miRNA repertoire and host immune factor regulation upon avian coronavirus infection in eggs.

Avian infectious bronchitis virus (IBV) is a coronavirus with great economic impact on the poultry industry, causing an acute and highly contagious disease in chickens that primarily affects the respiratory and reproductive systems. The cellular regulation of IBV pathogenesis and the host immune responses involved remain to be fully elucidated. MicroRNAs (miRNAs) have emerged as a class of crucial regulators of numerous cellular processes, including responses to viral infections. Here, we employed a high-throughput sequencing approach to analyze the miRNA composition of the spleen and the lungs of chicken embryos upon IBV infection. Compared to healthy chicken embryos, 13 and six miRNAs were upregulated in the spleen and the lungs, respectively, all predicted to influence viral transcription, cytokine production, and lymphocyte functioning. Subsequent downregulation of NFATC3, NFAT5, SPPL3, and TGFB2 genes in particular was observed only in the spleen, demonstrating the biological functionality of the miRNAs in this lymphoid organ. This is the first study that describes the modulation of miRNAs and the related host immune factors by IBV in chicken embryos. Our data provide novel insight into complex virus-host interactions and specifically highlight components that could affect the host's immune response to IBV infection.

Deletion of accessory genes 3a, 3b, 5a or 5b from avian coronavirus infectious bronchitis virus induces an attenuated phenotype both in vitro and in vivo.

Avian coronavirus infectious bronchitis virus (IBV) infects domestic fowl, resulting in respiratory disease and causing serious losses in unprotected birds. Its control is mainly achieved by using live attenuated vaccines. Here we explored the possibilities for rationally attenuating IBV to improve our knowledge regarding the function of IBV accessory proteins and for the development of next-generation vaccines with the recently established reverse genetic system for IBV H52 based on targeted RNA recombination and selection of recombinant viruses in embryonated eggs. To this aim, we selectively removed accessory genes 3a, 3b, 5a and 5b individually, and rescued the resulting recombinant (r) rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b. In vitro inoculation of chicken embryo kidney cells with recombinant and wild-type viruses demonstrated that the accessory protein 5b is involved in the delayed activation of the interferon response of the host after IBV infection. Embryo mortality after the inoculation of 8-day-old embryonated chicken eggs with recombinant and wild-type viruses showed that rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b had an attenuated phenotype in ovo, with reduced titres at 6 h p.i. and 12 h p.i. for all viruses, while growing to the same titre as wild-type rIBV at 48 h p.i. When administered to 1-day-old chickens, rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b showed reduced ciliostasis in comparison to the wild-type viruses. In conclusion, individual deletion of accessory genes in IBV H52 resulted in mutant viruses with an attenuated phenotype.

A comparison of AMPV subtypes A and B full genomes, gene transcripts and proteins led to reverse-genetics systems rescuing both subtypes.

Avian metapneumovirus (AMPV) infection of poultry causes serious disease in most countries and subtype A reverse-genetic (RG) systems have allowed a generation of viruses of known sequence, and proved useful in developments towards better control by live vaccines. While subtype B viruses are more prevalent, bacterial cloning issues made subtype B RG systems difficult to establish. A molecular comparison of subtype A and B viruses was undertaken to assess whether subtype A RG components could be partially or fully substituted. AMPV subtype A and B gene-end sequences leading to polyadenylation are, to our knowledge, reported for the first time, as well as several leader and trailer sequences. After comparing these alongside previously reported gene starts and protein sequences, it was concluded that subtype B genome copies would be most likely rescued by a subtype A support system, and this assertion was supported when individual subtype A components were successfully substituted. Application of an advanced cloning plasmid permitted eventual completion of a fully subtype B RG system, and proved that all subtype-specific components could be freely exchanged between A and B systems.

Supporting Measures to Improve Biosecurity within Italian Poultry Production.

This paper describes the selection and validation of supporting measures (SMs) aimed at enhancing biosecurity compliance within Italian poultry farms. A tailored methodology, based on a stakeholders' survey involving farmers and advisors, included a virtual farm tour, group discussion, and farmer coaching. Virtual farm tours and group discussions were delivered during two meetings targeting meat and egg production stakeholders, separately. Coaching was validated in 26 pilot farms (PFs) by assessing farmers' attitudes towards change (i.e., ADKAR®) and farms' biosecurity score (i.e., Biocheck.UgentTM) before and after a minimum six-month period. A total of 20 out of 26 farmers agreed to implement at least one action plan (AP). Full implementation of the agreed APs was observed in ten farms, while others only partially implemented (n = 7) or did not implement (n = 3) the improvement. Most APs focused on enhancing house hygiene locks (n = 7), followed by bacterial auto-control after cleaning and disinfection (n = 4). Scoring tools indicated minimal or no variations in farmers' attitudes towards change and farm biosecurity. Virtual farm tours and group discussions were found to be effective in fostering interaction and facilitating the exchange of experiences and knowledge among farmers and stakeholders of poultry production. Coaching indicated that farmers might prefer implementing minor changes possibly influenced by time and cost constraints associated with structural interventions. These limitations could have also impacted the scores of the farmer/farm. The findings of this study provide a foundation for further application of SMs to improve biosecurity in Italian poultry farms.

Biofilm Formation Ability of ESBL/pAmpC-Producing Escherichia coli Isolated from the Broiler Production Pyramid.

Escherichia coli able to produce extended spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (pAmpCs) represents a serious threat to public health, since these genes confer resistance to critically important antimicrobials (i.e., third generation cephalosporins) and can be transferred to non-resistant bacteria via plasmids. E. coli are known to be able to form a biofilm, which represents a favorable environment for the exchange of resistance determinants. Here, we assessed the ability of 102 ESBL/pAmpC-producing E. coli isolated from the broiler production pyramid to form a biofilm and to identify genetic factors involved in biofilm formation. All but one of the ESBL/pAmpC-producing E. coli were able to form a biofilm, and this represents a great concern to public health. E. coli belonging to phylogroups D, E, and F, as well as strains harboring the blaCTX-M-type gene, seem to be associated with an increased biofilm capability (p < 0.05). Furthermore, virulence genes involved in adherence and invasion (i.e., csgBAC, csgDEFG, matABCDEF, and sfaX) seem to enhance biofilm formation in E. coli. Efforts should be made to reduce the presence of ESBL/pAmpC- and biofilm-producing E. coli in the broiler production pyramid and, therefore, the risk of dissemination of resistant bacteria and genes.

Association between ability to form biofilm and virulence factors of poultry extra-intestinal Campylobacter jejuni and Campylobacter coli.

Campylobacter species are known to be able to produce biofilm, which represents an ideal protective environment for the maintenance of such fragile bacteria. Since the genetic mechanisms promoting biofilm formation are still poorly understood, in this study we assessed the ability of C. jejuni (n = 7) and C. coli (n = 3) strains isolated from diseased poultry, and previously characterized by whole genome sequencing, to form biofilm. The in vitro analyses were carried out by using a microtiter based protocol including biofilm culturing and fixation, staining with crystal violet, and measurement of the optical density (OD570). The ability to form biofilm was categorized into four classes (no, weak, moderate, and strong producers). Potential correlations between OD570 and the presence/absence of virulence determinants were examined. The C. jejuni were classified as no (n = 3), weak (n = 2), and moderate (n = 2) biofilm producers; however, all possessed genes involved in chemotaxis, adhesion, and invasion to the host cells. No genes present exclusively in biofilm producers or in non-biofilm producers were identified. All C. coli were classified as weak producers and showed a similar set of virulence genes between each other. A trend of increased mean OD570 was observed in the presence of flaA and maf7 genes. No association between biofilm production classes and the explanatory variables considered was observed. The results of this study suggest that further investigations are needed to better identify and characterize the genetic determinants involved in extra-intestinal Campylobacter biofilm formation.

Stakeholders' Perceptions of Biosecurity Implementation in Italian Poultry Farms.

The level of implementation of biosecurity measures (BMs), the reasons for not implementing BMs and the effectiveness of BMs were assessed according to the perceptions of stakeholders (i.e., farmers and advisors) in Italian poultry farms. For this purpose, data were collected using a questionnaire administered to advisors (n = 37) and farmers (n = 30) of conventional broiler (n = 13) and layer (n = 13), free-range broiler (n = 8) and layer (n = 10), turkey (n = 13), duck (n = 3) and breeder (n = 7) farms between April and September 2021. The frequency of the implementation of BMs was 66.97% and 81.14% according to the answers provided by the advisors and farmers, respectively, with the breeder sector showing the highest level of implementation (85.71%). "Not knowing advantages" (21.49% for advisors) and "other/specific reasons" (21.49% for advisors and 38.32% for farmers) were the most common answers regarding the lack of implementation of BMs for all poultry sectors. Only 31.09% of farmers acknowledged the effectiveness of not-implemented BMs in contrast to 61.02% of advisors, with the layers' stakeholders being the most aware. The findings of this study may be useful for identifying failures in biosecurity and failures to develop intervention strategies to fulfil the biosecurity gaps still present in Italian poultry farms.

SARS-CoV-2 and Companion Animals: Sources of Information and Communication Campaign during the COVID-19 Pandemic in Italy.

This study analyzed data on the sources and the level of Italians' awareness on the risk of infection by SARS-CoV-2 at the human-animal interface. Data were collected through a survey-type investigation on a representative sample of the Italian population. Forty-five percent of the interviewees were aware that companion animals could be infected by SARS-CoV-2. However, 29.8% were familiar with preventive measures to adopt to avoid viral transmission between infected humans and companion animals, and only 20.7% knew which companion animals could be at risk of infection. Higher awareness regarding the risk of SARS-CoV-2 transmission between animals and humans (51.7%) and the measures to prevent it (33.3%) was detected among companion animals' owners. Notably, 40.4% of interviewees were not informed at all. Television broadcasts (26.4%) represented the main source of information, while only 3.5% of the interviewees relied on veterinarians, of which 31.9% considered this source of information as the most trustworthy. Overall, 72.4% of Italians recognized that the communication campaign on COVID-19 and companion animals was inadequate. This survey highlights the need for increasing the public awareness of the risk of companion animals being infected with SARS-CoV-2 and the involvement of professionals in the public communication on zoonoses.

A novel array of real-time RT-PCR assays for the rapid pathotyping of type I avian paramyxovirus (APMV-1).

Newcastle disease (ND) caused by virulent avian paramyxovirus type I (APMV-1) is a WOAH and EU listed disease affecting poultry worldwide. ND exhibits different clinical manifestations that may either be neurological, respiratory and/or gastrointestinal, accompanied by high mortality. In contrast, mild or subclinical forms are generally caused by lentogenic APMV-1 and are not subject to notification. The rapid discrimination of virulent and avirulent viruses is paramount to limit the spread of virulent APMV-1. The appropriateness of molecular methods for APMV-1 pathotyping is often hampered by the high genetic variability of these viruses that affects sensitivity and inclusivity. This work presents a new array of real-time RT-PCR (RT-qPCR) assays that enable the identification of virulent and avirulent viruses in dual mode, i.e., through pathotype-specific probes and subsequent Sanger sequencing of the amplification product. Validation was performed according to the WOAH recommendations. Performance indicators on sensitivity, specificity, repeatability and reproducibility yielded favourable results. Reproducibility highlighted the need for assays optimization whenever major changes are made to the procedure. Overall, the new RT-qPCRs showed its ability to detect and pathotype all tested APMV-1 genotypes and its suitability for routine use in clinical samples.

Swine Norovirus: Past, Present, and Future.

Norovirus, an ssRNA + virus of the family Caliciviridae, is a leading disease burden in humans worldwide, causing an estimated 600 million cases of acute gastroenteritis every year. Since the discovery of norovirus in the faeces of swine in Japan in the 1990s, swine norovirus has been reported in several countries on several continents. The identification of the human-associated GII.4 genotype in swine has raised questions about this animal species as a reservoir of norovirus with zoonotic potential, even if species-specific P-types are usually detected in swine. This review summarises the available data regarding the geographic distribution of norovirus in swine, the years of detection, the genotype characterisation, and the prevalence in specific production groups. Furthermore, we discuss the major bottlenecks for the detection and characterisation of swine noroviruses.

Assessing Biosecurity Compliance in Poultry Farms: A Survey in a Densely Populated Poultry Area in North East Italy.

Biosecurity in poultry farms represents the first line of defense against the entry and spread of pathogens that may have animal health, food safety, and economic consequences. The aim of this study was to assess biosecurity compliance in poultry farms located in a densely populated poultry area in North East Italy. A total of 259 poultry farms (i.e., broilers, turkeys, and layers) were surveyed between 2018 and 2019 using standardized checklists, and differences in biosecurity compliance between the poultry sectors and years (only for turkey farms) were tested for significance. Among the three sectors, turkey farms showed the highest compliance. Farm hygiene, infrastructure condition, cleaning and disinfection tools, and procedures were the biosecurity measures most complied with. Some deficiencies were observed in the cleanliness of the farm hygiene lock in broiler farms, as well as the presence of the house hygiene lock in broiler and layer farms and an adequate coverage of built-up litter in turkey and broiler farms. In conclusion, this study highlighted a generally high level of biosecurity in the visited poultry farms (probably due to the stringent national regulation and the integration of the poultry industry) and identified some measures that still need to be improved.

Amoxicillin and thiamphenicol treatments may influence the co-selection of resistance genes in the chicken gut microbiota.

The aim of this study was to assess the dynamics of microbial communities and antimicrobial resistance genes (ARGs) in the chicken gut following amoxicillin and thiamphenicol treatments and potential co-selection of ARGs. To this purpose, the microbial community composition, using 16S rRNA NGS, and the abundance of ARGs conferring resistance to ß-lactams and phenicols, using qPCRs, were determined. Results revealed that the administered antimicrobials did not significantly reduce the gut microbiota diversity, but changed its composition, with taxa (e.g. Gallibacterium and Megamonas) being enriched after treatment and replacing other bacteria (e.g. Streptococcus and Bifidobacterium). Positive correlations were found between ARGs (e.g. cmlA, blaCMY-2, and blaSHV) and the relative abundance of specific taxa (e.g. Lactobacillus and Subdoligranulum). The selective pressure exerted by both amoxicillin and thiamphenicol resulted in an increased abundance of ARGs conferring resistance to ß-lactams (e.g. blaTEM-1, blaSHV, and blaCTX-M1-like) and phenicols (e.g. floR and cmlA). These findings, together with the co-occurrence of genes conferring resistance to the two antimicrobial classes (e.g. blaTEM-1 and cmlA), suggest a possible interaction among antimicrobials on resistance emergence, possibly due to the presence of mobile genetic elements (MGEs) carrying multiple resistance determinants.

Beehive products as bioindicators of antimicrobial resistance contamination in the environment.

The use of antimicrobials in agricultural, veterinary and medical practice exerts selective pressure on environmental microbiota, promoting the emergence and spread of antimicrobial resistance (AMR), a global concern for the One Health Initiative Task Force (OHITF). Honeybees have been studied as bioindicators of AMR in the environment, but little is known about beehive products like honey and pollen. The aim of this study was to assess the prevalence of AMR genes (ARGs) in beehive products and investigated their origins. Specifically, possible associations between ARGs, microbiota and other characteristics of different honey and pollen samples, including country of origin, flower type, type of commercial distribution and environmental factors, such as land use, weather and composition of the environment surrounding the beehives were investigated. We found that beehive products harboured ARGs conferring resistance to ß-lactams, macrolides, (fluoro)quinolones and polymyxins. Most samples possessed resistance to multiple antimicrobial classes, with honey and pollen showing similar ARG profiles. Even if Lactobacillus and Acinetobacter genera were common in the microbial communities of both honey and pollen, Bacillus, Clostridium, and Bombella defined honey microbiota, while Pseudomonas and Vibrio were enriched in pollen. ErmB and blaTEM-1 co-occurred with Lactobacillus and Fructobacillus, while positive associations between ß-lactams and macrolides and anthropogenic environments (i.e. industrial and commercial areas and non-irrigated arable lands) were found. Altogether, our findings suggest that ARGs in honey and pollen might originate from the honeybee foraging environment, and that the beehive products can be used as bioindicators of the AMR environmental contamination.

Optimization of five qPCR protocols toward the detection and the quantification of antimicrobial resistance genes in environmental samples.

Here, we describe the optimization and validation of five quantitative PCR (qPCR) assays by employing the SYBRGreen chemistry paired with melting curve analysis to detect and quantify clinically relevant antimicrobial resistance genes (ARGs) (i.e. ermB, blaCTXM1-like, blaCMY-2, qnrA and qnrS) from environmental samples (i.e. soil and manure). These five protocols accurately detected and quantified the aforementioned ARGs in complex environmental matrices and represent useful tools for both diagnostic and monitoring activities of resistant bacteria and ARGs into the environment.

Occurrence of Colibacillosis in Broilers and Its Relationship With Avian Pathogenic Escherichia coli (APEC) Population Structure and Molecular Characteristics.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the disease with the highest economic loss for the broiler industry. However, studies focusing on the prevalence and population structure of APEC in the broiler production pyramid are scarce. Here, we used genotyping and serotyping data to elucidate the APEC population structure and its changes in different broiler production stages along with whole-genome sequencing (WGS) in a subset of APEC isolates to determine transmission patterns amongst dominant APEC sequence types (STs) and characterize them in detail. Comparison of genotypes encountered in both APEC and avian fecal E. coli (AFEC) provided further insights. Overall, APEC-related mortality, as the proportion of the total sampled mortality in the broiler production, was high (35%), while phylogroup C and serogroup O78 were predominant amongst APEC isolates. We found a low (34.0%) and high (53.3%) incidence of colibacillosis in chicks and end-cycle broilers, respectively, which may be related to a shift in APEC genotypes, suggesting a trend from commensalism to pathogenicity across different broiler production stages. Despite considerable APEC genotypic diversity, there was substantial genotype overlap (40.9%, overall) over the production stages and convergence of STs to the four clusters. Within these clusters, WGS data provided evidence of clonal transmission events and revealed an enriched virulence and resistance APEC repertoire. More specifically, sequenced APEC were assigned to defined pathotypes based on their virulence gene content while the majority (86%) was genotypically multi-drug resistant. Interestingly, WGS-based phylogeny showed that a subset of APEC, which are cephalosporin-resistant, may originate directly from cephalosporin-resistant AFEC. Finally, exploration of the APEC plasmidome indicated that the small fraction of the APEC virulome carried by IncF plasmids is pivotal for the manifestation of the APEC pathotype; thus, plasmid exchange can promote pathogenicity in strains that are at the edge of the commensal and pathogenic states.

Genomic analysis of extra-intestinal Campylobacter jejuni and Campylobacter coli isolated from commercial chickens.

Campylobacter jejuni and Campylobacter coli have commonly been considered harmless commensal inhabitants of the chicken gut; however, these Campylobacter spp. are known to be able to multiply in the gut and invade other tissues, negatively affecting host health and performance. In this study, fourteen Campylobacter spp. were isolated from chickens showing foci of necrosis on the liver surface resembling lesions observed in cases of avian vibrionic hepatitis/spotty liver disease. The whole genome sequences of the fourteen isolates were analysed and their virulomes compared to those of Campylobacter reference sequences, aiming to investigate the possible association between virulence genes and the observed pathological lesions. Nine C. jejuni and five C. coli were studied. These Campylobacter shared twelve virulence factors with other isolates originated from chicken livers and hosted a higher number of virulence-associated genes in comparison to the reference genomes, including genes encoding for factors involved in adherence to and invasion of the intestinal epithelial cells. Our findings seem to point out that these twelve common virulence-associated genes, together with the presence of a high number of virulence factors involved in adherence, invasion and motility, might be responsible for the extra-intestinal spread of our isolates and the colonization of parenchymatous tissues, possibly causing the pathological lesions observed.

Microbial community composition and antimicrobial resistance in agricultural soils fertilized with livestock manure from conventional farming in Northern Italy.

Antimicrobials are commonly used in conventional livestock production and manure is widely applied to agricultural lands as fertilizer. This practice raises questions regarding the effects of fertilization on (i) soil microbiota composition and (ii) spread of antimicrobials and antimicrobial resistance (AMR) in the environment. This study was conducted in a high-density farming area of Northern Italy and aimed at assessing the impact of (dairy cattle, chickens and swine) manure application on soil microbiome, antimicrobial concentrations and antimicrobial resistance gene (ARG) abundance. We found the microbial community composition in manure to be different and less diverse than in soil, with manure application altering only marginally the soil microbiome. Exceptions were the phyla Firmicutes, Tenericutes and Cloacimonetes, which significantly enriched in fertilized soil. Of the antimicrobials investigated, only flumequine concentrations increased after manure application, albeit non-significantly. ARGs were more abundant in manure, with ermA, ermB, blaOXA-1 and oqxA being significantly enriched in fertilized soil. Positive correlations between oqxA and qnrS abundances and flumequine concentrations were observed, together with the co-occurrence of some ARGs and microbial taxa (e.g. oqxA correlated with Acidobacteria and Gemmatimonadetes). This study showed that manure application has little effect on soil microbiome but may contribute to the dissemination of specific ARGs into the environment. Moreover, flumequine residues seem to enhance the emergence of oqxA and qnrS in soil.

Infectious bronchitis virus Mass-type (GI-1) and QX-like (GI-19) genotyping and vaccine differentiation using SYBR green RT-qPCR paired with melting curve analysis.

Infectious Bronchitis Virus (IBV) is a highly contagious virus of chicken, causing huge economic losses in the poultry industry. Many genotypes circulate in a given area, and optimal protection relies on vaccination with live attenuated vaccines of the same genotype. As these live vaccines are derived from field viruses and circulate, understanding the prevalence of different IBV genotypes in any area is complex. In a recent study, the genome comparison of an IBV QX vaccine and its progenitor field strain led to the identification of vaccine markers. Here we developed a simplex SYBRgreen RT-qPCR assay for differentiation between QX-like field and vaccine strains and a multiplex SYBRgreen RT-qPCR assay for IBV genotyping with melting curve analysis, as each virus produced distinct and reliable melting peaks. Both the simplex and the multiplex assays showed excellent efficiency, sensitivity and specificity representing a low cost diagnostic tool for IBV genotyping and vaccine differentiation.

Identification of two divergent swine Noroviruses detected at the slaughterhouse in North East Italy.

Norovirus (NoV) has emerged as one of the major causative agents of non-bacterial, food- and water-borne gastroenteritis in humans, with the main genogroup involved in human outbreaks (GII), which has been detected worldwide in different animal species including swine. A four-month investigation at the slaughterhouse aiming to examine the presence of NoV in the swine in North-Eastern Italy, enabled the detection of two divergent Noroviruses (NoVs) (GII.P11) in two swine farms. This represents the first study in the swine population of North-Eastern Italy, which has paved the way for future integrated virological and epidemiological investigations on swine NoVs.

Detection of avian influenza virus: a comparative study of the in silico and in vitro performances of current RT-qPCR assays.

Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; hence, an accurate and rapid AIV diagnosis is essential. To date AIV detection relies on molecular methods, mainly RT-qPCR directed against AIV M gene segment. The evolution of AIV represents a relevant issue in diagnostic RT-qPCR due to possible mispriming and/or probe-binding failures resulting in false negative results. Consequently, RT-qPCR for AIV detection should be periodically re-assessed both in silico and in vitro. To this end, a specific workflow was developed to evaluate in silico the complementarity of primers and probes of four published RT-qPCR protocols to their target regions. The four assays and one commercially available kit for AIV detection were evaluated both for their analytical sensitivity using eight different viral dilution panels and for their diagnostic performances against clinical specimens of known infectious status. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances. This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions.