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SEROLOGICAL OBSERVATIONS ON AN EPIDEMIC OF EQUINE INFLUENZA IN INDIA

Gurkirpal Singh, MVSc, PhD

S U M M A R Y

During the epidemic of equine influenza which oc- curred in India in 1987, serum samples were collected at late acute/early convalescent phase (7-9 days), at 5 weeks and at 18-23 weeks after onset of illness, from six affected horses from the Union Territory of Chandigarh, and Nawanshahr, Punjab State, India, and were examined for antibodies to A/eq-1 and A/eq-2 influenza viruses by hemagglutination inhibition (HI) tests. It was found that the antibody response to A/eq- 1 virus strains, Ludhiana/87 and Prague/56, was stronger and antibodies persisted at high levels in four animals. The fifth animal showed a diagnostic decrease in HI titers while the sixth animal seroconverted. The corresponding HI titers to A/eq-2/ Ludhiana virus showed a 4-fold decrease in all six animals.

Another nine equine animals in the single convales- cent serum samples had detectable or high HI titers against A/eq-I and A/eq-2 viruses.

In serum samples from horses and ponies, taken 5 weeks to 9 months after onset of illness, little or no difference in antibody titers to A/eq-2/Miami/63 and A/eq- 2/Fontainebleau/79 strains was found.

It seems clear that the antibody titers that ensued were indicative of recent influenza infections. Apparently, two distinct equine influenza viruses, A/eq- 1 and A/eq-2, were involved during the epidemic, infecting the equine ani- mals simultaneously in the region.

I N T R O D U C T I O N

Equine influenza is a viral disease affecting the upper respiratory tract of horses, mules and donkeys. It is Author's address: Virus Laboratory, Department of Veterinary Public Health and Epidemiology, Punjab Agricultural University, Ludhiana - 141 004, India

caused by two subtypes of the equine influenza A virus, A/ eq-1 (H7N7) and A/eq-2 (H3N8)J The A/eq-1 virus iso- lated by Sovinov~i et al.,2 although probably present for decades, has not been reported anywhere in the world since 1980. The A/eq-2 virus isolated in 1963 has been respon- sible for widespread epidemics and has become endemic in many countries. 3 Periodic antigenic drift of subtype 2 virus strains from prototype Miami/63 strain has been frequently demonstrated, 4 and outbreaks of A/eq-2 viruses often oc- cur. Epidemics arise when infected animals are introduced into a susceptible group assembled for showing, racing, breeding and sale. Serological diagnosis of equine in- fluenza is based on the appearance or increase in the titer of specific antibodies, to the serotype(s), in the convales- cent phase serum, when compared with the titer of antibod- ies in the acute phase serum.

This communication will present some serological observations, at the late stage of illness and at later inter- vals, on the 1987 epidemic of equine influenza in North India. The epidemic is of interest in that A/eq-1 virus, in addition to A/eq-2 virus, was isolated from the clinical material of affected animals. 5,6 The retrospective serology is especially important because it will yield antibody pat- tern and epidemiological information of influenza infec- tions under conditions of natural exposure to both the viral agents.

M A T E R I A L S A N D M E T H O D S

T h e e p i d e m i c The outbreak of equine influenza began in and around

Delhi in the last week of January, 1987 and subsequently spread over wide areas in North and Central India. It was seen in the Union Territory of Chandigarh in the spring (later part of February and early part of March) of 1987.

380 JOURNAL OF EQUINE VETERINARY SCIENCE

Within a few weeks, influenza infections were seen in a fairly dispersed population of equine animals in the Punjab State and in adjoining areas.

It is likely that horses which were imported from abroad to Delhi in January, 1987 were the source of infection. The subsequent assembly of these horses and other horses of different organizations at horse show or sport events at Delhi and their transport/return to various places could have led to the introduction of the disease in the region.

The clinical symptoms observed were a rectal tem- perature of 103~ to 105~ nasal discharge and conjunc- tival exudation. A dry cough was present, which lasted for 3 to 10 days. The disease was reported to have reoccurred in a few animals as per enquiries made in the region and as reported. 7

S e r a

Serum samples were collected at the late acute/early convalescent phase (7-9 days), at 5 weeks and at 18-23 weeks after the tentative date of onset of illness, from six affected horses from the Union Territory of Chandigarh, and Nawanshahr, Punjab, India. Single convalescent se- rum samples were also taken at about 4 weeks of onset of illness from another nine equine animals. Serum samples taken 5 weeks to 9 months after onset of illness from horses and ponies from various areas of the Punjab State and the Union Territory of Chandigarh were also used. Serum samples were pretreated with sodium periodate M/908 to remove non-specific inhibitors.

V i r u s s t r a i n s

The virus strains used in the hemagglutination inhibi- tion (HI) tests were :

A/eq- 1/Ludhiana/5/87 A/eq- 1/Prague/I/56 a A/eq-2/Ludhiana/8/87

In addition, A/eq-2/Miami/1/63 a and A/eq-2/ Fontainebleau/I/79 a strains were also employed.

All these strains were propagated in the allantoic cavity of embryonated eggs and pooled allantoic and amniotic fluids were clarified by low speed centrifugation for use as antigens. The A/eq-2 virus antigens were ether- treated 9 prior to use in HI tests.

H e m a g g l u t i n a t i o n i n h i b i t i o n (H I ) t e s t s

The HI tests were performed in round-bottomed microtitre perspex plates on all serum samples using the procedures advocated by W.H.O. 8 All the serum samples of animal(s) were examined together. The sera diluted in a two-fold series were incubated with 4 hemagglutinating units of virus antigen for 30 minutes at room temperature before adding the fowl red blood cells (0.5 per cent). The

aThe virus strains were supplied by Dr. B.L. Rao of the National Institute of Virology, Pune, India,

Table 1 . Serological response to natural equine influenza infections in horses.

Horse Test virus Serum titres

Days or weeks after onset of illness

7-9 5 18-23 days weeks weeks

1 Bina A/eq-1/Ludhiana/87 160 80 160 A/eq-1/Prague/56 80 80 80

Neq-2/Ludhiana/87 80 40 20

2 Kiran

3 Padmini

4 Rajni

5 Bulbul

6 Jagga

A/e( -1/Ludhiana/87 A/eq- 1/Prague/56

A/ec -2/LudhianaJ87

A/er -1/Ludhiana/87 A/eq- 1/Prague/56

A/er -2/Ludhiana/87

A/ec -1/Ludhiana/87 A/eq- 1/Prague/56

A/ec -2/Ludhiana/87

A/er -1/Ludhiana/87 A/eq-1/Prague/56

A/ec -2/Ludhiana/87

Ner -1/Ludhiana/87 A/eq-1/Prague/56

A/er -2/Ludhiana/87

320 160 160 160 160 160 160 80 40

160 ND 160 80 ND 160 80 40 20

80 80 80 40 80 20

320 320 40 160 320 40 80 40 20

<20 160 160 <20 160 80

80 40 20

Note: All the titres were expressed as reciprocal of initial dilution.

tests were repeated if controls or titers of sera examined earlier did not reproduce. A two-fold variation in titer was tolerated on repetition of the test.

R E S U L T S

The antibody response in serum samples collected at different times from affected animals are shown in Table 1. With A/eq-1 viruses, Ludhiana/87 and Prague/56, the HI antibody titers in late acute/early convalescent phase se- rum samples ranged from 80 to 320 in five animals. In four of the five animals, the titers were maintained at similar levels in 5 weeks and 18-23 weeks serum samples. In the fifth animal, however, a four-fold or greater decrease in titer from 160/320 to 40 was demonstrable between the early convalescent phase serum and the 22 weeks serum. In the remaining one animal, no A/eq-1 antibodies (<20) could be detected in serum taken at the late acute stage of illness. However, HI antibodies were demonstrated in 5 weeks serum at a titer of 160. In the 19 weeks serum, the HI antibody titer remained at the same level as the Ludhiana/ 87 virus while a two-fold decline was noticed against the Prague/56 virus antigen.

The corresponding HI antibody titers to A/eq-2/ Ludhiana/87 virus were 160/80 in late acute/early conva-

Volume 17, Number 7, 1997 381

Table 2 , Late post- infect ion HI ant ibody titers to standard A/eq-2 viruses in sera from horses and ponies in Punjab State and Chandigarh.

Days/ No. of weeks after

Place animals onset of illness

HI titer +

A/eq-2/ A/eq-2/ Miami/63 Font.179 (H3N8) (H3N8)

Jalandhar 2 5 months 20 40 20 20

Nawanshahr 2 5 weeks 80 160 40 20

3 2 months 40 20 80 40 40 40

Ludhiana 3 6 weeks 80 40 6 months 20 20

40 40

Chiniwal 2 6 weeks 80 80 80 80

Bhatinda 2 9 months 20 20 20 40

Chandigarh 1 5 weeks 80 (Union Territory)

80

+All the titers were expressed as reciprocal of initial dilution. An HI titer of 20 or greater was considered as positive evidence of infection. The ether-treated antigens of the same batch were used in the HI tests.

lescent phase serum samples (Table 1). The titers declined gradually to 40/20 in 18-23 weeks serum samples in all the six animals and the decrease was in regular fashion.

Seven of the nine single convalescent phase serum samples showed HI antibodies with titers varying from 40 to 160 for A/eq- 1 and A/eq-2 serotypes. The remaining two serum samples with detectable A/eq-1 antibodies did not show A/eq-2 antibodies (<20) (Data not tabulated).

To further investigate the antibody level to A/eq-2 viruses, 15 serum samples taken from equine animals at varied times were tested using ether-treated Miami/63 and Fontainebleau/79 virus antigens. The results showed that HI antibodies were demonstrable at titers varying from 20 to 160 (Table 2) with little or no difference in antibody levels to two standard viruses used. The antibody titers to A/eq-2/Ludhiana virus, although not depicted, were found to be similar.

D I S C U S S I O N

The results described here have indicated that HI antibody response to AJeq-1 viruses was stronger. It was demonstrable at high levels in all the samplings in four animals. On the basis of high titered antibodies, these

animals could be regarded as seropositive. The presence of high titered A/eq- 1 antibodies in these animals with little or no decrease during the period of observation is of interest. This suggests the possibility of persistence of antibodies or reinfection with the virus. In the fifth animal, the A/eq-1 antibodies showed a marked fall from a high titer proving that infection with the A/eq-1 virus has occurred. A sero- conversion for A/eq-1 viruses was noticed in the sixth animal. This indicated that the animal was infected with the A/eq-1 virus. The infection seemed to have occurred at or after the convalescent stage. This observation corroborates with the recurrence of clinical influenza in few animals.

Interestingly, significant decreases in corresponding HI antibody titers to A/eq-2 virus were seen between the late acute/early convalescent phase serum and the serum at 18-23 weeks in all six animals, indicating infection with A/ eq-2 virus.

Significant decreases in antibody titer of specific antibodies to equine influenza serotype(s), in the 18-23 weeks serum, when compared with the titer of antibodies in the late acute/early convalescent phase serum, together with a solitary case of seroconversion served as the confir- mation of the etiology of the epidemic from which both the serotypes of equine influenza virus have been isolated. 6 Therefore, they are useful for retrospective serodiagnosis of the disease. The demonstration of such antibodies may become necessary when the serum samples at the com- mencement of illness could not be obtained, or where the affected animals are first seen only in the late stage of illness.

The findings that single convalescent serum samples from nine animals showed a detectable or a high level of HI antibodies for A/eq-2 and/or A/eq- 1 virus could be consid- ered as indicative of equine influenza infections since equine animals have never been vaccinated.

The antibody level/response to both the serotypes as determined by HI tests was typical of postinfection titers as reported. 10,1 t This finding indicated that the host response was not appreciably altered by the simultaneous infection by viruses of serotype 1 and serotype 2. In other words, the affected animals could support infection with equine influ- enza A virus of distinct antigenic types at the same time, presumably competing for the same sites of replication.

The finding that horses and ponies could produce adequate antibody titers to A/eq-1 and A/eq-2 infections going simultaneously may be of interest. It could mean that natural infections in equine animals would lead to an efficient immunity to viruses of both groups.

It has been reported that in many cases, the A/eq-2 infection stimulated a rise in the HI antibody to A/eq-1 virus together with increase in antibody titer against the infecting virus. 12,13,14,15 This has been found only in ani- mals which had pre-existing antibodies against the A/eq- 1 virus.13,14 The horses recovering from the A/eq-2 infection may have antibody rises to A/eq-1 strains as well as to

382 JOURNAL OF EQUINE VETERINARY SCIENCE

certain human A-2 strains suggesting existence of shared minor antigens. 16 In the present investigation, however, such a recall of antibody to the A/eq-1 virus was not expected since there was no record of influenza vaccina- tion or previous infection in any of these animals and the serotype A/eq-1, in addition to A/eq-2, has been isolated during the epidemic. 6,~7

For several years, circulation of the A/eq-1 virus has not been demonstrated virologically and the question was asked about the serotype persistence or disappearance, ls,b Therefore, the involvement of the A/eq-1 virus in the Indian epidemic is interesting. The pattern of HI antibody level/response in affected animals in this study, the demon- stration of antibodies to A/eq- 1 and A/eq-2 viruses in the equine population (which had not been vaccinated against influenza) postepidemically 5 together with the isolation of A/eq- 1 and A/eq-2 viruses from equine animals in the acute phase of illness 6,17,19 have established that during the equine influenza epidemic, two antigenically distinct equine influenza subtype 1 and subtype 2 viruses circulated simul- taneously in different areas of North India. This is an interesting observation and is of benefit to clinical veteri- narians and virologists who deal with respiratory disease outbreaks.

With A/eq-2/Miami/63 and A/eq-2/Fontainebleau/79 antigens, serum samples taken 5 weeks to 9 months of onset of illness showed a varied HI antibody response with little or no difference in HI titers to two standard viruses. In other words, sera from naturally infected equine animals did not distinguish the two standard A/eq-2 strains used, although the virus isolates related to them were reported active at that time in the equine population. ~9,s,2~

Considerable differences in HI titers have been found with the ether-treated antigens of two A/eq-2 virus iso- lates. 21 The variations in mean HI titers with antigens prepared from either virus were as great as those found between the two viruses. However, in the present study, little or no variation in HI titers with the ether-treated antigens of two standard Miami/63 and Fontainebleau/79 strains was noticed.

Since the international spread of the virus is caused by year-round transport of horses for racing and breeding purposes, it is advisable that the animals being imported have a certificate of vaccination against equine influenza, by an authorized laboratory of the country of origin, which may include vaccinations undertaken and the immune response developed. This can be made the vital component of health certification. While under quarantine, serum HI titers to vaccination or previous infection, if any, which are

22 considered a direct reflection of the immune state maybe measured. This will minimize variations due to the tech- niques used and the subjective differences in reading HI reactions, thus ensuring accuracy. If need be, revaccination could be undertaken. At times, diagnosis by virus isolation, bpilet CH. Personal Communication. 1993.

indirect immunofluorescene 23 and enzyme immunoas- says z4,25 may be necessary for detecting subclinical infec- tion in vaccinated individuals.

The findings above greatly impact the formulation of an equine influenza vaccine containing both the serotypes and control strategies against the disease.

C O N C L U S I O N

In naturally infected horses and ponies, the HI anti- body level/response to A/eq- 1 and A/eq-2 serotypes showed a distinctive pattern. It was stronger with A/eq-1 viruses. Four-fold or greater decrease in HI titers, against one or both serotypes, from the previous titers, together with a seroconversion against equine 1 serotype was of value for retrospective diagnostic serology of equine influenza. It also is clear that the antibody titers demonstrated were apparently due to concurrent infections with A/eq- 1 and A/ eq-2 viruses.

R E F E R E N C E S

1. McQueen JL, Steele JH, Robinson RQ: Influenza in animals. Ad in Vet Sci 1968;12:285-335.

2. Sovinova 0, T&mova. B, Poutska F, Nemec J: Isolation of a virus causing respiratory disease in horses. Acta Virol 1958; 2:52-61.

3. Waddell GH, Teigland MB, Sigel MM: A new influenza virus associated with equine respiratory disease. J Amer Vet IVied Assoc 1963;143:587-590.

4. HinshawVS, Naeve CW, Webster RG, Douglas A, Skehel J J, Dryans J: Analysis of antigenic variation in equine 2 influenza viruses. Bull WId Hlth Org 1983;61:153-158.

5. Singh G: Equine inf luenza 1987, postepidemic serological study in North India. J Equine Vet Sci 1992;12:342- 344.

6. Singh G: A note on the concurrent isolation, from horses and ponies, of influenza A/eq-1 and A/eq-2 viruses from an epidemic of equine influenza in India. Comp Immunol Microbiol Infect Dis 1995; 18:73-74.

7. Uppal PK, Yadav MP, Sharma SN: Occurrence of equine influenza outbreaks in India. Indian J Comp Microbiol Immunol Infect Dis 1987;8:91-94.

8. W.H.O. Concepts and Procedures for Laboratory Based Influenza Surveillance. U.S. Department of Health and Human Services~Public Health Service~Center for Disease Control 1211, Geneva 27, Switzerland B 17-29, 1982.

9. Berlin BS, McQueen JL, Minuse E, Davenport FM: A method for increasing the sensitivity of the haemagglutination inhibition test with equine influenza virus. Virology 1965; 21:665- 666.

10. Powell DG, Thomson GR, Spooner P, Plowright W, Burrows R, Schild GC: The outbreak of equine influenza in England April/May 1973. Vet Rec 1974;94:282-287.

11. Burrows R: Equine influenza viruses - Field and experimental observations of infection and vaccination. Proc Amer Assoc Equine Pract, 1979;p37-48.

12. Lief FS, Cohen D: Equine influenza. Studies of the virus and of antibody patterns in convalescent, inter-epidemic and post- vaccination sera. Amer J Epiderniofogy 1965;82:225-246.

13. Minuse E, McQueen JL, Davenport FM, Francis T: Studies of antibodies to 1956 and 1963 influenza viruses in horses and man. J Immunol 1965; 94:563-566.

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14. Paccaud MF, Couard M, B~rki E, Gerber H, L6hrer J: Outbreak of influenza A/eq-2 in Switzerland. Nature London 1966; 211:101-102.

15. Rose MA: Serological studies with equine influenza viruses. Brit Vet J 1966; 122:435-442.

16. Gillespie JH, Timoney JF: Hagan and Bruner's Infectious Diseases of Domestic Animals with Special Reference to Etiology, Diagnosis, & Biological Therapy. 7th Ed 1981; Comstock Publishing Associates, Cornell University Press, Ithaca. p 709.

17. Singh G: Characterization of A/eq-1 virus isolated during the equine influenza epidemic in India. Acta Virol 1994; 38:25-26.

18. BLirki F: Equine influenza undated. 1 lth Internat'l Symp World Assoc Vet Microbiologists Immunologists & Specialists in Infectious Diseases, held in Perugia - Montova, Italy Oct. 2-6, 1989; 279-284.

19. Singh G, Oberoi MS, Kwatra MS, Gill SS: Isolation of

influenza virus from horses in the equine influenza outbreak of 1987. Curr Sci 1987;56:1285-1286.

20. Uppal PK, Yadav MP, Oberoi MS: Isolation of A/eq-2 virus during 1987 equine influenza epidemic in India. Equine Vet J 1989;21:364-366.

21. Burrows R, Denyer M, Goodridge D, Hamilton F: Field and laboratory studies of equine influenza viruses isolated in 1979. Vet Rec 1981 ; 109:353-356.

22. Bryans JT, Doll ER, Wilson JC, Zent WW: Epizootiologic features of disease caused by Myxovirus inf/uenzae A equine. Amer J Vet Res 1967;28:9-17.

23. Anestad G, Maagaard 0: Rapid diagnosis of equine influenza. Vet Rec 1990;126:550-551.

24. Mumford JA: Equine Infectious Diseases V/. Eds W PIowright, PD Rossdale, JFWade. Newmarket, R&W Publications 1992;p207.

25. Chambers TM, Shortridge KF, Li PH, Powell DG, Watkins KL: Rapid diagnosis of equine influenza by the Directigen FLU-A enzyme immunoassay. Vet Rec 1994;135:275-279.

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