Relevance of detection of immunoglobulin M antibody response in birds used for arbovirus surveillance.

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 correlation 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.     

Test of recrudescence hypothesis for overwintering of eastern equine encephalomyelitis virus in gray catbirds

Eastern equine encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) epizootics are infrequent, but they can lead to high mortality in infected horses and humans. Despite the importance of EEEV to human and animal health, little is known about how the virus overwinters and reinitiates transmission each spring, particularly in temperate regions where infected adult mosquitoes are unlikely to survive through the winter. One hypothesis to explain the mechanism by which this virus persists from year to year is the spring recrudescence of latent virus in avian reservoir hosts. In this study, we tested the recrudescence hypothesis with gray catbirds (Dumatella carolinensis) captured in northern Ohio (July-August 2007). Birds were experimentally infected with EEEV on 1 October 2007. In January 2008, they were then exposed to exogenous testosterone and/or extended photoperiod to initiate reactivation of latent EEEV infection. All birds became viremic with EEEV, with mean viremia of 6.0 log10 plaque-forming units/ml serum occurring at 1 d postinoculation. One male in the testosterone, long-day treatment group had EEEV viral RNA in a cloacal swab collected on 18 January 2008. Otherwise, no other catbirds exhibited reactivated infections in cloacal swabs or blood. Antibody titers fluctuated over the course of the study, with lowest titers observed in January 2008, which corresponded with the lowest mean weight of the birds. No EEEV viral RNA was detected in the blood, kidney, spleen, brain, liver, and lower intestine upon necropsy at 19 wk postinfection.     

Avian serology in a St. Louis encephalitis epicenter before, during, and after a widespread epidemic in south Florida, USA.

Blood and serum from 3,915 wild and domestic birds (2,590 resident, 139 migrant, and 1,186 captive), representing 56 species collected in central Florida from 1989 through 1997, were analyzed for evidence of St. Louis encephalitis (SLE) virus transmission. All sera were tested for SLE hemagglutination inhibition (HI) antibody. Selected sera and bloods were tested for SLE neutralizing (NT) antibody and virus. The reproductive success of resident birds was highest from 1990-1992 and lowest from 1994-1997. Transmission of SLE to resident birds, especially mourning doves (Zenaida macroura), peaked during the summer of 1990, a year during which a widespread SLE epidemic was recorded in central Florida. The SLE antibody-positive resident birds 1st appeared during September of the epidemic year. Some SLE, HI antibody-positive resident birds were captured throughout 1991, but only 5% were yearlings, compared with 36% in 1990. By 1993, wild resident birds expressing HI and NT antibodies to SLE had nearly disappeared. None of the migrant birds tested were SLE-positive. Sentinel chickens maintained in Indian River County during the epidemic year seroconverted to SLE starting in early July with peak seroconversion rates in August, September, and October. High (> or = 50%) SLE seroconversion rates in sentinel chickens preceded those in wild birds by 10 wk and preceded peak human SLE transmission by at least 8 wk. Major SLE epidemics in south Florida depend on abundant wild bird populations, especially during the amplification phase of the transmission cycle. We propose that hard winter freezes along the temperature-subtropical climatic zone interface in central Florida, at approximately 27 degrees 30' North Latitude, opens foraging and nesting habitats for ground-feeding birds, resulting in high reproductive success and an abundance of seronegative individuals that rapidly amplify the SLE later in the year.     

Characteristics of the ecology of the eastern equine encephalomyelitis virus in the Republic of Cuba]

Virologic and serological surveys of wild vertebrates carried out in various provinces of Cuba demonstrated definitely that birds were the main hosts of eastern equine encephalomyelitis (EEE) virus in this territory. Fifteen strains of this virus were isolated from 8 species of birds belonging to 5 orders. Isolation of EEE virus from the blood of the endemic genus of iguanas indicates a certain role of cold-blooded animals in the ecology of this agent. Active EEE virus foci have been found in 4 provinces of the Republic of Cuba: Pinar del Rio, Havana, Matanzas and Las Villas. Isolation of a number of EEE virus strains from sick horses during an epizootic in the latter province confirmed the importance role of this agent in the infectious pathology of domestic animals in Cuba. The experimental results suggest that in Cuba there occur at least two types of foci of this infection: forest and water-littoral (fresh-water swamps and lakes, and sea coast with mangrove forests).     

Host (avian) biting preference of southern California Culex mosquitoes (Diptera: Culicidae)

The host preference of a vector mosquito species plays a significant role in determining human and animal risk of infection with mosquito-transmitted pathogens. Host preferences of common southern California Culex species for four bird species, American crow (Corvus brachyrhynchos), house sparrow (Passer domesticus), house finch (Carpodacus mexicanus), and mourning dove (Zenaida macroura), were examined by determining the proportion of each mosquito species that successfully engorged on each of the four bird species presented equally within a net trap to wild host-seeking mosquitoes. Bloodmeals in engorged mosquitoes captured within the net trap were identified to avian species by using a multiplex polymerase chain reaction assay targeting the cytochrome b gene sequence. There were significant differences in host selection by all three Culex species captured in numbers sufficient for analysis, with Culex erythrothorax Dyar preferentially biting American crows, Culex tarsalis Coquillett preferentially biting house sparrows, and Culex quinquefasciatus Say preferentially biting house finches. All three Culex species demonstrated more frequent engorgement on passerine birds (sparrows, finches, and crows) than the nonpasserine mourning dove. A greater preference for passerine birds might be expected to increase the transmission of pathogens, such as West Nile virus, to which passerine birds are particularly competent hosts.     

Arboviral infection in two species of wild jays (Aves: Corvidae): evidence for population impacts

We examined the prevalence of antibodies to three mosquito-borne arboviruses in blue jays, Cyanocitta cristata, and Florida scrub-jays, Aphelocoma coerulescens, to identify the effects on host survival, the influence of sex and age on infection, and the temporal patterns of antibody prevalence. Blood samples from 306 blue jays and 219 Florida scrub-jays were collected at Archbold Biological Station (Lake Placid, FL) from April 1994 through December 1995. Sera were analyzed for hemagglutination-inhibition antibody to eastern equine encephalitis (EEE) and St. Louis encephalitis (SLE) viruses, and neutralizing antibodies to EEE, Highlands J (HJ), and SLE viruses. Overall, 31.4% of blue jay samples and 22.1% of scrub-jay samples had antibodies to EEE. Antibodies to HJ were detected in slightly >15% of samples in each jay species, and SLE was detected in <3% of the samples in each jay species. A single EEE virus isolation was made from the blood of an 11-d-old scrub-jay nestling. Survival of adult blue jays seropositive to EEE was significantly lower than that of seronegative birds based on resight rates, but infection did not seem to affect survival of adult or juvenile Florida scrub-jays.     

Comprehensive serological analysis of two successive heterologous vaccines against H5N1 avian influenza virus in exotic birds in zoos

In 2005, European Commission directive 2005/744/EC allowed controlled vaccination against avian influenza (AI) virus of valuable avian species housed in zoos. In 2006, 15 Spanish zoos and wildlife centers began a vaccination program with a commercial inactivated H5N9 vaccine. Between November 2007 and May 2008, birds from 10 of these centers were vaccinated again with a commercial inactivated H5N3 vaccine. During these campaigns, pre- and postvaccination samples from different bird orders were taken to study the response against AI virus H5 vaccines. Sera prior to vaccinations with both vaccines were examined for the presence of total antibodies against influenza A nucleoprotein (NP) by a commercial competitive enzyme-linked immunosorbent assay (cELISA). Humoral responses to vaccination were evaluated using a hemagglutination inhibition (HI) assay. In some taxonomic orders, both vaccines elicited comparatively high titers of HI antibodies against H5. Interestingly, some orders, such as Psittaciformes, which did not develop HI antibodies to either vaccine formulation when used alone, triggered notable HI antibody production, albeit in low HI titers, when primed with H5N9 and during subsequent boosting with the H5N3 vaccine. Vaccination with successive heterologous vaccines may represent the best alternative to widely protect valuable and/or endangered bird species against highly pathogenic AI virus infection.     

A study of ELISA systems incorporating pooled viral and Mycoplasma antigen preparations for antibody screening of chicken sera

Enzyme linked immunosorbent assays (ELISAs) incorporating up to three different antigens for screening of avian sera for antibodies to several viruses or mycoplasma are described. A triple antigen test comprising Newcastle disease virus (NDV), infectious laryngotracheitis (ILT) virus and avian influenza (AI) antigens for screening sera normally negative for antibodies to these viruses, was shown to be as sensitive as the corresponding single antigen ELISA in detecting seroconversion in experimentally inoculated birds and was also as sensitive as the haemagglutination-inhibition (HI) test for NDV and AI, and the serum neutralisation test for ILT virus. Sensitivity was also demonstrated by comparison of end-points in serially diluted NDV, ILT or AI positive sera. A Mycoplasma synoviae ELISA was shown to be as sensitive as HI test for detection of MS and M. gallisepticum (MG) antibodies in experimentally inoculated birds and in field sera, and this antigen combined with NDV detected antibodies to MG, MS and NDV with sensitivity equivalent to the HI test in each case. The advantages of using pooled ELISA preparation for screening large numbers of sera which are normally negative for the pathogens concerned are discussed.     

Isolation of the influenza virus from the tree sparrow and a study of the infectivity of this virus in wild birds of the central Dnieper River area

An influenza virus belonging to the serovariant A/H3N2 and registered as A/sparrow/Ukraine/83 was isolated from a member of synanthropic birds, a tree sparrow, near Kanev. This virus showed low pathogenicity and immunologic activity in experimental infection of sparrows and other birds. Sera from a number of avian and mammal species had antibodies to this virus which indicates that synanthropic and semi-synanthropic birds may be a connecting link in spread of influenza virus.     

Retroviral expressed hemagglutinin-neuraminidase protein protects chickens from Newcastle disease virus induced disease

The hemagglutinin-neuraminidase (HN) gene and the phosphoprotein (P) gene of Newcastle disease virus (NDV) were inserted into a replication competent avian leukosis virus vector. The expression of the HN gene from this vector in chick embryo cells has been previously reported. The P gene is also expressed from this vector in chick embryo cells. The retroviruses were used to immunize 4-week-old chickens. Birds receiving the virus containing the HN gene developed low levels of serum HI titers and NDV neutralization titers. Upon challenge, all birds vaccinated with the HN gene containing virus were protected from disease but not viral infection and replication. In contrast, birds immunized with the P gene containing retrovirus developed more severe clinical signs of disease earlier than birds receiving no immunization or retrovirus alone. The results obtained with the HN gene may have potential application to reducing disease due to NDV genetically engineered vaccines.     

Enzyme immunoassay for detection of antibodies against eastern equine encephalomyelitis virus in sentinel chickens

We developed an enzyme immunoassay (EIA) for the detection of immunoglobulin M (IgM) and IgG subclass antibodies directed against eastern equine encephalomyelitis (EEE) virus in chickens. The assays were compared with the serum plaque reduction neutralization test (PRNT) and the hemagglutination inhibition (HI) test for ability to detect antibodies against EEE virus in laboratory-infected birds. No cross-reactivity was detected in serum from chickens inoculated with St. Louis encephalitis or Highlands J virus. The interval after infection when EEE virus-specific antibodies were first detected by IgM and IgG EIAs was found to be similar to that determined by the PRNT and HI tests: 2 to 4 days. The IgG EIA, PRNT, and HI test detected antibodies to EEE virus for at least 27 to 30 days after inoculation. In contrast, serum from five of seven chickens did not contain detectable IgM 30 days after infection. Similarly, in all three naturally infected sentinel chickens from Maryland, IgM class antibody was undetectable 1 to 5 weeks after IgM was initially detected. EIAs provide simple and rapid alternatives to traditional tests for monitoring EEE virus infections in sentinel chicken flocks. Moreover, the IgM EIA provides a means to separate recently infected chickens from those infected greater than or equal to 1 month earlier.     

Serological investigation of highly pathogenic avian influenza H5N2 in ostriches (Struthio camelus)

An ostrich farm of 929 birds that tested polymerase chain reaction-positive for highly pathogenic avian influenza H5N2 in a single sample was designated for culling, despite no evidence of sero-conversion as assessed by haemagglutination inhibition (HI) tests. A month later and immediately prior to culling, all birds were bled and tested with an IDEXX avian influenza virus (AIV) nucleoprotein (NP)-specific enzyme-linked immunosorbent assay (ELISA) and a high sero-prevalence was detected. To address the question of whether the NP-specific antibodies detected indicated exposure to H5 or non-H5 subtypes (H6N2 and H1N2 strains were also circulating regionally at the time), we developed two H5-specific ELISAs, both based on a recombinant H5 HA1 antigen. The H5 indirect ELISA used a horseradish peroxidase ostrich IgY conjugate that we produced in chicken eggs. The single-chain variable fragment (scFv) competitive ELISA (H5 scFv cELISA) used a scFv derived from an H5-immune chicken scFv library. By comparing IDEXX AIV ELISA results with those of the two H5-specific ELISAs and HI tests, we determined that up to 89% of the flock had been exposed to H5N2 AIV. We also detected evidence of suspected vaccination, since 17% of sera contained antibodies against the H5 glycoprotein but not the NP protein. Comparative analytical sensitivity indicated that HI tests are likely to miss up to 35% of H5-positive samples, and thus we consider that H5/H7-specific ELISAs should replace HI tests for ostrich testing in future.     

Virus serological diagnosis in two Avian species based on binding of human C1q to Avian antibody

Diagnosis of infectious diseases in birds using direct assays such as conventional ELISA or immunofluorescence require antibody directed to IgG of each avian species. In this study, we tested binding of human C1q to different antigen-antibody complexes of two avian species in a sandwich immunofluorescent complement fixation test (S-ICFT). The reaction was as follows: virus (in cells) + decomplemented avian serum + human-C1q + goat anti-human-C1q + fluorescein isothiocyanate rabbit anti-goat-IgG. Positive and negative chicken (order Galliformes) sera against chicken anaemia virus (CAV) and sera against avian pox virus as well as positive and negative sera against chicken pox virus raised in a milvago chimango (order Falconiformes) were used. Positive sera of either avian species demonstrated clear fluorescent staining of infected cells while negative sera did not show any reaction. This demonstrated that both chicken and milvago chimango antibodies were able to bind human C1q. Since both avian species tested belonged to different orders, we believe that antibodies of other avian species will also bind human Clq allowing serological surveys in feral birds through S-ICFT.     

Serum chemistry and antibody status to some avian pathogens of free-living and captive condors (Vultur gryphus) of central Chile.

This study provides information on serology and serum chemistry of the Andean condor (Vultur gryphus). Twenty condors living under natural conditions were captured, blood sampled, measured, and released. In addition, 12 captive condors maintained at the Metropolitan Zoo of Santiago, Chile, were included as a comparison with free-living birds. All sera were negative to antibodies against reference strains of avian paramyxoviruses of serotypes 1 to 9, avian influenza and avian pox virus. Free-living condors had total protein, albumin, globulin and Mg values significantly (P< 0.05) lower than those of captive birds. The haematological values obtained in free-living condors are of particular interest since they may correspond to the optimum values of the species.     

Serologic cross-reactivity among humans and birds infected with highly pathogenic avian influenza A subtype H5N1 viruses in China

To study immunogenicity and serologic cross-reactivity of hemagglutinins (HAs) among humans and birds infected with highly pathogenic avian influenza (HPAI) H5N1, four representative H5N1 HA genes from humans and birds infected with distinct genetic clusters of H5N1 viruses in China were cloned, and several H5N1 infected human serum and H5N1 positive bird serum samples were used. Recombinant HA proteins were generated for ELISA assays and pseudotype viruses containing HAs were produced for neutralization assays and hemagglutination inhibition (HI) tests. We found significant differences among clades compared to species in binding, neutralization and HI activity of H5N1 strains isolated from birds. While significant differences were observed among species in H5N1 isolated from humans, investigation of H5N1 infected human and avian sera provided evidence that the pressure from nAb may be a driving force for positive selection. Therefore, improved anti-viral nAb therapies could block avian influenza transmission in humans.     

Potency of an inactivated avian influenza vaccine prepared from a non-pathogenic H5N1 reassortant virus generated between isolates from migratory ducks in Asia

A reassortant influenza virus, A/duck/Hokkaido/Vac-1/2004 (H5N1) (Dk/Vac-1/04), was generated between non-pathogenic avian influenza viruses isolated from migratory ducks in Asia. Dk/Vac-1/04 (H5N1) virus particles propagated in embryonated chicken eggs were inactivated with formalin and adjuvanted with mineral oil to form a water-in-oil emulsion. The resulting vaccine was injected intramuscularly into chickens. The chickens were challenged with either of the highly pathogenic avian influenza virus strains A/chicken/Yamaguchi/7/2004 (H5N1) or A/swan/Mongolia/3/2005 (H5N1) at 21 days post-vaccination (p. v.), when the geometric mean serum HI titers of the birds was 64 with the challenge virus strains. The vaccinated chickens were protected from manifestation of disease signs upon challenge with either of the highly pathogenic avian influenza viruses. However, challenge virus was recovered at low titers from the birds at 2 and 4 days post-challenge (p.c.). All 3 chickens challenged at 6 days p.v. died, whereas 3 chickens challenged at 8 days p.v. survived. These results indicate that the present vaccine confers clinical protection and reduction of virus shedding against highly pathogenic avian influenza virus challenge and should be useful as an optional tool in emergency cases.     

Identification of measles virus-specific hemolysis-inihibiting antibodies separate from hemagglutination-inhibiting antibodies.

The occurrence of antibodies giving hemolysis inhibition (HLI) but not hemagglutination inhibition (HI) was examined in human convalescent and rabbit hyperimmune sera. HI antibodies, which through their interaction with hemagglutinin components display HLI activity, were removed by absorption with Tween 80-ether (TE)-treated measles virus material. This absorption did not change the titer of non-HI HLI antibodies. After removal of HI antibodies from 16 late measles convalescent sera and three batches of gamma globulin. HLI antibody titers showed a two- to eightfold reduction. The titers of neutralizing antibodies were reduced from 1/4 to 1/20 of the original titers. There was a good correlation between the titers of neutralizing and HLI antibodies both in sera from which HI antibodies had been removed by absorption and in sera spontaneously showing markedly higher HLI than HI antibody titers. HLI antibodies with these characteristics could be identified in HI tests when whole virus instead of TE-treated material was used an antigen and anti-antiserum was added to the tests. In contrast to the situation in human sera, antibodies remaining after removal of HI antibodies from rabbit hyperimmune sera against purified virus particles were detectable in neutralization and HLI tests only in the presence of anti-antiserum. However, virus particles from which the major fraction of all envelope projections had been removed by treatment with 0.004% trypsin induced the production of non-HI HLI antibodies active also in the absence of anti-antiserum. TE and formalin treatment destroyed the hemolytic activity of virus preparations and also their capacity to induce a production of non-HI HLI antibodies.     

An ELISA blocking test using a peroxidase-labelled anti-HN monoclonal antibody for the specific titration of antibodies to avian paramyxovirus type 1 (PMV1)

Summary This report describes an ELISA blocking test using a peroxidase-labelled monoclonal antibody which binds to the HN protein of Newcastle disease (NDV).This test allows specific detection of type 1 avian paramyxovirus (PMV1) antibodies but does not detect other avian paramyxovirus (PMV2-9) antibodies recognized by the usual serological NDV tests (HI, Orgenics, and Agritech ELISA tests). Furthermore, swollen head syndrome and influenza antibodies were also not detected. ELISA blocking and HI titers of sera collected from SPF chickens immunized with 18 different PMV1 strains (including pigeon isolates) were the same; the correlation between ELISA blocking and HI titers was highly significant (P<0.001).In comparison with ELISA tests available commercially at the present time, the ELISA blocking test can be performed more quickly and is applicable without modification to sera from different species of fowls. For this reason, the test appears to be useful for determining the immunity and sanitary status of fowls. When recombinant or deleted vaccines become available, the test should make it possible to demonstrate with confidence any infection of fowls by wild type PMV1.     

Type a influenza viruses in birds in southern Spain: serological survey by enzyme-linked immunosorbent assay and haemagglutination inhibition tests

Of 927 sera taken from poultry from 84 flocks, 33% proved seropositive. Sixty-three per cent of flocks were found to be seropositive to ELISA (almost all situated within areas where there were waterfowl). The comparable figures, using an HI test, were 16% and 47%, respectively. These data suggest that the influenza A viruses may be enzootic in this area. Of 331 wild birds tested, belonging to 18 species of nine families, 40% proved seropositive to ELISA. Notable high infection rates were found among Anatidae (43%), flamingoes (43%) and sparrows (31%); the latter species may play an important role in carrying the disease from its natural reservoirs to domestic farms. Antibody titres found in wild birds were considerably higher than those found in poultry.     

Antibodies to type A influenza viruses in sera from nonhuman primates

Summary The results of serological testing of nonhuman primate sera obtained over a four-year period showed a high incidence of antibodies to influenza virus strains of the H2 and H3 hemagglutinin sub-types. This would indicate that outbreaks of type A influenza virus infection occurred in certain primate species and suggest another possible reservoir for influenza virus in nature. Hemagglutination-inhibition (HI) testing of sera collected from African green monkeys captured in the Kenya-Tanzania area of East Africa demonstrated significant antibody titers to A/Hong Kong (HK)/68 (H3N2) virus in serum samples obtained 8 to 10 months prior to the first report of influenza-like illness in East Africa, and 3 to 5 months prior to the first report of outbreaks of acute respiratory disease in southeastern China due to A/HK/68 (H3N2). The results suggest that certain species of nonhuman primates may be involved in the epidemiology of influenza due to their close association with human living areas.