Antigenic and biological characterisation of avian paramyxovirus type I isolates from pigeons - an international collaborative study

During 1981-1983 a disease of pigeons (Columba livia), characterised predominantly by nervous signs, spread across Europe. In the present study 57 viruses isolated from pigeons from 15 countries (12 European, Japan, Israel and Sudan) were characterised. All were shown to be avian paramyxoviruses of the A/PMV-1 serotype. Monoclonal antibody binding tests showed 53 of the viruses to be identical. The virus from Sudan was similar to these viruses but showed distinguishable variation. One vaccinal virus from France and two virulent viruses from Czechoslovakia were unrelated to the other pigeon A/PMV-1 isolates.     

Matrix Protein Gene Sequence Analysis of Avian Paramyxovirus 1 Isolates Obtained from Pigeons

The matrix protein gene was cloned and sequenced for several recent isolates of avian paramyxovirus type 1 (APMV-1). Specifically, isolates from pigeons and doves, members of the Columbidae family were examined. APMV-1 is the causative agent of Newcastle disease and the virus is associated with disease among a diverse number of avian species. Newcastle disease virus (NDV) isolates from pigeons have also been classified as pigeon paramyxovirus type 1 (PPMV-1). Matrix protein gene sequences for PPMV-1 isolates clustered together as a group relative to isolates from other species phylogenetically. However, there were also isolates from pigeons or doves that grouped with APMV-1 isolates from other species. This indicates that PPMV-1 may be circulating among Columbidae members as a distinct lineage, but that these avian species may also harbor other NDV strains as well. Of particular interest was a dove isolate from Europe that had an aberrant fusion protein cleavage site and was an outlying member phylogenetically between the two major groups of APMV-1 isolates.     

Quantitative estimation of Newcastle disease virus antibody levels in chickens and turkeys by ELISA

A quantitative single-well ELISA for estimation of Newcastle disease (ND) virus antibodies in chickens and turkeys was developed using purified antigen from PMV-1/Chicken/Ulster 2C/71. The test was standardised using sera from 20-week-old chickens or 20- 30-week-old turkeys. Absorbance values for negative sera in chickens increased with the age of the birds but overall was lower than the cut-off for the test. ND haemagglutination inhibition (HI) positive field sera were always positive by ELISA and the mean was significantly higher than that of the negative population. Standard antisera to four of seven of the other PMV serotypes (including PMV-3) gave positive reactions in ELISA and three were also positive at low level by PMV-1 HI test. Absorbance values remained negative in turkeys given two inoculations of PMV-3 vaccine in spite of good PMV-3 HI responses. Doubling dilutions of chicken and turkey sera were tested by ELISA and end-point titres calculated. Standard curves relating ELISA titre and absorbance of each sample at 1/100 dilution were constructed and used to determine titres of test samples from single well absorbance values. A significant positive correlation between ELISA titre and HI titre for chicken and turkey sera was demonstrated. Sensitivity of the test was investigated using birds experimentally infected with PMV-1/Chicken/Ulster 2C/71 or a pigeon PMV-1 isolate. Seroconversion was detected at the same time by ELISA and HI. In experiments to estimate the ELISA titre required to protect birds against virulent ND, five groups of chickens were vaccinated twice with one of four commercial ND vaccines (three inactivated; one live) on two occasions and challenged with a virulent ND strain (PMV-1/ Chicken/Antrim/73). Two of the groups vaccinated with inactivated vaccines were protected against challenge. In another group also given inactivated vaccine, clinical signs were seen in one bird and ELISA proved a better indicator of immune status than HI. In the groups given living vaccine, no signs of ND were seen and ELISA indicated a much higher level of vaccinal antibody than HI test. In turkeys given two inoculations with inactivated vaccine, antibody levels were boosted to acceptable levels by ELISA and HI indicating that vaccinal antibody levels were adequate.     

Characterization of avian paramyxovirus type 1 strains isolated in Germany during 1992 to 1996.

In Germany all avian paramyxoviruses (APMV) isolated in regional laboratories are collected and characterized by the National Reference Laboratory. From 1992 until 1996, 635 APMV-1 virus isolates were submitted from almost all regions. Of these viruses, 371 were isolated from chickens, 39 from other poultry, 171 from pigeons and 54 from exotic birds. All isolates were examined for virulence in intracerebral pathogenicity index (ICPI) tests, for their ability to react with a panel of monoclonal antibodies (mAb) and their thermostability. In addition, the nucleotide sequences of the cleavage site of the fusion protein of a few virus isolates were determined. Most isolates from chickens and other poultry were of the velogenic pathotype. This virus was responsible for the epizootic in 1993 to 1995 in many small flocks. The same virus was obtained from some pigeons and some exotic birds. The pathogenicity of the velogenic/epizootic virus was high with most viruses giving ICPI values of 1.8 to 1.9, and the sequences of the cleavage site of all velogenic isolates tested were closely related. However, viruses isolated at the beginning of the epizootic period differed from viruses isolated towards the end in their reaction with some mAbs. 149 virus isolates were identified as pigeon variant PMV-1 (PPMV-1). Most of these were obtained from pigeons but a few were isolated from chickens and other birds. Most lentogenic isolates proved to be vaccine virus strains.     

Failure of a low virulence Streptococcus gallolyticus serotype 1 strain to immunize pigeons against streptococcosis

Ten pigeons were inoculated intravenously with the low virulence Streptococcus gallolyticus strain PDH 827, which belongs to serotype 1, supernatant phenotype A - T2. The birds did not develop clinical disease but shed S. gallolyticus in their faeces, and antibodies against the bacterium were detected in post-inoculation plasma samples of all birds. Seven weeks later, these pigeons, as well as 14 control pigeons, were challenged intravenously with the highly virulent S. gallolyticus strain STR 357, which also belongs to serotype 1 but to the supernatant phenotype A + T1. Post-inoculation morbidity in the immunized group amounted to 90%, demonstrating that no protective immunity had been built up after the first infection. These findings indicate that serotype-specific antigens are not, or at least not solely, involved in induction of protection against S. gallolyticus septicaemia in pigeons.     

Assessment of vaccination of pigeons against paramyxovirus type 1 infection with inactivated aqueous-suspension or oil-emulsion vaccines

The immune response and resistance to PMV1 challenge of pigeons vaccinated with inactivated oil-emulsion (OE) or aqueous-suspension (AS) vaccines were compared. ASa was based on NDV LaSota strain, ASb on NDV Terumo strain, OEa on NDV Poletti strain and OEb on NDV Ulster 2C strain. Groups were each vaccinated subcutaneously with one of ASa (0.2ml), ASb (0.2ml), OEa (0.5ml), OEa (0.2ml) and OEb (0.5ml). An unvacci-nated group was kept as control. The immune response measured by HI tests was the highest with the single 0.2ml dose of ASa vaccine. The single 0.2ml dose of OEa vaccine produced a poor HI response. Challenge with 'pigeon' PMV1 (intramuscular + intranasal/ocular) one month after vaccination resulted in the vast majority of unvaccinated pigeons excreting virus in the laryngeal secretions and the faeces and all . birds died. In comparison, virus shedding and morbidity-mortality were significantly reduced in the five challenged vaccinated groups. However, morbidity-mortality was higher in the OE groups than in the AS groups. No ASa vaccinated pigeon developed clinical signs and only one ASb vaccinated pigeon presented nervous signs. In contrast, morbidity-mortality reached 30 and 35% in the OE vaccinated groups. Only the inactivated aqueous-suspension vaccines, especially that prepared from NDV LaSota strain, gave, after one dose injection (0.2ml), high resistance to a severe challenge with 'pigeon' PMV1.     

Newcastle disease in the European Union 2000 to 2009

Newcastle disease (ND) is a devastating disease of poultry that has to some extent been neglected by those working in the field in the past 10 to 15 years while attention has been focused on the emergence and spread of highly pathogenic avian influenza caused by a H5N1 subtype virus. During 2000 to 2009 in the European Union (EU) member states, ND viruses virulent for chickens have been detected in wild birds, domesticated pigeons and poultry. Based on these isolations it appears that the epizootic in racing pigeons caused by the variant viruses termed pigeon avian paramyxovirus type 1, which form the genetic group 4b(VIb) first seen in Europe in 1981, continued during 2000 to 2009, and the virus is probably enzootic in racing pigeons in some EU countries. This virus appears to have spread regularly to wild birds, especially those of the Columbidae family, and has been the cause of significant outbreaks in poultry. Other avian paramyxovirus type 1 viruses responsible for ND outbreaks in the EU during 2000 to 2009 have been those from genetic groups 5b(VIIb) and 5d(VIId). There is evidence that the former may well represent spread from a wild bird source and these viruses have also been isolated from wild birds, while the latter represents continuing spread from the East. Future legislation or recommendations aimed at the control and eradication of ND will need to encompass these three sources of virulent ND viruses.     

Characterization of two pigeon paramyxovirus type 1 isolates in China

For over three decades, there has been a continuing panzootic caused by a virulent variant avian paramyxovirus type 1 strain, the so-called pigeon paramyxovirus type 1. It is found primarily in racing pigeons, but it has also spread to wild birds and poultry. In this study, two pigeon paramyxovirus type 1 strains, SD12 and BJ13, obtained from diseased pigeons in China, were characterized. Phylogenetic analysis based on complete sequences allowed characterization of both strains as genotype VI, class II. Further phylogenetic analysis of a 374-nucleotide section of the fusion gene showed that SD12 fell into lineage VIbii-d and BJ13 into VIbii-f. The deduced amino acid sequence of the cleavage site of the fusion protein confirmed that both isolates contained the virulent motif ¹¹²K/RRQKR↓F¹¹⁷ at the cleavage site. Nevertheless, the values of intracerebral pathogenicity indices showed the SD12 isolate to be a velogenic strain and BJ13 isolate to be a mesogenic strain. The SD12 isolate was further investigated via clinical observation, RNA detection, histopathology and viral serology in experimentally infected 3-week-old chickens. It showed a mild pathological phenotype in chickens, with viral replication restricted to a few tissues. The molecular mechanism for the SD12 isolate to have a virulent motif but low levels of virulence for chickens requires further study.     

A molecular epidemiological investigation of isolates of the variant avian paramyxovirus type 1 virus (PPMV-1) responsible for the 1978 to present panzootic in pigeons.

A sequence of 375 nucleotides, which included the region encoding the cleavage activation site and signal peptide of the fusion protein gene, was determined for 178 isolates of the pigeon variant strain of Newcastle disease virus (PPMV-1). These were compared with the sequences of 47 similar isolates published by GenBank, which included 30 isolates from pigeons and 17 representatives from each sublineage of avian paramyxovirus type 1. The resulting alignment was analysed phylogenetically using maximum likelihood and the results are presented as unrooted phylogenetic trees. By phylogenetic analysis all the PPMV-1 isolates except one were placed in lineage 4b (VIb). Within this lineage there was considerable genetic heterogeneity, which appears to be predominantly influenced by the date of isolation and, to a lesser extent, geographical origins of the isolates. There were two large distinguishable groups, 4bi and 4bii. The earliest isolate available, PIQPI78442, isolated in 1978 in Iraq, was situated at the node from which the two groups diverge.     

Relationship between several criteria of challenge-immunity and humoral immunity in chickens vaccinated with avian infectious bronchitis vaccines

Several methods were compared for assessing the immunity of chickens vaccinated with avian-infectious bronchitis virus (IBV) by observing tracheal ciliary activity, respiratory signs and virus recovery from several organs following challenge with virus which had not been passaged in embryos. All chickens vaccinated twice with live Kita-1 strain were protected according to all criteria. However, in chickens vaccinated with killed IBV and with H-120 strain 13 weeks earlier, respiratory signs were present and tracheal virus was isolated from the birds with ciliostasis regardless of their having significant levels of haemagglutination inhibiting (HI) antibody. This indicated that only observing the ciliary activity is insufficient for assessing complete immunity and no direct correlation exists between the serum HI titre and the presence of ciliostasis, respiratory signs and virus recovery from tracheas, excluding those from kidneys and genitalia. Regardless of the serum HI titres at challenge, full protection based on the six criteria was found in some birds at 1 week and in most chickens from 2 to 6 weeks after intraocular vaccination with live Kita-1 strain.     

Vaccination trials against pigeon herpesvirus infection (Pigeon herpesvirus 1)

Vaccination trials were made with live attenuated or inactivated vaccines to control pigeon herpesvirus infection (Pigeon herpesvirus 1, PHV₁).Both kinds of vaccine gave similar results. Both of them were able to reduce primary viral excretion and clinical signs after challenge. Nevertheless, neither attenuated nor inactivated vaccines were able to prevent the appearance of carriers since most of the pigeons re-excreted virus after cyclophosphamidetreatment. However, vaccination would help to prevent spontaneous viral reexcretion and therefore to control viral dissemination. When animals were vaccinated with inactivated vaccine after challenge with a virulent strain, re-excretion was also reduced.     

Health status of free-living pigeons in the city of Santiago

A total of 100 free-living urban pigeons (Columba livia) were captured in the city of Santiago, Chile, in order to evaluate, for the first time, their health status. Negligible antibody titres (1 to 3 log2) were detected in 22% of the birds against a strain of the paramyxovirus (PMV) serotype 1. No pigeons had antibodies against PMV serotype 7 and avian influenza. Salmonella sp. belonging to serogroups B and D were isolated from the intestinal tract of three pigeons (3%). The protozoa Haemoproteus columbae, Plasmodium sp., and Leucocytozoon sp. were not detected in any pigeons. Trichomonas gallinae was detected in 11%, without observation of either clinical signs or gross pathological changes at necropsy. Sixty-seven percent of the birds showed the presence of the chewing lice Columbicola columbae and Campanulotes bidentatus compar, and 1% harboured the mite Laminosioptes cysticola. Seven species of nematodes were identified. The frequency at which each species was detected was; Tetrameres sp. (14%), Capillaria annulata (1%), Capillaria columbae (11%), Capillaria obsignata (1%), Ascaridia columbae (5%), Dispharynx spiralis (2%), and Gongylonema ingluvicola (2%). The class Cestoda, found in one pigeon, was represented by the species Aporina delafondi. No trematodes were detected in the sampled birds.     

A serological survey of chickens, Japanese quail, pigeons, ducks and crows for antibodies to chicken anaemia virus (CAV) in Japan.

The chicken has been considered as the natural host for chicken anaemia virus (CAV). To examine the prevalence of CAV in domestic and wild birds in Japan, we analyzed serum samples collected from 211 chickens, 168 Japanese quail, 105 pigeons, 113 ducks and 116 crows for the presence of antibodies to CAV by a micro-scale virus neutralization (VN) test. Nine of the 13 chicken flocks (69.2%) were found to be positive for VN antibodies to CAV. Of the 211 individual chicken serum samples, 127 (60.2%) were positive. VN antibodies were detected in 103 of the 168 (61.3%) quail samples with titres ranging from 1:20 to >/= 1:2560. Serum samples collected from quail in 1992 were found to possess a lower rate of antibody-positive sera (7.7%) and lower antibody titres (1:20 to 1:80) than those collected in 1995 (90.2% and 1:20 to >/= 1:2560, respectively). None of the pigeon, duck and crow samples neutralized CAV at a 1:20 dilution. These results indicate that quail might be one of the hosts of CAV or CAV-like virus in Japan. This is the first report of VN antibodies to CAV in a species other than chicken.     

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.     

Natural infection with torque teno sus virus 1 (TTSuV1) suppresses the immune response to porcine reproductive and respiratory syndrome virus (PRRSV) vaccination

To evaluate the effect of natural infection with TTSuV1 on the antibody response to vaccination with PRRS vaccine and clinical signs when co-infected with virulent PRRSV, 15 4-week-old TTSuV1-positive piglets and 20 TTSuV1-negative piglets were selected by PCR from two pig farms in Jiangsu province. TTSuV1-negative pigs were divided into four groups, and TTSuV1-positive pigs were divided into three groups. Experimental pigs were vaccinated with a PRRSV modified live virus (MLV) at 6 weeks of age and subsequently challenged with a virulent strain of PRRSV at 10 weeks of age. A TTSuV1-negative control group and an unvaccinated PRRS MLV control group were tested at the same time. The levels of antibody/cytokine and protective efficiency against PRRS MLV vaccine were evaluated. TTSuV1-infected/PRRSV-vaccinated pigs had lower levels of PRRSV antibody, as well as IFN-γ, IL-10 and T lymphocyte proliferation, than the TTSuV1-uninfected/PRRSV-vaccinated group (P < 0.05, except IL-10) after vaccination at only one time point. TTSuV1-infected/PRRS MLV-vaccinated/PRRSV-challenged pigs had more severe clinical signs (P > 0.05), more macroscopic lung lesions (P < 0.05) and lower levels of PRRSV antibody (P < 0.05 at 7 to 14 days post-PRRSV-challenge) than TTSuV1-uninfected/PRRSV-vaccinated/PRRSV-challenged pigs. These data indicate that TTSuV1 natural infection has an adverse effect on the development of host immune responses, suppresses immunization by the PRRS MLV vaccine, and exacerbates PRRS to a certain extent in pigs.     

Evolution of pigeon Newcastle disease virus strains

Twenty-seven Newcastle disease virus isolates obtained during the years 1998 and 1999 from racing pigeons were shown to be antigenically indistinguishable from the pigeon paramyxovirus type 1 (PPMV-1) viruses isolated in the years 1983 and 1984. Partial sequencing of 240 base pairs of the F gene demonstrated at least 94.7% identity at the nucleotide level between isolates from 1983 and 1984, and more recent viruses isolated in 1998 and 1999. Most of the nucleotide changes observed were silent mutations as only six amino acid changes were observed. Three amino acid substitutions were observed in the F2/F1 cleavage site. The sequence of the F2/F1 cleavage site of all isolates was typical for pathogenic paramyxovirus 1 viruses. Amino acids at the F2/F1 cleavage site changed from 112 GRQKRF 117 to 112 RRQKRF 117 , 112 RRKKRF 117 or 112 RRRKRF 117 . The motif 112 RRQKRF 117 was present in the majority of the isolates but the intracerebral pathogenicity indexes of PPMV-1 isolates having this motif was highly variable but largely lower (mean, 0.69) than that reported for PPMV-1 viruses isolated in the years 1983 and 1984 (mean, 1.44).