Development of an M2e-specific enzyme-linked immunosorbent assay for differentiating infected from vaccinated animals.

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.

Inclusion body disease in snakes: a review and description of three cases in boa constrictors in Belgium.

Inclusion body disease, a fatal disorder in Boidae, is reviewed, and three cases in boa constrictors, the first reported cases in Belgium, are described. The snakes showed nervous signs, and numerous eosinophilic intracytoplasmic inclusions, which are considered to be characteristic of the disease, were found in the liver and pancreas. The disease is suspected to be caused by a retrovirus, but transmission electron microscopic examinations of several tissues from one of the snakes did not reveal particles with a typical retroviral morphology.

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.

Significance of interactions between Escherichia coli and respiratory pathogens in layer hen flocks suffering from colibacillosis-associated mortality.

This study aimed to examine the significance of interactions between Escherichia coli and various respiratory pathogens during outbreaks of colibacillosis-associated mortality in layer hen flocks under field conditions. For this purpose, a case-control study involving 20 control flocks with baseline mortality and 20 flocks with increased mortality due to E. coli septicaemia and polyserositis, was conducted. In each colibacillosis flock, blood samples were taken from 20 hens at the onset of clinical disease and three times thereafter at 2-week intervals. Control flocks of comparable ages were sampled in the same way. Pooled sera, taken at the first and last sampling, were examined for antibody titres against infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), and the individual sera from all four samplings were examined for the presence and/or titres of antibodies against avian pneumovirus (APV), Mycoplasma gallisepticum, Mycoplasma synoviae and Ornithobacterium rhinotracheale. Titre increases were seen for IBV D274 (one control flock) and O. rhinotracheale (one control and one colibacillosis flock). An increase in per cent reactors was seen for APV (one control flock), and for M. synoviae (one control and two colibacillosis flocks). The study failed to detect any consistent interactions between E. coli and the aforementioned pathogens. These results indicate that, at least as observed in this study, outbreaks of increased mortality resulting from colibacillosis are not necessarily associated with IBV, NDV, APV, M. gallisepticum, M. synoviae or O. rhinotracheale infections.

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).

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.

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.

Infectious bursal disease (Gumboro disease).

Infectious bursal disease (IBD) (Gumboro disease) has been described throughout the world, and the socio-economic significance of the disease is considerable world-wide. Various forms of the disease have been described, but typing remains unclear, since antigenic and pathotypic criteria are used indiscriminately, and the true incidence of different types is difficult to determine. Moreover, the infection, when not fatal, leads to a degree of immunosuppression which is often difficult to measure. Finally, the control measures used are subject to variations, and seldom follow a specific or standardised plan. In the context of expanding international trade, the authors provide an overview of existing knowledge on the subject to enhance available information on the epidemiology of IBD, the identification of reliable viral markers for diagnosis, and the implementation of specific control measures to ensure a global and co-ordinated approach to the disease.

Production of antibodies against chicken interferon-gamma: 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.

[Significance of paramyxovirinae protein F in physiopathology and immunity]. / Importance de la protéine F des paramyxovirinae dans la physiopathologie et l'immunité.

The Paramyxovirinae sub-family contains viruses of major importance for humans (measles and mumps viruses) and animals (Newcastle disease virus, canine distemper virus, rinderpest virus) but also zoonotic viruses (Hendra and Nipah viruses). Newcastle disease virus, a Rubulavirus, which is specific of birds, can be used as a model for understanding the pathogenicity mechanisms of Paramyxovirinae and for the study of vaccination against the diseases they cause. The F protein of pathogenic strains of Newcastle disease virus have a polybasic cleavage site (K/R-Q-K/R-R) which is specific of furine, a cellular enzyme present in all cellular types, what allows viral multiplication in all cells of the host. Such cleavage site is also present in the F protein of measles and mumps viruses. Antibodies against the F protein are the most effective in the immune response against Newcastle disease virus. The major role played by the F protein in the immunity against Paramyxovirinae arises also from the research performed on other animal (rinderpest and canine distemper viruses) and human (measles virus) viruses. Recombinant and sub-unit vaccines as well as DNA vaccination based on the expression of viral glycoproteins allow effective vaccination of chickens against Newcastle disease. The use of a similar approach could lead to the development of new vaccines against measles or mumps and if necessary to produce vaccines against zoonotic Paramyxovirinae.

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.

[Inter-species transmission of the influenza virus]. / Transmission inter-espèces du virus de la grippe.

Influenza is an infection of human beings and several animal species. It is caused by influenza viruses which belong to the Orthomyxoviridae family. Type A influenza viruses are the most important as they cause severe epidemics and are responsible of important pathological troubles. Type A influenza viruses are classified in different sub-types depending of the nature of their surface glycoproteins: haemagglutinin (H) and neuraminidase (N). The nature of the genome and the mode of replication of influenza viruses account for the high variability of these two proteins which are responsible for the immunity to the virus. The continuous appearance of point mutations in the gene coding for the H protein, leads to the progressive emergence of new viral strains. This event which is called antigenic drift makes it necessary to annually assess the composition of the human flue vaccine. Genetic reassortment is another mechanism of antigenic variation. When the gene coding for the H protein, or when both genes coding for H and N proteins are involved in genetic reassortment, a new viral sub-type occurs which replace the precedent. This event, which is termed antigenic shift, occurs occasionally every 10 to 30 years, and it is responsible of the great human pandemics. The role of the animals and particularly the importance of pigs and poultry in the emergence of these new viruses is discussed.

Acute pancreatitis in chickens due to non-virulent Newcastle disease virus.

A non-virulent Newcastle disease virus (strain APMV-1 96/89 VB) was isolated from a broiler chicken from a backyard flock. Using monoclonal antibodies, the virus was shown to be different from the vaccinal virus strains Hitchner, La Sota and Ulster. The virus was shown to replicate in the pancreas of one-day-old specific pathogen-free chickens infected orally, and the histological lesions observed in the pancreas of chickens inoculated with the fourth chicken passage of the virus five to nine days after infection were consistent with an acute pancreatitis.

Newcastle disease outbreaks in recent years in western Europe were caused by an old (VI) and a novel genotype (VII).

Newcastle disease virus (NDV) strains, isolated from outbreaks during epizootics between 1992 and 1996 in Western European countries, were compared by restriction enzyme cleavage site mapping of the fusion (F) protein gene between nucleotides 334 and 1682 and by sequence analysis between nucleotides 47 and 435. Both methods revealed that NDV strains responsible for these epizootics belong to two distinct genotypes. Strains derived from sporadic cases in Denmark, Sweden, Switzerland and Austria were classified into genotype VI [6], the same group which caused outbreaks in the Middle East and Greece in the late 1960's and in Hungary in the early 1980's. In contrast, viruses that caused epizootics in Germany, Belgium, The Netherlands, Spain and Italy could be classified into a novel genotype (provisionally termed VII), hitherto undetected in Europe. It is possible that the genotype VII viruses originated in the Far East because they showed a high genetic similarity (97%) to NDV strains isolated from Indonesia in the late 1980's.

Antigenic and phylogenetic studies on a variant Newcastle disease virus using anti-fusion protein monoclonal antibodies and partial sequencing of the fusion protein gene.

A virulent Newcastle disease virus (NDV) isolate, 34/90, reported to show considerable antigenic diversity from more classical strains of NDV, including vaccine strains, was evaluated phylogenetically and for the presence of neutralizing epitopes on the fusion protein. Comparison of a 309 nucleotide sequence of the fusion protein gene of 34/90 with other viruses confirmed the diversity of this virus, placing it in a discrete fifth genetic lineage with an avirulent virus isolated from waterfowl and genetically quite distant from other strains and isolates. The virus-neutralizing mAbs used in the present study were directed against at least seven distinct epitopes on the fusion protein. Of these seven, five are shared by 34/90 and the live vaccine virus Hitchner B1 and these plus an additional epitope are shared by 34/90 and strain Ulster 2C, which is used in inactivated vaccines. Two potential distinct epitopes were also shared by these three viruses. The results suggest that despite the detected antigenic and genetic variation of 34/90, it is unlikely that mutants which fail to be neutralized by antibodies induced by conventional vaccines would arise readily.

Acute infectious bursal disease in poultry: Immunological and molecular basis of antigenicity of a highly virulent strain.

This study has confirmed, by the use of immunological and molecular tools, that the recent failures of vaccination against infectious bursal disease (IBD) encountered in Europe were not related to antigenic variation, but to increased virulence of the circulating IBD virus strains. Neutralizing monoclonal antibodies (Mabs) showed that the vaccines of intermediate virulence and the pathogenic strain 849VB had a similar pattern of reactivity in ss neutralization tests. Four distinct epitopes could be defined in seroneutralization and addition ELISA tests. All neutralizing Mabs bound to the structural VP2 protein only in its native form. Moreover, Mabs which did not neutralize some strains precipitated well the VP2 protein from extracts of cells infected with the same virus. This suggests that slight changes in the conformation of the epitope were sufficient to allow the virus to escape to neutralization. VP2 sequencing results confirmed that the neutralizing epitopes are clustered in the variable domain which is highly hydrophobic and flanked by two major hy-drophilic peaks. Three potential 'minor' antigenic sites were identified within the hydrophobic region. Comparison of the VP2 sequence of 849VB strain with other highly virulent isolates showed that they are close together and clearly distinct from 'classical' strains. Moreover, sequencing of IBD vaccines revealed that some of them had not been cloned.

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.

An ELISA for the detection of antibodies to avian nephritis virus and related entero-like viruses.

An ELISA for the detection of antibodies against avian nephritis virus (ANV) and related entero-like viruses was developed. Different antigenic preparations made from chicken kidney cells infected with the G-4260 strain of ANV were compared. Crude antigen obtained by fluorocarbon treatment of infected cells was found to be appropriate and to give reproducible results with antisera directed against ANV and three entero-like particles (ELPs): the Belgian entero PV2 and entero 3, and the Irish ELP-1. A cut-off value was determined using 30 specific pathogen-free (SPF) sera and the absence of antigenic relationships with nine different reference sera directed against avian viruses was demonstrated. The cross antigenic relationships between avian encephalomyelitis virus (AEV), ANV and the three ELPs was investigated by ELISA and confirms the classification of those fowl enteroviruses into two serotypes: the first including ANV and ELPs and the second including AEV. A strong correlation (94%) was demonstrated between ELISA and a seroneutralisation test. Using ELISA, antibodies against ANV and related ELPs were demonstrated in 13/14 breeding and 8/10 broiler flocks from Belgium.

Acute infectious bursal disease in poultry: Isolation and characterisation of a highly virulent strain.

A highly virulent strain of infectious bursal disease virus (IBDV) was isolated from the field and propagated in SPF chickens, causing up to 100% mortality. Although it still belongs to the standard serotype 1 IBD viruses, serological typing with monoclonal antibodies showed an antigenic drift in this pathogenic strain. Conventional 'intermediate' IBD vaccines are probably more antigenically related to the pathogenic strain than the mild ones and were effective in protecting SPF chickens against challenge.