returned for modification 10 August 1994/Accepted 9 September 1994 The detection of foot-and-mouth disease virus (FMDV)-persistent carriers among convalescent ruminants is of paramount importance in the aftermath of a field outbreak. To this purpose, FMDV-specific antibody should be investigated first, since virus isolation procedures from such carriers are seriously constrained. The complexity of the overall picture may be compounded by possible emergency vaccinations in the affected areas at the beginning of the outbreak. In this case, it is suggested that mucosal rather than serum antibody be investigated. In fact, we showed that FMDV-infected cattle regularly mount an antibody response in oropharyngeal fluids, in contrast to vaccinated cattle. Antibody could be revealed by neutralization assays and/or an immunoglobulin A (IgA)-specific kinetic enzyme-linked immunosorbent assay (ELISA). Cattle vaccinated once seldom showed a mucosal antibody response, which could be only detected by a total immunoglobulin-specific kinetic ELISA. Very few, if any, cattle showed a mucosal IgA response after repeated vaccinations. Our kinetic, IgA-specific ELISA generally allowed an early detection of FMDV-infected cattle; in particular, it proved to be more sensitive than the usual indirect, antigen-trapping ELISA in experiments on saliva samples. Foot-and-mouth disease (FMD) is a highly contagious disease that affects both domestic and wild cloven-hoofed animals. Infection of susceptible animals with FMD virus (FMDV) leads to the appearance of vesicles on the feet, in and around the oral cavity, and on the mammary glands of females. Mortality from myocarditis is most commonly seen in young animals, and healing of vesicular lesions usually takes place in 2 to 3 weeks. The economic consequences of an outbreak of FMD in intensively farmed regions can be devastating. Costs accrue from lost production and interference with international trade. Prophylactic vaccination against FMD within the European Union (EU) countries ceased during For the preservation of its FMD-free status, the EU currently relies upon a policy of importation control for livestock and livestock products. In the first instance, a future FMD outbreak within the EU would be controlled by stamping out, which would involve the slaughter of all affected and in-contact susceptible livestock, and the imposition of strict movement control. Under specific circumstances, such as FMD occurrence in a densely populated livestock area, emergency vaccination around a future outbreak would be resorted to as an aid to disease control, while keeping, of course, all of the above measures. The EU has consequently established an FMDV antigen bank which will be situated at four sites around the EU. FMDV vaccine could be formulated from antigen held in these reserves and used in an emergency vaccination campaign. Vaccinated and protected ruminants could subsequently become subclinically infected with FMDV following exposure to the virus during the height of an outbreak. In view of the high proportion of these cattle that would be expected to become * Corresponding author. Mailing address: Department of Vaccine Research and Development, Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia, via A. Bianchi 7, Brescia, Italy. Phone: Fax: persistently infected with FMDV, it would be necessary to treat all vaccinated animals as potential FMDV carriers and, as such, a source of FMDV for further elaboration of the outbreak. Therefore, in the aftermath of such an outbreak, one of the immediate considerations would be to determine the existence of FMDV carriers among the vaccinated population. There is currently no available diagnostic test able to detect FMDV carriers reliably. If a test which allowed the identification of individual vaccinated animals or herds subclinically infected with FMDV were available, it would be of great value for future FMD control within the EU following an emergency vaccination campaign. In general, the detection of FMDV carriers among convalescent ruminants is pursued by virus isolation procedures from samples of oropharyngeal (OP) fluids, collected with a probang cup (6). This approach suffers some major constraints: (i) virus excretion is not continuous and titers are usually low (21); (ii) FMDV is often complexed with antibodies (21); (iii) falsenegative results can be obtained due to virus inactivation during sampling and shipment to the laboratory; and (iv) the carrier state might be feigned, in that (according to experimental evidence) FMDV RNA can be encapsidated into coat proteins of bovine enteroviruses (10). Even a serological approach to the detection of infected but not necessarily carrier cattle by the demonstration of antibodies to nonstructural viral proteins such as 3D is actually cumbersome; in fact, multivaccinated cattle can be 3D antibody positive (15) and concentrated, inactivated FMDV antigen (to be used for an emergency vaccination) can be heavily contaminated by both 3D and 3A/B proteins (3). Finally, serological cross-reaction with bovine enterovirus-specific antibodies to viral proteins can be expected. Owing to the above, we wondered whether the detection of mucosal antibodies in OP fluids could provide more useful information for revealing previously infected, possibly carrier cattle. The rationale for that would be the following: (i) an 79

2 80 ARCHETTI ET AL. J. CLIN. MICROBIOL. antibody response has been clearly documented in OP fluids of FMD-convalescent cattle (8, 9, 11, 12, 21); (ii) this response would not take place in once-vaccinated cattle (8); and (iii) the virus-specific immunoglobulin (Ig) A response would be peculiar to the infected animals (8). Of course, this approach could be compromised by the very low antibody titers in OP fluids (9) or accidental plasma contamination during sampling. This paper deals with the mucosal antibody response in probang and saliva samples collected from FMD-vaccinated and/or experimentally infected cattle. Our study aimed at defining a reliable procedure for detecting FMDV-infected, possibly carrier animals after a disease outbreak and an emergency vaccination in the field. MATERIALS AND METHODS Samples. Probang and saliva samples were taken from cross-bred cattle after single or multiple injections of monovalent or trivalent, O/A/C, Al(OH) 3 FMD vaccines. Probang samples consisted of OP fluid collected with a probang sampling cup. Saliva samples were obtained by either the use of cotton swabs or the collection of free saliva following salt stimulation. The same samples were obtained at different times from O 1 Lausanne or O 1 BFS-convalescent cattle, infected either intranasally or by contact exposure to pigs in the early acute phase of FMD. Virus from carrier animals was isolated on primary bovine thyroid cells in roller tubes and subsequently typed as FMDV type O in the standard FMDV antigen detection enzyme-linked immunosorbent assay (ELISA) (16). Probang and saliva samples were homogenized by adding 3-mm glass beads and vortexing; samples were centrifuged (10,000 rpm, 20 min) before further freezing at 20 C. Cattle. The following groups of cross-bred cattle were included in our study: (A) 9 calves, neither infected nor vaccinated; (B) 6 steers, infected with O 1 BFS FMDV by contact exposure; (C) 5 steers, 2 of which had been vaccinated once with a monovalent O 1 Lausanne vaccine (8 days later, all were infected with O 1 Lausanne FMDV by contact exposure, together with group B cattle); (D) 10 calves, 5 of which had been vaccinated once with a trivalent, O/A/C, FMD vaccine (the other five served as nonvaccinated, control animals); (E) 40 cows, repeatedly injected (two shots or more) with trivalent, O/A/C, FMD vaccines; (F) 5 calves, vaccinated once with a monovalent, very potent ( 20 50% protective doses) O 1 Lausanne vaccine; and (G) 8, 8- to 12-month-old steers vaccinated with a polyvalent FMD vaccine, including a type O 1 vaccine strain (all were challenged intranasally with O 1 BFS FMDV 6 months later). Neutralizing antibody. Samples were inactivated (56 C, 30 min) and neutralizing antibody was assessed in a microtiter neutralization assay on IBRS-2 cells or in a plaque reduction assay on BHK-21 cells, as previously described (1). ELISAs. Samples were usually tested at 1:5 to 1:10 final dilutions. An alkaline phosphatase-based kinetic ELISA () (4) in which A 405 was automatically recorded at 4-min intervals at room temperature with intermittent shaking was used. The following parameters were taken into account: milli-od/s 10 3 (time/od slope), where OD is optical density; final OD and OD slopes (antigen-coated versus blank wells); and correlation coefficients (r; time versus OD). The system was applied in three distinct formats. (i) Purified, inactivated O 1 FMDV 146S antigen (5 g/ml) was absorbed onto ELISA plates. Samples were incubated at 4 C overnight, and antibody was revealed by an alkaline phosphatase-conjugated, rabbit anti-bovine antiserum (total Ig, 146S ). (ii) Inactivated O 1 FMDV antigen was trapped onto ELISA plates by a hyperimmune, type-specific rabbit antiserum. Samples were incubated at 4 C overnight; antibody was revealed by an alkaline phosphatase-conjugated, rabbit anti-bovine Ig antiserum (trapping, total Ig ). (iii) Inactivated O 1 FMDV antigen was trapped onto ELISA plates by a hyperimmune, type-specific rabbit antiserum. Samples were incubated at 4 C overnight; antibody was revealed by anti-bovine IgA monoclonal antibody 2H4 plus an alkaline phosphatase-conjugated, goat anti-mouse Ig antiserum (trapping, IgA ). Blocking of ELISA plates was performed with either phosphate-buffered saline (PBS) 2% bovine serum albumin or PBS 2% skim milk (1 h at 37 C) after addition of first-step reagents. To discriminate between antibody-negative and -positive samples, OD thresholds of 3 and 2 milli-od/s 10 3 were used for the total Ig and IgA tests, respectively; these values represent the difference between the time/od slopes on antigen-coated and blank wells, observed with the same saliva or probang sample. As a further control of specificity, minimum r values of 0.9 plus final OD values of 0.05 (146S and IgA trapping ELISAs) and 0.1 (total Ig trapping ELISA) were required to identify samples as antibody positive. RESULTS Group A. Saliva and probang samples from two cattle were antibody negative in all tests. Samples from the remaining B TABLE 1. Antibody response in OP fluids from FMDV-infected cattle (groups B and C) a Cattle group Convalescent, carriers (1 8 wk p.i.) No. of Ab-positive cattle/no. tested Total Ig Ab IgA Ab Saliva Probang Saliva Probang Neutral Ab (probang) 0/1 6/6 1/1 6/6 5/6 Total 0/1 6/6 1/1 6/6 5/6 C Convalescent, carriers (1 8 wk p.i.) Convalescent, noncarriers (1 6 wk p.i.) Vaccinated, convalescent, carriers (1 6 wk p.i.) ND b 2/2 ND 1/2 1/1 ND 1/1 ND 0/1 0/1 c ND 2/2 ND 2/2 0/2 c Total 5/5 3/5 1/4 a Ab, antibody. ND, not done. Not tested after day 26 p.i. seven cattle in this group were shown to be antibody negative in the trapping, total Ig. Groups B and C. Results for groups B and C are shown in Table 1 and Fig. 1. All cattle became carriers after infection, except one nonvaccinated steer of group C (S62). Control samples from two steers of group B were antibody negative in all tests at day 1 postinfection (p.i.). Five of six carrier cattle of group B developed neutralizing antibody in probang samples by day 35 p.i., the earliest positive reaction being detected at day 14 p.i. The only carrier animal without neutralizing antibody in probang samples was not checked after day 28 p.i. Titers varied between 0.5 and 1.0 log 10. As for group C, the convalescent noncarrier and the two vaccinated carrier steers showed no neutralizing antibody over the same period, as opposed to one convalescent carrier steer of the same group (group C); the latter showed detectable neutralizing antibody at day 26 p.i. Neutralizing antibody was not investigated in probang and saliva samples of the other convalescent carrier steer of group C. The total Ig, 146S proved to be less sensitive, and data are not shown for this reason. With respect to the trapping s, a few results are shown in Fig. 1. Antibody was detected in the first week p.i. in two cattle or, more often, at days 13 and 14 p.i.; a certain delay was evident in the two steers of group C, vaccinated 8 days before challenge, which remained antibody negative until days 19 and 26 p.i., respectively. Group D. All 10 saliva samples were antibody negative in the trapping, total Ig at day 40 postvaccination, regardless of the previous single shot of vaccine. No other tests were performed on the above samples. All calves had detectable, FMDV-specific, maternally derived serum antibody at the beginning of the experiment. Group E. Results for group E (Table 2) refer to saliva and probang samples, collected up to 10 months after the last vaccination. No animal was antibody positive in the total Ig, 146S and in plaque reduction assays. Two saliva and one probang sample was IgA positive (), whereas seven saliva and six probang samples were positive in the trapping, total Ig.

3 FIG. 1. experiments on probang and saliva samples of FMDV-infected cattle (groups B and C). Saliva (steers SA68 and SA71) and probang (remaining cattle) samples were tested in trapping s for total Ig and IgA antibody, respectively, at different times after contact exposure of cattle to FMDV-infected pigs. The y axes represent the OD slopes, expressed as milli-od/s

4 82 ARCHETTI ET AL. J. CLIN. MICROBIOL. TABLE 2. Antibody response in OP fluids from multivaccinated cattle (s; group E) a 2 FMD vaccine shots 3 FMD vaccine shots Total Ig Ab, Total Ig Ab, IgA Ab IgA Ab trapping trapping Sal Pr Sal Pr Sal Pr Sal Pr ND ND ND a Probang (Pr) and saliva (Sal) samples were collected from multivaccinated cows and tested in s for total Ig and IgA antibody (Ab)., positive sample;, weakly positive sample;, negative sample. ND, Not done. Group F. Results for group F are shown in Table 3. Saliva and probang samples at day 60 postvaccination were antibody negative in all tests except for total Ig (both 146S and trapping assays), in which some samples were weakly positive. Group G. Steers RW15 and RW16 were defined as noncarriers and the remaining six cattle were defined as carriers of FMDV (Table 4). The results obtained in the antibody tests are shown in Table 5. In four of eight animals, saliva samples showed FMDV-specific antibody in the trapping, total Ig at day 0 p.i. Neutralizing antibody was always detected after infection, the earliest positive result being obtained at day 7 p.i. on animal RW15, i.e., the only steer showing overt signs TABLE 3. Antibody response in OP fluids of O 1 Lausannevaccinated cattle (group F) a Samples designation Plaque reduction, 70% Antibody response Total Ig, 146S Total Ig, trapping IgA Saliva 3 S 7S ND 9S 21 S 22 Probang 3 S ND 7S ND 9S ND 21 S ND 22 ND a Saliva and probang samples were collected at day 60 postvaccination., negative sample;, positive sample; ND, not done. TABLE 4. FMDV isolations from probang samples collected from group G cattle following challenge with FMDV type O 1 BFS a Days p.i. b Virus isolation from samples from steer: RW15 RW16 RW17 RW18 RU22 RV27 RV28 RV29 0 c 2 d a Samples were assayed for cytopathic effects in bovine thyroid cells and confirmatory serotyping was performed by ELISA. b Days postchallenge with FMDV type O 1 BFS. c Negative cytopathic effect after 72 h. d Positive cytopathic effect for type O FMDV. of FMD after challenge. Also, IgA antibody appeared only after infection, by day 22 p.i. DISCUSSION The assessment of FMDV-specific mucosal antibody confirmed that vaccination is a very inefficient means of stimulating such a response (8), which would be normally lacking in noninfected, once-vaccinated cattle; thus, in the latter animals inhibition of primary virus replication in the upper respiratory tract would be mainly due to cell-mediated immune mechanisms, as previously suggested (2). Owing to the above, it is conceivable to investigate antibody in probang and saliva samples in order to detect infected, possibly carrier cattle after an outbreak has been stamped out and emergency ring vaccination has been adopted. Some distinct features of these antibody responses should be put forward. First, serum IgG antibodies in vaccinated animals can delay the mucosal responses after infection (8, 11), as also evidenced in our experiments. Such a delay could be appreciated in the two animals of group C, vaccinated 8 days before challenge, as opposed to animals of group G, vaccinated 6 months before challenge. However, the group C response is more relevant to a scenario of emergency vaccination after an FMD outbreak. With regard to group G, the earliest neutralizing antibody response was seen in steer RW15, showing clinically overt FMD; this result is consistent with the massive exudation of plasma proteins following oral vesicular lesions. This assumption was confirmed by the time kinetics of the total Ig and IgA responses after infection in groups B and C (Fig. 1); the IgA response, peaking between weeks 4 and 8 p.i. (i.e., after healing of the vesicular lesions), should be viewed as a genuine mucosal response, sustained by resident antibodysecreting B cells (7). In this respect, it has been shown that the secretory immune system in the respiratory and digestive tracts of cattle shows a complex architecture, with a combination of different immunoglobulin profiles in nasal and buccal secre-

5 VOL. 33, 1995 MUCOSAL ANTIBODY RESPONSE OF CATTLE TO FMDV 83 TABLE 5. Antibody response in saliva of FMDVinfected cattle (group G) Days p.i. Plaque reduction, 70% (titer) Antibody response a Total Ig, 146S total Ig IgA RW / /45 ND ND 22 1/ / / / /90 ND RW / / / /10 RW ND ND 22 1/ / / /10 RW / / / /49 ND RU ND ND ND ND 13 ND ND ND ND 22 1/10 ND ND ND 29 1/10 ND ND ND 36 1/ / /10 RV / / / / / /90 RV /10 ND 22 1/ / /10 ND 44 1/ /90 Continued Days p.i. TABLE 5 Continued Plaque reduction, 70% (titer) Antibody response a Total Ig, 146S total Ig IgA RV ND ND ND 22 1/ / / /90 a, Negative sample;, positive sample; ND, not done. tions (11) and areas of prevalent IgA or IgG1 responses (14). Besides, plasma contamination in samples may arise due to inflammatory processes or accidental tissue damage. However, by diluting minute amounts of antibody-positive sera in probang or saliva samples, a dramatic reduction of reactivity could be shown in the above-mentioned ELISAs which would mainly reveal secretory, protease-resistant antibody molecules (data not shown). Our results show that the neutralizing antibody and IgA () assays are suitable for detecting FMDV-infected, possibly carrier cattle after an emergency vaccination in the field; in fact, these tests did not reveal antibody induced by the previous, single shot of vaccine. After repeated injections of FMD vaccines (group E), no animal showed detectable neutralizing antibody in saliva and probang samples; very few, if any, showed an FMDV-specific IgA response. No correlation was found between antibody levels in and neutralizing activity, which could possibly derive from an affinity maturation of the antibody response (11). The rationale for adopting a procedure has already been illustrated (18). In fact, such a procedure is suited to detect minute amounts of specific antibody in mucosal secretions; these samples are usually characterized by high contents of detached cells, proteases, and tissue particles, which often contributes to considerable background reactions in the ELISA. Thus, low-titered, low-affinity, specific antibody may only be detected by comparative evaluation of the reaction kinetics in antigen-coated and blank wells. This assay system proved to be more sensitive for the detection of IgA in saliva samples from group G cattle than an indirect, antigen-trapping ELISA (Table 6). Furthermore, antibody-negative and -positive samples beyond the defined threshold levels have been consistently scored as such on repeated testing in IgA. The choice of investigating genuine mucosal responses, sustained by resident IgA-secreting cells, is also meant to avoid the detection of the serum IgG subtypes, which are submitted to passive mucosal transudation (20). With regard to cattle, transfer of circulating IgG1 to the intestinal lumen has been clearly documented (5). As opposed to other experimental procedures (19), our anti-iga monoclonal antibody was not absorbed onto the solid phase as a catcher of mucosal IgA. Our approach has been criticized on the basis of competition between the sample isotypes for available antigen, with the suggested loss of the IgA component due to predominant IgG antibody. However, in our experience, IgA is the main antibody isotype in probang and saliva samples and FMDV antigen is in excess in the ELISA. For these reasons, we do not think competition between antibody isotypes is of concern. This was further

6 84 ARCHETTI ET AL. J. CLIN. MICROBIOL. TABLE 6. FMDV-specific IgA titers in saliva samples collected from group G cattle following challenge with FMDV type O 1 BFS a Days p.i. b IgA titer RW15 RW16 RW17 RW18 RU22 RV27 0 c a Titers were measured in an isotype-specific indirect antigen-trapping ELISA (13) and calculated as the reciprocal dilution with an OD reading of 1.0. b Days postchallenge with FMDV type O 1 BFS. c Value below the detection limit of the assay; the starting dilution of the sample in this assay was 1/10. substantiated by control experiments on a few saliva samples of group G cattle, in which monoclonal antibody 2H4 and a mouse anti-bovine IgA serum were used as catchers in the antibody capture assay, as previously described (19); in fact, under these conditions, fewer antibody-positive samples were detected (data not shown). After an FMD outbreak has been stamped out, extensive surveillance on a herd-by-herd basis should be implemented in the affected areas to detect virus-carrying ruminants. The possible emergency vaccination raises the threshold for animals to be infected and become carriers; nevertheless, whenever this happens, the recognition of these asymptomatic virus carriers has considerable hurdles. According to our results, epidemiological surveys should be implemented about 4 weeks after the last disease case in the field. Both saliva and probang samples should be collected and kept refrigerated and frozen, respectively. First, the antibody tests (neutralization and/or IgA-) should be carried out on 1:5- to 1:10-diluted saliva samples; then, the probang samples of antibody-positive animals should be investigated by virus isolation and/or PCR procedures (17). Concerning neutralizing antibody, the plaque reduction assay is certainly more sensitive than the microplate assay; in our experience, a 70% threshold can be used without detrimental effects on specificity. In a possible field scenario after an FMD outbreak, mucosal antibody-positive animals somehow became infected. If samples are antibody negative, the carrier state is extremely unlikely. Conversely, a persistent mucosal antibody response (beyond 3 to 4 months) is strongly indicative of a carrier state (12). ACKNOWLEDGMENTS We thank L. Capucci for providing monoclonal antibody 2H4 and for useful suggestions. The skillful technical assistance of M. G. Bertoni is gratefully acknowledged. REFERENCES 1. Ahl, R., R. J. Lorenz, and G. 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