Relevance of Detection of Immunoglobulin M Antibody Response in Birds Used for Arbovirus Surveillance

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1986, p. 770-774 0095-1137/86/1770-05$02.00/0 Copyright C) 1986, American Society for Microbiology Vol. 24, No. 5 Relevance of Detection of Immunoglobulin M Antibody Response in Birds Used for Arbovirus Surveillance CHARLES H. CALISHER,l* HENRY N. FREMOUNT,2 WENDY L. VESELY,' AHMED 0. EL-KAFRAWI,3 AND MOHAMMED I. AL-DEEN MAHMUD4 Division of Vector-Borne Viral Diseases, Center for Infectious Diseases, Centers for Disease Control, Fort Collins, Colorado 522'; Department of Biology, East Stroudsburg University, East Stroudsburg, Pennsylvania 183012; Faculty of Medicine, Al-Azhar University, Cairo, Egypt3; and College of Medicine, University of Basrah, Basra, Iraq4 Received 9 September 1985/Accepted 11 August 1986 Young chickens were inoculated with 5,000 PFU of eastern equine encephalitis (EEE) virus and bled at intervals thereafter for determinations of hemagglutination-inhibiting (HI), neutralizing (N), immunoglobulin M (IgM), and IgG antibodies. HI, N, and IgM antibodies were first detected 4 days after infection, and IgG was detected 7 days after infection. All four antibodies persisted through day 90 after infection. HI, N, and IgM antibody titers remained elevated and were not cross-reactive with the related alphavirus western equine encephalitis (WEE) virus. IgG antibody titers also remained high, but heterologous reactivity to WEE virus increased with time after infection. Serum samples from sentinel chickens and wild birds infected in nature with EEE, WEE, or St. Louis encephalitis virus and submitted to this laboratory from state and local health departments were tested for IgM antibody by using anti-chicken IgM for capture and for IgG antibodies to the EEE and WEE viruses. There was essentially complete torrelation between HI, N, and either IgM (indicating recent infections) or IgG (indicating more remote infections) antibody. We conclude that the IgM antibody capture enzyme immunoassay can be used as a specific and sensitive assay to replace the routinely used HI test for detecting antibody in sentinel chickens and in young, wild birds used for arbovirus surveillance. The test is rapid and relatively inexpensive and can be performed in essentially all adequately supplied laboratories. Amplification of mosquito-borne viruses causing eastern say (MAC ELISA) for use in rapidly detecting specific IgM equine encephalitis (EEE), western equine encephalitis antibody to the EEE and WEE viruses. The results reported (WEE), and St. Louis encephalitis (SLE) is coincident withi here demonstrate the specificity and sensitivity of the MAC both amplification of the vectors carrying these viruses andi ELISA and suggest its usefulness in arbovirus surveillance amplification of populations of the primary avian hosts of the systems that use chickens as sentinel vertebrate hosts. viruses (4). Protecting humans and equines from these Assays for IgG-class antibody in birds were also performed, diseases depends, in part, on preventing, or at least reducing, contact between the susceptible mammalian hosts andi with the MAC ELISA. and the results were compared for rapidity and specificity the virus-infected vectors. The logistics and attendant costs of such programs make it impractical to continue them on a routine basis. Therefore, to determine the presence and MATERIALS AND METHODS magnitude of virus build-up in nature, state and local health departments use a variety of surveillance systems. One such system uses chickens as sentinels. Chickens born in the Serum samples. Serum samples from sentinel chickens and current year, so that antibody detected in them can be from wild birds in Florida, Alabama, Mississippi, Pennsylvania, and Minnesota were from the serum bank of the ascribed to infection in the current year, are placed at strategic locations in a given area. The birds are then bled at Division of Vector-Borne Viral Diseases, Centers for Disease Control, Fort Collins, Colo. They were selected from regular intervals, and a determination is made of their serum antibody titers to viruses of particular epidemiologic interest. Most commonly, hemagglutination inhibition (HI) tests larger collections on the basis of prior HI positivity, titer, and the volume available. are used. When antibody prevalence in a given flock or in In a preliminaty study, 21 White Leghorn-Grey Leghornbirds from a specific area is found to increase from an Auracana Blue hybrid chickens 3 to 4 weeks old were acceptably low level to 5 or % or greater, this is seen as an inoculated subcutaneously with 5,000 PFU of EEE virus, indication of virus amplification, and mosquito control is strain NJO. At 0, 4, 7,, 14, 21, 30, and 90 days after implemented in an attempt to reduce the populations of inoculation, three chickens were exsanguinated by cardiac infected vector species. puncture, and serum was separated from the clot by centrifugation, divided into aliquots, and held at -70 C until tested. Among the drawbacks of such a surveillance system are the lack of rapidity with which HI tests can be performed (to Subsequently, four chickens of the same crossbreed were maintain the relevance of the results) and the crossreactivity of HI antibody to the antigenically related inoculated subcutaneously with approximately 5,000 PFU of EEE virus, strain NJO. These chickens were bled 31, 75, alphaviruses EEE, WEE, and Highlands J viruses. We 121,, 2, and 250 days after inoculation, and the serum therefore attempted to adapt an accepted immunoglobulin M samples were separated and stored as described above. (IgM) antibody capture enzyme-linked immunosorbent as- HI and N tests and ELISAs. For HI tests, sertm samples were acetone extracted (5), treated with protamine sulfate (7), and tested by the method of Clarke and Casals (5) with * Corresponding author. microtiter plates. Serum samples were also tested by the 770 Downloaded from http://jcm.asm.org/ on September 16, 18 by guest

VOL. 24, 1986 IgM IN SENTINEL CHICKENS 771 TABLE 1. Results of HI test, serum dilution-plaque reduction N test, MAC ELISA and IgG assay with sera from chickens experimentally infected with EEE virus Days Chicken after HI test N test MAC ELISA IgG assay infection EEE WEE EEE WEE EEE WEE EEE WEE 1 0 _a _ -_- - - -- 2 0 3 0 - - 4 4-225,600 5 4-2.3-225,600 - - 6 4-225,600 7 7-23 -2.25,600 3,0 8 7.6-23 -225,600 25,600 9 7 3 23 -.25,600 3,0.6.3 -.25,600-251,0 0 11.6-2.3-25,600-251,0 12 26-23 ->25,600 - >51,0 0 13 14 26-23 -2.25,600-251,0 14 14 26-23 -225,600-251,0 3,0 15 14.6 - -3 ->25,600 - >51,0 16 21.6-23 ->25,600 2>51,0 17 21.6-23 12,0 25,600 18 21 -.3 3,0 25,600 19 30.6 -.3-25,600 ->51,0 0 30-23 -225,600-25,600 21 30.6-2.3-225,600 2>51,0 3,0 22 90 6-2.3-25,600 ->51,0 3,0 23 90 < -23 3,0 3,0 0 24 90 6-23 12,0 ->51,0 6,0 a -, < in HI and N tests and < in MAC and IgG ELISAs. a_< in HI and N tests and < in MAC and IgG ELISAs. serum dilution-plaque reduction neutralization (N) method published previously (3). MAC ELISAs were done by a modification of a standard method (11). Briefly, pretitrated goat anti-chicken IgM (Cooper Biomedical, Inc., West Chester, Pa.) was diluted in carbonate-bicarbonate buffer (ph 9.6) and used to coat Immuilon 2 (Dynatech Laboratories, Inc., Alexandria, Va.) flat-bottom polystyrene plates. The plates were then washed mnechanically (Titertek Microplate washer; Flow Laboratories, Inc., McLean, Va.) times with phosphate-buffered saline (PBS) containing 0.05% Tween. With intervening washes with PBS-Tween after each step, the following reagents were diluted with PBS containing 5% fetal bovine serum and added sequentially: 1: and 1:0 dilutions of test sera, 0 ng of gradient-purified EEE (strain NJO) or WEE (strain Fleming) virus, a 1:,000 dilution of ascitic fluid from mice hyperimmunized () with EEE or WEE virus, and pretitrated goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, Inc., Avondale, Pa.). After more washes with PBS-Tween, we added substrate (ABTS; Kirkegaard and Perry Laboratories, Gaithersburg, Md.). Coating (capture) antibody and AJ3TS substrate were added in 75-pl volumes and incubated at room temperature overnight and for 15 min, respectively. All other reagents were added in 50-,ul volumes and incubated at 37 C for 1 h. IgG assays were performed by coating plates with 500 ng of purified EEE or WEE virus in carbonate-bicarbonate buffer overnight at room temperature. Then, 1: and 1:0 dilutions of chicken sera, a pretitrated goat anti-chicken IgG conjugated to horseradish peroxidase (Kirkegaard and Perry Laboratories), and ABTS substrate were added. Again, the coating antigen and substrate were added in 75-,ul volumes, and the other reagents were added in 50-,ul volumes; all reagents except the coating antibody and substrate were incubated at 37 C for 1 h, and the addition of each reagent was followed by washes with PBS-Tween. Plates were mechanically scanned for determination of the optical density of the wells at 9 nm (OD9) in an automatic device (Titertek Multiskan; Flow Laboratories); the results were recorded by the same instrument. Controls included sera from nonimmunized chickens and from immunized chickens tested with and without antigen; wells were coated and treated as described above for the test samples, except that no serum was added. If the OD of the control (serum tested without antigen) did not exceed 0.0 and the test sample OD exceeded 0.350, the results were accepted and the ratio of the OD of the test sample with antigen to the OD of the test sample without antigen was calculated. When such ratios were.2.0, the test serum sample was considered positive for IgM or IgG antibody at that dilution. Serum samples considered positive by either the MAC or IgG ELISA were then titrated by testing twofold dilutions essentially as described above. Once the reagents and tests were standardized, infectious supernatant fluids from cell cultures infected with EEE or Downloaded from http://jcm.asm.org/ on September 16, 18 by guest

772 CALISHER ET AL. WEE virus and betapropiolactone-inactivated, sucroseacetone-extracted antigens prepared from the brains of infected suckling mice were substituted for purified virus in the MAC ELISA. Controls were supernatant fluids from uninfected cell cultures or sucrose-acetone extracts of the brains of uninfected suckling mice. IgG assays were likewise modified to reduce the quantity of purified virus necessary for coating plates; plates were coated with EEE or WEE virus-immune rabbit sera diluted in carbonate-bicarbonate buffer, and either supernatant fluids from cell cultures infected with EEE or WEE virus or inactivated antigens from brains of infected suckling mice were then added. RESULTS Purified virus, supernatant fluids from infected cell cultures, and inactivated sucrose-acetone-extracted antigens provided essentially equivalent results in the MAC ELISA and IgG assay. The results reported here are those with purified virus. Serum samples from chickens immunized with EEE virus and exsanguinated were tested by HI, N, and MAC ELISA for IgG antibody (Table 1). HI, N, and IgM antibodies to the infecting virus were detected within 4 days after inoculation, and IgG antibodies were detected within 7 days. N, IgM, IgG and, with one exception (chicken 23), HI antibodies persisted through the 90 days of the study. Heterologous IgG antibody titers to WEE virus were present but were much lower than homologous titers. HI, N, and IgM antibodies were essentially specific for the infecting virus. A direct relationship was not seen in the MAC ELISA between the titer and either the OD of the test serum or the test/control OD ratio in the screening dilution. In four chickens inoculated with EEE virus and bled sequentially, IgM antibody persisted for at least 250 days after infection (Table 2). Homologous MAC ELISA titers decreased and then increased over the more than 8-month observation period and were relatively high 250 days after inoculation. IgG titers were higher to the EEE virus than to the heterologous WEE virus but also fluctuated. Likewise, HI antibody to the EEE virus fluctuated in two of four birds, but titers remained stable and low or decreased in the other two chickens. Two chickens inoculated with supernatant fluid from uninfected Vero cell cultures (controls) were consistently negative (titers <0 in tests for IgM, < in tests for IgG, and < in tests for HI antibodies). Of four sentinel chickens, three (no. 1 to 3) were considered positive by HI and N tests and MAC ELISA for antibody to the EEE virus (Table 3). Chicken no. 4 was HI positive, but the results were unconfirmed by N testing. Of 13 serum samples from wild birds positive in HI tests (no. 27 to 39), 9 were confirmed by N tests, and 7 of the 9 were MAC ELISA positive. The other two (no. 27 and 32) had IgG antibody. Six of seven serum samples from sentinel chickens (no. 5 to 11) positive by HI for antibody to the WEE virus were confirmed by the N and IgG antibody assays and by MAC ELISA. The remaining chicken (no. 11) was not positive in other tests. None of 12 sentinel chickens (no. 12 to 23) or three wild birds (no. to 42) with HI and N antibody to the SLE virus were positive by the MAC ELISA or IgG assay for antibody to the EEE or WEE virus. Serum samples from three chickens (no. 24 to 26) and three wild birds (no. 43 to 45) with no HI antibody to the EEE, WEE, or SLE virus were negative in the N and IgG assays and in the MAC ELISA. TABLE 2. Results of MAC ELISA, IgG assay, and HI test with sera from chickens experimentally infected with EEE virus and bled serially Chicken Days after MAC ELISA IgG ELISA HI testa infection EEE FEE WEE EEE 1 31 51,0 2,560 75 51,0 6 121 1,600 6 6,0 < 2 1,600 1,2 250 6,0 3 < 2 31 51,0,2 3 75 51,0 5,1 3 121.25,600 5,1 3 6,0 2,560 2.25,600 2,560 250.25,600 2,560 3 31 51,0,2 3 75 51,0 2,560 121 6,0 6 1,600 2,560 2 6,0 2,560 < 250 6,0 2,560 4 31 51,0,2 3 75 51,0 2,560 < < 121 6,0 2,560 0 5,1 2 1,600 2,560 < 250 12,0 1,2 < a Antibody titers to WEE virus in HI test were < for all chickens at all times after infection. DISCUSSION J. CLIN. MICROBIOL. That chickens experimentally infected with EEE virus produced IgM antibody detectable by day 4 and IgG antibody detectable by day 7 after inoculation indicates the usefulness of the assays in rapid, sensitive, and specific serologic determinations of recent infections with the virus. The methods we used for detecting IgM- and IgG-class antibodies are not comparable. The capture assay preferentially selects IgM antibody from a serum sample, whereas the assay for IgG antibody is essentially generic, allowing for competition between all classes of antibodies in their attachment to the antigen on the solid phase. Whereas falsepositives are not likely, false-negatives can occur because IgM and other immunoglobulin-class antibodies may be present in large quantities and IgG only in small and, therefore, undetected quantities. However, the persistence of IgM antibody for at least 250 days indicates a need to further define the duration and cause of persistent IgM antibody responses in chickens and, in particular, in wild birds. The persistence of IgM antibody for 8 months or longer in wild birds would be less meaningful than the absence of IgM antibody in such birds sampled for surveillance, because it could not be stated with certainty that the IgM antibody detected was a result of recent (4 days to a few weeks) or remote (a few weeks to 6 to 12 months) infection. That is, IgM antibody can result from infection in the previous arbovirus season and, therefore, not be relevant to current concerns. However, the absence of IgM antibody does provide a piece of negative information with respect to Downloaded from http://jcm.asm.org/ on September 16, 18 by guest

VOL. 24, 1986 IgM IN SENTINEL CHICKENS 773 TABLE 3. Results of HI test, serum dilution-plaque reduction N test, MAC ELISA, and IgG assay with sera from sentinel chickens and wild birds naturally infected with EEE, WEE, or SLE virus Bird Species HI test N test MAC ELISA IgG assay 1 Chicken 2 Chicken 3 Chicken 4 Chicken 5 Chicken 6 Chicken 7 Chicken 8 Chicken 9 Chicken Chicken 11 Chicken 12 Chicken 13 Chicken 14 Chicken 15 Chicken 16 Chicken 17 Chicken 18 Chicken 19 Chicken Chicken 21 Chicken 22 Chicken 23 Chicken 24 Chicken 25 Chicken 26 Chicken 27 Bobwhite 28 Blue jay 29 Blue jay 30 Blue jay 31 Blue jay 32 Blue jay 33 Blue jay 34 Blue jay 35 Mourning dove 36 House sparrow 37 Cardinal 38 Cardinal 39 Cardinal Cormorant 41 Mourning dove 42 Blue jay 43 Gull 44 Gull 45 Gull EEE WEE SLE EEE WEE SLE EEE WEE EEE WEE.. _a 2 2 2-2 a, < in HI and N tests and < in MAC and IgG ELISAs. the absence of virus in an area. That is, when positive, the MAC ELISA provides at least as much information as does either the HI or N test and, when negative, provides more information than does the HI or N test. Therefore, the MAC ELISA is recommended for surveillance of wild adult birds in which EEE virus infection is possible. Immature wild birds and sentinel chickens with antibody, whether determined by the MAC ELISA or by the HI or N test, obviously have acquired their infections within the previous few months, so all of these tests are adequate. The MAC ELISA requires no treatment of the serum, and plates may be precoated with capture (anti-igm) antibody and stored for later use. Since only 2 of the 28 experimentally infected chickens had heterologous HI antibody to the WEE virus, the HI test seems to be sufficiently sensitive and specific to be useful in testing sera from sentinel chickens. 2 2. 3 > > 2 2 2 2 2-2 -2 -.0.0.0.0.0.0.0.0.0.0.0.0 -.0 _.0-2.0 - - - However, heterologous reactivity is not usually a problem, except in the central United States where the possibility of both EEE and WEE virus infections exist. In the eastern United States, an antigenic relative of the WEE virus, the Highlands J virus, coexists with the EEE virus. Serum samples 27 to 30 (Table 3), from wild birds collected in Alabama, were probably from birds with EEE virus infections; significant heterologous HI reactivity to the WEE virus was not detected by the MAC ELISA. With one exception (no. 7, Table 3), the MAC ELISA was specific in samples from WEE virus-infected chickens. Together, the data suggest that the MAC ELISA is at least as sensitive as either the HI or N test and is sufficiently specific to distinguish between EEE and WEE virus infection in sentinel chickens and wild birds. This has been shown to be the case for EEE, WEE, and other alphavirus infections in Downloaded from http://jcm.asm.org/ on September 16, 18 by guest

774 CALISHER ET AL. humans (2) and may depend on the same antigenic complex specificity as that in birds (8). From our results, we conclude that nonspecific HI reactivity appears to have no parallel in the MAC ELISA; serum samples from birds infected with the SLE virus do not react with EEE or WEE virus in the MAC ELISA, and serum samples without HI antibody to the EEE, WEE, or SLE virus are likewise devoid of IgM antibody to the first two viruses. Homologous IgG antibody to the EEE virus in experimentally infected chickens was much higher than heterologous reactivity to the WEE virus. IgG antibody was, however, detectable within 1 week after infection and could be used as a primary test if endpoints were determined, the serum samples were not simply screened at low dilutions, and the sentinel chickens were birds hatched that year. That anti-chicken IgM antibody can be used to capture IgM antibody from other avian species suggests that the sensitivity of this assay could be improved by using antispecies-specific IgM or anti-family (i.e., anti-passerine) capture antibody. We do not understand why the ELISA specifically detected IgG antibody to WEE virus in WEE virus-infected birds (no 5 to 7, Table 3) but was not as specific in detecting antibody to EEE virus in birds experimentally infected with EEE virus (Tables 1 and 2). One possible explanation would be the presence of higher homologous titers in experimentally infected as opposed to naturally infected birds. Byrne and Robbins (1) detected N antibody much later than HI antibody in chickens experimentally infected with the EEE virus, whereas we were able to detect both HI and N antibodies as early as day 4 after infection. This difference probably reflects the greater sensitivity of the serum dilution-plaque reduction N test we used, in contrast to the virus dilution N test they used. Like Byrne and Robbins, however, we detected HI antibody within a few days after infection, suggesting that the timely collection, processing, and testing of sera from sentinel birds can be quite relevant in an early-warning surveillance system. Two reports of ELISAs with sera from experimentally infected chickens were found and contain some puzzling results. One, by Slaght et al. (9), indicates that avian antibodies exhibit generally lower affinity constants than do mammalian antibodies, require unusually high salt concentrations or low ph for precipitin assays, and have a high affinity for plastic. In the MAC ELISA and IgG assay reported here no attempts were made to address such problems; we simply adapted standard ELISAs used for the detection of IgM and IgG antibodies to the EEE, WEE, and other viruses in sera from humans and equines. Perhaps differences between the conclusions drawn by Slaght et al. and our conclusions could be accounted for by differences in the quality of the plastic phase or the number of washes after each incubation step. The other report was by Griffiths and McClain (6), who suggested that IgG antibody is short-lived in chickens naturally infected or vaccinated with EEE virus, peaking 3 to 4 weeks after immunization and declining thereafter. They further stated that IgM antibody peaks at to 11 weeks J. CLIN. MICROBIOL. after inoculation and declines subsequently. Most of the differences between the results of Griffiths and McClain and our results could be ascribed to their use of killed virus and an intravenous route for immunization, the low dilution of test serum they used, and the considerable differences between their method and ours for IgM antibody assays. Our results indicate that the MAC ELISA can be used as a sensitive and specific assay to replace the routinely used HI test for detecting antibody in sentinel chickens and young, wild birds used for arbovirus surveillance. The MAC ELISA can be standardized with purified virus, but this expensive reagent can be easily replaced with either infectious supernatant fluid from infected cell cultures or with inactivated antigens such as are routinely available in arbovirus laboratories or from the Centers for Disease Control. Precoating plates with anti-igm or with purified virus for IgM and IgG antibody assays, respectively, can save considerable time and effort and reduce the response time needed by vector control authorities to begin implementing control measures. LITERATURE CITED 1. Byrne, R. J., and M. L. Robbins. 1961. Mortality patterns and antibody response in chicks inoculated with eastern equine encephalomyelitis virus. J. Immunol. 86:13-16. 2. Calisher, C. H., A. 0. El-Kafrawi, M. I. A.-D. Mahmud, A. P. A. Travassos da Rosa, C. R. Bartz, M. Brummer-Korvenkontio, S. Haksohusodo, and W. Suharyono. Complex-specific immunoglobulin M antibody patterns in humans infected with alphaviruses. J. Clin. Microbiol. 23:155-159. 3. Calisher, C. H., T. P. Monath, N. Karabatsos, and D. W. Trent. 1981. Arbovirus subtyping: applications to epidemiologic studies, availability of reagents, and testing services. Am. J. Epidemiol. 114:619-631. 4. Chamberlain, R. W. 1982. Arbovirology-then and now. Am. J. Trop. Med. Hyg. 31:430-437. 5. Clarke, D. H., and J. Casals. 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7:561-573. 6. Griffiths, B. B., and 0. McClain. 1985. Immunological response of chickens to eastern equine encephalomyelitis virus. Vet. Sci. 38:65-68. 7. Holden, P., D. Muth, and R. B. Shriner. 1966. Arbovirus hemagglutination-inhibiting in avian sera: inactivation with protamine sulfate. Am. J. Epidemiol. 84:67-73. 8. Ogata, M., and R. J. Byrne. 1961. Relationships between eastern and western equine encephalomyelitis viruses as demonstrated by the hemagglutination-inhibition antibody response of experimentally infected chickens. Am. J. Vet. Res. 22:266-270. 9. Slaght, S. S., T.-J. T. Yang, and L. Van Der Heide. 1979. Adaptation of enzyme-linked immunosorbent assay to the avian system. J. Clin. Microbiol. :698-702.. Tikasingh, E. L., L. Spence, and W. G. Downs. 1966. The use of adjuvant and sarcoma 1 cells in the production of mouse hyperimmune ascitic fluids to arboviruses. Am. J. Trop. Med. Hyg. 15:219-226. 11. Voller, A., D. Bidwell, and A. Bartlett. 1976. Microplate enzyme immunoassays for the immunodiagnosis of virus infections, p. 506-512. In N. R. Rose and H. Friedman (ed.), Manual of clinical immunology. American Society for Microbiology, Washington, D.C. Downloaded from http://jcm.asm.org/ on September 16, 18 by guest