Ludovica Curcio

Novel plasmid-mediated colistin resistance mcr-4 gene in Salmonella and Escherichia coli, Italy 2013, Spain and Belgium, 2015 to 2016

A novel mcr colistin resistance gene was identified in a strain of Salmonella enterica, monophasic variant of serovar Typhimurium (4,5,12:i:- ), isolated from a pig at slaughter in Italy in 2013, and in Escherichia coli strains collected during routine diagnostic of post-weaning diarrhoea in pigs from Spain and Belgium in 2015 and 2016. Immediate implementation of mcr-screening including this novel gene variant is required for Salmonella and E. coli from humans and food-producing animals in Europe.

Review: A review on classical and atypical scrapie in caprine: Prion protein gene polymorphisms and their role in the disease.

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goat. It has been known for ~250 years and is characterised by the accumulation of an abnormal isoform of a host-encoded prion protein that leads to progressive neurodegeneration and death. Scrapie is recognised in two forms, classical and atypical scrapie. The susceptibility to both types of scrapie is influenced by polymorphisms of the prion protein gene (PRNP). Sheep susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP. The genetic role in atypical scrapie in sheep has been defined by polymorphisms at codons 141, 154 and 171, which are associated with different degrees of risk in the occurrence of the ovine disease. Progress has been achieved in the prevention of scrapie in sheep due to efficient genetic breeding programmes based on eradication and control of the disease. In Europe, the success of these programmes has been verified by applying eradication and genetic selection plans. In general terms, the ovine selection plans aim to eliminate and reduce the susceptible allele and to enrich the resistant allele ARR. During outbreaks all susceptible animals are slaughtered, only ARR/ARR resistant rams and sheep and semi-resistant females are preserved. In the occurrence of scrapie positive goats a complete cull of the flock (stamping out) is performed with great economic loss and severe risk of extinction for the endangered breeds. The ability to select scrapie-resistant animals allows to define new breeding strategies aimed to boost genetic progress while reducing costs during scrapie outbreaks. Allelic variants of PRNP can be protective for caprine scrapie, and the knowledge of their distribution in goats has become very important. Over the past few years, the integration of genetic information on goat populations could be used to make selection decisions, commonly referred to as genetic selection. The objective of this review was to summarise the main findings of polymorphisms of the caprine prion protein (PrP) gene and to discuss the possible application of goat breeding schemes integrating genetic selection, with their relative advantages and limitations.

Detection of the colistin resistance gene mcr-1 in pathogenic Escherichia coli from pigs affected by post-weaning diarrhoea in Italy

OBJECTIVES The aim of this study was to investigate the presence of plasmid-mediated colistin resistance genes in Escherichia coli from pigs affected by post-weaning diarrhoea (PWD). METHODS DNA samples collected from 51 E. coli isolates from Italian pigs affected by PWD in 2015-2016 were studied. Isolates were classified as presumptively resistant to colistin by routine susceptibility testing and were investigated for the presence of the mcr-1 gene of plasmid origin by PCR. E. coli isolates testing negative for mcr-1 were analysed for the presence of a novel plasmid-mediated gene, mcr-2. Isolates were characterised for fimbrial [F4 (k88), F5 (k99), F6 (987P), F18 and F41] and toxin (LT, STa, STb and Stx2e) determinants by PCR as well as for the occurrence of haemolysis by phenotypic observation. Susceptibility to apramycin, cefquinome, enrofloxacin, florfenicol, gentamicin, tetracycline and trimethoprim/sulfamethoxazole (SXT) was also determined by disk diffusion. RESULTS Most of the isolates showed the presence of at least one virulence factor, confirming their pathogenic potential. The presence of mcr-1 was shown in 37 (72.5%) of the 51 isolates. All of the mcr-1-negative isolates tested negative for the mcr-2 gene. Moreover, 80.4% of the isolates were resistant to apramycin, 9.8% to cefquinome, 54.9% to enrofloxacin, 52.9% to florfenicol, 76.5% to gentamicin, 96.1% to tetracycline and 78.4% to SXT. CONCLUSIONS This is the first report documenting the presence of the mcr-1 gene in pathogenic E. coli isolated from pigs affected by PWD in Italy.

In-house Validation of a DNA Extraction Protocol from Honey and Bee Pollen and Analysis in Fast Real-Time PCR of Commercial Honey Samples Using a Knowledge-Based Approach

For consumers, honey is a natural product that should not be subjected to treatment or alteration. Since the question of the presence of genetically modified organisms concerned pollen in honey, the aim of this research was to find an alternative, however, practical and efficient method of honey and bee pollen DNA extraction for routine analysis application. Furthermore, to evaluate the extracted DNA, a real-time PCR system based on the actin gene was optimized and validated in fast mode for the first time to reduce analysis time. To develop an alternative DNA extraction protocol, two already published procedures were combined and tested with some variations, in particular without beads/filters used to grind/enrich pollen. The best approach found in terms of quantity and quality of extracted DNA was a combination of the pretreatment and the extraction method, described in the German guideline, with some modifications and the addition of a DNA purification kit. This protocol was validated with DNA extracts from honey and bee pollen and it was applied to 18 commercial honey samples. Furthermore, a sample proved positive in transgenic screening elements analysis and for transgenic event identification, a knowledge-based approach was adopted. Since the DNA extraction protocol proved suitable, it could be applied for other analysis such as molecular species characterization, the study of traceability, and environmental monitoring, considering honey as a vector of authorized and not authorized genetically modified organisms.

Characterization of Pasteurella multocida involved in rabbit infections

In rabbit, P. multocida is considered a predominant pathogenic agent; despite this, few data on the molecular epidemiology are available so far. The aim of this work was to characterize P. multocida isolates from rabbit affected by various diseases in Italy. Comparison was made to reference strains from other countries. Thirty-nine isolates were tested using PCRs to detect the genes coding capsular antigens, virulence factors and lipopolysaccharide structures (LPS). Multilocus sequence typing (MLST) was performed and 19 STs registered that belonged to 9 clonal complexes. Italian isolates were all related to P. multocida subsp. P. multocida. Three sequence types dominated (ST9, ST50 and ST74). The isolates were assigned to capsular types A (20/39), D (9/39) and F (10/39), to virulence genes pfhA (13/39), hgbB (21/39) and pfhA+hgbB (4/39) (one without virulence factors) and the isolates either belonged to the LPS genotypes 3 (22/39) or 6 (17/39). The clonal relationships of the Italian strains from rabbit had similarity to previously reported rabbit isolates that belonged to ST9, ST74, ST204 and ST206, however, they differed from other rabbit references strains that belonged to six other STs. In particular, ST9 with capsular type F has been previously reported from diseased rabbit in Czech Republic and ST74 has been observed for older rabbit isolates. ST50 has probably been reported from Spain. ST9 and ST50 have previously also been reported from birds and pig, respectively, whereas ST74 has exclusively been reported from pig. It remains to be investigated if the isolates obtained from diseased rabbit in Italy represent introductions from other host or they are primarily of rabbit origin.

Evaluation of Systems for Nopaline Synthase Terminator in Fast and Standard Real-Time PCR to Screen Genetically Modified Organisms

The increasing number and diversity of genetically modified organisms (GMOs) developed and commercialised forces laboratories to apply many different methods of analysis. Matrix-based approach helps to minimize analytical effort reducing the number of identifications. However, the correctness of screening phase needs efficient methods. In this paper, 15 systems for the nopaline synthase terminator (T-nos) from Agrobacterium tumefaciens, a genetic element present in several genetically modified (GM) plants, were tested. The systems were obtained from three methods, and their primers and probes were combined and tested in real-time polymerase chain reaction (PCR) in fast and standard mode. The results have showed that the fast mode presented the lowest mean quantification cycle (Cq) and the highest ∆Rn, that is, the difference between normalized reporter and baseline. These parameters of system efficiency prove that the fast real-time PCR is a possible approach to obtain good data in less than half the time compared to standard mode. The proposed approach allows a practical way to evaluate the most efficient set of oligonucleotides (e.g., primers and probe) in fast and standard real-time PCR before validation. This article describes also in-house validation of the best set oligonucleotides.

Biodiversity and Genetic Polymorphisms Against Scrapie in Sopravissana Sheep Breed

Scrapie is a neurodegenerative disease affecting ovine and it is one of several transmissible spongiform encephalopathies (TSEs). Scrapie is recognized as two forms – classical and atypical. Susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP gene. Genetic role in atypical scrapie has been described at codons 141 and 154, these ones are involved in the occurrence of the disease at different risk degrees. The aim of this study was to assess the allelic and genotypic frequencies in Sopravissana breed, an endangered autochthon breed of central Italy; in addition, the presence and the frequency of potential protective ARQ allele variants able to increase scrapie resistance by preserving a higher variability of PRNP were evaluated. Three alleles (ARQ, ARR and AHQ) and seven genotypes were observed based on codons 136, 154 and 171 with different frequencies. Moreover, the entire coding sequence of prion protein (PRNP) of the ARQ/ARQ was sequenced; two non-synonymous L141F (23.8%) and H143R (16.7%), also two synonymous polymorphisms (codons 231 and 237) were found. The results showed that the ARQ/ARQ sheep should be considered as a genetic class, which may potentially include animals with different scrapie susceptibility because of the presence of additional polymorphisms. This may allow the application of alternative strategies for breeding programmes in this endangered breed.

Chimeric DNA/LNA-based biosensor for the rapid detection of African swine fever virus

African swine fever (ASF) virus is a DNA virus responsible for a severe haemorrhagic fever in pigs, which (still in the absence of vaccination strategies) results in high mortality rates. Herein, we present a biosensor-based method for the detection of ASF viral DNA in the blood of pigs. The biosensor exploits a single-strand DNA probe with locked nucleic acid nucleotides (LNA) substitutions as the complementary recognition element for the conserved region of vp72 gene of ASF virus. The biosensor was calibrated using qPCR-quantified ASF viral DNA extracted from the blood of pigs experimentally infected with the virulent Italian isolate 49/08, genotype I. Globally, the proposed biosensor showed good sensitivity and specificity, with the limits of detection (LOD) and quantification (LOQ) being 178 and 245 copies/μL of genomic ASF viral DNA, respectively. The reversible nature of the interaction between the DNA/LNA probe and the target DNA sequence granted multiple rapid analyses, with up to 40 analyses per single surface possible, and a single test requiring approximately 5 min. When applied to non-amplified DNA extracts from the blood of field-infected pigs, the assay discriminated between ASFV-infected and ASFV non-infected animals, and allowed the rapid quantification of ASF viral DNA, with values falling in the range 373-1058 copies/μL of genomic ASFV DNA. In this range, excellent correlation was observed between the results of this biosensor and OIE-approved qPCR. This method represents a promising screening assay for preliminary ASF diagnosis, having the major advantages in the relative rapidity, ease-of-use, the reusability of the sensing surface, and low cost per single test.

A multi-screening Fast qPCR approach to the identification of abortive agents in ruminants

Abortion in ruminants represents an important economic concern for farmers. Microbial agents, such as Brucella spp., Chlamydia spp., Coxiella burnetii, Leptospira spp., Neospora caninum, Salmonella spp. and Toxoplasma gondii, are among the main infectious causes of abortion and require rapid and reliable diagnosis. This study describes the development of a multi-screening assay using Fast Real-Time PCR (Fast qPCR) that allows, in a single test, the simultaneous identification of the above-mentioned abortive agents. This multi-screening approach is characterized by a mean diagnostic sensitivity and specificity of 100% and 97%, respectively; it has a limit of detection (LOD) ranging from 5 × 103 to 4 × 104 genomic copies/g of tissue and a very good concordance with traditional end-point PCR assays used in routine diagnostic activity. The proposed method represents a rapid approach to the simultaneous detection of the main abortive agents in ruminants that allows to make an accurate diagnosis and to set up appropriate control measures in a short period of time.