Publications by authors named "Andrea Laconi"

11 Publications

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

Sci Total Environ 2021 Mar 6;760:143404. Epub 2020 Nov 6.

Department of Comparative Biomedicine and Food Science, University of Padua, Viale dell'Università 16, 35020 Legnaro, Padua, Italy. Electronic address:

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, bla 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.
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http://dx.doi.org/10.1016/j.scitotenv.2020.143404DOI Listing
March 2021

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

Sci Rep 2020 05 21;10(1):8441. Epub 2020 May 21.

Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università, 10, Legnaro, Padova, 35020, Italy.

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.
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http://dx.doi.org/10.1038/s41598-020-64003-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242438PMC
May 2020

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

Porcine Health Manag 2020 16;6. Epub 2020 Apr 16.

3Diagnostic Virology Laboratory, Department of Animal Health, Istituto Zooprofilattico Sperimentale Delle Venezie, Legnaro, Padua, 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.
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http://dx.doi.org/10.1186/s40813-020-00147-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160966PMC
April 2020

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

Arch Virol 2020 Apr 6;165(4):835-843. Epub 2020 Feb 6.

Faculty of Veterinary Medicine, Department Biomolecular Health Sciences, Division Pathology, Utrecht University, Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1007/s00705-020-04527-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086581PMC
April 2020

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.

J Virol Methods 2020 01 8;275:113771. Epub 2019 Nov 8.

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

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.
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http://dx.doi.org/10.1016/j.jviromet.2019.113771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113787PMC
January 2020

Two similar commercial live attenuated AMPV vaccines prepared by random passage of the identical field isolate, have unrelated sequences.

Vaccine 2019 05 16;37(21):2765-2767. Epub 2019 Apr 16.

Department of Infection Biology, University of Liverpool, Leahurst Campus, Neston, Cheshire CH64 7TE, United Kingdom.

Since late '80 s Avian metapneumovirus subtype A causes sufficient disease in Europe for commercial companies to have started developing live attenuated vaccines. Here, two of those vaccines were fully consensus sequenced alongside their progenitor field strain (#8544). Sequences comparison shows that the attenuation of field strain #8544 was associated with no common substitutions between the two derived vaccines. This finding suggests that the attenuation of field viruses via serial passage on cell cultures or tissues is the result of a random process, rather than a mechanism aiming to achieve a specific sequence. Furthermore, field vaccination strategies would greatly benefit by the unambiguous vaccine markers identified in this study, enabling a prompt and confident vaccines detection.
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http://dx.doi.org/10.1016/j.vaccine.2019.04.036DOI Listing
May 2019

Molecular characterization of whole genome sequence of infectious bronchitis virus 624I genotype confirms the close relationship with Q1 genotype.

Transbound Emerg Dis 2019 Jan 20;66(1):207-216. Epub 2018 Sep 20.

Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell'Emilia, BO, Italy.

Infectious Bronchitis virus (IBV) genotype Q1 was detected for the first time in China in 1996, and then spread worldwide. The first report of Q1 genotype in Italy occurred in 2011 and a deep molecular investigation of a Q1 isolated in Italy in 2013 has led to speculation regarding the origin of this genotype. Phylogenetic analysis of the S1 sequence of a Q1 Italian strain revealed a close relationship with sequences of the 624I strains circulating in Italy in the early 1990s and this led to the idea that 624I was an ancestor of the Q1 genotype. Despite the fact that most heterogeneity of IBVs occurs in the S1 gene, the sequence analysis of this gene alone was not sufficient to confirm or deny this hypothesis. In the present study, an Italian 624I (gammaCoV/AvCov/Ck/Italy/IP14425/96) was fully sequenced for the first time and compared to all available complete Q1 genome sequences. This analysis confirmed the genetic correlation between GammaCoV/AvCov/Ck/Italy/IP14425/96 and Q1 strains, suggesting a common origin between 624I and Q1 genotypes.
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http://dx.doi.org/10.1111/tbed.13000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168539PMC
January 2019

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.

J Gen Virol 2018 Aug 1. Epub 2018 Aug 1.

1​Faculty of Veterinary Medicine, Department Pathobiology, Pathology Division, Utrecht University, Utrecht, 3584CL, The Netherlands.

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.
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http://dx.doi.org/10.1099/jgv.0.001130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079694PMC
August 2018

Identification of IBV QX vaccine markers : Should vaccine acceptance by authorities require similar identifications for all live IBV vaccines?

Vaccine 2017 10 13;35(42):5531-5534. Epub 2017 Sep 13.

Department of Infection Biology, University of Liverpool, Leahurst Campus, Neston, Cheshire CH64 7TE, United Kingdom. Electronic address:

IBV genotype QX causes sufficient disease in Europe for several commercial companies to have started developing live attenuated vaccines. Here, one of those vaccines (L1148) was fully consensus sequenced alongside its progenitor field strain (1148-A) to determine vaccine markers, thereby enabling detection on farms. Twenty-eight single nucleotide substitutions were associated with the 1148-A attenuation, of which any combination can identify vaccine L1148 in the field. Sixteen substitutions resulted in amino acid coding changes of which half were in spike. One change in the 1b gene altered the normally highly conserved final 5 nucleotides of the transcription regulatory sequence of the S gene, common to all IBV QX genes. No mutations can currently be associated with the attenuation process. Field vaccination strategies would greatly benefit by such comparative sequence data being mandatorily submitted to regulators prior to vaccine release following a successful registration process.
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http://dx.doi.org/10.1016/j.vaccine.2017.06.021DOI Listing
October 2017

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

J Gen Virol 2016 Jun 9;97(6):1324-1332. Epub 2016 Mar 9.

Department of Infection Biology, University of Liverpool, Leahurst Campus, Neston, Cheshire, CH64 7TE, UK.

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.
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http://dx.doi.org/10.1099/jgv.0.000450DOI Listing
June 2016

Molecular investigation of a full-length genome of a Q1-like IBV strain isolated in Italy in 2013.

Virus Res 2015 Dec 17;210:77-80. Epub 2015 Jul 17.

Department of Animal Medicine, Production and Health, University of Padua, Viale dell'università, 16, 35020, Legnaro (PD), Italy.

Since 1996 a new Infectious Bronchitis virus (IBV) genotype, referred to as Q1, circulated in China and was reported for the first time in Italy in 2011, associated with an increase of mortality, kidney lesions and proventriculitis. During northern Italian outbreak of respiratory disease in a broiler flock in 2013, an IBV strain was detected by RT-PCR and characterized as Q1-like based on partial S1 sequence. The virus was isolated and named γCoV/Ck/Italy/I2022/13. All coding regions of the isolate were sequenced and compared with 130 complete genome sequences of IBV and TCoV, downloaded from ViPR. This showed the highest identity with a Chinese strain CK/CH/LDL/97I (p-distance=0.044). To identify potential recombination events a complete genome SimPlot analysis was carried out which revealed the presence of possible multiple recombination events, but the minor parent strains remained unknown. A phylogenetic analysis of the complete S1 gene was performed using all complete S1 sequences available on ViPR and showed the isolate clustered with an Q1-like strain isolated in Italy in 2011 (p-distance=0.004) and a group of Chinese Q1-like strains isolated from the mid 90's (p-distance equal or higher than 0.001). It could be hypothesized that the isolate descended from the Italian 2011 Q1-like strain or was the result of a separate introduction from China through commercial trade or migratory birds; but the data currently available does not distinguish between these possibilities.
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http://dx.doi.org/10.1016/j.virusres.2015.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114532PMC
December 2015