Neutralizing Activity in Bovine Secretions against Foot-and-Mouth Disease Virus

In 1976 and 1984, cattle and buffalo in northwestern India experienced major outbreaks of foot-and-mouth disease (FMD) due to type Asia 1. Normally, FMD virus (FMDV) type O is predominant in this region. Outbreaks of disease caused by FMDV Asia 1 show periods of low incidence and then major reemergence. Mechanisms of persistence of FMDV in ruminants remain unclear. The primary site for FMDV replication and persistence is the oropharyngeal area. Thus, from an epizootiologic point of view, neutralizing activity against FMDV in buccal secretions of ruminants is of special interest. In this study, neutralizing activity against FMDV type Asia 1 in nasal and buccal secretions of calves was measured after vaccination and/or infection with FMDV type Asia 1. Fourteen calves, 6-8 months old, were kept in an isolation facility. The calves were dewormed with fenbendazole and sprayed with malathion, and their peripheral blood smears were screened for hemoprotozoa. Esophageal-pharyngeal fluids were tested for persistent FMDV. Calves were randomly divided into 4 groups (3-4 calves/ group). Each calf in groups I and III was vaccinated with monovalent FMDV Asia 1 vaccine. Secretions were collected 0, 14, 21, and 28 days after vaccination. After 21 days, vaccinated calves in group I and unvaccinated calves in group II were challenged with 10,000 TCID50 of FMDV Asia 1 by the intradermolingual route, and secretions were collected 7, 14, and 21 days postchallenge. Calves in group IV were unvaccinated, uninfected controls. The seed stock of FMDV Asia l was passaged 4 times in baby hamster kidney-21 cell line and stored at -20 C. The calves were vaccinated with tissue-culture-propagated formalinand heat-inactivated aluminum-hydroxide-adsorbed FMDV Asia I vaccine (2.5 ml/calf). Before collection of secretions (nasal and oral), the calves were kept off feed overnight and were only offered drinking water. Secretions were collected from these calves by methods previously described. Secretions were centrifuged at 5,000 rpm for 20 minutes to sediment any feed particles. The samples were inactivated at 56 C for 30 minutes and dialyzed against isotonic saline (100 times the volume of the sample) for 18 hours at 4 C. Before testing, all the samples were checked for contamination with blood by Adler’s benzidine base test for hemoglobin. The samples were stored at -20 C. A neutralization test was performed using constant virus and varying secretion method. Serial dilutions of the samples were prepared in phosphate-buffered saline (pH 7.2) containing penicillin (1,000 IU/ml) and streptomycin (500 μg/ ml). An equal volume of virus dilution was added to each sample dilution to give a final virus concentration of 100 MLD50 (mouse lethal dose). The virus-sample mixture was incubated at 37 C for 30 minutes and inoculated intraperitoneally into suckling mice (50 μl/mouse). Neutralization titers were calculated by the Reed-Muench formula and expressed as the log,, of the reciprocal of the sample dilution protecting 50% of the mice inoculated with the sample-virus mixture. 10 Neutralization titers in pooled secretion samples (3-4 calves) are shown in Fig. 1. In uninfected, unvaccinated control calves, secretions (nasal and esophageal-pharyngeal fluid) showed no neutralizing activity. However, the saliva had a titer of log100.8-1.2. The neutralizing factor present in normal saliva has not been completely characterized. It is dialyzable, heat labile at 70 C, insensitive to sodium azide, insensitive to mercuric chloride, and insensitive to iodo acetamide. After vaccination, calves developed neutralizing activity against FMDV in the secretions. However, another research group failed to observe neutralizing activity up to 30 days after vaccination with an inactivated vaccine by subcutaneous or nasopharyngeal route. Reason for the failure to observe neutralizing activity in their study may be the diluted nasal secretions (total protein, 2 mg/ml) used, as compared with those of our study (total protein, 10 mg/ml). We observed neutralizing activity in nasal secretions collected 45 days postvaccination (log,, 1.05) and 60 days postvaccination (log100.6). Among the 4 groups of calves, the unvaccinated challenged group developed higher neutralizing activity than did vaccinated-challenged and vaccinated groups (Fig. 1). Among the 3 secretions, saliva had the highest neutralizing activity, followed by esophageal-pharyngeal fluid and nasal secretions. In our study, neutralizing activity was higher than that reported in European cattle for FMDV types O, A, and C. This higher activity could be due to differences in host genotype, in type of FMDV used, or in assay systems (in vivo mouse protection test vs. in vitro virus neutralization test in cell culture). Bos indicus responds better to keyhole limpet hemocyanin than does Bos taurus at the mucosal surfaces. Neutralization titers in our study did not correlate with immunoglobulin levels measured by single radial immunodiffusion (data not shown). Secretions tested by the mouse bioassay system showed From the Department of Veterinary Diagnostic Investigation, Colatypical neutralization activity with nonneutralized virus lege of Veterinary Medicine, Manhattan, KS 66506 (Kapil), and the fractions. In a few initial dilutions, although the antibody Department of Veterinary Microbiology, College of Veterinary Medconcentrations were high, FMDV was not neutralized. In the icine, CCS Haryana Agricultural University, Hisar, Haryana, India next few dilutions of the secretion samples, there was com(Ahuja, Prasad). Received for publication March 3, 1994. plete FMDV neutralization. On subsequent dilutions, there was no neutralization. Similar observations have been made