Hong Kong influenza infection in swine: experimental and field observations

Bull. Org. mond. Sante 1972, 47, 489-491 Buill. Wid Hith Org. Hong Kong influenza infection in swine: experimental and field observations W. D. KUNDIN I & B. C. EASTERDAY 2 The recovery of influenzaviruses indistinguishable from A/Hong Kong/68 virus from pigs in Taiwan in 1970 provided the first direct evidence of the interspecies transfer of influenzaviruses. Pigs were infected experimentally with that virus and transmitted the virus to pen mates. There was no evidence of A/Hong Kong/68 antibodies in serum col- lected from pigs prior to 1968. Such antibodies were detected in serum collected from pigs that lived during the period when Hong Kong influenza was epidemic in the human population. Because of the recent isolations of the A/Hong Kong/68 influenzavirus from naturally infected pigs and the subsequent appearance of antibodies in swine in Taiwan (Kundin, 1970) a study was initiated to observe the course of infection in experimentally infected pigs. Serological studies were also carried out to ascertain whether antibody against human influenzaviruses was present among pigs in the north-central area of the USA. MATERIALS ANID METHODS Experimental infection ofpigs Approximately 1 x 104 EID50 (embryo median infectious dose) of second-egg-passage A/Hong Kong influenzavirus, originally isolated from domestic pigs in Taiwan (A/swine/Taiwan/7251/70(H3N2)),3 were administered to 5 three-month-old pigs by intra- nasal instillation. Five uninfected contact pigs of the same age were kept in the same isolation room (floor area approximately 15 m2). Nasal swabs were taken prior to infection and on post-infection days 3, 5, 7, 9, 11, 13, and 15. Each swab was placed in 2 ml of broth containing peni- cillin and streptomycin. The broth was filtered through a membrane filter (average pore diameter 0.22 ,um), inoculated by the allantoic route into 10-day-old embryonated hens' eggs, and incubated for 40-44 hours at 33.5°C. The allantoic fluids were 1 Department of Veterinary Science, University of Wis- consin, Madison, Wis., USA (requests for reprints should be sent to this address). ' Present address: World Health Organization, Djakarta, Indonesia. then tested for the presence of virus by haemag- glutination techniques using chicken erythrocytes. The recovered viruses were identified by the haemag- glutination inhibition (HI) technique using appro- priate reference antisera. There were no blind passages of allantoic fluids that did not contain haemagglutinin. Serum was collected from all pigs prior to infection and on post-infection days 15, 22, and 33 and was tested for the presence of influenza antibodies by the HI test. Rectal temperatures of each pig were recorded daily from 1 day before until 10 days after exposure. Serum collection from pigs Sera were obtained at a local abattoir from sows (2-4 years of age) and " market " pigs (6-7 months of age) in October, November, and December 1970. Most of these pigs were from Wisconsin, but a small proportion may have come from adjacent states. These and other swine sera, collected from Wisconsin farms for other purposes in previous years, were tested for the presence of influenza antibodies by the HI test. Serological testing Sera were tested by standard HI techniques using plastic trays (WHO Expert Committee on Respi- ratory Virus Diseases, 1959). The antigens used were: human A/Hong Kong/1/68(H3N2) virus (inhi- bitor resistant), A/Singapore/1/57(H2N2), A/swine/ 3 Kindly provided by Dr Walter Dowdle, Center for Disease Control, Atlanta, Ga., USA. 2921 - 489 W. D. KUNDIN & B. C. EASTERPAY Wisconsin/68(HswlNl), and A/equine/l/Miami/ 63(Heq2Neq2). Sera were treated with trypsin and potassium periodate prior to testing. All sera that were positive against A/Hong Kong or A/Singapore viruses at a dilution of 1: 40 were retested after treat- ment with receptor-destroying enzyme (RDE) (WHO Expert Committee on Respiratory Virus Diseases, 1959). RESULTS Experimentally infected pigs Virus was recovered on the third day after in - fection from the 5 pigs that were exposed intra- nasally to A/swine/Taiwan/70 virus and from 2 of these pigs on post-exposure day 5. Virus was not demonstrated in any of the swabs collected from the 5 contact pigs. The recovered viruses reacted specifically against reference A/Hong Kong anti- serum in HI tests. HI antibody was detected in all of the contact pigs in the experiment, with titres ranging from 1: 40 to 1: 160, indicating that these animals be- came infected during the experiment. HI antibody against A/Singapore/1/57, A/swine/Wisconsin/68, and A/equine/l/Miami/63 was not detected in any of the sera. There were no overt signs of disease during the 15-day observation period. Occasional rectal tem- peratures of 40.5-41.50C were recorded but they appeared to be related more to the state of agitation of the pigs than to the viral infection. Thus, under the conditions of our test procedures, we found local pigs to be readily infected with the influenzavirus isolated from pigs in Taiwan that is antigenically indistinguishable from the human A/Hong Kong influenzavirus. The virus was re- covered from their nasal secretions, and although it was not possible to recover the organism from uninoculated contact pigs, such infection was demon- strated serologically. Field observations The results of the serological tests made on swine sera collected in Wisconsin are shown in Table 1. None of the 147 pigs bled prior to the emergence of the A/Hong Kong influenzavirus had antibodies against it or the A/Singapore/57 virus. None of the 6-month-old market pigs bled in the autumn of 1970 had antibodies against human influenza antigens. However, 8 of the older sows, bled at the same time, had antibody against the A/Hong Kong virus. The demonstration of A/ Singapore antibodies in two sera was probably a false positive result reflecting serological cross-reac- tions (see Table 2). The number of sera that were positive against swine influenza antigen is usual for this area. It was mentioned above that the sera were initially pretreated with trypsin and potassium periodate, and that all the sera that inhibited the A/Hong Kong and A/Singapore antigens were retested after RDE treatment. The results of the two tests are shown in Table 2. After the trypsin-periodate treatment, 13 of the 597 sera inhibited the A/Hong Kong antigen and 9 sera inhibited A/Singapore antigen. After RDE treatment 8 of 13 sera were still positive against A/Hong Kong. The five that became negative had previously had an HI titre Table 1. No. of swine sera with influenza antibodies in the haemagglutination inhibition test Antigens Dates of Age of Number Albleeding pigs tested A/Hong A/ A/ equine/ Kong/68 Singapore/57 swine/68 Mami/63 1963-1968 mixed 147 0 0 22 0 fall, 1970 % year 150 0 0 11 0 fall, 1970 2-4 years 300 8 2a 65 1 b a See table 4. b Endpoint of 1 : 80. Endpoint was 1 : 640 against A Hong Kong/68. 490 HONG KONG INFLUENZA INFECTION IN SWINE 491 Table 2. No. of swine sera positive in the haemag- glutination inhibition test: comparison of results after pretreatment with trypsin-periodate (T-P) and receptor destroying enzyme (RDE) Antigen and Pretreatment HI titre A/HK/68 A/Sing./57 T-P RDE T-P RDE 1:40 8 1 1:80 2 6 1a 1:160 3 1 8 1b 1 :320 2 2 1 1:640 3 total 13 8 16 2? a Also seropositive against A/Hong Kong/68 at 1: 640. b Also seropositive against A/swine/Wisconsin/68 at 1: 160. of 1: 40. Those that remained seropositive generally had a higher endpoint after RDE treatment. When the 9 sera with antibodies to A/Singapore/ 1/57 virus were retested after RDE treatment 2 re- mained positive. Because the two seropositive sera also reacted against other influenza antigens it is probable that the A/Singapore antibodies were more apparent than real. DISCUSSION The data presented indicate that pigs are readily infected experimentally with the swine/Taiwan/70 virus, a strain that is antigenically indistinguishable from the human A/Hong Kong/68 virus, and that infection can be transmitted to other pigs. Our failure to recover the virus from nasal secre- tions of contact infected pigs may have been occa- sioned by transient or low-level excretion of virus. The studies of swine sera collected from the abattoir and from farms provide no evidence of A/Hong Kong virus infections in Wisconsin pigs prior to the summer of 1968 or since the spring of 1970. However, 2.5% of the sera tested from sows indi- cate that natural infections have occurred since the appearance of the Hong Kong virus in July 1968. Although the evidence is strong that Wisconsin pigs were indeed naturally infected, the numbers involved were too small to suggest that the infection was anything more than a casual one in the area studied. The true role of domestic pigs in the epi- demiology of human influenza infections remains to be elucidated. ACKNOWLEDGEMENTS The authors thank Mrs Henrietta Peterson and Miss Susan Ayres for their invaluable technical assistance. The study was supported in part by funds provided by the World Health Organization. RESUMt INFECTION PAR LA GRIPPE DE HONG KONG CHEZ LE PORC: OBSERVATIONS EXPERIMENTALES ET DONNEES RECUEILLIES SUR LE TERRAIN On a reussi sans difficulte a infecterdes porcs A I'aide du virus A/swine/Taiwan/7251/70, souche recemment isol6e chez des porcs domestiques A Taiwan et antigenniquement indiscemable du virus de la grippe humaine A/Hong Kong/68. Le virus a e retrouve apres quelques jours chez tous les animaux inocules. Des porcs non infectes directement mais parques dans lememe enclos ont contracte l'infection par contact: chez eux, le virus n'a pu etre isole, mais tous ont elabore des anticorps inhibant l'hemagglutination actifs contre le virus A/Hong Kong/68. D'autre part, aucun des 147 serums preleves chez des porcs de 1963 A 1968, avant I'apparition du virus A/Hong Kong/68, ne contenait d'anticorps pour ce virus. En re- vanche, de tels anticorps ont et6 deceles dans 8 s6rums por- cins sur 300 examin6s en 1970. REFERENCES Kundin, W. D. (1970) Nature (Lond.), 228, 857-858 WHO Expert Committee on Respiratory Virus Diseases (1959) WId Hlth Org. techn. Rep. Ser. No 170