Identification of new populations of chicken natural killer (NK) cells

Natural killer (NK) cell activity is conserved throughout vertebrate development, but characterization of non-mammalian NK-cells has been hampered by the absence of specific mAbs for these cells.Monoclonal antibodies were generated against in vitro IL-2 expanded sorted CD3-CD8α+ peripheral blood lymphocytes, previously described to contain chicken NK-cells. Screening of embryonic and adult splenocytes with hybridoma supernatants resulted in five candidate NK markers.Activation of chicken NK-cells with PMA/Ionomycin or with the NK target cell-line LSCC-RP9 resulted in increased expression of CD107 (LAMP-1) and a newly developed flow cytometry based cytotoxicity assay showed that NK-cells were able to kill target cells. Combining NK markers with functional assays indicated that marker positive cells showed NK-cell function.In conclusion, we generated new monoclonal antibodies and developed two functional assays which will enhance our understanding of the role of NK-cells in healthy and diseased chickens

Specificity Protein 2 (Sp2) Is Essential for Mouse Development and Autonomous Proliferation of Mouse Embryonic Fibroblasts

BACKGROUND: The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with the phenotypes of Sp1, Sp3 and Sp4 knockout strains shows that, despite their structural similarity and evolutionary relationship, all four glutamine-rich members of the Sp family of transcription factors have distinct non-redundant functions in vivo

Epidemiology-driven approaches to surveillance in HPAI-vaccinated poultry flocks aiming to demonstrate freedom from circulating HPAIV

Incursion pressure of high pathogenicity avian influenza viruses (HPAIV) by secondary spread among poultry holdings and/or from infected migratory wild bird populations increases worldwide. Vaccination as an additional layer of protection of poultry holdings using appropriately matched vaccines aims at reducing clinical sequelae of HPAIV infection, disrupting HPAIV transmission, curtailing economic losses and animal welfare problems and cutting exposure risks of zoonotic HPAIV at the avian-human interface. Products derived from HPAIV-vaccinated poultry should not impose any risk of virus spread or exposure. Vaccination can be carried out with zero-tolerance for infection in vaccinated herds and must then be flanked by appropriate surveillance which requires tailoring at several levels: (i) Controlling appropriate vaccination coverage and adequate population immunity in individual flocks and across vaccinated populations; (ii) assessing HPAI-infection trends in unvaccinated and vaccinated parts of the poultry population to provide early detection of new/re-emerged HPAIV outbreaks; and (iii) proving absence of HPAIV circulation in vaccinated flocks ideally by real time-monitoring. Surveillance strategies, i.e. selecting targets, tools and random sample sizes, must be accommodated to the specific epidemiologic and socio-economic background. Methodological approaches and practical examples from three countries or territories applying AI vaccination under different circumstances are reviewed here

Risk for low pathogenicity avian influenza virus on poultry farms, The Netherlands, 2007–2013

Using annual serologic surveillance data from all poultry farms in the Netherlands during 2007–2013, we quantified the risk for the introduction of low pathogenicity avian influenza virus (LPAIV) in different types of poultry production farms and putative spatial-environmental risk factors: distance from poultry farms to clay soil, waterways, and wild waterfowl areas. Outdoor-layer, turkey (meat and breeder), and duck (meat and breeder) farms had a significantly higher risk for LPAIV introduction than did indoor-layer farms. Except for outdoor-layer, all poultry types (i.e., broilers, chicken breeders, ducks, and turkeys) are kept indoors. For all production types, LPAIV risk decreased significantly with increasing distance to medium-sized waterways and with increasing distance to areas with defined wild waterfowl, but only for outdoor-layer and turkey farms. Future research should focus not only on production types but also on distance to waterways and wild bird areas. In addition, settlement of new poultry farms in high-risk areas should be discouraged

Genetic analysis of infectious bronchitis virus (IBV) in vaccinated poultry populations over a period of 10 years

Infectious bronchitis virus (IBV) is an avian pathogen from the Coronavirus family causing major health issues in poultry flocks worldwide. Because of its negative impact on health, performance, and bird welfare, commercial poultry are routinely vaccinated by administering live attenuated virus. However, field strains are capable of rapid adaptation and may evade vaccine-induced immunity. We set out to describe dynamics within and between lineages and assess potential escape from vaccine-induced immunity. We investigated a large nucleotide sequence database of over 1700 partial sequences of the S1 spike protein gene collected from clinical samples of Dutch chickens submitted to the laboratory of Royal GD between 2011 and 2020. Relative frequencies of the two major lineages GI-13 (793B) and GI-19 (QX) did not change in the investigated period, but we found a succession of distinct GI-19 sublineages. Analysis of dN/dS ratio over all sequences demonstrated episodic diversifying selection acting on multiple sites, some of which overlap predicted N-glycosylation motifs. We assessed several measures that would indicate divergence from vaccine strains, both in the overall database and in the two major lineages. However, the frequency of vaccine-homologous lineages did not decrease, no increase in genetic variation with time was detected, and the sequences did not grow more divergent from vaccine sequences in the examined time window. Concluding, our results show sublineage turnover within the GI-19 lineage and we demonstrate episodic diversifying selection acting on the partial sequence, but we cannot confirm nor rule out escape from vaccine-induced immunity. RESEARCH HIGHLIGHTS Succession of GI-19 IBV variants in broiler populations. IBV lineages overrepresented in either broiler, or layer production chickens. Ongoing episodic selection at the IBV S1 spike protein gene sequence. Several positively selected codons coincident with N-glycosylation motifs

Specificity protein 2 (Sp2) is essential for mouse development and autonomous proliferation of mouse embryonic fibroblasts

Background: The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown. Methodology/Principal Findings: Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation. Conclusions/Significance: Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with th

Infectious Bronchitis Coronavirus: Genome Evolution in Vaccinated and Non-Vaccinated SPF Chickens

Infectious Bronchitis virus (IBV) continues to cause significant economic losses for the chicken industry despite the use of many live IBV vaccines around the world. Several authors have suggested that vaccine-induced partial protection may contribute to the emergence of new IBV strains. In order to study this hypothesis, three passages of a challenge IBV were made in SPF chickens sham inoculated or vaccinated at day of age using a live vaccine heterologous to the challenge virus. All birds that were challenged with vaccine heterologous virus were positive for viral RNA. NGS analysis of viral RNA in the unvaccinated group showed a rapid selection of seven genetic variants, finally modifying the consensus genome of the viral population. Among them, five were non-synonymous, modifying one position in NSP 8, one in NSP 13, and three in the Spike protein. In the vaccinated group, one genetic variant was selected over the three passages. This synonymous modification was absent from the unvaccinated group. Under these conditions, the genome population of an IBV challenge virus evolved rapidly in both heterologous vaccinated and non-vaccinated birds, while the genetic changes that were selected and the locations of these were very different between the two groups

The DECIDE project: from surveillance data to decision-support for farmers and veterinarians

Farmers, veterinarians and other animal health managers in the livestock sector are currently missing sufficient information on prevalence and burden of contagious endemic animal diseases. They need adequate tools for risk assessment and prioritization of control measures for these diseases. The DECIDE project develops data-driven decision-support tools, which present (i) robust and early signals of disease emergence and options for diagnostic confirmation; and (ii) options for controlling the disease along with their implications in terms of disease spread, economic burden and animal welfare. DECIDE focuses on respiratory and gastro-intestinal syndromes in the three most important terrestrial livestock species (pigs, poultry, cattle) and on reduced growth and mortality in two of the most important aquaculture species (salmon and trout). For each of these, we (i) identify the stakeholder needs; (ii) determine the burden of disease and costs of control measures; (iii) develop data sharing frameworks based on federated data access and meta-information sharing; (iv) build multivariate and multi-level models for creating early warning systems; and (v) rank interventions based on multiple criteria. Together, all of this forms decision-support tools to be integrated in existing farm management systems wherever possible and to be evaluated in several pilot implementations in farms across Europe. The results of DECIDE lead to improved use of surveillance data and evidence-based decisions on disease control. Improved disease control is essential for a sustainable food chain in Europe with increased animal health and welfare and that protects human health