Research Note: An overview on distribution of fowl adenoviruses.

Fowl adenoviruses (FAdV), detected during routine diagnostic investigations from 38 countries (5 continents) over a decade, were partially sequenced and grouped by phylogenetic analysis. The partial polymerase gene nucleotide sequences of the 365 fowl adenovirus isolates resulted in the following species distribution: 11% FAdV-A; 3% FAdV-B; 2% FAdV-C; 34% FAdV-D; and 50% FAdV-E. Noticeably, only 79 of the detected strains could be associated with adenovirus-specific pathologic conditions: 62 (79%) with inclusion body hepatitis; 9 (11%) with gizzard erosion; and 8 (10%) with hepatitis hydropericardium syndrome. The remainder of the FAdV strains was detected as concomitant infection from other disease conditions almost exclusively in boilers of 27 to 42 d of age: the majority of them was FAdV-E followed by FAdV-D, and to a lesser extent of FAdV-A, B, and C, the latter ones have not been associated with any of the established adenovirus-caused syndromes in our collection. The highest ratio of coinfections was observed for FAdV-B (62%), while it was about 30% for the rest of the FAdV species. The most frequent coinfection, in connection with all FAdV species, was with the avian infectious bronchitis virus. The presented database will serve as the basis for comparative whole genome and cross-neutralization analysis of selected FAdV isolates.

Protection Afforded by a Recombinant Turkey Herpesvirus-H5 Vaccine Against the 2014 European Highly Pathogenic H5N8 Avian Influenza Strain.

A highly pathogenic avian influenza (HPAI) H5N8 (clade 2.3.4.4) virus, circulating in Asia (South Korea, Japan, and southern China) since the beginning of 2014, reached the European continent in November 2014. Germany, the Netherlands, the United Kingdom, Italy, and Hungary confirmed H5N8 infection of poultry farms of different species and of several wild bird species. Unlike the Asian highly pathogenic (HP) H5N1, this HP H5N8 also went transatlantic and reached the American West Coast by the end of 2014, affecting wild birds as well as backyard and commercial poultry. This strain induces high mortality and morbidity in Galliformes, whereas wild birds seem only moderately affected. A recombinant turkey herpesvirus (rHVT) vector vaccine expressing the H5 gene of a clade 2.2 H5N1 strain (rHVT-H5) previously demonstrated a highly efficient clinical protection and reduced viral excretion against challenge with Asian HP H5N1 strains of various clades (2.2, 2.2.1, 2.2.1.1, 2.1.3, 2.1.3.2, and 2.3.2.1) and was made commercially available in various countries where the disease is endemic. To evaluate the protective efficacy of the rHVT-H5 vaccine against the first German H5N8 turkey isolate (H5N8 GE), a challenge experiment was set up in specific-pathogen-free (SPF) chickens, and the clinical and excretional protection was evaluated. SPF chickens were vaccinated subcutaneously at 1 day old and challenged oculonasally at 4 wk of age with two viral dosages, 10(5) and 10(6) 50% egg infective doses. Morbidity and mortality were monitored daily in unvaccinated and vaccinated groups, whereas viral shedding by oropharyngeal and cloacal routes was evaluated at 2, 5, 9, and 14 days postinoculation (dpi). Serologic monitoring after vaccination and challenge was also carried out. Despite its high antigenic divergence of the challenge H5N8 strain, a single rHVT-H5 vaccine administration at 1 day old resulted in a full clinical protection against challenge and a significant reduction of viral shedding in the vaccinated birds.

Quantification of rHVT-F genome load in feather follicles by specific real-time qPCR as an indicator of NDV-specific humoral immunity induced by day-old vaccination in SPF chickens.

The purpose of this study was to look for a reliable molecular method for confirmation of uptake of recombinant turkey herpesvirus vaccine against Newcastle disease (rHVT-F) and for use as a valuable prediction tool of Newcastle disease virus (NDV)-specific immune response in chickens deprived of maternally derived antibody (MDA). A quantitative real-time polymerase chain reaction (real-time qPCR) specific to rHVT-F was developed. The method was applied to various tissue samples taken from specific pathogen free (SPF) chickens experimentally inoculated at day-old with one dose of rHVT-F vaccine over a 6-week period. Among the tested tissues, the rHVT-F vaccine was detected predominantly in the bursa of Fabricius (BF) and the lung for the first week, followed by a progressive decline from 9 days onwards. Then, an increase of genome load was observed in the feather follicles (FF) with a peak at 2 weeks, rising to a level almost 10(3)-fold greater than in the other tissues. Importantly, the rHVT-F genome load in FF appeared to be strongly correlated to the humoral immunity specific to NDV as evaluated by haemagglutination inhibition (HI) test and NDV-specific IgG, IgM and IgA ELISAs. This is the first report of quantification of rHVT-F vaccine in FF and its correlation with the induction of ND-specific immune response in chickens with no MDA. Our data indicate that the application of this real-time qPCR assay on FF samples taken from chickens in the field may be used to confirm rHVT-F vaccine administration and uptake with the important added benefit of offering a non-disruptive sampling procedure.

The combination of attenuated Newcastle disease (ND) vaccine with rHVT-ND vaccine at 1 day old is more protective against ND virus challenge than when combined with inactivated ND vaccine.

The recurrent outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks throughout the world indicate that routine vaccinations are failing to sufficiently induce the high levels of immunity necessary to control ND. There is a need for vaccination programmes that could be initiated at 1-day-old for mass application and which would induce a long-lasting immunity, with no need for a booster vaccination at a later age. In this context, the duration of immunity delivered by a vaccination programme including a recombinant herpesvirus of turkeys expressing the F gene of ND virus (rHVT-ND) and live ND vaccine at 1-day-old was compared with a classical programme that included a conventional live and an inactivated ND vaccine at the same age in commercial layer chickens. The humoral, cell-mediated and local immunity were followed weekly and birds were challenged with a viscerotropic velogenic ND virus strain at 6 and 10 weeks of age. We determined that immunity induced by the vaccination programme involving the rHVT-ND vaccine was more protective than that provided by the conventional vaccine-based regime. This might be related to a T-helper type 1 (Th1) cellular-driven immunological response, in contrast to the T-helper type 2 (Th2) humoral-oriented immune response provided by the current conventional vaccine-based vaccination programmes.

Chicken dendritic cells and type II pneumocytes express a common intracellular epitope.

1. CVI-ChNL 74·3, a dendritic cell-specific monoclonal antibody (mAb) also identifies chicken lung granular pneumocytes (type II pneumocytes), which produce surfactant. 2. The 74·3 mAb does not cross-react with any other avian or mammalian granular pneumocyte, and provides a convenient tool for monitoring the status of type II pneumocytes in the chicken lung.

Advancement in vaccination against Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions.

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to the limitation in their efficacy, current vaccination strategies against ND need improvements. This study aimed to evaluate a new-generation ND vaccine for its efficacy in providing clinical protection and reducing virus shedding after challenge. Broiler chickens were vaccinated in ovo or subcutaneously at hatch with a turkey herpesvirus-based recombinant vaccine (rHVT) expressing a key protective antigen (F glycoprotein) of Newcastle disease virus (NDV). Groups of birds were challenged at 20, 27, and 40 days of age with a genotype V viscerotropic velogenic NDV strain. Protection was 57% and 81%, 100% and 95%, and 100% and 100% after the subsequent challenges in the in ovo and subcutaneously vaccinated chickens, respectively. Humoral immune response to vaccination could be detected from 3-4 wk of age. Challenge virus shedding was lower and gradually decreased over time in the vaccinated birds compared to the unvaccinated control chickens. In spite of the phylogenetic distance between the NDV F gene inserted into the vector vaccine and the challenge virus (genotype I and V, respectively), the rHVT NDV vaccine provided good clinical protection and significantly reduced challenge virus shedding.

Efficacy of rHVT-AI vector vaccine in broilers with passive immunity against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains.

In countries where avian influenza has become endemic, early vaccination of layer pullets or broilers with classical inactivated vaccines at the hatchery is no longer an option because of interference with passive immunity indirectly induced by the necessary vaccination of the breeders. On the other hand, injection of thousands of chicks from 7 to 10 days old on farms has been determined to be unreliable and, therefore, poorly efficacious. For these reasons, interest has arisen regarding a newly developed live recombinant vector vaccine based on a turkey herpesvirus (HVT) expressing the H5 gene from a clade 2.2 H5N1 highly pathogenic avian influenza virus (HPAIV) strain (rHVT-H5), which in theory is capable of breakthrough passive immunity to both the vector (HVT) and the insert (H5) and is consequently applicable at the hatchery. The objectives of this trial were to evaluate the impact of maternally derived antibodies (MDAs) specific to H5N1 on the immunity and the efficacy (protection and virus shedding) of different vaccination programs including rHVT-H5 and inactivated H5N1 and H5N2 vaccines applied alone or in combination. Therefore, broilers carrying MDAs against both HVT and Asian H5N1 HPAIV were vaccinated on the first day of age with rHVT-H5, with or without boosting vaccination by an inactivated vaccine after 10 days. The different groups were challenged with two antigenically highly divergent Egyptian dade 2.2.1 H5N1 HPAIVs at 4 wk of age. Protection against challenge was compared with unvaccinated birds or vaccinated birds without MDAs. Between 70% and 90% clinical protection could be observed in the vaccinated groups possessing MDAs, indicating no or very low interference of MDAs with vaccination. Results regarding clinical protection, humoral, cell-mediated, and mucosal immunity, as well as re-excretion of challenge virus are presented and discussed.

Origin of the chicken splenic reticular cells influences the effect of the infectious bursal disease virus on the extracellular matrix.

The effects of infectious bursal disease virus (IBDV) (strain F52/70) infection were studied by immunohistochemical methods on the splenic extracellular matrix (ECM). The major fibrillar components of the ECM, the type I and type III collagens and the main ECM organizing glycoproteins (laminin, tenascin and fibronectin) were monitored up to 11 days post-infection (d.p.i.). By 3 d.p.i., the collagens that form the basic scaffold of the antigen-trapping region of the spleen are destroyed, which is followed by deterioration of the glycoproteins. The ECM in the red pulp and the other regions of the white pulp (periarteriolar lymphatic sheath and germinal centre) seem to be normal. The reason for the significantly different pathological alterations in the ECM between the two regions of the spleen may be explained by the origin of the reticular cells. The reticular cells in the antigen-trapping zone and other splenic regions are of haemopoietic and mesenchymal origins, respectively. Possibly, the reticular cells of the haemopoietic origin are more susceptible for the IBDV infection than the mesenchymal ones. Development of the antigen-trapping, B-cell-dependent zone of the splenic white pulp precedes that of the periarteriolar lymphatic sheath and germinal centre, which suggests that this region may contribute to B-cell maturation. Damage of the ECM in the antigen-trapping zones results in impairment of tissue organization, which may contribute to the permanent immunosuppression.

Further evidence of antigenic drift and protective efficacy afforded by a recombinant HVT-H5 vaccine against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains.

In this study, we have compared the protection afforded by a recombinant turkey herpesvirus vaccine expressing the H5 gene from a clade 2.2 H5N1 strain (rHVT-H5) and a Mexican-origin H5N2 inactivated vaccine, alone or in combination, against two antigenically divergent H5N1 Egyptian strains isolated in 2007 and 2008. Our results confirm the existence of a major antigenic drift among the Egyptian H5N1 strains such that, although protection against the "classical" 2007 HPAI H5N1 Egyptian strain could be obtained with both types of vaccines, only vaccination with the rHVT-H5 vaccine protected against challenge with the "variant" 2008 HPAI H5N1 Egyptian strain.

Passive protection afforded by maternally-derived antibodies in chickens and the antibodies' interference with the protection elicited by avian influenza-inactivated vaccines in progeny.

Systematic vaccination can be applied when a disease has become enzootic in a country or region. The final goal of the approach is to control or eradicate the disease within the country. This is a long-term vaccination plan that could be applied nationwide to all commercial and backyard poultry. However, after several months of vaccination in enzootic areas, maternally derived antibody (MDA) is present in young chicks, providing some protection and/or interference with vaccination. The aim of this study was to evaluate the level of protection afforded by MDA against challenge with highly pathogenic avian influenza virus (HPAIV), and its suspected interference with current inactivated vaccines in broilers under controlled laboratory conditions. In the first set of experiments, broilers were vaccinated with inactivated vaccines containing H5N2 subtype antigens in the presence or absence of homologue MDAs and challenged with a clade 2.2 H5N1 HPAIV. In the second set of experiments, day-old broilers, either with or without avian influenza MDA, received a regular-type monovalent H5N2 AI vaccine (0.5 ml) or a concentrated (0.2 ml) AL-Newcastle disease virus combined inactivated vaccine subcutaneously. They were then challenged at 11 or 35 days of age. In conclusion, our results indicate that protection induced by day-old administration of inactivated vaccine (regular or concentrated) in the presence or absence of MDA to H5N2 AIV induces poor protection against challenge with H5N1 HPAIV and should not be recommended. Based on our results, vaccination of MDA-positive chickens at a later age (10 days) seems to be a valuable recommendation, although MDAs may still interfere with vaccination to a lesser extent because they are present up to 3 wk posthatch. Therefore, in areas with high infection pressure, when possible, two vaccinations are recommended for optimal protection. Also, it might be advisable to take into account day-old AI MDA titers when one is determining the optimal age of vaccination.

Comparative histopathology and immunohistochemistry of QX-like, Massachusetts and 793/B serotypes of infectious bronchitis virus infection in chickens.

The aim of this study was to compare experimentally the pathogenicity and tissue distribution of the recently emerged QX-like strain of infectious bronchitis virus (IBV) with the widespread M41 and 793/B serotypes of the virus. Histopathological and immunohistochemical methods were employed to define the main sites of virus replication. One-day-old specific pathogen free chickens were inoculated with five different QX-like strains, or with the M41 and 793/B IBV strains and monitored for 42 days post-infection. Tracheal lesions developed in all infected birds, confirming the ability of all of the tested strains to induce respiratory disease. Replication of the isolates in the alimentary tract was detected, but the infection did not cause significant gut lesions. Four of the five QX-like IBV strains induced severe kidney lesions. Dilation of the oviduct with accumulation of serum-like fluid in the lumen of this structure, reported previously from field cases of QX-like IBV infection, was observed following experimental infection with all of the five QX-like strains. Microscopical and immunohistochemical examination of the affected oviducts did not help to elucidate the pathogenesis of this lesion.

Investigation of field outbreaks of turkey haemorrhagic enteritis in Hungary.

Epidemiological, pathological, serological and virological investigations are reported on turkey haemorrhagic enteritis virus (THEV) infection in Hungarian turkey flocks. The pathogenesis of infection in experimentally infected turkeys and chickens, as well as the usefulness of polymerase chain reaction (PCR)/sequencing method for epidemiological investigation and for the differentiation of vaccine and field strains of THEV was also studied. Since the first recognition of the disease in Hungary in the late 1970s, until recently the disease has been diagnosed sporadically in its mild form. In the last few years (2000-2005), however, the number of outbreaks and the severity of the disease increased (9-23 affected flocks/year). Most of the outbreaks occurred at the age of 6 to 8 weeks and was complicated with Escherichia coli infection. The antibody levels to THEV in turkey flocks gradually declined till 5-7 weeks of age, and then they increased sharply due to natural infection with THEV. The immune response to vaccination (at 5 weeks of age) showed no significant antibody level increase one week postvaccination, but four weeks later the antibody level reached high values and then remained at this high level. The agar gel immunodiffusion (AGID) test to detect turkey adenovirus A (TAdV-A) antigen and PCR methods for THEV-specific DNA gave similarly positive results if spleens with pathognomonic lesions were tested; however, PCR proved to be more sensitive in cases with less characteristic pathological lesions. Nucleotide sequence alignment of PCR products amplified from Hungarian field strains and the Domermuth vaccine strain and that of the published THEV hexon sequences in GenBank database revealed slight differences between the sequences.

Oesophageal tonsil of the chicken.

The oesophageal tonsil of the chicken is a novel member of the mucosal-associated lymphoid tissue (MALT), which is located around the entrance of the proventriculus. It consists of 6 to 8 single units, which are surrounded by a thin fibrous capsule. Each one is organised around the bottom of the longitudinal folds of the oesophagus, and serves as a 'tonsillar crypt'. Stratified squamous epithelium is infiltrated by lymphoid cells, i.e. T cells, plasma cells, macrophages, and dendritic cells, but not B cells, to form lymphoepithelium (LE). In the LE vimentin-, MHC II- and ATPase-positive cells possibly represent Langerhans' cells, but the appearance of 74.3 positive cells in the LE is unusual, because the 74.3 monoclonal antibody (mAb) recognises chicken follicular dendritic cells in the germinal centre and medulla of the bursal follicles. The subepithelial lymphoid tissue is organised into T- and B-dependent regions, which are the interfollicular areas and the germinal centres, respectively. Existence of high-endothelial venules in the interfollicular region suggests an extensive cellular connection between the oesophageal tonsil and the other lymphoid organs. In the resting oesophagus the lumen is closed, but during swallowing a bolus the crypt opens and the lymphoepithelium can be exposed to undigested food, antigens, infectious agents and vaccines. The location of the oesophageal tonsil, cranial to the stomach, may provide this organ with a unique role as compared to the other parts of the MALT; namely, it may contribute to the replication of infectious bursal disease virus (IBDV) and/or the pathogenesis of infectious bursal disease.

Comparative pathological studies on domestic geese (Anser anser domestica) and Muscovy ducks (Cairina moschata) experimentally infected with parvovirus strains of goose and Muscovy duck origin.

Parvovirus infection of Muscovy ducks caused by a genetically and antigenically distinct virus has been reported from Germany, France, Israel, Hungary, some Asian countries and the USA. The pathological changes include those of degenerative skeletal muscle myopathy and myocarditis, hepatitis, sciatic neuritis and polioencephalomyelitis. In the study presented here, day-old and 3-week-old goslings and Muscovy ducks were infected experimentally with three different parvovirus strains (isolates of D-216/4 from the classical form of Derzsy's disease, D-190/3 from the enteric form of Derzsy's disease, and strain FM from the parvovirus disease of Muscovy ducks). All three parvovirus strains caused severe disease in both day-old and 3-week-old Muscovy ducks but in the goslings only the two strains of goose origin (D-216/4 and D-190/3) caused disease with high (90-100%) mortality when infection was performed at day old. Strain FM (of Muscovy duck origin) did not cause any clinical signs or pathological lesions in the goslings. In the day-old goslings and Muscovy ducks the principal pathological lesions were severe enteritis with necrosis of the epithelial cells (enterocytes) of the mucous membrane and the crypts of Lieberkühn, and the formation of intranuclear inclusion bodies. Other prominent lesions included hepatitis and atrophy (lymphocyte depletion) of the lymphoid organs (bursa of Fabricius, thymus, spleen). In goslings infected with the strain originating from the classical form of Derzsy's disease mild myocarditis was also detected. After infection at three weeks of age, growth retardation, feathering disorders, myocardial lesions (degeneration of cardiac muscle cells, lympho-histiocytic infiltration) and hepatitis were the most prominent lesions in both geese and Muscovy ducks. In addition to the lesions observed in the geese, muscle fibre degeneration, mild sciatic neuritis and polioencephalomyelitis were also observed in the Muscovy ducks infected with any of the three parvovirus strains.

Diagnosis of duck circovirus infections by conventional and real-time polymerase chain reaction tests.

Development of the first conventional and real-time polymerase chain reaction (PCR) tests for the diagnoses of duck circovirus (DuCV) infections is described. Both tests amplified a 230 bp fragment specific to the DuCV Rep gene. Although both tests had the same detection limit (13 x 10(3) target DNA/ml) when the target DNA was diluted in water, the detection limit of the real-time test (13 x 10(4) target DNA/ml) was 10-fold less than the conventional test (13 x 10(5) target DNA/ml) when the amplifications were performed in the presence of cellular DNA. Using the conventional PCR test, DuCV DNA was detected in 85 (84%) of 101 bursa of Fabricius samples from dead or sick ducks, aged between 1 and 12 weeks, and in samples from 35 (94%) of 37 flocks. Application of the SYBR Green-based real-time PCR test to 54 selected bursa of Fabricius samples indicated that more samples were positive by real-time PCR than by conventional PCR, allowed the numbers of genome copies to be estimated and showed that some bursa of Fabricius samples contained over 10(13) genome copies/g tissue. Although DuCV infections were detected in birds aged from 1 to 12 weeks, higher virus DNA levels were detected in ducks aged older than 5 weeks than in ducks younger than 5 weeks. An in situ hybridization method for the detection of DuCV in histological samples was also developed. Additional work is required to determine the clinicopathological significance of DuCV infections.

Antigenic and genetic diversity of early European isolates of Infectious bursal disease virus prior to the emergence of the very virulent viruses: early European epidemiology of Infectious bursal disease virus revisited?

Eleven Polish and Hungarian isolates of Infectious bursal disease virus (IBDVs) obtained in the 70/80s (early IBDV) and in the 90s (recent IBDV) were characterized in an Antigen-Capture-ELISA with a panel of neutralizing monoclonal antibodies (Mabs), and by nucleotide sequencing of the VP2 variable domain (vVP2). The viruses were compared with reference IBDV strains, among others with Faragher 52/70 (F52/70, classical, isolated 1970), 89163 (typical very virulent-vvIBDV, isolated 1989) and 91168 (antigenically modified vvIBDV, isolated 1991). Only one of the early isolates (Hungarian strain P1) proved antigenically and genetically similar to F52/70. Other early isolates exhibited no reactivity versus Mabs 3, 4, 5 and/or 8 and had a common previously unrecognized combination of amino acid changes in vVP2. The recent isolates all proved antigenically and genetically related to typical vvIBDV strain 89163, except the Polish isolate 93/35 which proved related to the 91168 strain although no epidemiological relationship had been documented between these viruses in the field. Phylogenetic analysis confirmed that the non-P1 early IBDVs represent a previously unrecognized group among serotype 1 IBDVs. It is discussed whether these early isolates are derivatives of the F52/70-like viruses that might still be present in the field, or whether they represent early IBDV strains that might have been present prior to and progressively replaced by the F52/70-like viruses, as the latter have been replaced by vvIBDVs in the late eighties.

Diagnosis of goose circovirus infection in Hungarian geese samples using polymerase chain reaction and dot blot hybridization tests.

A polymerase chain reaction (PCR) and dot blot hybridization (DBH) test have been developed for the diagnosis of infection by a novel circovirus of geese (GoCV). These tests were applied to samples of bursae of Fabricius from sick and dead birds from commercial goose farms in Hungary. In this second report of the occurrence of circovirus infection in diseased geese, 103 of 214 (48.1%) and 37 of 150 (24.6%) birds, and 49 of 76 (64.5%) and 18 of 76 (23.7%) flocks were positive by PCR and DBH respectively. The sensitivity of the PCR test was such that 0.10 fg of virus DNA was detectable. The DBH test was less sensitive, only detecting larger amounts (40 pg) of DNA, but was used as a semi-quantitative method for detecting the presence of virus. The incidence of infection was affected by factors such as the age of the birds and rearing methods.

Esophageal tonsil: a novel gut-associated lymphoid organ.

The esophageal tonsil of the chicken is a novel, significant element of the gut-associated lymphoid tissue (GALT). Its stable location and histological organization fulfills the meaning of the term "tonsil." The six-to-eight-isolated tonsillar units are located at the border of the esophagus and the proventriculus. The number of tonsillar units is identical with that of the esophageal folds. Each tonsillar unit consists of a crypt lined by lymphoepithelium and surrounded by dense lymphoid tissue, which is organized into T- and B-dependent regions, like peripheral lymphoid organs. The excretory ducts of the mucosal glands of the esophagus are frequently involved in the formation of the lymphoepithelium. The esophageal tonsil is anatomically located cranial to the stomach, unlike the other parts of the GALT. Therefore, it is continuously exposed to undigested environmental antigens, allergens, food, and infectious agents. To develop effective oral vaccines, the existence of the esophageal tonsil has to be taken into account.

The role of egg drop syndrome virus in acute respiratory disease of goslings.

An outbreak of severe acute respiratory disease characterized by tracheitis and bronchitis was observed in young goslings on a large-scale goose farm in Hungary. Histological examination revealed amphophilic intranuclear inclusion bodies in the superficial epithelial cells of the trachea and bronchi. Adenovirus-like particles were detected by electron microscopy, and the virus isolated from the trachea and the lungs was identified as egg drop syndrome (EDS) virus by serological and genomic examination. The clinical and pathological signs were reproduced by intratracheal administration of the virus isolate to 1-day-old goslings free of EDS antibodies. The presence of EDS virus DNA in different organs of the naturally and experimentally infected goslings was detected by polymerase chain reaction. This is the first report on the involvement of EDS virus in severe respiratory disease of geese.

Pathological and immunological study of an in ovo complex vaccine against infectious bursal disease.

The appearance of very virulent strains of infectious bursal disease (IBD) virus at the end of the 1980s made it necessary to develop more effective immunization procedures. To facilitate this, the immunogenicity and the immunosuppressive effect of a mild (G-87), an intermediate (LIBD) and an intermediate-plus (IBDV 2512) IBDV strain were tested after the in ovo inoculation of 18-day-old SPF and broiler chicken embryos. It was established that no noteworthy difference existed between the immunized and the control embryos in hatching rate and hatching weight. The higher the virulence of the vaccine virus strain, the more severe damage it caused to the lymphocytes of the bursa of Fabricius. In SPF chickens, the haemagglutination inhibition (HI) titres induced by a Newcastle disease (ND) vaccine administered at day old decreased in inverse ratio to the virulence of the IBD vaccine strain, while in broiler chickens this was not observed. Despite the decrease of the HI titre, the level of protection did not decline, or did so only after the use of the 'hot' strain. SPF chickens immunized in ovo with a complex vaccine prepared from strain IBDV 2512 and IBD antibody showed the same protection against Newcastle disease as the broilers. In broiler chicken embryos immunized in ovo, only strain IBDV 2512 induced antibody production, and such chickens were protected against IBD at 3 weeks of age. The complex vaccine administered in ovo has been used successfully at farm hatcheries as well.