The Gut Microbiota of Farmed and Wild Brook Trout (Salvelinus fontinalis): Evaluation of Feed-Related Differences Using 16S rRNA Gene Metabarcoding.

The gut microbiota has become a topic of increasing importance in various fields, including aquaculture. Several fish species have been the subject of investigations concerning the intestinal microbiota, which have compared different variables, including the intestinal portions, the environment, and diet. In this study, the microbiota of farmed and wild brook trout (Salvelinus fontinalis) were analyzed, in which the wall and content of the medial portion of the intestine were considered separately. A total of 66 fish (age class 2+) were sampled, of which 46 were wild and 20 were farmed brook trout, in two different years. Microbiota data were obtained using a 16S metabarcoding approach by analyzing the V3-V4 hypervariable regions of the corresponding 16S rRNA. The data showed that the core microbiota of these species consist of Proteobacteria (Alpha- and Gammaproteobacteria), Actinobacteria, Firmicutes (Bacilli and Clostridia), and, only for farmed animals, Fusobacteria. The latter taxon's presence is likely related to the fishmeal-based diet administered to farmed brook trout. Indeed, alpha and beta diversity analysis showed differences between wild and farmed fish. Finally, statistically significant differences in the microbiota composition were observed between the intestinal walls and contents of wild fish, while no differences were detected in reared animals. Our work represents the first study on the intestinal microbiota of brook trout with respect to both farmed and wild specimens. Future studies might focus on the comparison of our data with those pertaining to other fish species and on the study of other portions of the brook trout intestine.

Analysis of the bacterial communities associated with pupae and winged or wingless adults of Lipoptena fortisetosa collected from cervids in Italy.

The hippoboscid Lipoptena fortisetosa Maa, 1965 is a hematophagous ectoparasite of cervids that can bite humans. This fly is expanding its geographical range and is of concern for animal and human health since it can potentially harbour harmful microorganisms. This study was aimed at characterizing the bacterial communities of L. fortisetosa in its different life-cycle stages. Pupae and wingless adults were collected from cervids hunted in Tuscan-Emilian Apennines (central Italy) and pooled into groups of 10 by life stage (30 individual pupae; 1420 individual wingless adults). Winged flies were caught by sweep netting and separated into five pools of 10 insects. After DNA extraction, the bacterial content of each pool was analysed using 16 S metabarcoding. Results revealed that the composition and relative abundance of different taxa greatly differed in the three analysed groups. Wingless adults showed a high abundance of Bartonella (33.07%), which is almost absent in winged flies and pupae. Among the detected pathogens, four genera of concern for human health were found: Bartonella, Moraxella, Mycobacterium and Rickettsia. Interestingly reads similar to Bartonella bovis, Moraxella osloensis and Arsenophonus lipopteni Operational Taxonomic Unit (OTUs) were detected. These findings suggest the possible role of L. fortisetosa as a reservoir of pathogenic microorganisms, confirming the need for further investigation to ascertain its vectorial capacity.

Prokaryotic Diversity of Ruminal Content and Its Relationship with Methane Emissions in Cattle from Kazakhstan.

In this study, we analyzed the microbial composition of the rumen contents of cattle from Kazakhstan. Specifically, samples of the liquid and solid fractions of the rumen were collected to determine the quantitative and qualitative composition of methanogenic archaea. Cattle were six steers receiving hay-concentrate feeding. Methane emission was determined by repeated measurements for each animal. Rumen samples were then taken from fistulas and analyzed using 16S metabarcoding via Next-Generation Sequencing (NGS). The difference between the rumen fractions was investigated, resulting in differential distribution of the families Streptococccaceae, Lactobacillaceae, Desulfobulbaceae, and Succinivibrionaceae, which were more abundant in the liquid fraction, while Thalassospiraceae showed a higher presence in the solid fraction. These differences can be explained by the fact that fibrolytic bacteria are associated with the solid fraction compared to the liquid. A relationship between methane emission and methanogenic microbiota was also observed. Steers producing more methane showed microbiota richer in methanogens; specifically, most Mathanobacteriaceae resided in the liquid fraction and solid fraction of animals 1 and 6, respectively. The same animals carried most of the Methanobrevibacter and Methanosphaera genera. On the contrary, animals 2, 3, and 5 hosted a lower amount of methanogens, which also agreed with the data on methane emissions. In conclusion, this study demonstrated a relationship between methane emission and the content of methanogenic archaea in different rumen fractions collected from cattle in Kazakhstan. As a result of the studies, it was found that the solid fraction of the rumen contained more genera of methanogens than the liquid fraction of the rumen. These results prove that taking rumen contents through a fistula is more useful than taking it through a probe. The presented data may be of interest to scientists from all over the world engaged in similar research in a comparative aspect.

Hepatitis E Virus (HEV): Identification of Subtypes 3b and 3m in Wild Boar Population in Liguria Region, Italy.

The wild boar is an important natural reservoir for the zoonotic transmission of the hepatitis E virus (HEV) around the world. In particular, HEV genotypes 3 and 4 are an emerging problem in industrialized countries, as the number of wild boars has increased, and their territory is ever closer to farms and populated areas. This study describes the HEV prevalence and geographic circulation among wild boar populations in the Ligurian region (Italy) during the period 2019-2022. Liver samples from 849 wild boars were analyzed for HEV RNA using real-time RT-PCR; positive samples were then subjected to sequencing and phylogenetic analysis. Overall, 6.7% of the wild boars were positive for HEV RNA; however, in the last two years, the percentage of positive animals almost doubled. Phylogenetic analysis showed that wild boar HEV sequences belonged to genotype 3 and clustered within subtypes 3a and 3c, and, for the first time in Italy, subtypes 3b and 3m were identified. Interestingly, 13 sequences could not be assigned to a recognized subtype. Furthermore, the results showed different circulations of identified subtypes across the territory. These findings increase the knowledge of HEV-3 heterogeneity in Italy and describe the role of wild boars in maintaining an active viral circulation in the environment.

Study of cattle microbiota in different regions of Kazakhstan using 16S metabarcoding analysis.

Methane (CH4) is an important greenhouse gas (GHG). Enteric methane emissions from farmed ruminant livestock account for approximately 15% of global GHG emissions, with approximately 44% of livestock emissions in the form of methane. The purpose of the research is to study the influence of feeding types and regional characteristics of Kazakhstan on the microbiota of feces and the number of methane-forming archaea of beef and meat-and-dairy cattle productivity. For this purpose, fecal samples were taken rectally from 37 cattle heads from four regions of Kazakhstan (Western, Southern, Northern and Southeast). The taxonomic composition of the community in all samples was determined by 16S metabarcoding; additionally alpha and beta diversities were calculated. The dominant phyla were: Firmicutes (57.30%), Bacteroidetes (17.00%), Verrucomicrobia (6.88%), Euryarchaeota (6.49%), Actinobacteria (4.77%) and Patescibacteria (3.38%). Significant differences with regard to methanogens bacteria were found: Euryarchaeota were less present in animals from Western Kazakhstan (2.40%), while Methanobacteriales and Methanobrevibacter were prevalent in Southeast, and less abundant in Western region. Western Kazakhstan differs from the other regions likely because animals are mainly grazed in the pasture. Thus, grazing animals has an impact on their microbiota thus leading to a decrease in methane emissions.

Validation of suitable reference genes for quantitative expression analysis by qPCR in bovine terminal ileum and ileocecal valve.

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The aim of this study was to identify a set of reference genes suitable for gene expression analysis in the distal portion of small intestine and ileocecal valve in cattle. These sites of intestine are of interest in veterinary science as they are the main sites of inflammation caused by Mycobacterium avium subsp. paratuberculosis, agent of paratuberculosis. We employed ten PCR assays for commonly used reference genes belonging to various functional classes and then determined their expression stability. The most stable genes were RPL13A and HMBS, followed by TFRC and B-ACT. NormFinder analysis provided similar results with B-ACT as the best reference gene, followed by RLP13A and TFRC. This validated gene panel may be useful for studies on paratuberculosis aiming to identify genes differentially expressed by qRT-PCR.

First detection of an Italian human-to-cat outbreak of SARS-CoV-2 Alpha variant - lineage B.1.1.7.

The emergence of new SARS-CoV-2 variants and their rapid spread pose a threat to both human and animal health and may conceal unknown risks. This report describes an Italian human-to-cat outbreak of SARS-CoV-2 lineage B.1.1.7 (the Alpha variant) . On March 7th, 2021, approximately ten days after COVID-19 appeared in the family, the onset of respiratory signs in a cat by COVID-19-affected owners led to an in-depth diagnostic investigation, combining clinical and serological data with rt-qPCR-based virus detection and whole genome sequencing. The Alpha variant was confirmed first in the owners and a few days later in the cat that was then monitored weekly: the course was similar with one-week lag time in the cat. In addition, based on comparative analysis of genome sequences from our study and from 200 random Italian cases of Alpha variant, the familial cluster was confirmed. The temporal sequence along with the genomic data support a human-to-animal transmission. Such an event emphasizes the importance of studying the circulation and dynamics of SARS-CoV-2 variants in humans and animals to better understand and prevent potential spillover risks or unwarranted alerts involving our pet populations.

Regucalcin expression profiles in veal calf testis: validation of histological and molecular tests to detect sex steroids illicit administration.

BACKGROUND: Sex steroids administration in meat producing animals is forbidden within the EU to preserve consumers' safety, but continuous monitoring to identify resurgence of their misuse is needed. Among biomarkers related to sex steroids abuse in veal calves the regucalcin (RGN) mRNA perturbations in testis have been described in RNAlater samples. To setup novel diagnostic method, to update current tests available in National Residue Control Plans (NRCPs) and in legal dispute when illicit practices on farm animals are suspected, the reliability of RGN profiling was assessed by histological and molecular techniques. METHODS: Formalin fixed paraffin embedded (FFPE) testis samples, chosen being the most effective preservation strategy adopted by histological NRCPs and allowing easier retrospective analysis if required by legal disputes, were analyzed from veal calves treated with nandrolone, 17ß-estradiol and a cocktail of the two hormones. RGN levels were determined by quantitative Real Time PCR and Immunohistochemistry assays. Test performances were assessed and compared by multiple ROC curves. RESULTS: Both tests resulted sensitive and specific, allowing to enrich, in future field investigation, novel integrated diagnostic protocols needed to unveil sex steroid abuse. DISCUSSION: Developed RT-qPCR and IHC methods confirmed RGN as a useful and robust biomarker to detect illegal administration of sex steroid hormones in veal calves. The developed methods, successfully applied to ten years old FFPE blocks, could allow both retrospective analysis, when supplementary investigations are requested by authorities, and future implementation of current NRCPs.

Tissue and species identification in minced meat and meat products from Italian commercial markets by DNA microarray and histological approach.

Adequate testing and adulterant detection of food products are required to assure its safety and avoid fraudulent activities. Adulteration/substitution of costlier meat with a cheaper or inferior meat is one of the most common fraudulence in meat industry. Aim of this study was to check the correct labelling of meat and ready to cook bovine meat products, combining the DNA microarray approach to identify the animal species with the histological examination, to check the composition and safety of meat. One hundred and one samples of bovine minced meat (Group 1) and ready to cook meat products (Group 2) were collected from supermarkets in Turin, Italy. DNA microarray revealed that 25.7% of samples were positive for species not declared on the label, swine being the most common. Histology showed the presence of cartilage, bone and glandular tissue. A higher presence of bacteria and inflammatory cells was detected in Group 1. Bacterial cells associated to inflammatory cells were detected with a higher score in Group 2. Sarcocystis spp. were present in 83.3% samples of Group 1 and 49.1% of Group 2. This study confirmed that the mislabelling of meat products is not uncommon. The combination of DNA microarrays and histology can increase the monitoring capacity in bovine meat industry.

Specific capture and whole-genome phylogeography of Dolphin morbillivirus.

Dolphin morbillivirus (DMV) is considered an emerging threat having caused several epidemics worldwide. Only few DMV genomes are publicly available. Here, we report the use of target enrichment directly from cetacean tissues to obtain novel DMV genome sequences, with sequence comparison and phylodynamic analysis. RNA from 15 tissue samples of cetaceans stranded along the Italian and French coasts (2008-2017) was purified and processed using custom probes (by bait hybridization) for target enrichment and sequenced on Illumina MiSeq. Data were mapped against the reference genome, and the novel sequences were aligned to the available genome sequences. The alignment was then used for phylogenetic and phylogeographic analysis using MrBayes and BEAST. We herein report that target enrichment by specific capture may be a successful strategy for whole-genome sequencing of DMV directly from field samples. By this strategy, 14 complete and one partially complete genomes were obtained, with reads mapping to the virus up to 98% and coverage up to 7800X. The phylogenetic tree well discriminated the Mediterranean and the NE-Atlantic strains, circulating in the Mediterranean Sea and causing two different epidemics (2008-2015 and 2014-2017, respectively), with a limited time overlap of the two strains, sharing a common ancestor approximately in 1998.

Gait analysis: Comparative evaluation of conventional total knee replacement and modular distal femoral megaprosthesis.

BACKGROUND: Gait alterations have been studied with computer-assisted gait analysis after megaprosthetic replacement for tumors around the knee. It has never been proven that megaprostheses affects gait more than total knee arthroplasty (TKA); this study aims to compare via gait analysis patients who underwent megaprosthesis with patients with TKA. METHODS: We analyzed 26 patients with a megaprosthetic replacement of the distal femur and 21 patients with a standard TKA. For each subject computerized gait analysis was performed. Range of motion (ROM) of the knee was recorded, Quality of Life and functional evaluation in the oncologic group were assessed with the Musculoskeletal Tumor Society (MSTS) questionnaire, while Short Form-36 (SF-36) scores were calculated for both groups. RESULTS: All patients walked slower than healthy people (P < 0.05). Gait analysis showed a lower cadence than in the healthy population but no significant difference between the two groups. A longer swing and a shorter stance phase were detected in the megaprosthetic sample. The osteoarthritis group showed greater flexion during the phase of loading response, even if this was lower than the contralateral limb or healthy population. There was a statically significant difference between the healthy limb and the operated one in both groups regarding ROM, but no significant difference was registered between the two implants. MSTS score and most of SF-36 parameters showed no significant differences compared with literature data. CONCLUSIONS: Gait analysis shows little discrepancy between the two groups; gait pattern abnormalities do not affect patients with a megaprosthetic replacement more significantly than patients undergoing TKA.

Polymorphisms associated to bovine paratuberculosis: investigation of their role in DNA-protein interactions and transcriptional regulation.

Previous studies led to identify SNPs in putative regulatory regions of the SLC11A1 and CARD15 genes with association to paratuberculosis in cattle. Aim of this study was to investigate the role of these mutations at the regulatory level by DNA-protein interaction analyses and transcriptome comparison between wild-type and mutated animals. Gene regions carrying the SNPs of interest were analysed by bioinformatic tools to predict allele-dependent binding sites for transcription factors (TFBS). Putative TFBS were in vitro explored by Electrophoretic Mobility Shift Assays (EMSA). EMSA did not show specific gel shifts for any allele indicating that these SNPs may eventually influence gene transcription without altering TFBS. Whole transcriptome expression analysis was performed on intestinal tissues of wild-type and mutated cattle by RNA-Seq. Differential regulation of five genes involved in innate immune system was detected. Specifically, ULBP3 was down-regulated, while S100A8, S100A12, LOC510860, and IFI27 were up-regulated. In previous studies, ULBP3, S100A8, and S100A12 resulted differentially expressed in cattle affected by paratuberculosis, suggesting a possible implication in the pathogen response. Further investigations are needed to elucidate the functional role of these SNPs and to understand the gene network involved in the interactions between non-coding SNPs and other genome regions.

Real-time PCR assay for detecting illicit steroid administration in veal calves allows reliable biomarker profiling of formalin-fixed, paraffin-embedded (FFPE) archival tissue samples.

In the context of mRNA biomarker profiling, formalin fixed paraffin embedded (FFPE) samples represent an interesting source for retrospective analysis. However, the implementation in routine analysis of FFPE samples, following legislation demands for validated and accredited methods, often requires critical revision and optimization. In the frame of an official control program the validation study of a molecular test for detection of sex steroids administration in calves, based on quantification of progesterone-Receptor mRNA in bulbo-urethral gland samples, was performed: analyses were made on FFPE tissues sections routinary used for histological investigations and compared with RNAlater tissue preservation. To overcome the limitations of original assays several modifications were tested. Obtained results confirmed how Progesterone-Receptor assay represent a useful tool to study suspected cases of sex steroid illicit administration in veal calves, complementary to histological and/or immune histochemical investigation, overcoming the limitation of field studies, where optimal pre-analytical condition cannot always be guarantee.

A single nucleotide variant in the promoter region of the CCR5 gene increases susceptibility to arthritis encephalitis virus in goats.

BACKGROUND: The small ruminant lentiviruses (SRLVs) are a heterogeneous group of viruses that includes caprine arthritis encephalitis virus (CAEV) and Maedi-Visna virus (MVV). SRLVs affect the production and welfare of sheep and goats worldwide. There is currently no effective treatment. Their high mutation rate precludes vaccine development, making innovative control measures necessary. A variant of the chemokine (C-C motif) receptor 5 (CCR5) gene is reportedly involved in resistance to human immunodeficiency (HIV) infection in humans and to SRLV in sheep. The aim of this study was to analyse the genetic structure and variability of the CCR5 gene in goats and to carry out a cross-sectional study to investigate the role of CCR5 genetic variants in controlling susceptibility/resistance to CAEV. RESULTS: The variant g.1059 T located in the promoter region revealed an interesting association with high proviral loads (a 2.8-fold increased risk). A possible explanation could be an alteration of the transcriptional level. Overexpression of the CCR5 receptor on the cell surface may increase virus internalization and proviral load as a consequence. CONCLUSIONS: Our findings could be advantageously used to reduce the susceptibility of goat herds to CAEV by negatively selecting animals carrying the g.1059 T mutation. Eliminating animals predisposed to high proviral loads could also limit the development of clinical signs and the spread of the virus, since these animals are also highly efficient in shedding the virus.

A case study on the labeling of bottarga produced in Sardinia from ovaries of grey mullets (Mugil cephalus and Mugil capurrii) caught in Eastern Central Atlantic coasts.

The aim of this case study is to show how traditional and molecular methods can be employed to identify the Mugilidae species currently used in Sardinia (Italy) to produce the traditional bottarga for the processing of their ovaries. A total of six specimens of Mugil cephalus (n=3) and Mugil capurrii (n=3) were subjected to external morphology and meristic measurements. Subsequently, tissue samples of white muscle and ovaries from three individuals per species were underwent PCR-sequencing assay of mitochondrial DNA cytochrome oxidase subunit I (COI). The external morphology and meristic characters showed a sufficient level of reliability in the identification between the two species. At the same time, the molecular techniques showed the discriminatory power and confirmed the correct species identification in all the sampling units. DNA barcoding may be an effective aid to traditional taxonomy and can facilitate accurate species identification among the Mugilidae.

High density SNP mapping and QTL analysis for time of leaf budburst in Corylus avellana L.

The growing area of European hazelnut (Corylus avellana L.) is increasing, as well as the number of producing countries, and there is a pressing need for new improved cultivars. Hazelnut conventional breeding process is slow, due to the length of juvenile phase and the high heterozygosity level. The development of genetic linkage maps and the identification of molecular markers tightly linked to QTL (quantitative trait loci) of agronomic interest are essential tools for speeding up the selection of seedlings carrying desired traits through marker-assisted selection. The objectives of this study were to enrich a previous linkage map and confirm QTL related to time of leaf budburst, using an F1 population obtained by crossing Tonda Gentile delle Langhe with Merveille de Bollwiller. Genotyping-by-Sequencing was used to identify a total of 9,999 single nucleotide polymorphism markers. Well saturated linkage maps were constructed for each parent using the double pseudo-testcross mapping strategy. A reciprocal translocation was detected in Tonda Gentile delle Langhe between two non-homologous chromosomes. Applying a bioinformatic approach, we were able to disentangle 'pseudo-linkage' between markers, removing markers around the translocation breakpoints and obtain a linear order of the markers for the two chromosomes arms, for each linkage group involved in the translocation. Twenty-nine QTL for time of leaf budburst were identified, including a stably expressed region on LG_02 of the Tonda Gentile delle Langhe map. The stability of these QTL and their coding sequence content indicates promise for the identification of specific chromosomal regions carrying key genes involved in leaf budburst.

Isolation of a gene encoding for a class III peroxidase in female flower of Corylus avellana L.

Hazelnut is a monoecious species characterized by mid-winter blooming and sporophytic incompatibility. The molecular mechanisms at the basis of the female flower development and of the pollen-stigma interaction are little known, although pollination in this species is a critical factor to ensure good yield. Differential display technique was used to study genes expressed during the female flower development, comparing styles before emergence from the bud and styles at full bloom. The full-length cDNA clone, designated CavPrx (Corylus avellana peroxidase) and isolated in mature styles, was characterized as a sequence encoding for a 330 amino acids protein, containing all the conserved features of class III peroxidases. CavPrx resulted expressed only in styles, with a peak in mature styles pollinated with compatible pollen. Class III peroxidases are expressed in several different plant tissue types and are involved in a broad spectrum of physiological processes. Until now, four peroxidases expressed in the stigma were identified in Arabidopsis thaliana and Senecio squalidus: they were assumed to be possibly involved in pollen-pistil interaction, pollen tube penetration/growth and/or in defence against pathogens. CavPrx is the first gene for a floral peroxidase isolated in hazelnut and its expression pattern suggests a possible role in the pollination process.