Experimental infection of young and laying geese with egg drop syndrome 1976 adenovirus strain B8/78

Of 19 large flocks of geese in Hungary 15 were serologically positive to the egg drop syndrome 1976 (EDS-76) avian adenovirus strain B8/78. Sixty-five to 100% of the samples taken from each flock were positive with haemagglutination inhibition (HI) titres between 1:8 and 1:256. All the progeny of geese with HI antibodies had maternally derived antibodies. After infection per os of 8-, 19- and 29-day-old goslings with the B8/78 strain of EDS-76 adenovirus clinical signs of disease and mortality were not seen although the birds developed antibodies and shed the virus in the faeces. No lesions were noted using light microscopy. Egg production was unaffected in geese experimentally infected with B8/78 virus strain and there was no change in egg quality. The layers developed antibodies to EDS-76 adenovirus and shed the virus in the faeces. Contact-controls responded in the same way. The results of these experiments suggest that when investigating the origin of EDS-76 avian adenovirus infection not only ducks but also geese should be considered as a potential source of infection.     

Results of a serological survey made in France (Vendeé region) in domestic ducks

The sera of domestic ducks were examined for antibodies to several infectious agents of palmipeds during the winter of 1982 in the abattoirs in la Vendée, an important region of duck production in the West of France. The performance of each batch and their antecedents was also studied. In Barbary ducks and crossbred ducks (from male wild ducks and female domestic ducks), antibodies were found to the virus of egg drop syndrome 16 (EDS 76), to Newcastle disease virus (NDV), to duck hepatitis virus and to chlamydia psittici. In Nantais ducks (resulting from a cross between Khaki Campbell and Pekin ducks), antibodies were detected against EDS 76 virus, duck hepatitis virus and chlamydia. The frequency and levels of antibodies varied between types or strains of ducks. About 50% of the Barbary ducks had high levels of antibodies to EDS 76 virus, whereas 70 to 80% of crossbred and Nantais ducks had these antibodies but at very low levels. Virtually 100% of Barbary ducks had high levels of antibodies against Derzsy's disease virus whereas only 10% of the batches of crossbred ducks had antibodies at varying levels. The proportion of batches with antibodies against NDV was higher in crossbred ducks (25%) than in Barbary ducks (2%) and the titres were low. Antibodies to duck hepatitis virus, when present, were of a high titre irrespective of type or strain of duck. Infection by chlamydia was suspected in 3% of lots of Barbary ducks, in 33% of lots of crossbred ducks, and in 3.7% of lots of Nantais ducks. These findings are discussed and considered in relation to breeding history and performance of the flocks. The EDS 76 virus could in certain circumstances cause weight loss in male Barbary ducks. These epidemiological observations need to be confirmed experimentally.     

Serological survey of wild birds in Australia for the prevalence of antibodies to egg drop syndrome 1976 (EDS-76) and infectious bursal disease viruses

Serum samples from 11 species of seasonal or sedentary wild water birds in Western Australia were examined for antibodies to viruses endemic in commercial chickens in Australia. Antibodies to two serotypes of infectious bursal disease virus, and to egg drop syndrome 1976 (EDS-76) virus were demonstrated in these sera.     

Dropped egg production in ducks associated with adenovirus infection

Six thousand laying ducks were kept on a large-scale farm, half of which were home-reared ducks, and the other half were imported birds. At the beginning of the laying period 70% of the ducks reared on the farm possessed EDS antibodies in titres of 1:32 to 512, whereas only 20% of the imported ducks were found to possess haemagglutination inhibition (HI) antibodies in titres of 1:16 to 1:32. When the ducks reached the peak of egg production, a severe diarrhoea was observed and egg production decreased by 50% and 10% in the imported ducks and the home-reared ducks, respectively. The decrease in egg production lasted only for 1 week, and production then returned to a normal level. Shell-less and soft-shelled eggs were not recorded, but sometimes misshapen eggs were found. From small intestinal contents of ducks with diarrhoea a haemagglutinating virus identical with McFerran's adenovirus 127 was isolated. HI antibodies appeared in 100% of the convalescent sera. Hatching losses exceeded those observed in previous year by 20%. It may be presumed that the drop in egg production of laying ducks was caused by EDS virus infection.     

Studies on EDS-76 virus infection in laying chickens

A new strain (D61) of EDS-76 virus associated with an egg-drop syndrome in chickens was compared by neutralisation and haemagglutination-inhibition (HI) tests with two recognised strains (127, BC14). All three strains were serologically indistinguishable. Each strain lacked any demonstrable ability to lyse the red cells of chickens or ducks. To investigate its pathogenicity and immunogenicity, chickens of different ages were inoculated with the D61 strain per os. All birds produced eggs with aberrant shells in varying numbers, but internal quality was not affected. There were no other constitutional effects and no obvious clinical signs throughout. Fertility and hatchability remained unaffected. At 19 weeks, onset of laying was delayed by 9 days, and total daily egg production was eventually 15 to 20% lower than that of uninfected controls of the same age. The total daily egg production of older chickens was unaltered, except for approximately 2 weeks after infection at 26 weeks of age and for 5 weeks at 47 weeks of age. The highest number of eggs with abnormal shells was laid by chickens infected at 33 weeks (up to 38%) or 47 weeks of age (up to 40%). Infective virus was successfully recovered from the livers of chicks immediately after hatching from eggs laid by hens 7 to 11 days following infection. Neutralising, HI and precipitating antibodies developed within 6 days of infection, and were associated with the IgG class of immunoglobulins. Chicks hatched from eggs of infected hens possessed maternal antibodies with a half-life of 3 days; these antibodies conferred a passive immunity to challenge for approximately the first 4 weeks of life, based on the evidence of HI responses after challenge.     

Development of a recombinant ELISA using yeast (Pichia pastoris)-expressed polypeptides for detection of antibodies against avian influenza A subtype H5

Two truncated sequences (designated P1 and rHA1) of influenza A virus subtype H5 haemagglutinin (HA) were cloned and expressed in yeast Pichia pastoris (P. pastoris). These polypeptides were used in an indirect recombinant ELISA (rELISA) for detection of H5 antibodies in poultry. Serum samples obtained from broiler chickens vaccinated with commercial inactivated vaccine (H5N2) and control negative sera from non-vaccinated chickens against influenza were tested using rP1-ELISA, rHA1-ELISA, whole H5N1-ELISA, Western blot, agar gel immunodiffusion (AGID) and haemagglutination inhibition (HI) tests. The rHA1-ELISA proved to be highly sensitive and specific. To study the validity of rHA1-ELISA, a total of 179 serum samples obtained from commercial broiler chickens vaccinated previously with commercial H5N2 inactivated vaccines, were tested by rHA1-ELISA, commercial ELISA (cELISA) and HI. The relative sensitivity and specificity between rHA1-ELISA, and HI tests were 100% and 70%, respectively, and between cELISA and HI were 100% and 57%, respectively. The agreement ratio between rHA1-ELISA and HI was 84.9% and between cELISA and HI tests was 76.5%. Serum samples obtained from ducks vaccinated with commercial inactivated H5N2 were tested by rHA1-ELISA and the results showed significant reactivity with duck sera. In conclusion, the results demonstrate the potential applicability of the rELISA for the determination of antibodies to H5 influenza virus in chickens and ducks.     

Natural infection of ducks with Mycoplasma synoviae and Mycoplasma gallisepticum and Mycoplasma egg transmission

Mycoplasma gallisepticum (MG), M. synoviae (MS), M. cloacale (MC) and M. anatis were isolated from ducks kept in a yard in close contact with chickens that were infected with MG, MS and some other avian Mycoplasma species. MG, MS and MC were isolated also from embryonated duck eggs and from infertile duck eggs laid during the first four weeks of egg production. Infected ducks did not show clinical signs of MG or MS infection in chicken. Detectable MG and MS agglutinating antibodies were not present in duck sera. However, they were found in two yolks of 10 tested from embryonated eggs. In the haemagglutination - inhibition (HI) tests yolks from embryonated eggs yielded significantly higher (P<0.01) titres of MS antibodies than duck sera. Geometric mean value of MS HI titres in tested duck sera was 20, while those of yolks from embryonated eggs was 333. It is probably the first report concerning isolation of MS from the naturally infected ducks and furthermore, concerning isolation of MG, MS and MC from naturally infected embryonated eggs.     

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.     

BK virus: I. Seroepidemiologic studies and serologic response to viral infection

Sera of 656 patients in seven age groups were tested by hemagglutination=inhibition (HI) and complement=fixation (CF) tests for antibodies to BK virus. Both tests revealed that BK virus antibodies are common in the population of southern Germany, and that primary infection is apparently acquired during childhood. The highest rate of positive sera, 71% in the HI test and 56% in the CF test, was found in the 15–30 year old group. The antibody titers correlated in both serologic tests, but HI tests were more sensitive and reliable than CF tests. An indirect immunofluorescence assay for the detection of BK virus-specific IgM antibodies was also developed. Studying umbilical cord blood sera from 846 healthy donors, BK virus IgM antibodies were detected in 77 sera (9.1%).     

Humoral immunity in natural infection by tick‐borne encephalitis virus

Tick‐borne encephalitis (TBE) virus is one of the most important flaviviruses associated with neurological disease in Europe. Cross‐reactive antibodies elicited by different flaviviruses can make difficult the interpretation of ELISA and hemagglutination‐inhibition (HI) tests for the diagnosis of TBE. Neutralization tests, which are more specific, are not in common use because they are difficult to perform and standardize. A plaque reduction neutralization test (PRNT), optimized previously in vaccinated children, was evaluated in sera from acute cases of TBE, collected for diagnostic purposes, and from healthy human population and wild ruminants, collected for serosurvey purposes. The PRNT results were compared with the results of ELISA and HI tests. In acute TBE disease, most sera were positive for IgM antibodies by ELISA and with high HI antibody titers; neutralizing antibodies were detected in 71.4% of patients, at a very low titer (1:10 NT₅₀) in almost all cases. Seroprevalences of 8% and 6.5% for anti‐TBE ELISA antibodies were found in healthy subjects and wild ruminants, respectively. Among anti‐TBE positive healthy subjects, a very low 1:10 NT₅₀ titer was detected in 17.4% of cases, while NT₈₀ titers ranging from 1:10 to 1:80 were detected in 65.2% of cases. Among wild ruminants, 90.9% of ELISA and HI positive samples showed a positive, ≥1:10 NT₈₀ titer. In conclusion, neutralization assays can be useful for the diagnosis and serosurveys of TBE. J. Med. Virol. 81:665–671, 2009 © 2009 Wiley‐Liss, Inc.     

Antibody responses to mumps virus proteins in natural mumps infection and after vaccination with live and inactivated mumps virus vaccines

Paired sera from 20 patients with acute mumps infection, 16 from persons vaccinated with live attenuated mumps virus vaccine, and 12 from persons vaccinated with formalin-inactivated virus vaccine were studied for mumps antibodies by single radial hemolysis (SRH), hemagglutination inhibition (HI), and by enzyme immunoassays (EIA) specific for whole virus, envelope glycoprotein, and nucleocapsid antibodies. Mumps patients had diagnostic rises in serum mumps antibodies in 90–100% of the cases depending on the method of assay. Vaccination resulted in seroconversion in 75–88% (live vaccine) and in 92% (inactivated vaccine) of the cases as detected by SRH or EIAs, whereas HI detected seroconversion only in 38% and 58% of the cases, respectively. Immunoprecipitation analyses revealed that all sera from mumps patients and nearly all postvaccination sera had antibodies against the main structural proteins of mumps virus. By immunoblotting, antibodies against denatured hemagglutinin-neuraminidase (HN) and fusion protein (F) were detected in 15–25 % of mumps patients and persons vaccinated with live vaccine, whereas most postvaccination sera from those vaccinated with inactivated vaccine had HN (92%) and F (83%) protein antibodies, suggesting that antibodies against the denatured form of proteins are formed.     

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.     

Comparison of immunofluorescence and hemagglutination inhibition tests and enzyme-linked immunosorbent assay for detection of serum antibody in rats infected with hemorrhagic fever with renal syndrome virus.

Serum antibodies against the B-1 strain of hemorrhagic fever with renal syndrome (HFRS) virus in wild and experimental rats were investigated by the indirect immunofluorescent antibody (IF) test, the hemagglutination inhibition (HI) test, and an enzyme-linked immunosorbent assay (ELISA). In the newly developed ELISA, monoclonal antibodies to the B-1 strain were applied as coating antibody. In sera from wild rats, the results obtained by the ELISA agreed quite well with those obtained by the HI test, but some serum samples that gave a negative reaction in the HI test gave a positive reaction in the IF test, although their IF titers were very low. In serum samples from experimental rats that had been kept in an animal house infected with HFRS virus, a group with high titers and a group with low or negative titers were clearly differentiated by all the tests.     

BK virus: II. Serologic studies in children with congenital disease and patients with malignant tumors and immunodeficiencies

Sera of 451 children with congenital diseases and of 185 tumor patients were tested for BK virus-specific antibodies by hemagglutination inhibition and IgM-immunofluorescence tests. Compared to age-matched control groups, higher percentages and significantly elevated geometric mean titers of HI antibodies were found in all patient groups tested. Of children under six months of age with congenital diseases such as dysplasia, cerebral defects, and hyperbilirubinemia and hepatosplenomegaly, 4.2% (17/402) had BK virus-specific IgM antibodies. No positive sera were found in 68 control sera. Of tumor patients 5–15 years of age, 8.6% (16/185) had IgM antibodies to BK virus. In the control group, 3% (3/99) had them. Serial serum samples from 76 tumor patients treated with cytostatic drugs showed seroconversion in three cases. No relationship between certain clinical features and BK virus infection was noted. Isolation of BK virus was successful from urines of two infants with connatal defects, six patients suffering from malignant tumors, and four patients with inherited immunodeficiencies.     

Influenza virus circulation in wild aquatic birds in Italy during H5N2 and H7N1 poultry epidemic periods (1998 to 2000).

Two epidemics of avian influenza due to H5 and H7 highly pathogenic viruses occurred in poultry in Italy in 1997/98 and 1999/2000, respectively. The circulation of these serotypes in wild aquatic birds was investigated examining 638 cloacal swabs and 621 sera collected from 150 gulls, 162 coots, and 326 ducks trapped in Italian wetlands from 1998 to 2000. Seroprevalences against influenza A viruses, detected by a double-antibody sandwich-blocking enzyme-linked immunosorbent assay (ELISA), were 11% in gulls, 16% in coots, and 45% in ducks. Among the Anatidae group, duck species wintering in Mediterranean areas showed significantly higher values than ducks wintering in South-Saharan areas of Africa. In order to detect H5 and H7 antibodies, the haemagglutination-inhibition assay and two competitive ELISA tests (H5-ELISA and H7-ELISA) using monoclonal antibodies specific for H5 and H7 subtypes were performed. None of the aquatic bird species were found seropositive for H7 subtype, whereas H5-positive sera were found by both the haemagglutination-inhibition and ELISA assays in ducks only. The highest H5 seroprevalences were detected by H5-ELISA; overall, 5% (10/201) of duck species wintering in Mediterranean areas tested positive by this assay, with annual seroprevalences ranging from 2% (2/123) to 12% (6/51). In the present study, only five viruses belonging to H1N1, H11N6, and H2N3 subtypes were isolated from ducks. However, the H5 seroconversion observed in one mallard duck at the beginning of 1998 indicates that H5 virus circulation also occurred in the study area.     

Comparison of enzyme-linked immunosorbent assay and haemagglutination inhibition test for the detection of Newcastle disease virus antibodies in human sera.

A comparison of haemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) techniques for the detection of antibodies against Newcastle disease virus in sera from persons working in poultry farms and veterinary vaccine institutes and from the general population revealed that 22% more sera were positive by ELISA compared to HI. No samples were negative by ELISA but positive by HI. While HI titres of positive sera were found in the range 8-64, ELISA titres were between 16 and 512. It was interesting that though 78% sera had concordant results by the two tests, titres obtained by ELISA were nearly six times higher than those by HI.     

Antibody response of wild birds to natural infection with alphaviruses

From 1986 to 1990, we conducted our second longitudinal study in the central (upstate) New York (CNY) area on the wild avian hosts of eastern equine encephalomyelitis (EEE) virus. Field-collecting methods mirrored a study conducted from 1978 to 1980 at the same endemic focus. Over the 5-yr study period, we captured 6,296 birds representing 99 species and took 4,174 blood samples from representatives of 83 species. Gray catbirds, song sparrows, and veerys were the three dominant species captured and bled, accounting for 40 and 55% of birds captured and bled. Blood clots were assayed for virus and sera tested for hemagglutination-inhibition (HI) antibodies to EEE and Highlands J virus. Virus isolations from birds defined two epiornitics of EEE virus in 1988 and 1990, and an epiornitic of HJ virus in 1986. Infected birds responded with the production of HI antibodies with titers indicative of recent infection (HI > or = 1:160), and titers of sera positive during the epiornitics were significantly higher than positive sera during nonepiornitics. The 1990 EEE epiornitic extended from mid-July to the end of September, providing data to compare infection rates among species, habitats, and combinations of species with habitats. Few significant differences were found. The HJ epiornitic was only the second time this virus has occurred in CNY. Song sparrows were identified as the primary amplifying avian host of both viruses, although our capture and serological data would suggest a role for gray catbirds as the species most likely involved in yearly virus reintroduction. However, the cryptic nature of enzootic virus maintenance remains unresolved for the CNY virus foci. The appearances of HJ and EEE viruses were not epidemiologically linked, and there were no virus isolations from adults returning on site or virus isolations without concurrent isolations from mosquito vectors. Whether EEE and/or HJ virus are consistently present in or sporadically introduced into the inland foci of CNY area still has not been determined.     

Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of Mycoplasma gallisepticum antibodies

Sensitivity and specificity of two commercial Mycoplasma gallisepticum (MG) enzyme-linked immunosorbent assay (ELISA) kits, rapid slide agglutination (SA) and haemagglutination inhibition (HI) tests were compared using sera from specific pathogen free chickens, turkeys or ducks which had been inoculated with various avian mycoplasmas, bacteria or with a reovirus. Results show that sensitivity of SA was superior to ELISA and HI tests in the ability to detect antibodies formed in early response to MG infection. However, both ELISA kits and HI tests had a higher degree of specificity.     

Dengue 2 in eastern Senegal: serologic survey in simian and human populations. 1974-85

After the previously reported isolations of dengue 2 virus in eastern Senegal in 1974 and 1981-1982, a retrospective serological study on simian and human populations was carried out in the same area. We investigated 1,095 simian sera collected at regular intervals between 1974 and 1984 from wild caught monkeys and 1,783 human sera from young children less than 11 years old collected during punctual surveys after the rainy season from 1976 to 1985. Sera were tested using HAI test, CF test and for someone's ELISA for specific IgM antibodies. Serological data from monkeys corroborated the virus isolations and demonstrated the existence of two epizootics in 1974-1975 and 1981-1982. No CF antibodies were detected in children sera up to 1981 epizootic when about 11% of tested sera showed a probable infection by dengue 2 virus, no clinical dengue infections were notified by the medical staff. After 1982, serological results showed that the virus maintained in the same area until 1985. The mechanism of the circulation of dengue 2 virus in eastern Senegal is discussed on the basis of these serological results.     

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.