.iournal of Virological Methods, 34 (1991) 221-225 0 1991 Elsevier Science Publishers B.V. / All rights reserved/0166-0934/91/%03.50 ADONIS 0166093491004315
221
VIRMET 01217
Short Communication
The use of ELISA for the detection of African horse sickness viruses in Culicoides midges C. Hamblin, P.S. Mellor and J. Boned RFRC Z~tit~te for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Waking, Surrey, U.K. (Accepted 9 May 1991)
The use of ELISA for the detection of African horse sickness viruses (AHSV) in midges preserved in 5.0% formahn was evaluate. No differences were detected by ELISA when testing AHSV infected batches of CuZicoide~midges collected in diluent with or without the addition of formalin. The ELISA was considered highly sensitive and easily distinguished between non-infected midges and batches containing varying numbers of infected and non-infected midges. Positive ELISA reactions were detected with formalin-preserved midges collected from the south of Spain during the 1988 AHSV epizootic. The assay, therefore, may be used in surveillance studies of either fresh or fo~alin-prese~ed midges to identify undisclosed and persistent AHSV foci. This information would be useful in helping to eradicate the virus from Europe and North Africa. ELISA; African horse sickness; Culicoides midge Insect surveillance studies have shown that ~~~ico~e~ imicoZa, the only proven field vector of African horse sickness virus (AHSV), is present in a number df European countries including Spain, Greece and Portugal (Mellor et al., 1983, 1985; Boorman and Wilkinson 1983; Boorman 1986). However, during the 1988 epizootic in southern Spain AHSV was isolated from two other species of biting midge, C. pulicaris and C. obsoletes (Mellor et al., 1990). Both of these species are common in the cooler regions of Europe including France and the U.K. No information is yet available to show how effective these Correspondenceto: C. Hamblin, Department of Virus Diagnosis, AFRC Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surr’ey GU24 ONF, U.K.
222
species might be in transmitting AHSV. If these species of midges are able to transmit the virus, even at a low frequency, there is a possibility that an epizootic of AHS could occur in a number of European countries or regions usually considered unable to support the disease. The appearance of African horse sickness (AHS) in Spain for four successive years (1987-1990) and in Morocco for two successive years (1989-1990) has emphasised the di~culties of eradicating the virus despite vigorous and widespread vaccination strategies, slaughter of infected animals, control of animal movement and insect control. The isolation and identification of AHSV from very large numbers of midges collected from different locations is labour intensive and time consuming and therefore has not been used to provide information on virus distribution. The development of an ELISA for AHSV detection (Hamblin et al., 1991), however, may now permit the rapid examination of large numbers or collections of fresh or formalin-preserved midges which could disclose previously unidenti~ed foci of the virus. Czdicoides variipennis were inoculated experimentally with 0.1 &fly of AHSV serotype 9 (MB3 BHK3) containing 105.4 LDse/ml (Boorman, 1975). Eight days after infection three groups, each consisting of 10 midges, were prepared. Group A was used as the control and insects were killed and immersed in 2.0 ml of distilled water (DW), group B was preserved in 2.0 ml of DW containing 0.1% detergent (Hederol) and group C was preserved in 2.0 ml of DW containing 5.0% formalin and 0.1% detergent. After 24 hours each group of insects was washed twice with 1.0 ml amounts of phosphate buffered saline (PBS). Samples were homogenised in Griffith’s tubes with 1.0 ml of Eagle’s medium (Glasgow modification) containing 30 mM Hepes (Flow Laboratories, Irvine, Scotland, U.K.). A tenfold dilution series of each midge suspension was prepared in the same Eagle’s/Hepes medium and assayed for infectious virus by intracerebral inoculation of one day old suckling mice (Mellor et al., 1974). A twofold dilution series of each midge suspension was also prepared in PBS containing 0.05% Tween 20 and neutral red indicator and tested by ELISA using methods described by Hamblin et al. (1991). Groups A and B both contained 105.6 LDSO/ml of infectious virus. No infectious virus could be detected in the homogenised insects of group C. The titration curves obtained by ELISA for all three groups, however, were identical (Fig. 1). Twenty-one batches, each containing 50 C. variipennis midges, were preserved in PBS containing 5.0% formalin and 0.1% detergent. Seven of these batches had been mixed with varying numbers of midges inoculated eight days previously with 0.1 $/fly of AHSV serotype 9 containing 105.4LDSe/ml. Each batch was washed twice with 1.0 ml of PBS, homogenised in Griffith’s tubes with 0.5 ml of PBS containing 0.05% Tween 20 and examined by ELISA. Table 1 identifies the individual batches, the number of insects in each batch, the batches which had been mixed with infected midges, the number of infected insects added and the corresponding ELISA titre. The ELISA titres were based on the threshold for positivity [(optical density (OD) 2 0.15)] as determined by Hamblin et al. (1991). All seven ‘spiked’ batches of midges were strongly
223
1.6
-
0.6
-
0.6
-
l,,,,,,,,‘,,, 0.3
0.9 LOGto
1.5
2.1
2.7
3.31
DILUTION
Fig. 1. Titration of experimentally infected Culicoidesvariipennis by ELISA. a-0 DW diluent (control), m--m DW containing 0.1% detergent, &--A DW containing 0.1% detergent and 5.0% formalin.
positive. The remaining 14 batches of insects were below OD 0.15 and therefore negative. Retrospective studies were carried out with batches of Culicoides midges which had previously been identified and sorted. The numbers of insects in each batch were estimated to be between 20 and 100. These midges were collected from the Provinces of Cadiz and Malaga in southern Spain during the 1988 epizootic and had been kept at ambient temperatures for two years in PBS containing 5.0% formalin and 0.1% detergent. After washing, the insects were TABLE 1 Detection of AHSV in Culicoides variipennispreserved in PBS containing formalin Batch no.
No. of Culicoides Non-infected
1,3,4,5,6,7,8,11,13,14,15,16,17,20 2 9 10 12 18 19 21
Infected 0
: 44 47 40 ::
2 3 10 :
10
40
0.1% detergent and 5.0% ELISA titre (loglo) GO.0 1.95 1.95 1.5 22.55 1.5 1.8 32.55
224 TABLE 2 Detection
of AHSV in midges collected from Cadiz and Malaga in southern Spain in 1988
Batch no.
Species
Origin
Date
ELISA
1 2 and 3 4 and 5 67 and 8 9 10
Culicoides imicola C. imicoln C. imicola C. ~mi~olu C. imicola C. imicoia C. obsoletus C. imicola C. obsoletus C. species mixed C. pulicaris C. species mixed C. species mixed C. species mixed C. imieofa
Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Las Chapos, Cadiz Las Chapos, Cadiz Las Chapos, Cadiz Las Chapos, Cadiz Castellor, Malaga Castellor, Malaga Los Pinos, Cadiz Los Pinos, Cadiz
17-11-88 17-l l-88 17-l I-88 17-1 l-88 17-11-88 6-11-88 17-11-88 7-11-88 7-11-88 7-11-88 7-11-88 14-11-88 17-10-88 31-10-88 31-10-88
+ + + -
t;’ 12 13 16 15, 18 and I9 17 20 21
homogenised and examined by ELBA as described above. Table 2 shows the origin and date of each collection of Culicoides midges and the ELBA results. Positive ELISA reactions were recorded with seven batches of insects collected in Cadiz. The origin of the positive batches of midges corresponded to those areas where AHS had been reported in 1988 and from where isolations of AHSV had been made (Mellor et al., 1990). The survival of AHSV in Spain for the last four years indicates that the climate and habitat in some parts of the country have been suitable for vector midges to remain active throughout the year (Mellor et al., 1990). Persistence of the virus, however, also depends on the interaction between invertebrate and susceptible vertebrate hosts. The data presented here show the potential value of ELBA in insect surveillance studies for identifying undisclosed and persisting foci of AHSV. Such information may be of value in helping to control and eradicate AHS from Europe and North Africa.
Acknowledgements The authors would like to thank Mr E. Denison, Mrs SD. Graham and Mr L. Pullen for their technical assistance. We would also like to thank Mr D. Panyai for the photography.
References Boorman, J. (1975) Semi-automatic 24, 90.
device for inoculation
of small insects with viruses. Lab. Praet.
22s Boorman, J.P.T. frsas) Presence of b~uetong~e virus vectors in Rhodes. Vet. Rec. 118, 2t. Boorman, J.P.T. and Wilkinson, P.J. (1983) Pateutiaf vectors of b~ueton~~ in Lesbos Greece. Vet. Rec. 113,395-396. Namblin, C., Me&ens, P,P.C., Melfor, P.S., Graham, SD., Burroughs, J.N. and Crowtber, J,C. (1991) A serogoup specific euz~a-~~uked immunosorbent assay for the detection and iden~~~tion of African horse sickness viruses. J. ViroI. Methods 3 1, 285-292. Meiior, P.S., coolant J.P.T. and Martinez Gomez, F. (1983) Potential vectors ofbi~eto~g~ and AfticaB horse sickness viruses in Spain. Vet. Rec. If2,229-~3~. M&or, PS,, Jen~~n~* D.M., ~~~k~nson, P-J. and coolant J.P.T. (1985) C~ljco~~~ j~~~~~~:a b~uetou~ vector in Spain and Portugal. Vet. Rec. It& 589-590. Metlor, P.S., Boned, J., ~ambiin, C. and Grabam, S.D. (1990) Isolationsof African horse sickness virus from vector insects made during the 1988 epizootic in Spain, ~~~dern. Infst. 105,447-454.
221
VIRMET 01217
Short Communication
The use of ELISA for the detection of African horse sickness viruses in Culicoides midges C. Hamblin, P.S. Mellor and J. Boned RFRC Z~tit~te for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Waking, Surrey, U.K. (Accepted 9 May 1991)
The use of ELISA for the detection of African horse sickness viruses (AHSV) in midges preserved in 5.0% formahn was evaluate. No differences were detected by ELISA when testing AHSV infected batches of CuZicoide~midges collected in diluent with or without the addition of formalin. The ELISA was considered highly sensitive and easily distinguished between non-infected midges and batches containing varying numbers of infected and non-infected midges. Positive ELISA reactions were detected with formalin-preserved midges collected from the south of Spain during the 1988 AHSV epizootic. The assay, therefore, may be used in surveillance studies of either fresh or fo~alin-prese~ed midges to identify undisclosed and persistent AHSV foci. This information would be useful in helping to eradicate the virus from Europe and North Africa. ELISA; African horse sickness; Culicoides midge Insect surveillance studies have shown that ~~~ico~e~ imicoZa, the only proven field vector of African horse sickness virus (AHSV), is present in a number df European countries including Spain, Greece and Portugal (Mellor et al., 1983, 1985; Boorman and Wilkinson 1983; Boorman 1986). However, during the 1988 epizootic in southern Spain AHSV was isolated from two other species of biting midge, C. pulicaris and C. obsoletes (Mellor et al., 1990). Both of these species are common in the cooler regions of Europe including France and the U.K. No information is yet available to show how effective these Correspondenceto: C. Hamblin, Department of Virus Diagnosis, AFRC Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surr’ey GU24 ONF, U.K.
222
species might be in transmitting AHSV. If these species of midges are able to transmit the virus, even at a low frequency, there is a possibility that an epizootic of AHS could occur in a number of European countries or regions usually considered unable to support the disease. The appearance of African horse sickness (AHS) in Spain for four successive years (1987-1990) and in Morocco for two successive years (1989-1990) has emphasised the di~culties of eradicating the virus despite vigorous and widespread vaccination strategies, slaughter of infected animals, control of animal movement and insect control. The isolation and identification of AHSV from very large numbers of midges collected from different locations is labour intensive and time consuming and therefore has not been used to provide information on virus distribution. The development of an ELISA for AHSV detection (Hamblin et al., 1991), however, may now permit the rapid examination of large numbers or collections of fresh or formalin-preserved midges which could disclose previously unidenti~ed foci of the virus. Czdicoides variipennis were inoculated experimentally with 0.1 &fly of AHSV serotype 9 (MB3 BHK3) containing 105.4 LDse/ml (Boorman, 1975). Eight days after infection three groups, each consisting of 10 midges, were prepared. Group A was used as the control and insects were killed and immersed in 2.0 ml of distilled water (DW), group B was preserved in 2.0 ml of DW containing 0.1% detergent (Hederol) and group C was preserved in 2.0 ml of DW containing 5.0% formalin and 0.1% detergent. After 24 hours each group of insects was washed twice with 1.0 ml amounts of phosphate buffered saline (PBS). Samples were homogenised in Griffith’s tubes with 1.0 ml of Eagle’s medium (Glasgow modification) containing 30 mM Hepes (Flow Laboratories, Irvine, Scotland, U.K.). A tenfold dilution series of each midge suspension was prepared in the same Eagle’s/Hepes medium and assayed for infectious virus by intracerebral inoculation of one day old suckling mice (Mellor et al., 1974). A twofold dilution series of each midge suspension was also prepared in PBS containing 0.05% Tween 20 and neutral red indicator and tested by ELISA using methods described by Hamblin et al. (1991). Groups A and B both contained 105.6 LDSO/ml of infectious virus. No infectious virus could be detected in the homogenised insects of group C. The titration curves obtained by ELISA for all three groups, however, were identical (Fig. 1). Twenty-one batches, each containing 50 C. variipennis midges, were preserved in PBS containing 5.0% formalin and 0.1% detergent. Seven of these batches had been mixed with varying numbers of midges inoculated eight days previously with 0.1 $/fly of AHSV serotype 9 containing 105.4LDSe/ml. Each batch was washed twice with 1.0 ml of PBS, homogenised in Griffith’s tubes with 0.5 ml of PBS containing 0.05% Tween 20 and examined by ELISA. Table 1 identifies the individual batches, the number of insects in each batch, the batches which had been mixed with infected midges, the number of infected insects added and the corresponding ELISA titre. The ELISA titres were based on the threshold for positivity [(optical density (OD) 2 0.15)] as determined by Hamblin et al. (1991). All seven ‘spiked’ batches of midges were strongly
223
1.6
-
0.6
-
0.6
-
l,,,,,,,,‘,,, 0.3
0.9 LOGto
1.5
2.1
2.7
3.31
DILUTION
Fig. 1. Titration of experimentally infected Culicoidesvariipennis by ELISA. a-0 DW diluent (control), m--m DW containing 0.1% detergent, &--A DW containing 0.1% detergent and 5.0% formalin.
positive. The remaining 14 batches of insects were below OD 0.15 and therefore negative. Retrospective studies were carried out with batches of Culicoides midges which had previously been identified and sorted. The numbers of insects in each batch were estimated to be between 20 and 100. These midges were collected from the Provinces of Cadiz and Malaga in southern Spain during the 1988 epizootic and had been kept at ambient temperatures for two years in PBS containing 5.0% formalin and 0.1% detergent. After washing, the insects were TABLE 1 Detection of AHSV in Culicoides variipennispreserved in PBS containing formalin Batch no.
No. of Culicoides Non-infected
1,3,4,5,6,7,8,11,13,14,15,16,17,20 2 9 10 12 18 19 21
Infected 0
: 44 47 40 ::
2 3 10 :
10
40
0.1% detergent and 5.0% ELISA titre (loglo) GO.0 1.95 1.95 1.5 22.55 1.5 1.8 32.55
224 TABLE 2 Detection
of AHSV in midges collected from Cadiz and Malaga in southern Spain in 1988
Batch no.
Species
Origin
Date
ELISA
1 2 and 3 4 and 5 67 and 8 9 10
Culicoides imicola C. imicoln C. imicola C. ~mi~olu C. imicola C. imicoia C. obsoletus C. imicola C. obsoletus C. species mixed C. pulicaris C. species mixed C. species mixed C. species mixed C. imieofa
Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Sotogrande, Cadiz Las Chapos, Cadiz Las Chapos, Cadiz Las Chapos, Cadiz Las Chapos, Cadiz Castellor, Malaga Castellor, Malaga Los Pinos, Cadiz Los Pinos, Cadiz
17-11-88 17-l l-88 17-l I-88 17-1 l-88 17-11-88 6-11-88 17-11-88 7-11-88 7-11-88 7-11-88 7-11-88 14-11-88 17-10-88 31-10-88 31-10-88
+ + + -
t;’ 12 13 16 15, 18 and I9 17 20 21
homogenised and examined by ELBA as described above. Table 2 shows the origin and date of each collection of Culicoides midges and the ELBA results. Positive ELISA reactions were recorded with seven batches of insects collected in Cadiz. The origin of the positive batches of midges corresponded to those areas where AHS had been reported in 1988 and from where isolations of AHSV had been made (Mellor et al., 1990). The survival of AHSV in Spain for the last four years indicates that the climate and habitat in some parts of the country have been suitable for vector midges to remain active throughout the year (Mellor et al., 1990). Persistence of the virus, however, also depends on the interaction between invertebrate and susceptible vertebrate hosts. The data presented here show the potential value of ELBA in insect surveillance studies for identifying undisclosed and persisting foci of AHSV. Such information may be of value in helping to control and eradicate AHS from Europe and North Africa.
Acknowledgements The authors would like to thank Mr E. Denison, Mrs SD. Graham and Mr L. Pullen for their technical assistance. We would also like to thank Mr D. Panyai for the photography.
References Boorman, J. (1975) Semi-automatic 24, 90.
device for inoculation
of small insects with viruses. Lab. Praet.
22s Boorman, J.P.T. frsas) Presence of b~uetong~e virus vectors in Rhodes. Vet. Rec. 118, 2t. Boorman, J.P.T. and Wilkinson, P.J. (1983) Pateutiaf vectors of b~ueton~~ in Lesbos Greece. Vet. Rec. 113,395-396. Namblin, C., Me&ens, P,P.C., Melfor, P.S., Graham, SD., Burroughs, J.N. and Crowtber, J,C. (1991) A serogoup specific euz~a-~~uked immunosorbent assay for the detection and iden~~~tion of African horse sickness viruses. J. ViroI. Methods 3 1, 285-292. Meiior, P.S., coolant J.P.T. and Martinez Gomez, F. (1983) Potential vectors ofbi~eto~g~ and AfticaB horse sickness viruses in Spain. Vet. Rec. If2,229-~3~. M&or, PS,, Jen~~n~* D.M., ~~~k~nson, P-J. and coolant J.P.T. (1985) C~ljco~~~ j~~~~~~:a b~uetou~ vector in Spain and Portugal. Vet. Rec. It& 589-590. Metlor, P.S., Boned, J., ~ambiin, C. and Grabam, S.D. (1990) Isolationsof African horse sickness virus from vector insects made during the 1988 epizootic in Spain, ~~~dern. Infst. 105,447-454.
