Appendix 71 Secretory IgA as an indicator of oropharyngeal FMDV replication Abstract Introduction Materials and methods

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1 Appendix 71 Secretory IgA as an indicator of oropharyngeal FMDV replication Satya Parida, David Paton*, Sarah Cox, Paul Barnett, John Anderson Pirbright Laboratory, Ititute for Animal Health, Ash Road, Woking, Surrey, GU4 NF, UK. Abstract Introduction: FMDV-specific IgA in oronasal secretio has been reported as a useful indicator of in vaccinated cattle. We have developed an ELISA for the detection of IgA to FMDV structural protei in saliva and evaluated it using sera from cattle following or vaccination plus. The results have been compared to those of other tests for virus replication and antibody development. Materials and Methods: Samples came from naïve cattle and from three cattle experiments involving 66 animals and serotype O FMDV. Vaccinated, multiply vaccinated or unvaccinated cattle were challenged by contact with virus-donor cattle. Animals were monitored for clinical sig, virus replication and serological respoes and kept for 1-6 months after challenge. An ELISA was developed to detect FMDV-specific, salivary IgA using a rabbit capture antibody, whole virus antigen and a commercially available polyclonal anti-iga conjugate. Results: The IgA assay on saliva was specific for infected animals. Vaccination alone, even after two repeat vaccinatio rarely resulted in significant levels of IgA being detected. Acute lead to the development of elevated salivary IgA and there was a correlation between the continued presence of FMD virus in the oropharynx and elevated levels of salivary IgA. Discussion: This new assay has promise for detecting animals that have become persistently infected with FMDV following vaccination. Introduction Garland (1974) studied levels of FMDV-specific antibodies in the oral and nasal secretio of FMD vaccinated or infected cattle and found that these were mainly IgA and IgG1 and that they appeared 3-5 days after, peaking by 1-8 days. In contrast, killed parenterally administered FMD vaccines produced little secretory antibody that was mainly IgG1, although successive doses of vaccine could elicit IgA. Archetti et al. (1995); Salt et al. (1996) and Amadori et al. () coidered that mucosal IgA detection in oesophago-pharyngeal fluids had potential as an indicator of. However, Moonen et al., (4) did not find IgA detection to be a reliable indicator of carrier status in a longitudinal study of FMDV vaccinated and subsequently FMDV inoculated cattle. Here, we report on the development and evaluation of an IgA ELISA with coiderable promise for the diagnosis of persistently infected cattle. After initial tests on saliva, nasal and oropharyngeal fluids, saliva was coidered the best mucosal fluid to test on account of ease of collection and satisfactory test results. Materials and methods Saliva samples were collected from the cheek region of the mouth using cotton tampo predampened by the addition of phosphate buffered saline and held by forceps. In the laboratory, the saliva was extracted from the tampo by squeezing in the barrel of a syringe or by centrifugation. In order to test the specificity of the method, saliva samples were collected from 173 cattle that had not been vaccinated or infected with FMDV, shortly after arrival at our laboratory for a variety of experimental studies. Saliva was also collected at different time-points from three different cattle studies involving vaccination with oil adjuvanted O Manisa and subsequent challenge by contact with donor cattle that had been previously inoculated in the tongue with O UKG 1. Two of these studies were as described by Cox et al., (this proceedings), except that some animals were retained beyond 8 days for up to 168 days post challenge (dpc, study 1) or up to 15 dpc (study ). In each case, twenty recipient cattle were vaccinated, whilst 5 remained unvaccinated and all were challenged 1 days after vaccination by 5 days of co-mingling with 5 donor cattle. The vaccinated and unvaccinated recipients and the donors were then split into three separately housed groups. The difference between studies 1 and was that a x1 payload of vaccine was used in study. In a third study, six calves were vaccinated on three occasio at 1 day intervals with the x1 payload of vaccine. They were challenged 35 days after the last vaccination by 5 days of co-mingling with 4 donor cattle derived from the unvaccinated control group of study, at the point when these calves had been removed after their own 5 days exposure to. In each study, samples of blood and oesophago-pharyngeal (OP) fluid were collected periodically for serology and virus detection respectively. The IgA ELISA was based on a modification of the SPCE (Paiba et al., 4) using plates coated with rabbit anti-fmdv and subsequently loaded with O Manisa virus capsid antigen. Thereafter, a dilution of saliva was added and IgA was detected with a conjugated polyclonal antibody to bovine IgA. ELISA results were expressed as optical deity values. Serology was performed with three commercially available ELISA tests for antibodies to non-structural protei (NSP) of FMDV according to the manufacturers itructio. The three tests were the Cedi-Diagnostics FMDV-NS test, the UBI FMDV 44

2 NSP ELISA and the Bommeli CHEKIT-FMD-3ABC. Since the Cedi test detected the most infected vaccinates from study 1, it was the only method used to analyse sera from studies and 3. Results The frequency distribution of ELISA result values for the 173 saliva samples obtained from FMDV naïve cattle are presented in Fig 1. The median category value was the range from.11 to.. At a cut-off of.47 (mean plus three standard deviatio) the specificity of the test was 97.1%, whereas increasing the cut-off to.6 yielded a test specificity value of 99.4%. None of the vaccinated cattle from any of the challenge tests showed sig of clinical FMD, whereas the control, unvaccinated cattle were all severely affected. Similarly, virus was only detected in the blood of unvaccinated cattle. Results of virus isolation and RT-PCR on OP samples of the cattle from studies 1 and are summarised in Figs and 3 respectively. Despite the absence of clinical sig and viraemia, FMDV was detected in OP fluids from most of the vaccinated cattle as well as in all of the unvaccinated animals. Two groups of animal could be delineated; (1) those from which FMDV could not be detected or detected only traiently, and () those that became persistently infected. FMDV was detected in OP samples from nine unvaccinated cattle from study 1 up to and beyond 8 dpc, and from three such cattle in study. FMDV was also detected repeatedly in OP samples of two other vaccinated cattle from study, up to, but not beyond 8 dpc. FMDV persistence in OP fluids beyond 8 dpc was noted in three unvaccinated cattle from study, two regularly (UY95, UY96) and one on an isolated occasion (UY93). In Study 3, no virus was recovered from OP fluids collected at any time after challenge of the multiply vaccinated cattle. However, RT-PCR results are still awaited. NSP serology revealed seroconversion in all of the unvaccinated contact challenged cattle and in study 1, this result was obtained with all three tests. However, only two sera obtained from the study 1 vaccinates that did not become persistently infected were scored positive by NSP serology (UV at 16 dpc and UV8 at 4 dpc both by the Cedi test). The numbers of persistently infected study 1 cattle that were scored positive by NSP serology is summarised in Fig 4, showing a coistently superior seitivity with the Cedi test. The two persistently infected cattle that were never scored as NSP seropositive in any test were UV and UV14. In the second study, using the Cedi test, 6 animals were scored positive at or beyond 8 dpc on most occasio, namely UY7, UY76, UY79, UY83, UY87 and UY9.. From Fig 3, it can be seen that all of these cattle were subclinically infected with FMDV, but only UY76, UY83 and UY9 were found to be persistently infected by virus isolation beyond 8 dpc. Two of the multiply vaccinated cattle (VC14 and VC19) seroconverted in the Cedi test,after challenge. However these two animals revealed higher percentage of inhibition after their third vaccination. The numbers of persistently infected vaccinated cattle from study 1 scored positive by IgA ELISA, when.6 was used as the cut-off OD value, are summarised in Fig 4. An equal or greater proportion was scored positive as when the Cedi NSP serological test was used (up to 8 are positive for IgA and 7 positive for the Cedi test). One persistently infected vaccinated animal was completely undetected by IgA ELISA and this was UV5, which was detected by NSP serology, whilst two animals UV and UV14 were not detected at all by NSP serology. In study, the six persistently infected cattle (three vaccinates and three non-vaccinates) scored positive by IgA ELISA, although UY9 had a test OD value that fluctuated between.3 and.7 in the period between 8 and 15 dpc and in the nonvaccinated animal UY93, from which virus was only detected once after 8 dpc, the IgA respoe was not maintained. Many, but not all of the vaccinated and non-vaccinated cattle identified as traiently infected by virus isolation and RT-PCR were also scored positive in the IgA test; elevated IgA levels were also traient in most cases, but persisted longer in some individuals (UV18, UY7). Representative respoes from study are shown in Fig 5. IgA results for study 3 are depicted in Fig 6. A moderate elevation in IgA was found in some cattle after the second or third vaccination, but only in two cattle, VC15 and VC18, did the OD values exceed.5. Two of the cattle showed elevatio in FMDV-specific IgA between three and five weeks after challenge and these were also scored positive by the NSP Cedi test. Discussion A FMDV-specific IgA ELISA has been developed that gave strong OD signals with saliva from FMDVinfected cattle. Regardless of vaccination status, levels of virus-specific IgA became elevated after and persistence of IgA was correlated to viral persistence in OP fluids. Increases in IgA could be detected in vaccinated cattle that had become subclinically infected after exposure to FMD affected donor cattle. The IgA test therefore has coiderable potential for the detection of subclinical in vaccinated cattle and for identification of persistently infected cattle following the application of a vaccinate-to-live policy. Nevertheless, more work is needed to evaluate the method with larger numbers of samples from the different categories of naïve, vaccinated and vaccinated-and-infected 441

3 cattle as well as with samples from other species and after vaccination/ with other FMDV serotypes. Field testing is also required. These studies are currently underway. Coiderable fluctuation was sometimes noted in the OD values obtained with serial saliva samples obtained from the same animal. This may be related to physiologic changes in the concentration of salivary protei and requires further study. Test values could benefit from being normalised to the value obtained with a standard control sample. References Amadori, M., Haas, B., Moos, A. & Zerbini, I.. IgA respoe of cattle to FMDV in probang and saliva samples. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Borovets, Bulgaria, 5-8 September. Rome: FAO, Appendix 9: Archetti, I.L., Amadori, M., Donn, A., Salt, J. & Lodetti, E Detection of foot-and-mouth disease virus-infected cattle by assessment of antibody respoe in oropharyngeal fluids. J. Clin. Microbiol. 33: Garland, A.J.M The inhibitory activity of secretio in cattle agait foot and mouth disease virus. PhD thesis, London School of Tropical Hygiene and Medicine, London University and The Animal Virus Research Ititute, Pirbright, Surrey May Moonen, P., Jacobs, L., Crienen, A. & Dekker, A. 4. Detection of carriers of foot-and-mouth disease virus among vaccinated cattle. Vet Microbiol. 13: Paiba, G. A., Anderson, J., Paton, D. J., Soldan, A. W., Alexandersen, S., Corteyn, M., Wilsden, G., Hamblin, P., Mackay, D. K. J. & Donaldson, A. I. 4. Validation of a Foot-andmouth disease antibody screening Solid-phase competition ELISA (SPCE). J. Virol. Methods 115: Salt, J.S., Mulcahy, G. & Kitching, R.P Isotype-specific antibody respoes to foot-andmouth disease virus in sera and secretio of "carrier' and "non-carrier' cattle. Epidemiol. Infect. 117: Acknowledgements This work has been funded by the UK Department for Environment Food and Rural Affairs through grant SE918. Fig 1. Frequency distribution for ELISA results with saliva from 173 naïve cattle 1 1 Frequency <= Optical deity (FMDV IgA ELISA) 44

4 Fig. Results of virus isolation and RT-PCR on OP fluids from study 1 Animals No or traient Persistent Unvaccinated UV6 UV7 UV8 UV15 UV3 UV4 UV18 UV16 UV UV1 UV1 UV UV14 UV5 UV1 UV13 UV11 UV19 UV9 UV17 UV UV3 UV4 UV5 UV VI positive RT-PCR positive VI + RT-PCR positive Animal dead no sample Fig 3. Results of virus isolation and RT-PCR on OP fluids from study Animals No or traient Persistent Unvaccinated U7 78 UY 85 UY 88 UY 89 UY 87 UY 77 UY 73 UY 86 UY 74 UY 81 UY 7 UY 84 UY 79 UY 8 UY 9 UY 91 UY 8 UY 76 UY 83 UY 9 UY 93 UY 94 UY 95 UY 96 UY 97 VI positive RT-PCR positive VI + RT-PCR positive Animal dead no sample Results of RT-PCR testing after 8 days post challenge not yet available 443

5 Fig 4. Comparative detection of persistently infected vaccinates from study 1 Number of positive animals Weeks post challenge 4 IgA Cedi Bommeli UBI Fig 5. FMDV-specific IgA respoes detected in different categories of cattle from study Vaccinated cattle Optical Deity UY7 UY76 UY8 UY81 UY83 Non-vaccinated cattle Optical deity in ELISA UY93 UY96 UY97 444

6 Fig 6. FMDV-specific IgA respoes detected in cattle from study 3 Optical desnity in ELISA VC14 VC15 VC16 VC17 VC18 VC19 VD 7 445