Antibodies against human types A and B influenzaviruses in the sera of animals in Spain*

Bull. Org. mond. Santd 1 48 378-380 Bull. Wid Hith Org. Antibodies against human types A and B influenzaviruses in the sera of animals in Spain * M. VICENTE,' M. V. FERNANDEZ, M. P. PEREZ BRENA, E. VALLE, & R. NAJERA Abstract Sera from several animal species were subjected to serological tests for antibodies against types A and B influenzaviruses of human origin. Of the swine sera, 13% were positive by the haemagglutination-inhibi- tion test for the human A strain and, of those, 68% were also positive by the neuraminidase-inhibition test. None of the sera tested was positive for the B strain. Kaplan (1969) and Tumova & Easterday (1969) have suggested that genetic recombination of human and animal influenzaviruses could account for the antigenic variation that some of them show. Many studies of animal influenzaviruses and their relation- ship to human strains have since been undertaken in various countries. Especially relevant are the studies of Harkness et al. (1972) and Schild et al. (1972) on the relationship between human and porcine influenza. Webster & Laver (1972) have presented evidence that, in pigs, under natural conditions of transmission, human and swine influenzaviruses may recombine genetically. Similarly, the virus of fowl plague and turkey influenza may recombine in turkeys. The observations that we have made in Spain confirm those made in other countries. Materials and methods The presence of specific antibodies against human influenzaviruses of types A and B was studied in 497 sera from 4 animal species: (1) 57 quail sera, obtained between February and October 1970 from quail farms in the province of Madrid; (2) 40 sera from migrating wild pigeons caught in October 1971 at Echalar, province of Navarra; (3) 100 sera from healthy sheep, collected between November 1971 and April 1972 from the main slaughter-house in Madrid; and (4) 300 sera collected from pigs at the same time and place as the sheep sera. * From the National Centre for Public Health Virology and Ecology, Majadahonda, Madrid, Spain. 1 Present address: Instituto Nacional de Bacteriologia, Santiago, Chile. The techniques used were haemagglutination- inhibition (HI), neuraminidase-inhibition (NI), im- munoprecipitation (IP), and complement fixation (CF). For HI, the Takatsy micromethod (modified by Sever, 1962) in plastic plates and the Cooke Engineer- ing Co. microtitre technique were employed. Hen red cells in a 0.5% dilution and 4 HA units of influenzavirus, each in a volume of 0.025 ml, were used. The diluent was phosphate buffer saline O.1M with a pH of 7. NI was performed by the technique of Laver & Kilbourne (1966) with fetuin as the sub- strate and an incubation period of 18 h at 37°C. Titres were expressed as the serum dilution capable of inhibiting 50% of the enzymatic activity, accord- ing to Schild & Newman (1969). The neuraminidase source was the X-3 1 influenzavirus recombinant from A/Hong Kong/68 (H3N2) and A/PR/8/34 (HONI) (Kilbourne, 1969). The method of Schild & Pereira (1969) was used for IP, and CF was carried out with the micromethod described for HI, using Mayer-Croft solution as the diluent. Two different influenzaviruses of human origin were employed: A/Madrid/71 (H3N2)-a strain antigenically similar to A/Hong Kong/68 (H3N2)- and B/Barcelona/71 (similar to B/Italia/66), which Professor A. Pumarola sent to us from Barcelona. Both strains were used in infected chorioallantoic fluid and stored at -70°C. X-31-infected chorio- allantoic fluid, concentrated and purified according to the method of Schild et al. (1972), was used as the antigen for NI and IP. The treatments used to remove nonspecific in- hibitors were: (a) trypsin, heat, and periodate (Jensen, 1956), followed by a further periodate treat- ment (Kaplan & Payne, 1959) for 1 h at room tem- perature; (b) kaolin and kaolin-mercaptoethanol, as described by Stewart et al. (1967) and Banatvala et al. (1967) for rubella; and (c) receptor-destroying enzyme, as described by Robinson & Dowdle (1969). In order to remove nonspecific haemagglutinins, a 50% suspension of hen red cells in PBS was added to the individual sera in a proportion of 1: 10. 3020 378- BRIEF COMMUNICATIONS 379 Table 1. Results of the Hi test for influenzavirus in swine sera tested against A/Madrid/71 (H3N2), by age group No. of sera with: No. of TotalAge sea negative aoiietirst positiveof animal sera titres <8 8 16 32 64 128 No. % 45-65 days 100 100 - - - - - - 0 6-8 months 100 69 18 8 2 1 2 31 31 >2years 100 91 3 1 1 1 3 9 9 Total 300 260 21 9 3 2 5 40 13 a The titres are expressed as the reciprocal of the highest dilution giving 100 % inhibition. ,After having remained for 1.5 h at room temperature, the sera were centrifuged. Results The results obtained for the swine sera are shown in Table 1 for the A strain, by age groups. No positive results were found in the lower age groups; the proportions of positive results in the intermediate age group (6-8 months) and higher age group were 31 % and 9%, respectively; for all groups together, the proportion was 13% (confidence limits at P = 0.95: 9-17%). Of the 40 swine sera that were positive by HI, 27 (68%) were also positive by NI. IP and CF gave negative results for all 40 sera. The 97 sera from quail and pigeons were negative for both human influenzavirus strains. Of the 100 sheep sera, 6 were positive by HI for the A strain, although the titres were low. These 6 positive sera were negative by NI. All sera were negative for the type B influenzavirus strain. Discussion Only among the swine sera were positive results observed. The finding of 6 positive sera among the 100 sheep sera is most probably attributable to non- specific inhibitors. However, as all the sheep were more than 3 years of age, it is theoretically possible that there were cross-reactions with strains of the A type that had infected the sheep before A/Hong Kong/68 became prevalent. The sera from quail and pigeons were negative. It should be mentioned that the quail were raised on farms, whereas the pigeons were wild birds, captured while returning to their southern winter habitat from summer nesting areas in northern Europe. From the positive results in swine, it seems as if the younger animals (45-75 days of age) had had no contact with the human infiluenzavirus strains under study and had not received maternal antibodies. In the older swine, however, the presence of specific antibodies to viruses closely related to A/Madrid/71 (and therefore to A/Hong Kong/68) but not to B/Barcelona/71 would indicate such contact. The positive results confirmed by NI but not reconfirmed by IP and CF may have stemmed from less recent infections. The findings of the present study in Spain confirm similar observations made in other countries and suggest that, during the period 1969-72, type A influenzaviruses infected pigs in Spain and that these viruses were closely related to influenzavirus A/Hong Kong/68, which produced outbreaks in human beings during the same period. ACKNOWLEDGEMENTS The authors thank Dr G. C. Schild, Medical Research Council, London, for his constructive criticism; Dr J. J. Vifies, Provincial Chief of Health, Navarra, for having collected the sera of wild pigeons; and Professor A. Puma- rola, for supplying the influenza B strain isolated in his laboratory in Barcelona. One of the authors (M.V.) received a fellowship from the Institute of Hispanic Culture. REFERENCES Banatvala, J. E. et al. (1967) Brit. med. J., 3, 285-286 Harkness, J. W. et al. (1972) Bull. Wld Hith Org., 46, 709-719 380 BRIEF COMMUNICATIONS Jensen, K. E. (1956) Influenza viruses. 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