G. Meulemans

Acute infectious bursal disease in poultry: Protection afforded by maternally derived antibodies and interference with live vaccination

Maternally derived antibodies (MDA) were found insufficient to protect broiler chicks against a highly pathogenic strain of IBDV during the growing period even if the parent flocks had been boostered at point of lay by using oil emulsion vaccines. Whatever the vaccination scheme of the parent flocks, maximum mortality was observed after a challenge performed at 38 days of age in broiler and layer chicks, suggesting that the offspring need to be vaccinated with live vaccines before that age. MDA were demonstrated to interfere with live vaccination but strain D78 was shown to be more efficient as it could establish an infection even at higher antibody levels than the other strains. Given this pathogenicity and the lack of uniformity in the transmitted immunity, there is always a critical period where immune and susceptible birds coexist in the same flock. This period seems to be broader when parental flocks were vaccinated with an inactivated oil emulsion vaccine before lay.

Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses

A polymerase chain reaction combined with restriction enzyme analysis was developed for detection and differentiation of all 12 fowl adenovirus (FAdV) serotypes representing the five fowl adenovirus (A to E) species. For primer design, the published sequences of the hexon proteins of FAdV1, FAdV8 and FAdV9 were aligned and conserved regions in the two pedestal regions adjacent to the L1 loop region were determined. A primer pair (hexon A/hexon B) was constructed and was shown to amplify approximately 900 bp of the hexon gene including the L1 loop region. An amplification product was detected using supernatant of infected cell cultures from all FAdV1 to FAdV12 reference strains used in our study. The sequence and the restriction patterns of the hexon A/B fragments of the 12 FAdV strains were determined and compared. The successive use of four different endonucleases allowed the complete differentiation of the reference FAdV strains. Twenty-six fowl adenoviruses isolated during our routine virological diagnosis activities could all be amplified using hexon A/hexon B primers. Restriction analysis results showed that 8/26 adenovirus strains contained two different FAdV types. FAdV4, FAdV12, FAdV1, FAdV5 and FAdV6 were the most frequently isolated.

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).

Assessment of the cell-mediated immune response in chickens by detection of chicken interferon-γ in response to mitogen and recall Newcastle disease viral antigen stimulation

The potential of a capture enzyme-linked immunosorbent assay (ELISA) specific for chicken interferon-γ (ChIFN-γ) has been evaluated as a tool to assess cell-mediated immunity (CMI) in the chicken. In a first step, ChIFN-γ production and cell proliferation of mitogen-activated chicken splenocytes have been compared. In general, for each of the stimulation conditions where significant proliferation was observed, production of ChIFN-γ could be measured by ELISA. In our hands, the combination of ionomycin and phorbol-12-myristate 13-acetate or the use of recombinant chicken interleukin-2 gave the most satisfactory results. Then, the CMI response induced by live or killed Newcastle disease virus (NDV) vaccines has been evaluated sequentially by ex vivo antigen-specific ChIFN-γ production and cell proliferation of splenocytes from immune chickens. The ex vivo data showed that both types of NDV vaccines are capable of stimulating CMI responses to NDV in chickens as measured by the ChIFN-γ ELISA. However, most of the chickens vaccinated with the live vaccine produced ChIFN-γ after antigen recall stimulation, from 2 to 4 weeks after vaccination, when only some chickens vaccinated with the inactivated vaccine showed a specific response 4 weeks after vaccination. No significant proliferative responses to either NDV vaccine were detectable during the 4 weeks of the study. From our results, it appears that antigen-specific ChIFN-γ production can be used as a good indicator of actively acquired immunity to NDV and that the sensitivity range of the capture ELISA test is well adequate to measure ex vivo release of ChIFN-γ.

Significance of paroviruses, entero‐like viruses and reoviruses in the aetiology of the chicken malabsorption syndrome

Specific-pathogen-free White Leghorn chickens and commercial broilers were inoculated orally at 1 day of age with different intestinal preparations containing a chicken parvovirus, an entero-like virus associated with a reovirus from field materials, or the entero-like viruses and reovirus alone. Despite viral multiplication in inoculated birds, no clinical signs or growth retardation were observed in SPF and broiler chickens infected with the reo or parvoviruses. Abnormal faeces and reduction in weight gains were observed after infection with the field materials and the entero-like viruses. Some easily sedimentable particles could be involved with the entero-like virus in the aetiology of runting syndrome. Proventriculitis was present in chickens inoculated with one of the field materials and with the entero-like virus isolated from that material. Specific-pathogen-free White Leghorn chickens were as susceptible as commercial broiler chickens to weight gain depression after oral inoculation with crude homogenates at 1 day of age.

Epidemiology of infectious bronchitis virus in Belgian broilers: a retrospective study, 1986 to 1995.

Infectious bronchitis virus (IBV) was isolated from each of 236 broiler flocks that had respiratory infection (86%), impaired growth, enteritis and/or nephritis (14%), over a 10-year period from 1986 to 1995 in Belgium. Among them, 65% of the investigated flocks had not been vaccinated against infectious bronchitis. Type-specific reverse transcriptase polymerase chain reactions (RT-PCRs) were used after propagation of the isolates in embryonated eggs in order to detect and differentiate Massachusetts, D274, B1648 and 793/B types. The incidence of these types was approximately 50, 38, 11 and 1%, respectively. In 16% of cases, two or three types of IBV were detected, representing mostly combinations of Massachusetts and D274. The majority of the Massachusetts and D274 isolates (68 and 69%, respectively) were recovered from non-vaccinated flocks, confirming that such flocks are at greatest risk of infection by these types of IBV. Interestingly, the B1648 type was isolated from more vaccinated flocks (14%) than non-vaccinated flocks (7.6%). Most surprising was the very low incidence (1%) of the 793/B type, which was the dominant type in some neighbouring countries, during the period of investigation. The DNA derived by RT-PCR from 24 of the Massachusetts-type isolates from 12 vaccinated and 12 non-vaccinated flocks was sequenced and compared with the sequence of Massachusetts vaccines used in Belgium. This revealed that the sequence of four of the isolates (two from vaccinated and two from non-vaccinated flocks) was identical to that of a Massachusetts vaccine strain. Similar results were obtained for D274 isolates when compared with the sequence of D274 vaccines. These sequencing results demonstrate a co-circulation of vaccine and wild-type infectious bronchitis viruses in broilers, and are further justification for permanent monitoring of circulating strains in order to rationally modify vaccination strategies to make them appropriate to the field situation.

Ultrastructural Changes of the Tracheal Epithelium after Vaccination of Day-Old Chickens with the La Sota Strain of Newcastle Disease Virus

The progression of tracheal lesions induced by vaccination of day-old specific pathogen-free chicks with the La Sota strain of Newcastle disease virus (NDV) was examined by relating surface changes as observed by scanning electron microscopy with subcellular changes seen by transmission electron microscopy. NDV infection resulted in hypertrophy of goblet cells, their rupture, and the formation of excess mucus. Activation of goblet cells peaked within 4 days postvaccination. Afterward, the activation levels gradually decreased. At the level of the ciliated cells, a marked increase in the proportion of nonciliated to ciliated cells and later an almost complete deciliation of the tracheal surface were observed because a simple squamous to cuboidal epithelium replaced the original pseudostratified epithelium. Fifteen days postvaccination, all epithelial damage was restored. Because the observed vaccination-induced lesions are detrimental to epithelial integrity and function as a barrier against invading microorganisms, they might explain at the ultrastructural level the secondary complications of vaccination with the La Sota strain against NDV

Production of antibodies against chicken interferon-γ: demonstration of neutralizing activity and development of a quantitative ELISA

Four monoclonal antibodies (mAbs) specific for chicken interferon-gamma (ChIFN-gamma) were generated by gene gun immunization and were utilized to develop a mAb-based capture ELISA specific for ChIFN-gamma. Each mAb reacted specifically with both baculovirus and Escherichia coli-derived recombinant ChIFN-gamma in ELISA and Western Blot analysis or natural ChIFN-gamma in immunofluorescence experiments. As determined by competition ELISAs, mAbs 3D5, 4C6 and 3A3 recognized the same or adjacent epitopes on the ChIFN-gamma molecule, whereas mAb 1E12 recognized a distant epitope. Moreover, this latter mAb was able to highly neutralize the biological activities of both recombinant and natural ChIFN-gamma as measured by inhibition of viral replication and macrophage activation. To improve the detection of ChIFN-gamma, a capture ELISA was developed using mAb 1E12 as capture antibody and biotinylated mAb 4C6 as detection antibody. In addition to being more rapid and easier to perform than classical cell-mediated immunity tests, this ELISA has excellent sensitivity and improved specificity. The use of a specific rabbit polyclonal serum as revealing antibody further increased the sensitivity of the detection down to 0.5ng/ml of ChIFN-gamma. This ELISA would provide a sensitive tool to measure the in vitro release of ChIFN-gamma by T-cells in response to specific recall antigen.

Development of an M2e-Specific Enzyme-Linked Immunosorbent Assay for Differentiating Infected from Vaccinated Animals

Abstract Vaccination programs for the control of avian influenza (AI) in birds have restrictions because of some limited efficacy and the difficulty of discriminating between vaccinated and virus-infected poultry. We studied M2e, the highly conserved external domain of the influenza A M2 protein, as a potential differential diagnostic marker for influenza virus infection. The M2 protein is an integral membrane protein, scarcely present on virus particles, but abundantly expressed on virus-infected cells. M2e-specific enzyme-linked immunosorbent assays (ELISAs) for different avian influenza strains were developed by coating the peptides corresponding to the first 18 amino acids, without the first methionine, of the universal human consensus M2e sequence and the specific M2e sequence of two highly pathogenic AI (HPAI) strains, H7N7 and H5N1. Using the M2e ELISAs, M2e-specific antibodies were observed in chickens and ducks experimentally infected with H7 or H5 HPAI, respectively, that correlated well with hemagglutination inhibition (HI) antibodies. Conversely, sera from chicken and ducks inoculated with inactivated AI vaccines were positive for HI test but negative for the M2e ELISAs. Moreover, ducks inoculated with inactivated vaccine and challenged with a HPAI H5N1 seroconverted for antibodies to the M2e peptide, with significantly different levels from those measured between the vaccinated and infected groups. These results indicate the potential benefit of a simple and specific M2e ELISA in the assessment of the efficacy of vaccination as well as for diagnostic and survey applications.

Comparison of biological activities of natural and recombinant chicken interferon-gamma

In recent years, chicken interferon-gamma (ChIFN-gamma) has been identified and cloned from a chicken T cell line. In this study, recombinant ChIFN-gammma produced in the baculovirus and prokaryotic (Escherichia coli) expression systems were characterized and their activity was compared to that of naturally ChIFN-gamma produced by mitogen-activated splenic T cells. The baculovirus-derived ChIFN-gamma protein (Bac-ChIFN-gamma) proved to have physiochemical properties and biological activities similar to those of natural ChIFN-gamma. Indeed, Bac-ChIFN-gamma was able to inhibit the replication of cytolytic viruses in chicken embryo fibroblasts and to activate macrophages, as was determined by nitric oxide production. Levels ranging between 100 and 300 microg/ml of BacChIFN-gamma could be obtained in the supernatants of infected insect cells. On the other hand, yields of the E. coli produced ChIFN-gamma rarely exceeded 100 microg/ml after purification steps and although it was also able to activate the HD11 macrophage cell line in a specific manner, no anti-viral activity could be demonstrated. Therefore, the baculovirus expression system is an appropriate system for the high-level expression of biologically active ChIFN-gamma and will allow further studies of the immunomodulatory and therapeutic effects of this cytokine in vivo.