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.

Molecular Screening of Echinococcus spp. and Other Cestodes in Wild Carnivores from Central Italy.

Tapeworm infections are among the most relevant parasitic diseases in humans and animals. Tapeworms from the Genus Echinococcus are particularly important as they can cause cystic or alveolar echinococcosis. A molecular screening was performed on 279 fecal samples collected from carcasses of wild carnivores from Central Italy using PCR targeting diagnostic fragments of nad1, rrnS, and nad5 genes. Samples positive for either Taenia spp. or Echinococcus granulosus were sequenced to taxonomically identify the parasitic DNA. Of the 279 samples, 134 (48.0%) gave positive results in the multiplex PCR. Only one (0.4%) sample from an Apennine wolf tested positive for Echinococcus granulosus sensu stricto (genotype G3), whereas no sample tested positive for E. multilocularis. The most frequently detected tapeworms were: Mesocestoides corti (syn M. vogae) (12.9%), M. litteratus (10.8%), Taenia serialis (9.3%), and T. hydatigena (6.5%), other tapeworms were rarely detected. The results suggest that Echinococcus infections in Central Italy do not seem to be sustained by sylvatic cycles, confirming the absence of E. multilocularis in Central Italy. The survey corroborates, yet again, the importance of passive surveillance of wild animals that can serve as reservoirs for zoonotic pathogens, especially on wild canids that in other areas are strongly implicated in the transmission of E. granulosus and E. multilocularis.

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.

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.

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.

Molecular characterization and antimicrobial susceptibility of Pasteurella multocida strains isolated from hosts affected by various diseases in Italy.

Pasteurella multocida is a widespread pathogen associated with major animal diseases of economic significance. Despite this, little is known about the capsular types, virulence gene pattern, and antimicrobial susceptibility of isolates from hosts affected by different diseases, and no data are available in Italy. One hundred eighty six isolates of P. multocida, were taken from different species in different states of health in several Italian regions, and were tested for genes encoding for capsular types (cap) and major virulence factors (tbpA, toxA, hgbB and pfhA). Antimicrobial susceptibility was investigated with the agar diffusion test. The majority of isolates was capA+. However, the distribution differed according to species and disease of origin, with a greater heterogeneity in isolates from rabbits; capE was never found, while capB was detected once. Only capA+ and capF+ strains tested positive for pfhA. Conversely, almost all capD+ isolates were hgbB+. In bovine respiratory disease, pfhA+/tbpA+/capA+ isolates predominated, while tbpA+/toxA+/capD+ isolates predominated in sheep. Overall, low levels of resistance were found, with full susceptibility to ceftiofur and florfenicol. Lower susceptibility to older antimicrobials was recorded, since only approximately 1/3 of the isolates showed susceptibility to tylosin and erythromycin, and resistance to tetracycline (7.5%), and trimethoprim - sulphametoxazole (4.8%) was also observed.

Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair.

Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins.