MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in the post-transcriptional regulation of gene expression. Since miRNAs modulate host immune responses, they represent promising molecular biomarkers of paratuberculosis (PTB), particularly during early or subclinical stages, when conventional diagnostic tests may lack sensitivity. In this context, faecal miRNAs could provide valuable insights into intestinal immune responses and mucosal damage associated with Mycobacterium avium subsp. paratuberculosis (MAP) infection. Although miRNAs have been extensively investigated in serum, blood, and tissues, their detection and characterization in bovine faeces remain poorly explored. The aim of this study was to evaluate the expression profiles of selected candidate faecal miRNAs in Marchigiana beef cattle naturally exposed to MAP and to assess their association with different infection phenotypes. Thirty-four cows were classified into three phenotypic groups: healthy exposed, MAP-infected, and PTB-affected based on longitudinal diagnostic records including interferon-γ assay, serological testing (ELISA), and faecal qPCR. Five candidate miRNAs were selected from previous studies and quantified in faecal samples by RT-qPCR. Four of the five selected miRNAs were consistently detected across samples. Bta-miR-92a was significantly downregulated in both MAP-infected and PTB-affected animals compared with healthy cattle, suggesting early modulation during MAP infection. Bta-miR-223 was significantly upregulated in PTB-affected animals compared with both healthy and MAP-infected groups, consistent with its established role in the regulation of intestinal inflammation. The ortholog of hsa-miR-501-5p was significantly upregulated in MAP-infected cattle, potentially reflecting early host-pathogen interactions at the intestinal mucosal level, while bta-miR-24-3p showed no significant differences among groups. Overall, these findings support the feasibility of faecal miRNA analysis as a complementary non-invasive molecular approach to support traditional diagnostic tests for PTB, especially during the early and subclinical stages of MAP infection.

A certain number of inherited diseases and traits in cattle have been characterized at the molecular level, identifying the causative gene variant in the DNA locus. Also the three main native Italian beef cattle breeds located in the Apennine Mountains (Chianina, Marchigiana and Romagnola) are interested by this discovery. Genetic selection in Italian beef cattle is implemented by the National Association of Italian Beef Cattle Breeders, with the aim of improving meat production, growth ability and muscle development. Contextually, bovines, particularly those with pedigree registered in the genealogical book, have to be constantly monitored for the main congenital and hereditary disorders, among them "Paunch Calf syndrome" and "Congenital Ichthyosis". Paunch Calf Syndrome (PCS) is associated with a missense variant in the KDM2B gene on chromosome 17, while Congenital Ichthyosis (CI) is caused by an insertion of a cytosine in FA2H gene causing a frame-shift on chromosome 18. The aim of this work was to present an update of the prevalence of PCS in Marchigiana and Romagnola breeds and of CI in Chianina breed, in the Central Italy territory. In the period 2022-2024, a frequency of 6.67% heterozygous PCS carriers was registered, while for CI, in 2024, a prevalence of 5.67% heterozygous carriers was observed. These findings show that cattle should be constantly monitored and the genotyping of causal variant should be used, at least for excluding risk matings of carriers to prevent further animal losses due to these two congenital disorders.

European hedgehogs are an important reservoir for many pathogens of health interest. Since hedgehogs live in close contact with humans, potential zoonotic fungi raise significant public health concerns, especially in areas with a high hedgehog density. From 2020 to 2023, 134 hedgehogs were surveyed for potential zoonotic fungi. Non-invasive methods were used, such as brushing live animals with a sterile toothbrush and taking oral and rectal swabs from deceased ones (86 animals). Dermatophytes were cultured on Dermasel agar and identified using molecular tools, while yeasts were isolated on Sabouraud agar with chloramphenicol and determined using Candida Chromogenic agar (MicroBiolDiagnostici®, Cagliari, Italy) and MALDI-TOF (Microflex LT Smart Biotyper with FlexControlBiotyper 3.4 software, Bruker Daltonics, Bremen, Germany). Minimum inhibitory concentrations (MICs) were determined for Candida albicans isolates. Dermatophytes were found in just one hedgehog (0.8%, 95% C.I.: 0–0.04), identified as Paraphyton mirabile. Yeasts were detected in 22 of 86 hedgehogs (25.6%, 95% C.I.: 16.4–34.8), with 25 isolates obtained, including 21 Candida albicans, 2 Yarrowia lipolytica, 1 Rhodotorula mucilaginosa, and 1 Meyerozyma guilliermondii. All C. albicans isolates showed a high susceptibility to the antimycotic panel tested. Monitoring zoonotic fungi harbored by European hedgehogs, as well as raising public awareness on the topic, is of great importance for public health.

Antimicrobial resistance (AMR) among commensal Escherichia coli from poultry is a growing concern for food safety and public health. This study investigated AMR patterns in E. coli isolated from broiler neck skin at slaughter, comparing organic, antibiotic-free (ATB-free), and conventional production systems. A total of 375 samples were collected from two Italian slaughterhouses and tested by broth microdilution following EU protocols. E. coli was recovered from 358 samples, and 37.9% were presumptively positive for ESBL/AmpC-producing strains. Conventional broilers showed the highest resistance to ampicillin (73.8%), sulfonamides (72.5%), and fluoroquinolones (nalidixic acid, 62.5%; ciprofloxacin, 67.5%), while organic and ATB-free systems showed significantly lower levels. Intermediate resistance occurred for trimethoprim (21.4–47.9%) and tetracycline (36–54%), and low prevalence (<10%) was found for gentamicin, tigecycline, and third-generation cephalosporins. No relevant resistance was detected to colistin or carbapenems (≤1.2%). Total E. coli counts did not differ among systems, suggesting differences in resistant strain proportions rather than bacterial load. ATB-free flocks processed after conventional batches displayed higher resistance, indicating possible cross-contamination during slaughter. These results highlight the influence of farming practices and slaughterhouse hygiene on AMR dissemination, underscoring the need for integrated farm-to-slaughter control strategies.

Toxoplasmosis is a zoonosis of warm-blooded animals, affecting also humans, caused by Toxo-plasma gondii, an obligate intracellular parasite with a complex biological cycle. Toxoplasmosis is considered a re-emerging zoonosis and an important public health threat, also for the absence, up to now, of efficient vaccines and preventive measures. Sources of transmission to humans are environmental or food-borne, mainly through the consumption of raw and undercooked meat, predominantly of Sus scrofa, considered highly susceptible to T. gondii. Mainly in the last dec-ades, an effective strategy to counter animal infectious diseases was based on the discovery of resistance immuno-genetic markers, possibly to be used for association studies between geno-typic and phenotypic traits and consequently in Marker Assisted Selection. Until now, this gen-otyping approach has never been adopted for T. gondii infection, so this study aimed, for the first time, to fill this gap. In particular, pigs, reared in different farms of Central Italy, were serologically characterized by an ELISA assay performed on diaphragm meat juice. Out of 179 tested animals, 98 resulted seropositive (54.74%), 57 seronegative (31.84%) and 24 doubtful (10.6%), underlining a possible re-emerging diffusion of this protozoan in the investigated areas. Atten-tion was firstly focused on IL-18 gene, encoding for a pro-inflammatory cytokine that plays a key role starting from the upstream steps of the immunity pathway towards T. gondii. A RT-PCR assay followed by Sanger sequencing of IL-18 cDNA was de novo developed. For this interleukin, at this stage, genetic analysis has not highlighted significant polymorphic variations compared to the reference sequence, except for a seropositive animal. However, the outcomes of this pre-liminary innovative study will be investigated in depth analyzing other relevant genetic re-gions and other target genes.

In an era with an ever-growing population, sustainability and green transition are the main milestones to be considered within the current European Green Deal program, and the recovery of by-products for the integration of feed with bioactive molecules, that are sustainable and with high nutritional value, is an ambitious mission to be explored also in aquaculture. Olive oil extraction produces a range of solid and liquid by-products, in varying proportions depending on the utilized production techniques, all of which are considered as possible pollutants. However, these products are also rich of polyphenols, bioactive molecules with several and well-known beneficial properties (antimicrobic, anti-inflammatory, antioxidant, and immune-modulating). On this basis, this work aimed at evaluating the effects of dietary supplementation with polyphenols derived from olive mill wastewater on growth performance and on gene expression modulation, by means of RT-qPCR assays, in farmed Sparus aurata. Particularly, some target genes of metabolic, immunity, and oxidative stress pathways have been investigated in breeding gilthead seabream. Differential gene expression analysis was carried out, and differences between the control group (n = 9) and the treated one (n = 9) were computed with Student’s t test. The results have highlighted that supplemented feed enhanced fish growth, with a significant feed conversion ratio between the two groups. Furthermore, the polyphenol diet had a beneficial impact on gene expression fold with a level of significance for fatty acid binding protein 2, superoxide dismutase 1, and interleukin-12 genes at hepatic or intestinal district. These significant and promising preliminary findings promote, in the future, other investigations on polyphenolic by-products and on their putative or possible re-utilization in fish feeding.

Toxoplasmosis is a re-emerging food-borne zoonosis of warm-blooded animals caused by Toxoplasma gondii, an obligate intracellular parasite with a complex biological cycle. Until now, the genotypic approach for discovering putative resistance immuno-genetic markers has never been adopted for T. gondii infection. Thus, this study aimed, for the first time, to deepen these novel insights. In particular, pigs, reared in different farms in Central Italy, were phenotypically characterized by serological assays performed on diaphragm meat juice. Out of 179 tested animals, 98 resulted seropositive (54.74%), 57 seronegative (31.84%), and 24 doubtful (13.40%), underlining a possible re-emerging diffusion of this protozoan in the investigated areas. Contextually, an RT-PCR assay, followed by Sanger sequencing for IL-18, a pro-inflammatory cytokine with a key role upstream of the infection, was developed. At this stage, for this interleukin, significant polymorphic variations were not detected compared to the reference sequence, except in a seropositive animal. However, the starting outcomes of this novel and preliminary study will be investigated in depth with different approaches also on other target genes, with a crucial activity in the immunity pathway towards T. gondii, to unveil the possible presence of resistance genetic mechanisms and, finally, to make pork derived food safer.

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.

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.

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.

Many studies highlighted potential associations of β-casein A1 with specific human diseases and a minor digestibility of milk, due to the bioactive peptide β-casomorphin 7 (BCM-7) release during digestion. Conversely, the ancestral β-casein A2 variant seems to be a favorable trait because it is not associated with BMC-7 release. The aim of this work was to evaluate frequencies of β-casein variants in offspring of previously genotyped cows inseminated with A2 homozygous semen. The frequency of the A2/A2 animals has almost doubled from 37 to 69%. These are encouraging results with the perspective of reaching the goal of producing A2 milk.

A direct sandwich enzyme-linked immunosorbent assay (sELISA) was developed for the detection of the atypical β2-toxin (CPB2) of Clostridium perfringens. Polyclonal (PAbs) and monoclonal (MAbs) antibodies were previously obtained employing recombinant CPB2 produced in the baculovirus system as antigen. In the current study, PAbs were used as capture molecules, while purified MAbs conjugated to horseradish peroxidase (MAbs-HRP) were used for the detection of atypical CPB2 toxin. MAbs 5C11E6 and 2G3G6 showed high reactivity, sensitivity and specificity when tested on 232 C. perfringens cell culture isolates. In addition, a reactivity variation among different strains producing atypical CPB2 toxin was observed using the conformation-dependent MAb 23E6E6, suggesting the hypothesis of high instability and/or the existence of different three-dimensional structures of this toxin. Results obtained by sELISA and Western blotting performed on experimentally CPB2-contaminated feces revealed a time-dependent proteolytic degradation as previously observed with the consensus allelic form of CPB2. Finally, the sELISA and an end-point PCR, specific for the atypical cpb2 gene, were used to test field samples (feces, rectal swabs and intestinal contents) from different dead animal species with suspected or confirmed clostridiosis. The comparison of sELISA data with those obtained with end-point PCR suggests this method as a promising tool for the detection of atypical CPB2 toxin.

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.

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.

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.

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.

A 40 years old male Nile crocodile (Crocodylus niloticus) was diagnosed with pulmonary mycobacteriosis caused by a member of Mycobacterium chelonae/abscessus group. Post-mortem examination showed a severe systemic visceral granulomatous involvement, with lesions in lungs, heart, liver, spleen and kidneys. Histopathological examination of lung, spleen, heart and liver revealed multifocal to coalescing granulomas showing eterophils in central zone and outer rim of epithelioid histiocytes, multinucleated giant cells and lymphocytes. The Ziehl–Neelsen histological staining revealed rare vacuoles containing numerous alcohol-acid resistant bacteria. Mycobacterial infection was confirmed by culture and PCR targeting rRNA 16S gene. Sequence analysis of the DNA amplicon revealed a 100% homology with the M. chelonae/ abscessus group. Even if the classification of the memebrr of this group is still on updating, to the best of our knowledge, this is the first report of M. chelonae/abscessus member infection in a Nile crocodile species.

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.

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.

AimsTo evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis-infected dairy herds. Methods and ResultsBoth plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one-stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two-stage plant. ConclusionsMycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two-stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit. Significance and Impact of the StudyOur data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP.