Survey on resistance occurrence for F4+ and F18+ enterotoxigenic Escherichia coli (ETEC) among pigs reared in Central Italy regions.

Porcine Post Weaning Diarrhoea (PWD) is one of the most important swine disease worldwide, caused by Enterotoxigenic Escherichia coli (ETEC) strains able to provoke management, welfare and sanitary issues. ETEC is determined by proteinaceous surface appendages. Numerous studies conducted by now in pigs have demonstrated, at the enterocytes level, that, the genes mucin 4 (MUC4) and fucosyltransferase (FUT1), coding for ETEC F4 and F18 receptors respectively, can be carriers of single nucleotide polymorphisms (SNPs) associated with natural resistance/susceptibility to PWD. The latter aspect was investigated in this study, evaluating the SNPs of the MUC4 and FUT1 genes in slaughtered pigs reared for the most in Central Italy. Genomic DNA was extracted from 362 swine diaphragmatic samples and then was subjected to the detection of known polymorphisms on MUC4 and FUT1candidate target genes by PCR-RFLP. Some of the identified SNPs were confirmed by sequencing analysis. Animals carrying the SNPs associated with resistance were 11% and 86% for the FUT1 and MUC4 genes respectively. Therefore, it can be assumed that the investigated animals may be an important resource and reservoir of favorable genetic traits for the breeding of pigs resistant to enterotoxigenic E.coli F4 variant.

Porcine Reproductive and Respiratory Syndrome (PRRS) and CD163 Resistance Polymorphic Markers: What Is the Scenario in Naturally Infected Pig Livestock in Central Italy?

Porcine Reproductive and Respiratory Syndrome (PRRS) caused by the PRRS virus affects farmed pigs worldwide, causing direct and indirect losses. The most severe manifestations of PRRS infection are observed in piglets and pregnant sows. The clinical outcome of the infection depends on the PRRSV strain's virulence, the pregnancy state of the female, environmental factors, the presence of protective antibodies due to previous infections, and the host's genetic susceptibility. The latter aspect was investigated in this study, in particular, evaluating the most significant polymorphisms (SNPs) of the CD163 gene in slaughtered pigs reared in Central Italy. Total RNAs were extracted from 377 swine samples and subjected to RT-PCR targeted to the CD163 gene, followed by sequencing analysis. Contextually, the viral RNA was detected by RT-qPCR in order to phenotypically categorize animals into infected and not infected. In particular, 36 haplotypes were found, and their frequencies ranged from 0.13% to 35.15%. There were 62 resulting genotypes, three of which were associated with a putative resistance to the disease. Both the haplotypes and genotypes were inferred by PHASE v.2.1 software. To the best of our knowledge, this type of investigation was conducted for the first time on pig livestock distributed in different regions of Central Italy. Thus, the obtained findings may be considered very important since they add useful information about swine genetic background in relation to PRRS infection, from the perspective of adopting Marker-Assisted Selection (MAS) as a possible and alternative strategy to control this still widespread disease.

Tapeworms detected in wolf populations in Central Italy (Umbria and Marche regions): A long-term study.

Tapeworms are trophically-transmitted and multi-host parasites with a complex indirect life cycle, strictly depending on predator-prey interactions. Their presence in a free-living population, mainly definitive hosts, is arduous to study due to the complexity of collecting fecal samples. However, epidemiological studies on their frequency are crucial from a public health perspective, providing information on food habits and prey selection of predators. The present study aims to update the frequency of tapeworms detected in stool samples by molecular analysis in Italian wolf populations of Umbria and Marche regions collected from 2014 to 2022. Tapeworm's total frequency was 43.2%. In detail, Taenia serialis was detected in 27 samples (21.6%), T. hydatigena in 22 (17.6%), and Mesocestoides corti (syn. M. vogae) in 2 (1.6%). Three samples were identified as M. litteratus and E. granulosus s.s. (G3) and T. pisiformis, with a proportion of 0.8%, respectively. The low frequency of E. granulosus in a hyperendemic area is discussed. The results show for the first time a high frequency of Taenia serialis not comparable to other Italian studies conducted on wild Carnivora; thus, a new ecological niche is conceivable. These findings suggest a plausible wolf-roe deer cycle for T. serialisin the investigated area.

Evaluation of Single Nucleotide Polymorphisms (SNPs) Associated with Genetic Resistance to Bovine Paratuberculosis in Marchigiana Beef Cattle, an Italian Native Breed.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), a widespread chronic enteritis of ruminants. The progression of the infection depends on the containment action of innate and cell-mediated immunity (CMI), and it is related to environmental and genetic factors. In particular, PTB susceptibility seems to be associated with specific genes coding for immune regulators involved in the cell-mediated response during the infection. The aim of this preliminary study was to verify, in Italian beef cattle, an association between MAP infectious status and the presence of single nucleotide polymorphisms (SNPs) in candidate genes. To the best of our knowledge, this is the first investigation conducted on a native beef cattle breed, known as Marchigiana, reared in Central Italy. The present research, based on a longitudinal study, aimed to identify and correlate phenotypic and genetic profiles characteristic of the subjects potentially able to contrast or contain PTB. In a MAP-infected herd, ELISA, IFN-γ tests, qPCR, and cultures were performed at a follow-up, occurring within a period ranging from three to six years, to evaluate the individual state of infection. Animals testing positive for at least one test were considered infected. DNA samples of 112 bovines, with known MAP statuses, were analyzed to verify an association with SNPs in the genes encoding gamma-interferon (BoIFNG), interleukin receptor 10 (IL10RA), interleukin receptor 12 (IL12RB2), and toll-like receptors (TLR1, TLR2, TLR4). Regarding statistical analysis, the differences among target genes and pairs of alleles in the analyzed groups of animals, were evaluated at a significance level of p < 0.05. For IL10RA and for IL12RB2 genes, relevant differences in genotypic frequencies among the considered cattle groups were observed. For all candidate genes studied in this investigation, SNP genotypes already associated with PTB resistance were found more frequently in our population, suggesting potential resistance traits in the Marchigiana breed.

Genetic Characterization of Small Ruminant Lentiviruses (SRLVs) Circulating in Naturally Infected Sheep in Central Italy.

Small ruminant lentiviruses (SRLVs) represent a very heterogeneous group of ss-RNA viruses that infect sheep and goats worldwide. They cause important, deleterious effects on animal production and limit the animal trade. SRLVs show a high genetic variability due to high mutation rate and frequent recombination events. Indeed, five genotypes (A-E) and several subtypes have been detected. The aim of this work was to genetically characterize SRLVs circulating in central Italy. On this basis, a phylogenetic study on the gag-pol genetic region of 133 sheep, collected from 19 naturally infected flocks, was conducted. In addition, to evaluate the frequency of mutation and the selective pressure on this region, a WebLogo 3 analysis was performed, and the dN/dS ratio was computed. The results showed that 26 samples out of 133 were clustered in genotype A and 106 samples belonged to genotype B, as follows: A9 (n = 8), A11 (n = 10), A24 (n = 7), B1 (n = 2), B2 (n = 59), and B3 (n = 45). No recombination events were found. Mutations were localized mainly in the VR-2 region, and the dN/dS ratio of 0.028 indicated the existence of purifying selection. Since the genetic diversity of SRLVs could make serological identification difficult, it is important to perform molecular characterization to ensure a more reliable diagnosis, to maintain flock health status, and for the application of local and national control programs.

First Survey of SNPs in TMEM154, TLR9, MYD88 and CCR5 Genes in Sheep Reared in Italy and Their Association with Resistance to SRLVs Infection.

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV), referred to as small ruminant lentiviruses (SRLVs), belong to the genus Lentivirus of the Retroviridae family. SRLVs infect both sheep and goats, causing significant economic losses and animal welfare damage. Recent findings suggest an association between serological status and allelic variants of different genes such as TMEM154, TLR9, MYD88 and CCR5. The aim of this work was to investigate the role of specific polymorphisms of these genes in SRLVs infection in some sheep flocks in Italy. In addition to those already known, novel variants in the TMEM154 (P7H, I74V, I105V) gene were detected in this study. The risk of infection was determined finding an association between the serological status and polymorphisms P7H, E35K, N70I, I74V, I105V of TMEM154, R447Q, A462S and G520R in TLR9 gene, H176H* and K190K* in MYD88 genes, while no statistical association was observed for the 4-bp deletion of the CCR5 gene. Since no vaccines or treatments have been developed, a genetically based approach could be an innovative strategy to prevent and to control SRLVs infection. Our findings are an important starting point in order to define the genetic resistance profile towards SRLVs infection.

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.

PRNP Polymorphisms in Eight Local Goat Populations/Breeds from Central and Southern Italy.

In goats, as in sheep, genotypes of the prion protein gene (PRNP) can influence animals' susceptibility to scrapie. Since the polymorphic codons in sheep are well known, a genetic selection plan has been implemented in Europe, in order to reduce the prevalence of susceptible genotypes to scrapie. In Italy, no breeding plan for scrapie resistance in goats has been adopted, yet. Likewise, according to the most recent modification of Regulation EU 999/2001 (Regulation EU 772/2020) of the European Commission (EU), based on all the available experimental and in field data, K222, D146 and S146 polymorphisms could be used as scrapie resistance alleles in genetic management both in scrapie outbreaks and in disease prevention. In order to collect data on the variability of PRNP, the present study aimed to analyze the sequence of the PRNP gene in eight Italian local goat populations/breeds reared in central and southern Italy (Bianca Monticellana, Capestrina, Facciuta della Valnerina, Fulva del Lazio, Garganica, Grigia Ciociara, Grigia Molisana, and Teramana), some of which were investigated for the first time; moreover, two cosmopolitan breeds (Alpine and Saanen) were included. Blood samples were collected from 219 goats. Genomic DNA was extracted from whole blood. DNA was used as template in PCR amplification of the entire PRNP open reading frame (ORF). Purified amplicons have been sequenced and aligned to Capra hircus PRNP. Particularly, the alleles carrying the resistance-related 222 K polymorphism occurred in all populations with a frequency between 2.5% and 12.5%. An additional resistance allele carrying the S146 variant was observed with a frequency of 3.7% only in the Alpine breed. For three of the estimated alleles, we could not establish if the found double polymorphisms in heterozygosis were in phase, due to technical limitations. In this context, in addition to selective culling in scrapie outbreaks according to the European regulation in force, in the future, selection plans could be adopted to deal with scrapie and to control its diffusion, meanwhile paying attention to preserve a high variability of PRNP.

Sesame, Pistachio, and Macadamia Nut: Development and Validation of New Allergenic Systems for Fast Real-Time PCR Application.

Food allergy is a worldwide health problem that concerns infants to adults. The main health risk for sensitised individuals is due to the presence of traces of allergens as the result of an accidental contamination during food processing. The labelling of allergens such as sesame, pistachio, and macadamia nut on food products is mandatory according to Regulation (EU) N. 1169/2011; therefore, the development of suitable and specific analytical methodologies is advisable. The aim of this study was to perform a multi-allergen real-time PCR system that works well in fast mode at the same annealing temperature and with the same thermal profile. The real-time PCR was developed designing new, specific, and efficient primer and probe systems for the 2S albumingene for sesame and pistachio and for the vicilin precursorgene for macadamia nut. These systems were subjected to a robust intra-laboratory qualitative validation process prior to their application, by DNA extraction and fast real-time PCR, on some real market samples to reproduce a potential allergen contamination along the food chain. The developed system results were specific and robust, with a sensible limit of detection (0.005% for sesame; 0.004% for pistachio; 0.006% for macadamia nut). The performance and the reliability of the target systems were confirmed on commercial food samples. This molecular approach could be used as a screening or as a support tool, in association with the other widespread monitoring techniques (such as ELISA).

Frequencies Evaluation of ß-Casein Gene Polymorphisms in Dairy Cows Reared in Central Italy.

The majority of proteins in cow's milk are caseins, which occur in four groups (α-s1, α-s2, ß, and k) encoded by different genes (CSN1S1, CSN1S2, CSN2, and CSN3, respectively). In this study, we focused on the ß-casein allele variants A1 and A2 due to their influence on milk's technological characteristics and human health. Digestion of the ß-casein variant A1 leads to the formation of ß-casomorphin 7 (BCM-7), a bioactive peptide that has been suggested to be a possible cause of various human diseases and associated with low milk digestibility. The potential negative role of the ß-casein variant A1 in human health has stimulated the planning of cattle breeding programs based on genetic selection to increase the frequency of the A2 variant, which is associated with increased milk digestibility. The aim of this work was to evaluate the frequencies of the different ß-casein variants in Italian Holstein Friesian dairy cows from cattle farms located in central Italy to select a population of A2 homozygous animals. ß-casein genotypes were identified by evaluating the presence of single nucleotide polymorphisms (SNPs) of the CSN2 gene using PCR and sequencing analysis. The frequency of the desirable ß-casein variant A2 in the studied bovine population was 0.61. The frequency of the undesirable A1 variant in the studied bovine population was 0.30. The frequency of the A2 allele was higher than expected for the breed; therefore, genetic selection for the A2 variant in these animals could be achieved in a fairly short time using A2 homozygous bulls.

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.