Adenoviral gizzard erosion in broiler chickens in Germany.

Avian adenovirus infections cause important disease complexes in chickens, but many of the viruses also infect chickens without resulting in overt disease. Previously several outbreaks of gizzard erosions caused by a fowl adenovirus A serotype-1 (FAdV-1) were reported from Japan. Here we report an outbreak of gizzard erosions in 12 broiler flocks in Germany in 2011. Chickens had a reduced daily weight gain and a higher total mortality rate of up to 8%. The birds showed a severe detachment of the koilin layer and ulcerative to necrotizing lesions of the underlying mucosa. Histopathologically, necrotizing ventriculitis with basophilic, intranuclear inclusion bodies in epithelial cells was diagnosed. Immunohistochemistry, egg culture, and electron microscopic examination revealed adenovirus-like particles in the samples. No concurrent infectious agent could be identified. The virus was genotyped as FAdV-1 by PCR and subsequent sequencing. Phylogenetic analysis of the hexon loop L1 gene yielded 100% sequence identity to the chicken embryo lethal orphan strain. These findings suggest that outbreaks of adenoviral gizzard erosion can lead to significant economic losses in Germany and may be caused by an unusual virulent FAdV-1 strain.

Vaccinal control of Marek's disease: current challenges, and future strategies to maximize protection.

Marek's disease is an economically important lymphoid neoplasm of chickens, caused by oncogenic strains of Marek's disease herpesvirus. The disease can be successfully controlled by vaccination with attenuated or non-pathogenic MDV strains. However, vaccine failures do occur as field strains continue to evolve towards pathotypes of greater virulence, and this evolution is likely to be driven by the vaccines themselves. Two general strategies can be considered to improve protection by vaccination. Firstly by the development of novel vaccines, and secondly by maximizing the potential of existing vaccines. This second goal requires investigation of optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. Accurate quantitation of Marek's disease vaccine virus in vaccinated chicks will contribute significantly to our understanding of vaccinal protection. We recently developed a real-time polymerase chain reaction (PCR) assay for quantitation of CVI988 vaccine virus in the feather tips, a rich source of viral DNA which can easily be sampled in a non-invasive manner. This PCR test is now used commercially to confirm the successful vaccination of chicks. We have also used the PCR to examine various aspects of vaccination in experimental chicks and commercial chicks with a view to determining how vaccine level in feathers correlates with protection against challenge, and for identifying optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. In this article we review some aspects of the current vaccinal control of Marek's disease, before highlighting some of the problems associated with current vaccines and vaccination strategies, and the challenges for the future. We go on to discuss the development and use of our real-time PCR feather test, its current applications and potential opportunities in Marek's disease vaccine research.

Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR.

A real-time PCR method was developed, optimised and validated, to enable quantitation of Marek's disease virus genomes as copy number per million host cells. The duplex PCR measured the virus meq gene and host ovotransferrin gene in a single reaction enabling correction for differences in amount of sample DNA added. A bacterial artificial chromosome (BAC) clone of the virus genome, and a plasmid (pGEM-T-ovo) bearing a fragment of the chicken ovotransferrin gene, were used to quantify virus and host genomes respectively. This sensitive and reproducible assay was established initially using chicken lymphocyte DNA, then adapted for feather tip DNA by inclusion of bovine serum albumin in the reaction to overcome inhibition by melanin. The principal advantages are: (1) determination of absolute virus genome copy number enabling meaningful comparison between samples; (2) expression of copy number per million cells, allowing direct correlation with plaque assays; (3) using BAC-cloned whole virus genome as a standard potentially enables any virus gene to be used as the PCR target. This is the first report of quantitation of MDV genomes in feather tips, and application of this assay could significantly further our understanding of pathogenesis, spread, diagnosis, genetic resistance and vaccinal control of Marek's disease.

Comparative anatomy of the foramen ovale in the hearts of cetaceans.

The structure of the cardiac foramen ovale from 17 species representing six cetacean families, the Monodontidae, Phocoenidae, Delphinidae, Ziphiidae, Balaenidae and the Balaenopteridae, was studied using the scanning electron microscope. Eight white whale fetuses (Delphinapterus leucas) and a narwhal fetus (Monodon monoceros) represented the Monodontidae; one fetal and nine neonatal harbour porpoises (Phocoena phocoena) and a finless porpoise fetus (Neophocoena phocoenoides) represented the Phocoenidae; two white-beaked dolphin fetuses (Lagenorhynchus albirostris), four fetal and one neonatal Atlantic white-sided dolphins (Lagenorhynchus acutus), a Risso's dolphin fetus (Grampus griseus), two common bottle-nosed dolphin neonates (Tursiops truncatus), a female short-beaked common dolphin fetus (Delphinus delphis), four killer whale fetuses (Orcinus orca) and two long-finned pilot whale fetuses (Globicephala melas) represented the Delphinidae; two northern bottlenose whale fetuses (Hyperoodon ampullatus) represented the Ziphiidae; one bowhead whale fetus (Balaena mysticetus) represented the Balaenidae and five Common minke whale fetuses (Balaenoptera acutorostrata), one blue whale fetus (Balaenoptera musculus), nine fin whale fetuses (Balaenoptera physalus) and four humpback whale fetuses (Megaptera novaeangliae) represented the Balaenopteridae. The hearts of an additional two incompletely identified toothed and four baleen whale fetuses were also studied. In each species the fold of tissue derived from the cardiac septum primum and subtended by the foramen ovale had the appearance of a short tunnel or sleeve which was fenestrated at its distal end. In the toothed whales the tissue fold was tunnel-shaped with the interatrial septum as the floor whereas in baleen whales it was more sleeve-like. In toothed whales thin threads extended from the fold to insert into the interatrial septum whereas a network of threads covered the distal end of the sleeve in the baleen whales. Similar structures were present in the corresponding cardiac tissues of neonatal Hippopotamidae.

Correlation of Marek's disease herpesvirus vaccine virus genome load in feather tips with protection, using an experimental challenge model.

We previously developed a real-time polymerase chain reaction (PCR) assay for absolute quantitation of serotype 1 Marek's disease virus in feather tips of chickens, and this has been used clinically to monitor a flock's response following vaccination with CVI988, an attenuated serotype 1 strain. The level of vaccine virus in feather tips associated with protection against challenge by virulent virus is not known. Here, we used an experimental challenge model, in which one dose of vaccine gives over 90% protection against mortality, to investigate correlation between the CVI988 level in feathers and protection. One-day-old chickens were vaccinated with 1, 0.1 or 0.01 commercial dose of CVI988 vaccine, and were then challenged with a virulent strain (RB-1B) 14, 21 or 28 days later. Replication of CVI988 virus was followed in each bird by real-time PCR analysis of feather DNA samples. Since the PCR does not differentiate between CVI988 and RB-1B, samples were taken only prior to challenge to ensure that the virus being measured was CVI988. Administration of one dose of vaccine ensured a uniform, rapid and high replication amongst birds, while replication following administration of the 0.1 or 0.01 dose was very variable. However, given time, a low early level of vaccine virus eventually replicated to high levels in some birds. Both the dose of vaccine virus administered and the level of vaccine virus in feather tips at 13 days post vaccination showed significant correlation with protection against challenge. A level of CVI988 vaccine virus of 132 genome copies/10000 feather tip cells was calculated to be the level required for 90% protection in this experimental model. The potential of this assay, and its limitations for monitoring protection in the field, are discussed.

Replication kinetics of Marek's disease vaccine virus in feathers and lymphoid tissues using PCR and virus isolation.

CVI988 (Rispens), an avirulent strain of Marek's disease virus, is the most widely used vaccine against Marek's disease. The kinetics of replication of CVI988 was examined in tissues of chickens vaccinated at either 1 day or 14 days of age and sampled regularly up to 28 days post-vaccination. Age at vaccination had no significant effect on the kinetics of CVI988 virus replication. During the cytolytic phase of infection (1-7 days), virus levels peaked in the spleen, bursa and thymus with very close correlation among these organs. Virus load in peripheral blood lagged behind and did not reach high levels. Significant numbers of virus genomes were detected in the feather tips only after 7 days, but subsequently rose to levels almost 10(3)-fold greater than in the other tissues. This is the first accurate quantitative data for kinetics of CVI988 replication in a variety of tissues. There was good correlation between data from virus isolation and PCR, with real-time PCR being the preferred method for rapid, accurate and sensitive quantification of virus. Feathers were ideal for non-invasive sampling to detect and measure CVI988 in live chickens and, from 10 days onwards, virus load in feather tips was predictive of virus load in lymphoid tissues where immune responses will occur. The potential for real-time PCR analysis of feather samples for further investigation of the mechanism of vaccinal protection, and to assist optimization of vaccination regimes, is discussed.