ORIGINAL ARTICLES ISOLATION OF MURRAY VALLEY ENCEPHALITIS VIRUS FROM SENTINEL CHICKENS J. CAMPBELL, F.I.M.L.T., and D. E. HORE,B.V.Sc., Ph.D.
Attwood Veterinary Research Laboratory, Department of Agriculture, Westmeadows, Victoria, 3047 Epizootiological investigations on arboviruses of groups A and B were initiated in 1971 following reports of encephalitis in horses in northern Victoria. Although none of the group A viruses that have been isolated in Australia is known to cause encephalitis they are antigenically related to exotic equine pathogens and Murray Valley encephalitis (MVE) virus of group B has produced ataxia in an experimentally infected mare (Miles 1952). As part of this program and after discussions with medical authorities in Victoria and Queensland on the potential epidemiological value of sentinel flocks, 3 groups of chickens were established in the Murray Valley late in 1973. Serological tests and virus isolation attempts were carried out to monitor these chickens for evidence of infection by arboviruses of groups A and B over the following 5 months.
B!ood clots were homogenised and examined for virus by intracerebral inoculation of 2-day-old mice. Brains from mice showing signs of encephalitis were tested for agglutination of goose erythrocytes by the methods of Clarke and Casals (1958). Complement fixation tests were carried out at the Queensland Institute of Medical Research by courtesy of Dr R. L. Doherty.
Australian Veterinary Journal, Vol. 5 1 , January, 1975
1
Results
None of the blood samples collected from chickens on 20 December 1973 showed H I antibody to arboviruses of groups A and B. The results of subsequent tests for antibody and examinations of blood clots for virus are given in Table 1. Reactions to HI tests against Sindbis virus revealed 2 periods of group A arbovirus activity. Materials and Methods The first occurred in the chickens at Mildura and Eighteen-week-old White Leghorn chickens Kerang early in January and the second in all 3 from a hatchery in central Victoria were distri- groups early in March. buted in groups of 14 to Mildura, Kerang and Group B antibody reacting to MVE virus was Echuca on 20 December 1973. Chickens in each first demonstrated in blood collected on 13 Febgroup were individually identified and run in open ruary 1974 from a chicken at Echuca. Chickens pens within 100 metres of the Murray River near at Mildura and Kerang remained negative until Mildura, the Campaspe River at Echuca and in 14 March I974 when 4 and 5 chickens at the the residential area of the town of Kerang. The respective centres gave positive HI reactions. number in each group was maintained at 14 by No further change in the HI antibody status to replacement from the same source. Sindbis or MVE viruses occurred in chickens at Blood samples from each chicken collected on the 3 centres after 14 March 1974. 20 December 1973 and at intervals of 14 days After the demonstration of antibody reacting to thereafter until 9 May 1974, were held at 4°C Sindbis virus on 2 and 17 January 1974, suckling pending and during transport to the laboratory. mice were routinely inoculated with homogenates Separated serums and clots were stored at -20°C of the clots from each blood sample. Agents until they were tested. causing encephalitis in suckling mice were isolated The strains of Sindbis and MVE viruses, from b!ood samples collected from single chickens representing arbovirus groups A and B respect- in the groups at Echuca and Kerang on 31 and 27 February 1974 respectively. The ively, have been described previously (Hore et a1 Ja.;~~ai-y 1973). Antiserums against these viruses were pre- agents (designated E47 and K97) were re-isolated pared in mice and extracted with acetone prior from the clots and continued to cause a fatal to testing for haemagglutination inhibition (HI) encephalitis on passage in suckling mice. Antiantibody. Chicken serums were routinely absorbed body to MVE virus was found in the serum of with 25% acid-washed kaolin. The HI techniques each chicken 14 days after the demonstration of viraemia. were those of Clarke and Casals (1958).
TABLE 1 Antibody to Arbovirus Groups A and B and Viral Isolations in Sentinel Chickens Along the Murray Valley Distribution?
Date5
Number with HI antibody A-Sindbis
Mildura Kerang Echuca Mildura Kerang Echuca Mildura Kerang Echuca Mildura Kerang Echuca Mildura Kerang Echuca Mildura Kerang Echuca
* Not
2 January 1974
B-MVE
2
Virus Isolation Group A
Group B
*
*
17 January 1974 31 January 1974 I 3 February 1974
27 February 1974 14 March 1974
4
1
5 1
+
-
-
-
examined.
3 Samples collected 20 December 1973 prior to distribution from central Victoria .were all negative.
Results of tests on samples collected after I4 March 1974 showed no changes in antibody levels. t Groups of 14 chickens. X One of the positive chickens died between 31 January 1974 and 13 February 1974. No antibody or virus demonstrated. NO antibody o r virus demonstrated.
-
town with a population of 4,000 and was probably less favourably sited than the other groups. Although C. annulirostris has a wide distribution (Standfast and Dyce 1972) the range of vectors for MVE virus may include other species more closely associated with domestic habitats. In this context, McLean (1953a) has shown that Culex fatigans is capable of carrying MVE virus. The first case of Murray Valley encephalitis in man in the 1974 outbreak was reported early in January. In the following month further cases occurred in widely separated areas in the Murray Valley (W. J. Stevenson 1974, personal communiDiscussion cation). The sentinel flocks were established in Serum surveys (Doherty 1972; Hore et al 1973) the 3 centres on 20 December 1973 but antibody and epidemiological studies (Anderson 1954; reacting to MVE virus was not detected until 13 McLean 1953a) have suggested that birds act as February 1974. This delay in indicating virus major hosts for MVE virus and thus participate activity might reflect the small numbers of chickens in the local and distant spread of infection in in each group, deficiencies in siting or the need to Australia. The present paper reports the isolation establish sentinel flocks earlier than the latter of MVE virus from naturally infected chickens. third of December. On the other hand, group A Evidence of infection by MVE virus was antibody reacting to Sindbis virus was demondemonstrated in each of the sentinel flocks. Two strated as early as 2 January 1974 and, of the were located on the banks of large watercourses recorded group A isolates in Australia, Sindbis and were probably exposed to Culex amulirostris virus was probably responsible for the HI activity which is a known vector of MVE virus (Doherty detected. In view of the similarities in ecology 1972), and one that has a host range which in- between MVE and Sindbis viruses (Doherty 1972) cludes birds (Lee et a1 1954). The third group of the demonstration of recent infection with this chickens was run within the built-up limits of a member of arbovirus group A could be an indiA sucrose-acetone extract of brains from mice inoculated with strains E47 and K97 showed optimal agglutination of goose erythrocytes at pH 6.75. Antiserum to MVE virus inhibited agglutination by both agents and homologous virus to the same titre. No inhibition was demonstrated with antiserum to Sindbis virus. The results of complement fixation tests with the E47 and K97 agents and antiserum to MVE, Kunjin, Alfuy and Japanese B encephalitis viruses were consistent with the identification of both as strains of MVE virus.
2
Australian Veterinary fournal. V d . 51, January, 1975
cation for a wider regional search for MVE virus activity in chickens of a suitable age. Viraemia with titres up to EID,,/ml has been demonstrated in young chickens for 5 to 8 days after inoculation with MVE virus (McLean 1953b). Although there is no information on the duration of viraemia in natuarlly infected chickens, it is likely that virus would be present in the blood for several days. Murray Valley encephalitis virus was isolated from blood collected from a chicken in the Echuca group on 31 January 1974. In the presence of intensive mosquito activity at that time, the failure to demonstrate evidence of infection in any other member of this group and the delay in demonstration of antibody in the other groups raises the question of the efficiency of chickens as sentinels for MVE virus activity. Chickens are convenient for this purpose but it seems desirable that the use of other domestic and possibly native hosts of MVE virus in this capacity should be investigated. Summary
Sentinel chickens were established in 3 centres along the Murray Valley on 20 December 1973. The demonstration of antibody in the serum of chickens in the MiIdura and Kerang areas indicated Sindbis virus activity late in December 1973 and early in January 1974. Tests for antibody to MVE virus were negative until blood collected from one chicken at Echuca on 27 February 1974 and several chickens at Mil-
Austrulian Veterinary Journal. Vol. 5 1, January, 1975
dura and Kerang on 14 March 1974, showed positive HI reactions. Murray Valley encephalitis virus was isolated from blood collected from chickens at Echuca and Kerang respectively on 31 January I974 and 27 February 1974. Ackww1edgmen:s
We are grateful to Dr R. L. Doherty, Director, Queensland Institute of Medical Research, for his assistance in the further testing of the agents isolated in this investigation and to Dr W. J. Stevenson, Chief HeaIth Officer, Department of Health Victoria for the information given by personal communication. We wish to thank and acknowledge the co-operation over a long period of Messrs E. Brough, W. Powell and J. Pike of the Division of Animal Health. References Anderson, S. G . (1954)-J. Hyg. [Camb.) 52: 447. Clarke, D. H. and Casals, J. (1958)-Am. 1. trop. Med. Hyg. 7: 561. Doherty, R. L. (1972)-Aust. vet. 1. 48: 172. Hore, D. E., Campbell, J. and Turner, A. J. (1973)Aust. vet. J. 49: 238. Lee, D. J., Clinton, K. I. and O’Gower, A. K. (1954)Aust. J . biol. Sci. 7: 282. McLean, D. M. (1953a)-Aust. J . exp. Biol. med. Sci. 31: 481. McLean, D. M. (1953b)-Aust. J . exp. Biol. med. Sci. 31: 491. Miles, J. A. R. (1952)-Aust. J . exp. Bid. med. Sci. 30: 341. Standfast, H. A. and Dyce, A. L. (1972)-Aust. vet. J. 48: 77. [Received for publication 13 August 1974)
3
Attwood Veterinary Research Laboratory, Department of Agriculture, Westmeadows, Victoria, 3047 Epizootiological investigations on arboviruses of groups A and B were initiated in 1971 following reports of encephalitis in horses in northern Victoria. Although none of the group A viruses that have been isolated in Australia is known to cause encephalitis they are antigenically related to exotic equine pathogens and Murray Valley encephalitis (MVE) virus of group B has produced ataxia in an experimentally infected mare (Miles 1952). As part of this program and after discussions with medical authorities in Victoria and Queensland on the potential epidemiological value of sentinel flocks, 3 groups of chickens were established in the Murray Valley late in 1973. Serological tests and virus isolation attempts were carried out to monitor these chickens for evidence of infection by arboviruses of groups A and B over the following 5 months.
B!ood clots were homogenised and examined for virus by intracerebral inoculation of 2-day-old mice. Brains from mice showing signs of encephalitis were tested for agglutination of goose erythrocytes by the methods of Clarke and Casals (1958). Complement fixation tests were carried out at the Queensland Institute of Medical Research by courtesy of Dr R. L. Doherty.
Australian Veterinary Journal, Vol. 5 1 , January, 1975
1
Results
None of the blood samples collected from chickens on 20 December 1973 showed H I antibody to arboviruses of groups A and B. The results of subsequent tests for antibody and examinations of blood clots for virus are given in Table 1. Reactions to HI tests against Sindbis virus revealed 2 periods of group A arbovirus activity. Materials and Methods The first occurred in the chickens at Mildura and Eighteen-week-old White Leghorn chickens Kerang early in January and the second in all 3 from a hatchery in central Victoria were distri- groups early in March. buted in groups of 14 to Mildura, Kerang and Group B antibody reacting to MVE virus was Echuca on 20 December 1973. Chickens in each first demonstrated in blood collected on 13 Febgroup were individually identified and run in open ruary 1974 from a chicken at Echuca. Chickens pens within 100 metres of the Murray River near at Mildura and Kerang remained negative until Mildura, the Campaspe River at Echuca and in 14 March I974 when 4 and 5 chickens at the the residential area of the town of Kerang. The respective centres gave positive HI reactions. number in each group was maintained at 14 by No further change in the HI antibody status to replacement from the same source. Sindbis or MVE viruses occurred in chickens at Blood samples from each chicken collected on the 3 centres after 14 March 1974. 20 December 1973 and at intervals of 14 days After the demonstration of antibody reacting to thereafter until 9 May 1974, were held at 4°C Sindbis virus on 2 and 17 January 1974, suckling pending and during transport to the laboratory. mice were routinely inoculated with homogenates Separated serums and clots were stored at -20°C of the clots from each blood sample. Agents until they were tested. causing encephalitis in suckling mice were isolated The strains of Sindbis and MVE viruses, from b!ood samples collected from single chickens representing arbovirus groups A and B respect- in the groups at Echuca and Kerang on 31 and 27 February 1974 respectively. The ively, have been described previously (Hore et a1 Ja.;~~ai-y 1973). Antiserums against these viruses were pre- agents (designated E47 and K97) were re-isolated pared in mice and extracted with acetone prior from the clots and continued to cause a fatal to testing for haemagglutination inhibition (HI) encephalitis on passage in suckling mice. Antiantibody. Chicken serums were routinely absorbed body to MVE virus was found in the serum of with 25% acid-washed kaolin. The HI techniques each chicken 14 days after the demonstration of viraemia. were those of Clarke and Casals (1958).
TABLE 1 Antibody to Arbovirus Groups A and B and Viral Isolations in Sentinel Chickens Along the Murray Valley Distribution?
Date5
Number with HI antibody A-Sindbis
Mildura Kerang Echuca Mildura Kerang Echuca Mildura Kerang Echuca Mildura Kerang Echuca Mildura Kerang Echuca Mildura Kerang Echuca
* Not
2 January 1974
B-MVE
2
Virus Isolation Group A
Group B
*
*
17 January 1974 31 January 1974 I 3 February 1974
27 February 1974 14 March 1974
4
1
5 1
+
-
-
-
examined.
3 Samples collected 20 December 1973 prior to distribution from central Victoria .were all negative.
Results of tests on samples collected after I4 March 1974 showed no changes in antibody levels. t Groups of 14 chickens. X One of the positive chickens died between 31 January 1974 and 13 February 1974. No antibody or virus demonstrated. NO antibody o r virus demonstrated.
-
town with a population of 4,000 and was probably less favourably sited than the other groups. Although C. annulirostris has a wide distribution (Standfast and Dyce 1972) the range of vectors for MVE virus may include other species more closely associated with domestic habitats. In this context, McLean (1953a) has shown that Culex fatigans is capable of carrying MVE virus. The first case of Murray Valley encephalitis in man in the 1974 outbreak was reported early in January. In the following month further cases occurred in widely separated areas in the Murray Valley (W. J. Stevenson 1974, personal communiDiscussion cation). The sentinel flocks were established in Serum surveys (Doherty 1972; Hore et al 1973) the 3 centres on 20 December 1973 but antibody and epidemiological studies (Anderson 1954; reacting to MVE virus was not detected until 13 McLean 1953a) have suggested that birds act as February 1974. This delay in indicating virus major hosts for MVE virus and thus participate activity might reflect the small numbers of chickens in the local and distant spread of infection in in each group, deficiencies in siting or the need to Australia. The present paper reports the isolation establish sentinel flocks earlier than the latter of MVE virus from naturally infected chickens. third of December. On the other hand, group A Evidence of infection by MVE virus was antibody reacting to Sindbis virus was demondemonstrated in each of the sentinel flocks. Two strated as early as 2 January 1974 and, of the were located on the banks of large watercourses recorded group A isolates in Australia, Sindbis and were probably exposed to Culex amulirostris virus was probably responsible for the HI activity which is a known vector of MVE virus (Doherty detected. In view of the similarities in ecology 1972), and one that has a host range which in- between MVE and Sindbis viruses (Doherty 1972) cludes birds (Lee et a1 1954). The third group of the demonstration of recent infection with this chickens was run within the built-up limits of a member of arbovirus group A could be an indiA sucrose-acetone extract of brains from mice inoculated with strains E47 and K97 showed optimal agglutination of goose erythrocytes at pH 6.75. Antiserum to MVE virus inhibited agglutination by both agents and homologous virus to the same titre. No inhibition was demonstrated with antiserum to Sindbis virus. The results of complement fixation tests with the E47 and K97 agents and antiserum to MVE, Kunjin, Alfuy and Japanese B encephalitis viruses were consistent with the identification of both as strains of MVE virus.
2
Australian Veterinary fournal. V d . 51, January, 1975
cation for a wider regional search for MVE virus activity in chickens of a suitable age. Viraemia with titres up to EID,,/ml has been demonstrated in young chickens for 5 to 8 days after inoculation with MVE virus (McLean 1953b). Although there is no information on the duration of viraemia in natuarlly infected chickens, it is likely that virus would be present in the blood for several days. Murray Valley encephalitis virus was isolated from blood collected from a chicken in the Echuca group on 31 January 1974. In the presence of intensive mosquito activity at that time, the failure to demonstrate evidence of infection in any other member of this group and the delay in demonstration of antibody in the other groups raises the question of the efficiency of chickens as sentinels for MVE virus activity. Chickens are convenient for this purpose but it seems desirable that the use of other domestic and possibly native hosts of MVE virus in this capacity should be investigated. Summary
Sentinel chickens were established in 3 centres along the Murray Valley on 20 December 1973. The demonstration of antibody in the serum of chickens in the MiIdura and Kerang areas indicated Sindbis virus activity late in December 1973 and early in January 1974. Tests for antibody to MVE virus were negative until blood collected from one chicken at Echuca on 27 February 1974 and several chickens at Mil-
Austrulian Veterinary Journal. Vol. 5 1, January, 1975
dura and Kerang on 14 March 1974, showed positive HI reactions. Murray Valley encephalitis virus was isolated from blood collected from chickens at Echuca and Kerang respectively on 31 January I974 and 27 February 1974. Ackww1edgmen:s
We are grateful to Dr R. L. Doherty, Director, Queensland Institute of Medical Research, for his assistance in the further testing of the agents isolated in this investigation and to Dr W. J. Stevenson, Chief HeaIth Officer, Department of Health Victoria for the information given by personal communication. We wish to thank and acknowledge the co-operation over a long period of Messrs E. Brough, W. Powell and J. Pike of the Division of Animal Health. References Anderson, S. G . (1954)-J. Hyg. [Camb.) 52: 447. Clarke, D. H. and Casals, J. (1958)-Am. 1. trop. Med. Hyg. 7: 561. Doherty, R. L. (1972)-Aust. vet. 1. 48: 172. Hore, D. E., Campbell, J. and Turner, A. J. (1973)Aust. vet. J. 49: 238. Lee, D. J., Clinton, K. I. and O’Gower, A. K. (1954)Aust. J . biol. Sci. 7: 282. McLean, D. M. (1953a)-Aust. J . exp. Biol. med. Sci. 31: 481. McLean, D. M. (1953b)-Aust. J . exp. Biol. med. Sci. 31: 491. Miles, J. A. R. (1952)-Aust. J . exp. Bid. med. Sci. 30: 341. Standfast, H. A. and Dyce, A. L. (1972)-Aust. vet. J. 48: 77. [Received for publication 13 August 1974)
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