The feather epithelium contributes to the dissemination and ecology of clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in ducks.

Immature feathers are known replication sites for high pathogenicity avian influenza viruses (HPAIVs) in poultry. However, it is unclear whether feathers play an active role in viral transmission. This study aims to investigate the contribution of the feather epithelium to the dissemination of clade 2.3.4.4b goose/Guangdong/1996 lineage H5 HPAIVs in the environment, based on natural and experimental infections of domestic mule and Muscovy ducks. During the 2016-2022 outbreaks, H5 HPAIVs exhibited persistent and marked feather epitheliotropism in naturally infected commercial ducks. Infection of the feather epithelium resulted in epithelial necrosis and disruption, as well as the production and environmental shedding of infectious virions. Viral and feather antigens colocalized in dust samples obtained from poultry barns housing naturally infected birds. In summary, the feather epithelium contributes to viral replication, and it is a likely source of environmental infectious material. This underestimated excretion route could greatly impact the ecology of HPAIVs, facilitating airborne and preening-related infections within a flock, and promoting prolonged viral infectivity and long-distance viral transmission between poultry farms.

Persistence of low pathogenic avian influenza virus in artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate.

Avian influenza viruses (AIVs) can affect wildlife, poultry, and humans, so a One Health perspective is needed to optimize mitigation strategies. Migratory waterfowl globally spread AIVs over long distances. Therefore, the study of AIV persistence in waterfowl staging and breeding areas is key to understanding their transmission dynamics and optimizing management strategies. Here, we used artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate (day/night cycles of photosynthetic active radiation and temperature, low water velocity, and similar microbiome to lowland rivers and stagnant water bodies) and then manipulated temperature and sediment presence (i.e., 10-13 °C vs. 16-18 °C, and presence vs. absence of sediments). An H1N1 low pathogenic AIV (LPAIV) strain was spiked in the streams, and water and sediment samples were collected at different time points until 14 days post-spike to quantify viral RNA and detect infectious particles. Viral RNA was detected until the end of the experiment in both water and sediment samples. In water samples, we observed a significant combined effect of temperature and sediments in viral decay, with higher viral genome loads in colder streams without sediments. In sediment samples, we didn't observe any significant effect of temperature. In contrast to prior laboratory-controlled studies that detect longer persistence times, infectious H1N1 LPAIV was isolated in water samples till 2 days post-spike, and none beyond. Infectious H1N1 LPAIV wasn't isolated from any sediment sample. Our results suggest that slow flowing freshwater surface waters may provide conditions facilitating bird-to-bird transmission for a short period when water temperature are between 10 and 18 °C, though persistence for extended periods (e.g., weeks or months) may be less likely. We hypothesize that experiments simulating real environments, like the one described here, provide a more realistic approach for assessing environmental persistence of AIVs.

Corrigendum: Dual Host and Pathogen RNA-Seq Analysis Unravels Chicken Genes Potentially Involved in Resistance to Highly Pathogenic Avian Influenza Virus Infection.

[This corrects the article DOI: 10.3389/fimmu.2021.800188.].

Rapid SARS-CoV-2 Inactivation in a Simulated Hospital Room Using a Mobile and Autonomous Robot Emitting Ultraviolet-C Light.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has made mask-wearing, physical distancing, hygiene, and disinfection complementary measures to control virus transmission. Especially for health facilities, we evaluated the efficacy of an UV-C autonomous robot to inactivate SARS-CoV-2 desiccated on potentially contaminated surfaces. ASSUM (autonomous sanitary sterilization ultraviolet machine) robot was used in an experimental box simulating a hospital intensive care unit room. Desiccated SARS-CoV-2 samples were exposed to UV-C in 2 independent runs of 5, 12, and 20 minutes. Residual virus was eluted from surfaces and viral titration was carried out in Vero E6 cells. ASSUM inactivated SARS-CoV-2 by ≥ 99.91% to ≥ 99.99% titer reduction with 12 minutes or longer of UV-C exposure and onwards and a minimum distance of 100cm between the device and the SARS-CoV-2 desiccated samples. This study demonstrates that ASSUM UV-C device is able to inactivate SARS-CoV-2 within a few minutes.

A 10-Year Retrospective Study of Inclusion Body Hepatitis in Meat-Type Chickens in Spain (2011-2021).

A surge in fowl adenovirus (FAdV) causing inclusion body hepatitis (IBH) outbreaks has occurred in several countries in the last two decades. In Spain, a sharp increase in case numbers in broilers and broiler breeder pullets arose since 2011, which prompted the vaccination of breeders in some regions. Our retrospective study of IBH cases in Spain from 2011 to 2021 revealed that most cases were reported in broilers (92.21%) and were caused by serotypes FAdV-8b and -11, while cases in broiler breeder pullets were caused by serotypes FAdV-2, -11, and -8b. Vertical transmission was the main route of infection, although horizontal transmission likely happened in some broiler cases. Despite the inconsistent and heterogeneous use of vaccines among regions and over time, the number of cases mirrored the use of vaccines in the country. While IBH outbreaks were recorded year-long, significantly more cases occurred during the cooler and rainier months. The geographic distribution suggested a widespread incidence of IBH and revealed the importance of a highly integrated system. Our findings contribute to a better understanding of FAdV infection dynamics under field conditions and reiterate the importance of surveillance, serological monitoring of breeders, and vaccination of breeders against circulating serotypes to protect progenies.

Dual Host and Pathogen RNA-Seq Analysis Unravels Chicken Genes Potentially Involved in Resistance to Highly Pathogenic Avian Influenza Virus Infection.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe systemic disease and high mortality rates in chickens, leading to a huge economic impact in the poultry sector. However, some chickens are resistant to the disease. This study aimed at evaluating the mechanisms behind HPAIV disease resistance. Chickens of different breeds were challenged with H7N1 HPAIV or clade 2.3.4.4b H5N8 HPAIV, euthanized at 3 days post-inoculation (dpi), and classified as resistant or susceptible depending on the following criteria: chickens that presented i) clinical signs, ii) histopathological lesions, and iii) presence of HPAIV antigen in tissues were classified as susceptible, while chickens lacking all these criteria were classified as resistant. Once classified, we performed RNA-Seq from lung and spleen samples in order to compare the transcriptomic signatures between resistant and susceptible chickens. We identified minor transcriptomic changes in resistant chickens in contrast with huge alterations observed in susceptible chickens. Interestingly, six differentially expressed genes were downregulated in resistant birds and upregulated in susceptible birds. Some of these genes belong to the NF-kappa B and/or mitogen-activated protein kinase signaling pathways. Among these six genes, the serine protease-encoding gene PLAU was of particular interest, being the most significantly downregulated gene in resistant chickens. Expression levels of this protease were further validated by RT-qPCR in a larger number of experimentally infected chickens. Furthermore, HPAIV quasi-species populations were constructed using 3 dpi oral swabs. No substantial changes were found in the viral segments that interact with the innate immune response and with the host cell receptors, reinforcing the role of the immune system of the host in the clinical outcome. Altogether, our results suggest that an early inactivation of important host genes could prevent an exaggerated immune response and/or viral replication, conferring resistance to HPAIV in chickens.

Pathobiology of the highly pathogenic avian influenza viruses H7N1 and H5N8 in different chicken breeds and role of Mx 2032 G/A polymorphism in infection outcome.

Chickens are highly susceptible to highly pathogenic avian influenza viruses (HPAIVs). However, the severity of infection varies depending of the viral strain and the genetic background of the host. In this study, we evaluated the pathogenesis of two HPAIVs (H7N1 and H5N8) and assessed the susceptibility to the infection of local and commercial chicken breeds from Spain. Eight chicken breeds were intranasally inoculated with 105 ELD50 of A/Chicken/Italy/5093/1999 (H7N1) or A/Goose/Spain/IA17CR02699/2017 (H5N8 clade 2.3.4.4. B) and monitored during 10 days. Chickens were highly susceptible to both HPAIVs, but H7N1 was considerably more virulent than H5N8 as demonstrated by the highest mortality rates and shortest mean death times (MDT). Both HPAIVs produced severe necrosis and intense viral replication in the central nervous system, heart and pancreas; however, the lesions and replication in other tissues were virus-dependent. High levels of viral RNA were detected by the oral route with both viruses. In contrast, a low number of H5N8-inoculated chickens shed by the cloacal route, demonstrating a different pattern of viral shedding dependent of the HPAIV. We found a high variation in the susceptibility to HPAIVs between the different chicken breeds. The birds carrying the genotype AA and AG at position 2032 in chicken Mx gene presented a slightly higher, but not significant, percentage of survival and a statistically significant longer MDT than GG individuals. Our study demonstrated that the severity of HPAI infection is largely dependent of the viral isolate and host factors, underlining the complexity of HPAI infections.

Foraging at Solid Urban Waste Disposal Sites as Risk Factor for Cephalosporin and Colistin Resistant Escherichia coli Carriage in White Storks (Ciconia ciconia).

White stork (Ciconia ciconia) may act as a reservoir and vehicle of cephalosporin resistant (CR) Escherichia coli. Between 2011 and 2014, we sampled white storks from colonies exposed to different degrees of anthropic pressure across the major areas of natural distribution of white storks in Spain. Cloacal swab samples (n = 467) were obtained from individuals belonging to 12 different colonies from six different regions. Additionally, 70 samples were collected from recently deposited droppings at the base of nesting platforms. We phenotypically characterized E. coli isolates, confirmed presence of CR genes and classified plasmids. Risk factors for acquiring these genes were assessed. Overall, 8.8% (41 out of 467) storks carried CR E. coli in their cloaca and five (7.1%) were identified from recently deposited droppings; therefore, 46 isolates were further characterized. Of them, 20 contained bla CTX-M- 1, nine bla CMY- 2, six bla CTX-M- 14, four bla SHV- 12, three bla CTX-M- 15, two bla CTX-M- 32, one bla CTX-M- 1 together with bla CMY- 2, and one bla CTX-M- 1 together with bla SHV- 12. All were multidrug-resistant, and four harbored the plasmid-mediated colistin resistance mcr-1 gene. CR genes were associated with the presence of IncI1, IncFIB, and IncN replicon families. XbaI-macrorestriction analysis revealed a great diversity among most of the XbaI-PFGE types, but indistinguishable types were also seen with isolates obtained from different locations. Clonal complex 10 was the most common among CR E. coli and two bla CTX-M- 15 positive isolates were identified as B2-ST131. Carriage of CR E. coli was significantly higher in colonies located close to solid urban waste disposal sites in which foraging on human waste was more likely and in one case to cattle grazing. The co-occurrence of bla CMY- 2 and mcr-1 on plasmids of E. coli isolated from wild birds as early as 2011 is of note, as the earliest previous report of mcr-1 in wild birds is from 2016. Our study shows that foraging at landfills and in association with cattle grazing are important risk factors for the acquisition of CR E. coli in white storks.

Differential Viral-Host Immune Interactions Associated with Oseltamivir-Resistant H275Y and Wild-Type H1N1 A(pdm09) Influenza Virus Pathogenicity.

Oseltamivir is a common therapy against influenza A virus (IAV) infections. The acquisition of oseltamivir resistance (OR) mutations, such as H275Y, hampers viral fitness. However, OR H1N1 viruses have demonstrated the ability to spread throughout different populations. The objective of this work was to compare the fitness of two strains of OR (R6 and R7) containing the H275Y mutation, and a wild-type (F) pandemic influenza A (H1N1) 2009 (pdm09) virus both in vitro and in vivo in mice and to select one OR strain for a comparison with F in ferrets. R6 showed faster replication and pathogenicity than R7 in vitro and in mice. Subsequently, R6 was selected for the fitness comparison with the F strain in ferrets. Ferrets infected with the F virus showed more severe clinical signs, histopathological lung lesions, and viral quantification when compared to OR R6-infected animals. More importantly, differential viral kinetics correlated with differential pro-inflammatory host immune responses in the lungs of infected ferrets, where OR-infected animals developed a protective higher expression of type I IFN and Retinoid acid Inducible Gene I (RIG-I) genes early after infection, resulting in the development of milder disease. These results suggest the presence of early specific viral-host immune interactions relevant in the development of influenza-associated lung pathology.

Evaluation of dietary supplementation of a novel microbial muramidase on gastrointestinal functionality and growth performance in broiler chickens.

This study was conducted to assess the effect of dietary supplementation of Muramidase 007 to broiler chickens on gastrointestinal functionality, evaluating growth performance, apparent ileal digestibility, intestinal histomorphology, vitamin A in plasma and cecal microbiota. A total of 480 one-day male chicks (Ross 308) were distributed in 16 pens allocated in 2 experimental diets: the control diet (CTR) without feed enzymes, coccidiostat or growth promoters, and the experimental diet (MUR): CTR supplemented with 35,000 units (LSU(F))/kg of the Muramidase 007. Digesta and tissue samples were obtained on days 9 and 36 of the study. A lower feed conversion ratio was observed in the MUR treatment. Apparent ileal digestibility of DM, organic matter and energy were improved by Muramidase 007. It was also observed that MUR improved digestibility of total fatty acids, mono-unsaturated fatty acids and poly-unsaturated fatty acids, and content of vitamin A in plasma at day 9 (P < 0.05). Histomorphological analysis of jejunum samples revealed no differences in the villus height or crypt depth; but a higher number of goblet cells and intraepithelial lymphocytes at day 36 with MUR. No differences were observed in plate counts of enterobacteria or Lactobacillus along the gastrointestinal tract, neither on the cecal short-chain fatty acids. An statistical trend was observed for reduction of cecal clostridia at day 9 for MUR. Analysis of cecal microbiota structure by 16S rRNA gene sequencing revealed relevant changes correlated to age. At day 9, broilers receiving MUR showed decreased alpha diversity compared to CTR that was not detected at day 36. Changes in specific taxonomic groups with an increase in Lactobacillus genus were identified. In conclusion, evaluation of the variables in this study indicates that dietary Muramidase 007 contributes to improve feed conversation ratio and gastrointestinal function in broiler chickens. Effects could have been mediated by slight shifts observed in the intestinal microbiota. More studies are guaranteed to fully understand the mechanisms involved.

Pathological findings in genital organs of bulls naturally infected with Besnoitia besnoiti.

Bulls chronically affected by bovine besnoitiosis can suffer from sterility. There is limited information about the distribution of Besnoitia cysts and their associated lesions within the male genital organs. This work describes the gross and histological abnormalities in the genital organs of 6 bulls chronically infected with Besnoitia besnoiti, including both clinically (n = 4) and subclinically (n = 2) affected cases. Parasitic cysts were observed in the genital organs of all the clinically affected bulls. The tissue cysts were most commonly found within the pampiniform plexus (4/4), where they were often seen within venous vascular walls and associated with vasculitis, followed by epididymis (3/4), tunica albuginea (2/4), and penis (1/4). In decreasing order of their frequency, observed abnormalities included seminiferous tubule degeneration, testicular fibrosis, testicular necrosis, lack of/or diminished numbers of spermatozoa, testicular atrophy, and Leydig cell hyperplasia. Only one of the subclinically infected bulls had few Besnoitia cysts within the pampinoform plexus, which was associated to small areas of necrosis and mineralization in the ipsilateral testicle. Results indicate that Besnoitia cysts and genital abnormalities are frequent in bulls chronically affected by bovine besnoitiosis, while they are mild and scarce in subclinically affected ones. Moreover, present data show that Besnotia-associated testicular lesions can occur without the presence of cysts within the testicular parenchyma. B. besnoiti cysts seem to have a tropism for the vascular structures of the spermatic chord, which may cause testicular abnormalities via vascular damage, reduced blood flow, and/or impaired thermoregulation and subsequently lead to the observed testicular lesions.

Computed tomographic features of destructive granulomatous rhinitis with intracranial extension secondary to leishmaniasis in a cat.

A 5-year-old castrated male Domestic Shorthair cat presented for evaluation of chronic history of nasal discharge and nasal stridor. On computed tomography (CT), a destructive ill-defined mass of soft tissue attenuation was occupying the right nasal cavity and extending into the left nasal cavity, nasopharynx, and rostral cranial cavity. Histopathology of the rhinoscopically excised samples consisted with destructive granulomatous rhinitis secondary to Leishmania spp. Chronic granulomatous rhinitis with intracranial and nasopharyneal extension secondary to Leishmania spp. infection should be included as a differential diagnosis for a destructive nasal mass of soft tissue attenuation, especially in endemic regions for leishmaniasis.

Retrospective study on transmissible viral proventriculitis and chicken proventricular necrosis virus (CPNV) in the UK.

Chicken proventricular necrosis virus (CPNV) is a recently described birnavirus, which has been proposed to be the cause of transmissible viral proventriculitis (TVP). The understanding of the epidemiology of both the virus and the disease is very limited. A retrospective investigation on TVP and CPNV in broiler chicken submissions from the UK from between 1994 and 2015 was performed with the aims of assessing the longitudinal temporal evolution of TVP and CPNV, and to review the histological proventricular lesions in the studied chickens. Ninety-nine of the 135 included submissions (73.3%) fulfilled the TVP-diagnostic criteria, while the remaining 36 submissions (26.7%) displayed only lymphocytic proventriculitis (LP). The first detection of CPNV by PCR dated from 2009. Results showed a rise in the number of both TVP and positive CPNV RT-PCR submissions from 2009 with a peak in 2013, suggesting that they may be an emerging or re-emerging disease and pathogen, respectively. Twenty-two out of the 99 submissions displaying TVP lesions (22%) and four out of the 36 (11%) submissions with LP gave positive CPNV RT-PCR results, further supporting the association between CPNV and TVP and confirming that CPNV is present in a low proportion of proventriculi that do not fulfil the TVP-diagnostic criteria. In addition, intranuclear inclusion bodies were observed in 22 of the submissions with TVP. The vast majority of these cases (21 of 22, 96%) gave negative CPNV RT-PCR results, raising the question of whether a virus other than CPNV is responsible for some of these TVP-affected cases.RESEARCH HIGHLIGHTSTVP and CPNV have been present in British broilers since at least 1994 and 2009, respectively.TVP and CPNV seem to be an emerging and re-emerging disease and pathogen, respectively.CPNV was detected in proventriculi with both TVP and LP-lesions.Viruses other than CPNV may be responsible for some TVP-affected cases.

Myxomatosis and Rabbit Haemorrhagic Disease: A 30-Year Study of the Occurrence on Commercial Farms in Spain.

In this retrospective study, we describe the relative occurrence of clinical myxomatosis, and rabbit haemorrhagic disease (RHD), on 1714 commercial farms visited in Spain, between 1988 and 2018. We determined the annual prevalence based on 817 visits to 394 farms affected by myxomatosis. Myxomatosis was more prevalent from August to March, being lowest in June (3%) and highest in September (8.9%). With regard to RHD, we assessed 253 visits to 156 affected farms. We analyzed mean annual and monthly incidence. Two important RHD epidemics occurred; the first in 1988-1989 due to RHDV GI.1 (also known as RHDV), and the second from 2011 to 2013 due to RHDV GI.2 (RHDV2 or RHDVb). These epidemics occurred at times when effective vaccination had not been carried out. Relative monthly incidence in 2011-2018 was higher from April to August (p < 0.001). The results we obtained from 1404 necropsies on 102 farms did not clearly relate serosanguinous nasal discharge in rabbits with disease caused by GI.2 infection. We also assessed vaccination schedules used on 200 doe farms visited from the end of 2014 to 2018; 95.5% vaccinated against myxomatosis and 97.5% against RHD. Both diseases remain prevalent; however, effective vaccination has produced a steady decline in myxomatosis and RHDV GI.1 and GI.2 on-farm detection. The maintenance of high hygienic standards will be needed to continue and improve this control. However, further studies are required to investigate the causes of sustained virus presence and vaccine breaks.

Phylodynamic analyses of Brazilian antigenic variants of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is a very important pathogen to poultry production and it is classified into three main groups: classical virulent (cvIBDV), very virulent (vvIBDV) and antigenic variants (avIBDV). This last group is composed by five different genetic lineages (recently classified in genogroups G2, G4, G5, G6, and G7) distributed in specific regions around the world. Brazil is one of the biggest poultry producers in the world and the present study aimed to investigate the evolutionary history of avIBDVs of the genogroup G4 in Brazil. A total of 5331 IBDV positive bursa samples, from different Brazilian poultry flocks, were genotyped in a period of ten years (2005 to 2014) and 1888 (35.42%) were identified as local avIBDVs. The highly variable region of the viral protein 2 (hvvp2) gene of 28 avIBDVs was sequenced and used in phylogenetic analyses and evaluation of local amino acid signatures. In addition, all complete and partial IBDV vp2 gene sequences, with local and year of collection information available on GenBank, were retrieved. Phylogenetic analyses were carried out based on a maximum likelihood method for the classification of genogroups occurring in Brazil. Based on a Maximum Likelihood (ML) phylogenetic tree, all Brazilian avIBDVs grouped into the genogroup 4. Bayesian phylodynamics analysis demonstrated the ancestor virus of this group was probably introduced in South America in 1968 (1960 to 1974, 95% HPD) and in Brazil in 1974 (1968 to 1977, 95% HPD) and the most likely source was East Europe (Hungary or Poland). All Brazilian avIBDV sequences, as well as the other genogroup 4 sequences, showed a specific pattern of amino acid: S222, T272, P289, I290, and F296. This report brings new insights about the IBDV epidemiology in Brazil and South America.

Can genotype mismatch really affect the level of protection conferred by Newcastle disease vaccines against heterologous virulent strains?

Newcastle disease (ND), caused by virulent class II avian paramyxovirus 1 (Newcastle disease virus, NDV), occurs sporadically in poultry despite their having been immunized with commercial vaccines. These vaccines were all derived from NDV strains isolated around 70 years ago. Since then, class II NDV strains have evolved into 18 genotypes. Whether the vaccination failure results from genotype mismatches between the currently used vaccine strains and field-circulating velogenic strains or from an impaired immune response in the vaccination remains unclear. To test the first hypothesis, we performed a heterologous genotype II vaccine/genotype XI challenge in one-day old specific pathogen free (SPF) chicks and reproduced viral shedding. We then produced two attenuated strains of genotype II and XI by reverse genetics and used them to immunize two-week old SPF chickens that were subsequently challenged with velogenic strains of genotypes II, VII and XI. We found that both vaccines could induce antibodies with hemagglutination inhibition titers higher than 6.5 log2. Vaccination also completely prevented disease, viral shedding in swabs, and blocked viral replication in tissues from different genotypes in contrast to unvaccinated chickens that died shortly after challenge. Taken together, our results support the hypothesis that, in immunocompetent poultry, genotype mismatch is not the main reason for vaccination failure.

Detection of transmissible viral proventriculitis and chicken proventricular necrosis virus in the UK.

Increasing evidence suggests that a new birnavirus, named chicken proventricular necrosis virus (CPNV), is the aetiological agent of transmissible viral proventriculitis (TVP). The present work aimed to explore the possible presence of both TVP and CPNV in the UK. Forty-four chickens showing TVP-compatible gross lesions were classified into three groups based on the histological lesions: (i) TVP-affected chickens: lymphocytic infiltration and glandular necrosis (n = 15); (ii) lymphocytic proventriculitis (LP)-affected chickens: lymphocytic infiltration without necrosis (n = 18); and (iii) without proventriculitis (WP): no lymphocytic infiltration or necrosis (n = 11). Nine proventriculi (seven out of 15 corresponding to TVP, and two out of 11 corresponding to LP) were positive for CPNV by reverse transcriptase polymerase chain reaction (RT-PCR). These results support the previously suggested idea of CPNV as causative agent of TVP. Moreover, these data show that CPNV can also be detected in a number of cases with LP, which do not fulfil the histological TVP criteria. Phylogenetic analysis of partial sequences of gene VP1 showed that British CPNV sequences were closer to other European CPNV sequences and might constitute a different lineage from the American CPNV. TVP cases with negative CPNV PCR results may be due to chronic stages of the disease or to the reduced PCR sensitivity on formalin-fixed paraffin-embedded tissues. However, involvement of other agents in some of the cases cannot totally be ruled out. As far as the authors are aware, this is the first peer-reviewed report of TVP as well as of CPNV in the UK, and the first exploratory CPNV phylogenetic study.

Six-year surveillance of Newcastle disease virus in wild birds in north-eastern Spain (Catalonia).

Given that Newcastle disease (ND) is one of the major threats for the poultry industry, testing of Newcastle disease virus (NDV) has been carried out since 2010 in cases of mortality in wild birds (passive surveillance) in Catalonia. The objective is to provide an early warning system to prevent the infection of poultry. Since 2010, 35 episodes of mortality in wild birds were attributed to NDV infection. Throughout this period there was a progressive expansion of NDV to new areas, with an increase in the episodes of mortality, although it is not clear whether they were the result of the spread of the virus, or of the improvement of the surveillance. Phylogenetic analyses indicate that two distinct sublineages of NDV, 4a and 4b, were circulating in Catalonia. Both sublineages seem to be endemic in the wild bird population, affecting mainly Eurasian-collared doves, with a clear pattern in relation to its spatial distribution (coincident with the distribution of this species), and its temporal distribution (with the majority of cases between September and February). So far, endemicity in wild birds has not resulted in ND outbreaks in poultry. However, there are still many uncertainties about, for example, whether NDV may expand to new areas of Catalonia (with higher poultry density), or about the threat that the apparently more novel sublineage 4a may represent. Hence, efforts should be made so that measures to prevent infection of poultry farms (particularly in high-risk areas and periods) are encouraged, and surveillance is maintained.

Involvement of the different lung compartments in the pathogenesis of pH1N1 influenza virus infection in ferrets.

Severe cases after pH1N1 infection are consequence of interstitial pneumonia triggered by alveolar viral replication and an exacerbated host immune response, characterized by the up-regulation of pro-inflammatory cytokines and the influx of inflammatory leukocytes to the lungs. Different lung cell populations have been suggested as culprits in the unregulated innate immune responses observed in these cases. This study aims to clarify this question by studying the different induction of innate immune molecules by the distinct lung anatomic compartments (vascular, alveolar and bronchiolar) of ferrets intratracheally infected with a human pH1N1 viral isolate, by means of laser microdissection techniques. The obtained results were then analysed in relation to viral quantification in the different anatomic areas and the histopathological lesions observed. More severe lung lesions were observed at 24 h post infection (hpi) correlating with viral antigen detection in bronchiolar and alveolar epithelial cells. However, high levels of viral RNA were detected in all anatomic compartments throughout infection. Bronchiolar areas were the first source of IFN-α and most pro-inflammatory cytokines, through the activation of RIG-I. In contrast, vascular areas contributed with the highest induction of CCL2 and other pro-inflammatory cytokines, through the activation of TLR3.

Effect of different vaccination strategies on IBV QX population dynamics and clinical outbreaks.

The extreme variability and rapid evolution of Infectious bronchitis virus (IBV) has always represented the key challenge for its control because of the limited cross-protection among different strains. Several experimental trials have proven a broadening of the protection spectrum when animals are vaccinated with multiple genotypes. Nevertheless, the conditions of vaccine administration in field are so different that the generalization of experimental results is, at least, questionable. In the present study a large scale epidemiological-phylodynamic approach was used to reconstruct the demographic history of the major field genotype (i.e. the QX one) circulating in Italy and Spain. These two countries were selected because, even if they share a comparable epidemiological scenario, the implemented vaccination protocols did not vary in Spain while changed dramatically in Italy over the time period considered. One hundred and ninety-five Italian and 98 Spanish non-recombinant sequences of the hyper-variable region of the S1 gene obtained between 2012 and 2016 were analyzed using a serial coalescent-based approach to reconstruct viral population history over time. While the IBV QX population dynamics remained constant in Spain, a much more complex pattern was evidenced in Italy; both in terms of viral population size and clinical outbreak frequency. Remarkably, a strong association with changes in vaccination strategies was recognized. This allowed demonstrating, by accomplishing all Hill's criteria for causation, the cause-effect relationship between the vaccine administration/withdrawal and the variation in viral population dynamics and, above all, IBV related outbreaks. Thus, a robust confirmation about the efficacy of IBV vaccination in field conditions was provided. Additionally, the history herein reported testifies the primary importance of rigorously planning not only the intervention strategies but also their monitoring and evaluation.