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The majority of proteins in cow’s milk are caseins, which occur in four groups (α-s1, α-s2, β, and k) encoded by different genes (CSN1S1, CSN1S2, CSN2, and CSN3, respectively). In this study, we focused on the β-casein allele variants A1 and A2 due to their influence on milk’s technological characteristics and human health. Digestion of the β-casein variant A1 leads to the formation of β-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 β-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 β-casein variants in Italian Holstein Friesian dairy cows from cattle farms located in central Italy to select a population of A2 homozygous animals. β-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 β-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.

Host population genetics can shape disease spread in wildlife, yet it is rarely integrated into epizootic investigations. To explore whether connectivity patterns in wild boar populations may have influenced the spread of African swine fever (ASF) in north-western Italy, we characterised the genetic structure of the local population. Microsatellite genotyping was performed on 578 wild boar sampled from 26 hunting districts across thirteen loci and analysed using Bayesian clustering, correspondence analysis and spatial PCA. In parallel, 2,414 ASF detections recorded between December 2021 and March 2025 were examined through retrospective spatiotemporal scan statistics and directional spread analysis. We identified two main genetic clusters, one largely corresponding to Piedmont and the other more prevalent in Liguria regions, with zones of admixture along their border and a connectivity corridor through the Ligurian Apennines. Over the 38-month period, 16 significant ASF clusters were detected. The outbreak spread eastward and north-eastward from the initial focus at the Liguria-Piedmont border. Four clusters showed significant directionality, and recurrent clustering in certain areas suggested local persistence. Notably, several ASF clusters overlapped with genetic admixture zones and connectivity hubs. Our findings suggest two mechanisms underpinning disease spread: short-range transmission within genetically related groups and longer-range movement along ecological corridors. Embedding genetic monitoring into routine surveillance may enhance the effectiveness of ASF control by guiding carcass removal, search efforts and spatial prioritisation toward high-risk transition zones.

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.

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.

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.

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.

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.

Since the very beginning of the COVID-19 pandemic, SARS-CoV-2 detection has been described in several animal species. A total of 625 outbreaks in animals have been reported globally, affecting 17 species in 32 countries and the human source of infection has been recognized including pet owners, zookeepers, and farmers. In this report, we describe the case of a paucisymptomatic dog in Italy infected with SARS-CoV-2 from a household with three confirmed human cases of COVID-19 living in Pesaro (Marche region, Italy). The dog showed high viral RNA titers in the nasal and oropharyngeal swabs. In the nasal swab, SARS-CoV-2 RNA lasted for a least a week. By sequencing, the strain was assigned to the AY.23 lineage (PANGO), one of the sub-lineages of the major SARS-CoV-2 Delta variant of concern (VOC). Although we did not process the swabs of the three human cases, we strongly suspect a human origin for the dog infection. In this regard, AY.23 sequences, although never released thus far in the Marche region, were detected in the neighboring regions. Our findings highlight once more the need for a One Health approach for SARS-CoV-2 surveillance, management, and control, thus preventing viral spillover from animals to humans.

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.

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.