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In France during winter 2016-2017, 487 outbreaks of clade 2.3.4.4b H5N8 subtype high pathogenicity (HP) avian influenza A virus (AIV) infections were detected in poultry and captive birds. During this epizootic, HPAIV A/decoy duck/France/161105a/2016 (H5N8) was isolated and characterized in an experimental infection transmission model in conventional mule ducks. To investigate options to possibly protect such ducks against this HPAIV, three vaccines were evaluated in controlled conditions. The first experimental vaccine was derived from the hemagglutinin gene of another clade 2.3.4.4b A(H5N8) HPAIV. It was injected at three weeks of age, either alone (Vac1) or after a primer injection at day-old (Vac1 + boost). The second vaccine (Vac2) was a commercial bivalent adjuvanted vaccine containing an expressed hemagglutinin modified from a clade 2.3.2 A(H5N1) HPAIV. Vac2 was administered as a single injection at two weeks of age. The third experimental vaccine (Vac3) also incorporated a homologous 2.3.4.4b H5 HA gene and was administered as a single injection at three weeks of age. Ducks were challenged with HPAIV A/decoy duck/France/161105a/2016 (H5N8) at six weeks of age. Post-challenge virus excretion was monitored in vaccinated and control birds every 2-3 days for two weeks using real-time reverse-transcription polymerase chain reaction and serological analyses (haemagglutination inhibition test against H5N8, H5 ELISA and AIV ELISA) were performed. Vac1 abolished oropharyngeal and cloacal shedding to almost undetectable levels, whereas Vac3 abolished cloacal shedding only (while partially reducing respiratory shedding) and Vac2 only partly reduced the respiratory and intestinal excretion of the challenge virus. These results provided relevant insights in the immunogenicity of recombinant H5 vaccines in mule ducks, a rarely investigated hybrid between Pekin and Muscovy duck species that has played a critical role in the recent H5 HPAI epizootics in France., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

End-point and real-time avian metapneumovirus (AMPV) RT-PCRs have been developed to detect one or two of the four recognized subgroups (A,B,C, and D) simultaneously or for broad range AMPV detection. Current subgroup specific tests target variable areas of the genome which makes these PCRs sensitive to specificity defects as recently documented. In the current study, a single five-plex digital droplet RT-PCR targeting the conserved viral polymerase gene of AMPV, which is less prone to genetic drift, has been designed. This digital droplet RT-PCR was capable of identifying each of the four AMPV subgroups. Each subgroup was identified according to a specifically assigned fluorescent amplitude. Specificity, which was tested including 31 AMPV strains, non-AMPV avian viruses and closely related human respiratory viruses, was 100%. The specific limit of detection for extracted viral RNA was estimated between 1 and 3 copies/μl. This tool simplifies the number of tests required for AMPV genotype diagnostics and should be theoretically less effected by viral genome evolution due to its target region. Ultimately, application of this test will contribute to an improved understanding of the global geographic distribution and subgroup host range of field strains., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lemaitre, Bougeard, Allée, Eterradossi, Courtillon and Brown.)

During winter 2020-2021, France and other European countries were severely affected by highly pathogenic avian influenza H5 viruses of the Gs/GD/96 lineage, clade 2.3.4.4b. In total, 519 cases occurred, mainly in domestic waterfowl farms in Southwestern France. Analysis of viral genomic sequences indicated that 3 subtypes of HPAI H5 viruses were detected (H5N1, H5N3, H5N8), but most French viruses belonged to the H5N8 subtype genotype A, as Europe. Phylogenetic analyses of HPAI H5N8 viruses revealed that the French sequences were distributed in 9 genogroups, suggesting 9 independent introductions of H5N8 from wild birds, in addition to the 2 introductions of H5N1 and H5N3., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

An H3N1 avian influenza virus was detected in a laying hens farm in May 2019 which had experienced 25% mortality in Northern France. The complete sequencing of this virus showed that all segment sequences belonged to the Eurasian lineage and were phylogenetically very close to many of the Belgian H3N1 viruses detected in 2019. The French virus presented two genetic particularities with NA and NS deletions that could be related to virus adaptation from wild to domestic birds and could increase virulence, respectively. Molecular data of H3N1 viruses suggest that these two deletions occurred at two different times., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Currently, turkey coronaviruses (TCoV) are isolated from homogenized intestines of experimentally infected embryos to ensure a maximum recovery of viral particles from all components of the intestines. However, the process of homogenization also ensures release of a significant amount of cellular RNAs into the sample that hinders downstream viral genome sequencing. This is especially the case for next generation sequencing (NGS) which sequences molecules at random. This characteristic means that the heavily abundant cellular RNA in the sample drowns out the minority viral RNA during the sequencing process and, consequently, very little to no viral genome data are obtained. To address this problem, a method was developed, in which 10 descendent isolates of the European strain of TCoV were recovered uniquely from the intestinal lumen without homogenization of the tissue. For nine out of 10 samples, NGS produced viral RNA reads with good coverage depth over the entire TCoV genomes. This is a much-needed new, simple and cost effective method of isolating TCoV that facilitates downstream NGS of viral RNA and should be considered as an alternative method for isolating other avian enteric coronaviruses in the interest of obtaining full-length genome sequences.

Immunosuppression in poultry production is a recurrent problem worldwide, and one of the major viral immunosuppressive agents is Infectious Bursal Disease Virus (IBDV). IBDV infections are mostly controlled by using live-attenuated vaccines. Live-attenuated Infectious Bursal Disease (IBD) vaccine candidates are classified as "mild," "intermediate," "intermediate-plus" or "hot" based on their residual immunosuppressive properties. The immunosuppression protocol described by the European Pharmacopoeia (Ph. Eur.) uses a lethal Newcastle Disease Virus (NDV) infectious challenge to measure the interference of a given IBDV vaccine candidate on NDV vaccine immune response. A Ph. Eur.-derived protocol was thus implemented to quantify immunosuppression induced by one mild, two intermediate, and four intermediate-plus live-attenuated IBD vaccines as well as a pathogenic viral strain. This protocol confirmed the respective immunosuppressive properties of those vaccines and virus. In the search for a more ethical alternative to Ph. Eur.-based protocols, two strategies were explored. First, ex vivo viral replication of those vaccines and the pathogenic strain in stimulated chicken primary bursal cells was assessed. Replication levels were not strictly correlated to immunosuppression observed in vivo . Second, changes in blood leukocyte counts in chicks were monitored using a Ph. Eur. - type protocol prior to lethal NDV challenge. In case of intermediate-plus vaccines, the drop in B cells counts was more severe. Counting blood B cells may thus represent a highly quantitative, faster, and ethical strategy than NDV challenge to assess the immunosuppression induced in chickens by live-attenuated IBD vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Courtillon, Allée, Amelot, Keita, Bougeard, Härtle, Rouby, Eterradossi and Soubies.)

In 2021, France faced large avian influenza outbreaks, like in 2016 and 2017. Controlling these outbreaks required the preventive depopulation of a large number of duck farms. A previous study in 2017 showed that the quality of decontamination of trucks and transport crates used for depopulation was often insufficient. A new study was then set up to evaluate cleaning and disinfection (C&D) of trucks and crates used for duck depopulation and whether practices had changed since 2017. Three methods were used to assess decontamination: 1) detection of avian influenza virus (AIV) genome, 2) visual inspection of cleanliness, and 3) microbial counts, considering that 2 and 3 are commonly used in abattoirs. Another objective of the study was to evaluate the correlation between results obtained with the 3 methods. In 5 abattoirs, 8 trucks and their crates were sampled by swabbing to detect AIV genome by rRT-PCR before and after decontamination. Visual cleanliness scores and coliform counts were also determined on crates after C&D. Trucks and crates were decontaminated according to the abattoirs' protocols. Before C&D, 3 quarters of crates (59/79) and 7 of 8 trucks were positive for AIV genome. C&D procedures were reinforced in 2021 compared to 2017; use of detergent solution and warm water were more common. Nevertheless, 28% of the crates were positive for AIV genome after C&D, despite the fact that cleaning scores and microbiological counts were satisfactory for 84% and 91% of the crates, respectively. No correlation was observed between results for AIV genome detection and results from visual control or from coliform counts. Abattoirs are encouraged to use environmental sampling coupled with AIV genome detection to monitor the quality of cleaning and disinfection of trucks and crates during AI outbreaks. Reinforcement of biosecurity measures at abattoirs is still needed to avoid residual contamination of the equipment and cross-contamination during the decontamination process., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Guinea fowl fulminating enteritis has been reported in France since the 1970s. In 2014, a coronavirus was identified and appeared as a possible viral pathogen involved in the disease. In the present study, intestinal content from a guinea fowl involved in a new case of the disease in 2017 was analysed by deep sequencing, revealing the presence of a guinea fowl coronavirus (GfCoV) and a picornavirus (GfPic). Serial passage assays into the intra-amniotic cavity of 13-day-old specific pathogen-free chicken eggs and 20-day-old conventional guinea fowl eggs were attempted. In chicken eggs, isolation assays failed, but in guinea fowl eggs, both viruses were successfully obtained. Furthermore, two GfCoV and two GfPic isolates were obtained from the same bird but from different sections of its intestines. This shows that using eggs of the same species, in which the virus has been detected, can be the key for successful isolation. The consensus sequence of the full-length genomes of both GfCoV isolates was highly similar, and correlated to those previously described in terms of genome organization, ORF length and phylogenetic clustering. According to full-length genome analysis and the structure of the Internal Ribosome Entry Site, both GfPic isolates belong to the Anativirus genus and specifically the species Anativirus B . The availability of the first isolates of GfCoV and GfPic will now provide a means of assessing their pathogenicity in guinea fowl in controlled experimental conditions and to assess whether they are primary viral pathogens of the disease "guinea fowl fulminating enteritis". RESEARCH HIGHLIGHTS First isolation of guinea fowl coronaviruses and picornaviruses.Eggs homologous to the infected species are key for isolation.Isolates available to precisely evaluate the virus roles in fulminating enteritis.First full-length genome sequences of guinea fowl picornaviruses.

Avian metapneumovirus (aMPV) is a viral pathogen that causes respiratory signs and drops in egg production most notably in turkeys but also affects chickens, ducks and other species. Four subgroups (aMPV-A, -B, -C and -D) have been characterized to date, which differ genetically, antigenically and in terms of geographic distribution and host range. Despite the rising awareness towards aMPV, many aspects of its epidemiology are still poorly understood. In this pilot study, a serological survey was carried out to assess the possible circulation of subgroups A, B, and C in intensively raised ducks in Northern Italy. A total of 220 sera from 10 Pekin duck flocks and one mallard flock were collected at slaughter and tested by subgroup-specific aMPV-A, B, and C indirect ELISA assays. None of the tested birds had been vaccinated against aMPV and no symptoms had been reported. No aMPV-A, B or C antibodies were detected in any bird of the Pekin duck flocks, whereas the entire mallard flock tested positive for aMPV-C antibodies. This is the first report of aMPV-C antibodies in ducks in Italy, where only aMPV-B has been reported to circulate in recent years. Further research efforts will be targeted towards the screening of a larger panel of samples, in the hope of obtaining positive samples from which full length genome sequences and infectious viruses can be isolated for virus characterization. Finally, the demonstration of aMPV-C specific antibodies in the serum of all sampled mallards indirectly suggests that this species is readily infected by aMPV-C and supports a possible role of wild anatids as a transmission vector of the virus., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

We detected 3 genotypes of highly pathogenic avian influenza A(H5N8) virus in France during winter 2016-17. Genotype A viruses caused dramatic economic losses in the domestic duck farm industry in southwestern France. Our phylogenetic analysis suggests that genotype A viruses formed 5 distinct geographic clusters in southwestern France. In some clusters, local secondary transmission might have been started by a single introduction. The intensity of the viral spread seems to correspond to the density of duck holdings in each production area. To avoid the introduction of disease into an unaffected area, it is crucial that authorities limit the movements of potentially infected birds.

Avian influenza A viruses are a major threat to animal and public health. Since 1997, several highly pathogenic H5N1 avian viruses have been directly transmitted from poultry to humans, caused numerous human deaths, and had considerable economic impact on poultry markets. During 2015-2016, a highly pathogenic avian influenza outbreak occurred in southwestern France. Different subtypes circulated, including the A/chicken/France/150169a/2015 H5N1 highly pathogenic virus, which did not possess the full set of genomic determinants known to promote transmission to humans. In order to evaluate the predicted absence of zoonotic potential, a quick method based on in vitro tests was developed to analyze some genetic and phenotypic host restriction determinants. A receptor-binding assay showed that the virus preferentially recognizes avian cell receptors. Temperature sensitivity revealed a cold-sensitive phenotype of the virus at 33 C as virus replication was reduced in contrast with what is expected for human influenza viruses, according to their primary infection sites. Altogether, our quick evaluation method suggests that the A/chicken/France/150169a/2015 H5N1 highly pathogenic virus has an avian phenotype in vitro, in accordance with in silico predictions based on genomic markers.

To date, four subgroups of avian metapneumoviruses have been defined (AMPV-A, B, C and D) based on genetic and antigenic differences. The extent of infection in the three principal species (turkeys, chickens and ducks) by these subgroups is, however, not well defined. Here, a series of controlled and ethically approved experimental infections were performed in specific pathogen-free turkeys, chickens and ducks with each of the four AMPV subgroups. For subgroup C, one strain isolated from turkeys in the USA (turkey AMPV-C) and one isolated from ducks in France (duck AMPV-C) were compared. Globally, these extensive experimental trials demonstrated that AMPV-A, B, turkey C and D were well adapted to Galliformes, especially turkeys; however, chickens showed limited clinical signs and differences in seroconversion and transmission. Notably, chickens did not transmit AMPV-A to contacts and were shown for the first time to be susceptible to AMPV-D. The duck AMPV-C was well adapted to ducks; however, chickens and turkeys seroconverted and were positive by virus isolation. In addition, seroconversion of contact turkeys to duck AMPV-C demonstrated horizontal transmission of this virus in a non-palmiped species under our experimental conditions. Interestingly, in chickens and turkeys, duck AMPV-C isolation was possible despite a lack of detection of viral RNA. Likewise, the turkey AMPV-C virus was well adapted to turkeys yet was also isolated from chickens despite a lack of detection of viral RNA. These results would suggest a selection for viral genetic sequences that differ from the original strain upon adaptation to a 'non-conventional host'.

Numerous viruses, mostly in mixed infections, have been associated worldwide with poult enteritis complex (PEC). In 2008 a coronavirus (Fr-TCoV 080385d) was isolated in France from turkey poults exhibiting clinical signs compatible with this syndrome. In the present study, the median infectious dose (ID 50 ) , transmission kinetics and pathogenicity of Fr-TCoV were investigated in 10-day-old SPF turkeys. Results revealed a titre of 10 4.88 ID 50 /ml with 1 ID 50 /ml being beyond the limit of genome detection using a well-characterized qRT-PCR for avian coronaviruses. Horizontal transmission of the virus via the airborne route was not observed however, via the oro-faecal route this proved to be extremely rapid (one infectious individual infecting another every 2.5 hr) and infectious virus was excreted for at least 6 weeks in several birds. Histological examination of different zones of the intestinal tract of the Fr-TCoV-infected turkeys showed that the virus had a preference for the lower part of the intestinal tract with an abundance of viral antigen being present in epithelial cells of the ileum, caecum and bursa of Fabricius. Viral antigen was also detected in dendritic cells, monocytes and macrophages in these areas, which may indicate a potential for Fr-TCoV to replicate in antigen-presenting cells. Together these results highlight the importance of good sanitary practices in turkey farms to avoid introducing minute amounts of virus that could suffice to initiate an outbreak, and the need to consider that infected individuals may still be infectious long after a clinical episode, to avoid virus dissemination through the movements of apparently recovered birds., (© 2018 Blackwell Verlag GmbH.)

An avian influenza H3N2 virus was isolated from domestic ducks in France in 2016. Although this French H3N2 virus possesses traits of an avian virus, the genetic distances observed for hemagglutinin (HA) and neuraminidase (NA) show that these two genes most likely evolved independently from other avian influenza sequences.

Infectious bursal disease (IBD) is an immunosuppressive viral disease, present worldwide, which causes mortality and immunosuppression in young chickens. The causative agent, the Avibirnavirus IBDV, is a non-enveloped virus whose genome consists of two segments (A and B) of double-stranded RNA. Different pathotypes of IBDV exist, ranging from attenuated vaccine strains to very virulent viruses (vvIBDV). In Algeria, despite the prophylactic measures implemented, cases of IBD are still often diagnosed clinically and the current molecular epidemiology of IBDV remains unknown. The presence of the virus and especially of strains genetically close to vvIBDV was confirmed in 2000 by an unpublished OIE report. In this study, nineteen IBDV isolates were collected in Algeria between September 2014 and September 2015 during clinical outbreaks. These isolates were analyzed at the genetic, antigenic and pathogenic levels. Our results reveal a broad genetic and phenotypic diversity of pathogenic IBDV strains in Algeria, with, i) the circulation of viruses with both genome segments related to European vvIBDV, which proved as pathogenic for specific pathogen-free chickens as vvIBDV reference strain, ii) the circulation of viruses closely related - yet with a specific segment B - to European vvIBDV, their pathogenicity being lower than reference vvIBDV, iii) the detection of reassortant viruses whose segment A was related to vvIBDV whereas their segment B did not appear closely related to any reference sequence. Interestingly, the pathogenicity of these potentially reassortant strains was comparable to that of reference vvIBDV. All strains characterized in this study exhibited an antigenicity similar to the cognate reference IBDV strains. These data reveal the continuous genetic evolution of IBDV strains in Algerian poultry through reassortment and acquisition of genetic material of unidentified origin. Continuous surveillance of the situation as well as good vaccination practice associated with appropriate biosecurity measures are necessary for disease control., (Copyright © 2018. Published by Elsevier B.V.)

Several new highly pathogenic (HP) H5 avian influenza virus (AIV) have been detected in poultry farms from south-western France since November 2015, among which an HP H5N1. The zoonotic potential and origin of these AIVs immediately became matters of concern. One virus of each subtype H5N1 (150169a), H5N2 (150233) and H5N9 (150236) was characterised. All proved highly pathogenic for poultry as demonstrated molecularly by the presence of a polybasic cleavage site in their HA protein - with a sequence (HQRRKR/GLF) previously unknown among avian H5 HPAI viruses - or experimentally by the in vivo demonstration of an intravenous pathogenicity index of 2.9 for the H5N1 HP isolate. Phylogenetic analyses based on the full genomes obtained by NGS confirmed that the eight viral segments of the three isolates were all part of avian Eurasian phylogenetic lineage but differed from the Gs/Gd/1/96-like lineage. The study of the genetic characteristics at specific amino acid positions relevant for modulating the adaptation to and the virulence for mammals showed that presently, these viruses possess most molecular features characteristic of AIV and lack some major characteristics required for efficient respiratory transmission to or between humans. The three isolates are therefore predicted to have no significant pandemic potential., (This article is copyright of The Authors, 2017.)

Infectious bursal disease virus (IBDV) causes an economically significant disease of young chickens worldwide. The emergence of very virulent IBDV (vvIBDV) strains has brought more challenges for effective prevention and control of this disease. The aim of the present study was to characterize four IBDV isolates from various regions of China between late 1990s and recent years and to compare them with previously isolated European IBDV strains. In this study, one Chinese vvIBDV strain isolated in 1999 and three strains isolated between 2005 and 2011 were analyzed at the genetic, antigenic and pathogenic levels. Strain SH99 was closely related and clustered in the same genetic lineage as the typical vvIBDV based on the genomic sequences of segments A and B. However, the three more recent Chinese vvIBDV (HLJ0504, HeB10 and HuN11) showed several genetic changes in both segments and clustered in a distinct lineage from the typical vvIBDV and the previously known Chinese vvIBDV. Based on the binding to a panel of neutralizing monoclonal antibodies in antigen capture enzyme-linked immunosorbent assays, all Chinese vvIBDVs exhibited similar antigenicity with the European typical vvIBDV strains. Nonetheless, the pathogenicity caused by the recent Chinese vvIBDV was higher than that induced by the European typical vvIBDV. This study calls for a sustained surveillance of IBD situation in China in order to support a better prevention and control of the disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Four avian metapneumovirus (AMPV) subgroups (A-D) have been reported previously based on genetic and antigenic differences. However, until now full length sequences of the only known isolates of European subgroup C and subgroup D viruses (duck and turkey origin, respectively) have been unavailable. These full length sequences were determined and compared with other full length AMPV and human metapneumoviruses (HMPV) sequences reported previously, using phylogenetics, comparisons of nucleic and amino acid sequences and study of codon usage bias. Results confirmed that subgroup C viruses were more closely related to HMPV than they were to the other AMPV subgroups in the study. This was consistent with previous findings using partial genome sequences. Closer relationships between AMPV-A, B and D were also evident throughout the majority of results. Three metapneumovirus "clusters" HMPV, AMPV-C and AMPV-A, B and D were further supported by codon bias and phylogenetics. The data presented here together with those of previous studies describing antigenic relationships also between AMPV-A, B and D and between AMPV-C and HMPV may call for a subclassification of metapneumoviruses similar to that used for avian paramyxoviruses, grouping AMPV-A, B and D as type I metapneumoviruses and AMPV-C and HMPV as type II.

EU annual serosurveillance programs show that domestic duck flocks have the highest seroprevalence of H5 antibodies, demonstrating the circulation of notifiable avian influenza virus (AIV) according to OIE, likely low pathogenic (LP). Therefore, transmission characteristics of LPAIV within these flocks can help to understand virus circulation and possible risk of propagation. This study aimed at estimating transmission parameters of four H5 LPAIV (three field strains from French poultry and decoy ducks, and one clonal reverse-genetics strain derived from one of the former), using a SIR model to analyze data from experimental infections in SPF Muscovy ducks. The design was set up to accommodate rearing on wood shavings with a low density of 1.6 ducks/m(2): 10 inoculated ducks were housed together with 15 contact-exposed ducks. Infection was monitored by RNA detection on oropharyngeal and cloacal swabs using real-time RT-PCR with a cutoff corresponding to 2-7 EID50. Depending on the strain, the basic reproduction number (R0) varied from 5.5 to 42.7, confirming LPAIV could easily be transmitted to susceptible Muscovy ducks. The lowest R0 estimate was obtained for a H5N3 field strain, due to lower values of transmission rate and duration of infectious period, whereas reverse-genetics derived H5N1 strain had the highest R0. Frequency and intensity of clinical signs were also variable between strains, but apparently not associated with longer infectious periods. Further comparisons of quantitative transmission parameters may help to identify relevant viral genetic markers for early detection of potentially more virulent strains during surveillance of LPAIV., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The compression of a capsule between two plates is considered. The problem is solved numerically for a capsule made of an incompressible liquid drop surrounded by a thin elastic membrane which has a negligible bending stiffness. Numerical results are provided for three different mechanical laws of the membrane. By considering elastic moduli independent of the deformation, we show that the isotropic dilation plays the major role. In particular, an asymptotic behavior independent of the shear modulus is found for large deformations. For more complex models, the deformation limits beyond which the variation of elastic moduli starts to play a role are examined. The results indicate that the distinction between the different models requires a careful inspection of both small and large deformations. The theoretical predictions are compared with experimental results. For millimetric capsules with membranes made of covalently linked human serum albumin and alginate, the best agreement is obtained by considering that the elastic moduli are independent of the deformation and range from 0.1 to 4 N/m.

Virgin olive oil is made from diverse cultivars either mixed or single. Those ensure different tastes and typicity, and these may be also enhanced by the region of production of cultivars. The different olive oil labels correspond to their chemical composition and acidity. Labels also may correspond to a protected origin indication, and thus, such oils contain a given composition in cultivars. To verify the main cultivars used at the source of an olive oil sample, our method is based on DNA technology. DNA is present in all olive oil samples and even in refined oil, but the quantity may depend on the oil processing technology and oil conservation conditions. Thus, several supports were used to retain DNA checking different techniques (silica extraction, hydroxyapatite, magnetic beads, and spun column) to prepare DNA from variable amounts of oil. At this stage, it was usable for amplification through PCR technology and especially with the magnetic beads, and further purification processes were checked. Finally, the final method used magnetic beads. DNA is released from beads in a buffer. Once purified, we showed that it did not contain compounds inhibiting PCR amplification using SSR primers. Aliquot dilution fractions of this solution were successfully routinely used through PCR with different SSR primer sets. This enables confident detection of eventual alien alleles in oil samples. First applied to virgin oil samples of known composition, either single cultivars or mixtures of them, the method was verified working on commercial virgin oil samples using bottles bought in supermarkets. Last, we defined a protocol starting from 2 x 40 mL virgin olive oil, and DNA was prepared routinely in about 5 h. It was convenient to genotype together several loci per sample to check whether alleles were in accordance with those of expected cultivars. Thus, forensic applications of our method are expected. However, the method needs further improvement to work on all oil samples.