Early Detection of Mycobacterium avium subsp. paratuberculosis Infected Cattle: Use of Experimental Johnins and Innovative Interferon-Gamma Test Interpretative Criteria.

Paratuberculosis (PTB), also known as Johne's disease, is a chronic proliferative enteritis of ruminants caused by Mycobacterium avium subsp.paratuberculosis (MAP). To date, PTB diagnosis, based on serology, fecal culture, and real-time polymerase chain reaction, has identified animals in advanced stages of infection. To detect MAP infection in animals earlier, the interferon-gamma (IFN-γ) test may be applied. This assay detects cytokines produced by T-lymphocytes of infected subjects after stimulation with purified protein derivatives (PPDs), extracted from Mycobacterium bovis (MB) and from M. avium (MA). The study involved three bovine herds: one PTB-infected herd, one PTB-free herd, and one with an outbreak of bovine tuberculosis. The IFN-γ test was performed on 235 animals, using bovine PPD (PPDB), avian PPD (PPDA), and three experimental PPD Johnins (PPDJs) extracted from a synthetic liquid medium culture of MAP (PPDJ A, B, and C), to assess early MAP detection and avoid false reactions to MB. Furthermore, IFN-γ results were evaluated using 12 interpretative criteria (ICs), based on the differences and ratio between PPD optical density (OD) and IFN-γ basal OD values after lymphocytic stimulation. IC accuracy was expressed as area under the receiver operating characteristic curve. Through a longitudinal study, PPDJs proved to be specific and sensitive in the detection of MAP-infected animals. Among the evaluated ICs, six showed the best performance in terms of accuracy (p < 0.0001), highlighting PTB subclinical infections. In particular, the two best criteria reached sensitivity values of 100% [confidence interval (CI) 95%, 94.1-100%] with a specificity of 91.8% (CI 95%, 81.9-97.3%) and sensitivity levels of 80.6% (CI 95%, 69.1-89.2%) with a specificity of 100% (CI 95%, 94.1-100%). Thus, the IFN-γ assay proved to be a useful diagnostic tool to identify early subclinical MAP-infected animals, in order to manage infected cattle or those exposed to MAP and to monitor younger calves within a herd. Furthermore, the IFN-γ test can be considered an additional test to avoid the introduction of MAP-infected animals, especially in herds where disease has already been eradicated and preservation of the health status is required to maintain the PTB certification level.

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.

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.

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.

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.

Uniparental genetic systems: a male and a female perspective in the domestic cattle origin and evolution

Over the last 20 years, the two uniparentally inherited marker systems, namely mitochondrial DNA and Y chromosome have been widely employed to solve questions about origin and prehistorical range expansions, demographic processes, both in humans and domestic animals. The mtDNA and the Y chromosome, with their unique patterns of inheritance, continue to be extremely important source of information. These markers played significant roles in farm animals in the evaluation of the genetic variation within- and among-breed strains and lines and have widely applied in the fields of linkage mapping, paternity tests, prediction of breeding values in genome-assisted selection, analysis of genetic diversity within breeds detection of population admixture, assessment of inbreeding and relationships between breeds, and assignment of individuals to their breed of origin. This approach offers a unique opportunity to save genetic resources and achieving improved productivity. In the past years, significant progress was achieved in reconstructing detailed cattle phylogenies; many studies indicated multiple parental sources and several levels of phylogeographic structuring. More detailed researches are still in progress in order to provide a more comprehensive picture of such extant variability. This paper is focused on reviewing the use of the two uniparental markers as valuable tool for the characterization of cattle genetic diversity. Furthermore, their implications in animal breeding, management and genetic resources conservation are also reported.