The main findings of the post-mortem examination of poultry infected with highly pathogenic avian influenza viruses (HPAIV) include necrotizing inflammation and viral antigen in multiple organs. The lesion profile displays marked variability, depending on viral subtype, strain, and host species. Therefore, in this study, a semiquantitative scoring system was developed to compare histopathological findings across a wide range of study conditions. Briefly, the severity of necrotizing lesions in brain, heart, lung, liver, kidney, pancreas, and/or lymphocytic depletion in the spleen is scored on an ordinal four-step scale (0 = unchanged, 1 = mild, 2 = moderate, 3 = severe), and the distribution of the viral antigen in parenchymal and endothelial cells is evaluated on a four-step scale (0 = none, 1 = focal, 2 = multifocal, 3 = diffuse). These scores are used for a meta-analysis of experimental infections with H7N7 and H5N8 (clade 2.3.4.4b) HPAIV in chickens, turkeys, and ducks. The meta-analysis highlights the rather unique endotheliotropism of these HPAIV in chickens and a more severe necrotizing encephalitis in H7N7-HPAIV-infected turkeys. In conclusion, the proposed scoring system can be used to condensate HPAIV-typical pathohistological findings into semiquantitative data, thus enabling systematic phenotyping of virus strains and their tissue tropism.

Highly contagious Newcastle disease (ND) is associated with devastating outbreaks with highly variable clinical signs among gallinaceous birds. In this study we aimed to verify clinical ND suspicions in poultry holdings in Egypt suffering from respiratory distress and elevated mortality, comparing two groups of ND-vaccinated poultry holdings in three governorates. Besides testing for Newcastle disease virus (NDV), samples were screened for infectious bronchits virus (IBV) and avian influenza virus (AIV) by RT-qPCR as well as by non-directed cell-culture approach on LMH-cells. Virulent NDV was confirmed only in group A (n = 16) comprising small-scale holdings. Phylogenetic analysis of the fusion protein gene of 11 NDV-positive samples obtained from this group assigned all viruses to genotype 2.VIIb and point to four different virus populations that were circulating at the same time in one governorate, indicating independent epidemiological events. In group B, comprising large commercial broiler farms (n = 10), virulent NDV was not present, although, in six farms NDV vaccine type virus (genotype 2.II) was detected. Besides, in both groups, co-infections by IBV (n = 8), AIV H9 (n = 3) and/or avian reovirus (ARV) (n = 5) and avian astrovirus (AastVs) (n = 1) could be identified. Taken together, the study confirmed clinical ND suspicion in small scale holdings, pointing to inefficient vaccination practice in this group A. However, it also highlighted that even in an endemic situation like ND in Egypt, in cases of suspected ND vaccine failure, clinical ND suspicion has to be verified by pathotype-specific diagnostic tests.

Outbreaks of highly pathogenic avian influenza A virus (HPAIV) subtype H5N8, clade 2.3.4.4, were first reported in January 2014 from South Korea. These viruses spread rapidly to Europe and the North American continent during autumn 2014 and caused, in Germany, five outbreaks in poultry holdings until February 2015. In addition, birds kept in a zoo in north‐eastern Germany were affected. Only a few individual white storks (Ciconia ciconia) showed clinical symptoms and eventually died in the course of the infection, although subsequent in‐depth diagnostic investigations showed that other birds kept in the same compound of the white storks were acutely positive for or had undergone asymptomatic infection with HPAIV H5N8. An exception from culling all of the 500 remaining zoo birds was granted by the competent authority. Restriction measures included grouping the zoo birds into eight epidemiological units in which 60 birds of each unit tested repeatedly negative for H5N8. Epidemiological and phylogenetical investigations revealed that the most likely source of introduction was direct or indirect contact with infected wild birds as the white storks had access to a small pond frequented by wild mallards and other aquatic wild birds during a period of 10 days in December 2014. Median network analysis showed that the zoo bird viruses segregated into a distinct cluster of clade 2.3.4.4 with closest ties to H5N8 isolates obtained from mute swans (Cygnus olor) in Sweden in April 2015. This case demonstrates that alternatives to culling exist to rescue valuable avifaunistic collections after incursions of HPAIV.

Infections with pigeon circovirus (PiCV) (also termed columbid circovirus) occur in meat and racing pigeons (Columba livia) of all ages and have been reported worldwide. A PiCV infection is associated with immunosuppression and the development of young pigeon disease syndrome. An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of virus-specific serum antibody was developed for research purposes. In the absence of a method to propagate PiCV in cell culture, the assay was based on a recombinant truncated capsid protein (rCapPiCV) produced by overexpression in Escherichia coli. A 6×His-Tag was fused to the N-terminus of the protein to facilitate purification by metal affinity chromatography and detection by anti-His antibody. PiCV-negative and PiCV-positive control sera were generated by inoculation of pigeons with tissue homogenate containing PiCV, followed by five weekly blood sample collections. Western blotting of the immune serum revealed a specific protein band of approximately 32 kDa, which was absent in the pre-immune sera. Using rCapPiCV as antigen in an indirect ELISA, PiCV-specific antibody was detected in sera of the experimentally PiCV-infected pigeons collected at 1 to 5 weeks post infection. By testing 118 field sera collected in the years 1989, 1991, 1994 and 2008 in the rCapPiCV ELISA, virus-specific antibody was detected in 89 (75%) of the sera. The results obtained demonstrate that the rCapPiCV-based indirect ELISA is able to detect PiCV-specific antibodies in pigeon sera and may be a useful tool for PiCV serodiagnosis.

Newcastle disease (ND), caused by avian orthoavulavirus type‐1 (NDV), is endemic in poultry in many regions of the world and causes continuing outbreaks in poultry populations. In the Middle East, genotype XXI, , used to be present in poultry in Egypt but has been replaced by genotype VII. We investigated whether virus evolution contributed to superseding and focused on the antigenic sites within the Hemagglutinin‐Neuraminidase (HN) spike protein. Full length sequences of an NDV genotype VII isolate currently circulating in Egypt was compared to a genotype XXI isolate that was present as co‐infection with vaccine type viruses (II) in a historical virus isolated in 2011. Amino acid differences in the HN glycoprotein for both XXI and VII viruses amounted to 11.7% and 11.9 %, respectively, compared to the La Sota vaccine type. However, mutations within the globular head (aa 126‐570), bearing relevant antigenic sites, were underrepresented (aa divergence of 8.8% and 8.1 % compared to 22.4% and 25.6% within the protein domains encompassing cytoplasmic tail, transmembrane part and stalk regions (aa 1‐125) for genotypes XXI and VII, respectively). Nevertheless, reaction patterns of HN‐specific monoclonal antibodies inhibiting receptor binding revealed differences between vaccine type viruses and genotype XXI and VII viruses for epitopes located in the head domain. Accordingly, compared to Egyptian vaccine type isolates and the La Sota vaccine reference strain, single aa substitutions in 6 of 10 described neutralizing epitopes of HN were found. However, the same alterations in neutralization sensitive epitopes were present in old genotype XXI as well as in newly emerged genotype VII isolates. In addition, isolates were indistinguishable by polyclonal chicken sera raised against different genotypes including vaccine viruses. These findings suggest that factors other than antigenic differences within the HN protein account for facilitating the spread of genotype VII versus genotype XXI viruses in Egypt.