The effects of intradermal M. bovis and M. avium PPD test on immune-related mRNA and miRNA in dermal oedema exudates of water buffaloes (Bubalus bubalis).

Tuberculosis (TB) is a zoonotic disease primarily caused by pathogens belonging to the genus of Mycobacterium. Programs of control and eradication for bovine TB include a screening using single intradermal tuberculin (SIT) test with Mycobacterium bovis (M. bovis)-purified protein derivatives (PPD-B) single or concurrent with Mycobacterium avium (M. avium)-purified protein derivatives (PPD-A). This study aimed to determine the effects of intradermal PPD-B and PPD-A test on immune-related mRNA and microRNAs in dermal oedema exudates of water buffaloes (Bubalus bubalis). The investigation was carried out on RNA extracted from dermal oedema exudates of 36 animals, of which 24 were M. bovis positive (M. bovis+) and 12 M. avium positive (M. avium+). The lymphocyte polarization toward Th1, Th2, TReg, and Th17 lineages was addressed by measuring the abundance of the respective cytokines and transcription factors, namely TBET, STAT4, IFNγ, and IL1ß for Th1; STAT5B, and IL4 for Th2; FOXP3 and IL10 for TReg; and RORC, STAT3, and IL17A for Th17. Due to the very low abundance of Th17-related genes, a digital PCR protocol was also applied. The abundance of microRNAs involved in the immune response against PPDs, including miR-122-5p, miR-148a-3p, miR30a, and miR-455-5p, was equally measured. Results showed that IFNγ (fold change = 2.54; p = 0.037) and miR-148a-3p (fold change = 2.54; p = 0.03) were upregulated in M. bovis+ as compared to M. avium+ samples. Our preliminary results supported the pivotal role of IFNγ in the local immune response related to PPD-B and highlighted the differential expression of miR-148a-3p, which downregulates the proinflammatory cytokines and the TLR4-mediated NF-κB activation, providing an anti-inflammation modulator in responses to mycobacterial infection.

Short communication: Milk microbiota profiling on water buffalo with full-length 16S rRNA using nanopore sequencing.

The identification of milk microbial communities in ruminants is relevant for understanding the association between milk microbiota and health status. The most common approach for studying the microbiota is amplifying and sequencing specific hypervariable regions of the 16S rRNA gene using massive sequencing techniques. However, the taxonomic resolution is limited to family and, in some cases, genus level. We aimed to improve taxonomic classification of the water buffalo milk microbiota by amplifying and sequencing the full-length 16S rRNA gene (1,500 bp) using Nanopore sequencing (single-molecule sequencing). When comparing with short-read results, we improved the taxonomic classification, reaching species level. We identified the main microbial agents of subclinical mastitis at the species level that were in accordance with the microbiological culture results. These results confirm the potential of single-molecule sequencing for in-depth analysis of microbial populations in dairy animals.

MicroRNA Expression in Formalin-Fixed, Paraffin-Embedded Samples of Canine Cutaneous and Oral Melanoma by RT-qPCR.

MicroRNAs (miRNAs) are a class of small, noncoding RNA that post-transcriptionally regulate protein expression. miRNAs are emerging as clinical biomarkers of many diseases, including tumors. The aim of this study was to investigate whether miRNA expression could vary in melanoma samples derived from formalin-fixed, paraffin-embedded (FFPE) tissues. The study included 4 groups: (1) 9 samples of oral canine malignant melanoma, (2) 10 samples of cutaneous malignant melanoma, (3) 5 samples of healthy oral mucosa, and (4) 7 samples of healthy skin. The expression levels of 6 miRNAs-miR-145, miR-146a, miR-425-5p, miR-223, miR-365, and miR-134-were detected and assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using TaqMan probes. Cutaneous canine malignant melanoma showed a decrease of the expression level of miR-145 and miR-365 and an increase of miR-146a and miR-425-5p compared to control samples. MiR-145 was also downregulated in oral canine malignant melanoma. The miRNAs with decreased expression may regulate genes involved in RAS, Rap1, and transforming growth factor ß (TGF-ß) signaling pathways, as well as upregulated genes associated with phosphatidylinositol signaling system, adherens junction, and RAS signaling pathways. In conclusion, miR-145, miR-365, miR-146a, and miR-425-5p were differentially expressed in canine malignant melanoma and healthy FFPE samples, suggesting that they may play a role in canine malignant melanoma pathogenesis.

Short communication: Intra- and inter-individual milk microbiota variability in healthy and infected water buffalo udder quarters.

The concept that ruminant mammary gland quarters are anatomically and physiologically unrelated has been recently challenged by immunological evidence. How this interdependence reflects on individual quarter milk microbiota is unknown. The aim of the present study was to cover this gap by investigating the interdependence of quarters among the same mammary gland at the milk microbiota level using next-generation sequencing of the V4-16S rRNA gene. A total of 52 samples were included in this study and classified as healthy or affected by subclinical mastitis. Extraction of DNA, amplification of the V4-16S rRNA gene, and sequencing using Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA) were carried out. We found that the intra-individual variability was lower than the inter-individual one. The present findings further support at milk microbiota level the hypothesis of the interdependence of quarters, as previously demonstrated following immunological studies, suggesting that individual factors (e.g., immunity, genetics) may have a role in modulating milk microbiota.

Circulating biomarkers in familial cerebral cavernous malformation.

BACKGROUND: Cerebral Cavernous Malformation (CCM) is a rare cerebrovascular disease, characterized by the presence of multiple vascular malformations that may result in intracerebral hemorrhages (ICHs), seizure(s), or focal neurological deficits (FND). Familial CCM (fCCM) is due to loss of function mutations in one of the three independent genes KRIT1 (CCM1), Malcavernin (CCM2), or Programmed Cell death 10 (PDCD10/CCM3). The aim of this study was to identify plasma protein biomarkers of fCCM to assess the severity of the disease and predict its progression. METHODS: Here, we have investigated plasma samples derived from n = 71 symptomatic fCCM patients (40 female/31 male) and n = 17 healthy donors (HD) (9 female/8 male) of the Phase 1/2 Treat_CCM trial, using multiplexed protein profiling approaches. FINDINGS: Biomarkers as sCD14 (p = 0.00409), LBP (p = 0.02911), CXCL4 (p = 0.038), ICAM-1 (p = 0.02013), ANG2 (p = 0.026), CCL5 (p = 0.00403), THBS1 (p = 0.0043), CRP (p = 0.0092), and HDL (p = 0.027), were significantly different in fCCM compared to HDs. Of note, sENG (p = 0.011), THBS1 (p = 0.011) and CXCL4 (p = 0.011), were correlated to CCM genotype. sROBO4 (p = 0.014), TM (p = 0.026) and CRP (p = 0.040) were able to predict incident adverse clinical events, such as ICH, FND or seizure. GDF-15, FLT3L, CXCL9, FGF-21 and CDCP1, were identified as predictors of the formation of new MRI-detectable lesions over 2-year follow-up. Furthermore, the functional relevance of ang2, thbs1, robo4 and cdcp1 markers was validated by zebrafish pre-clinical model of fCCM. INTERPRETATION: Overall, our study identifies a set of biochemical parameters to predict CCM progression, suggesting biological interpretations and potential therapeutic approaches to CCM disease. FUNDING: Italian Medicines Agency, Associazione Italiana per la Ricerca sul Cancro (AIRC), ERC, Leducq Transatlantic Network of Excellence, Swedish Research Council.

Location and condition based reconstruction of colon cancer microbiome from human RNA sequencing data.

BACKGROUND: The association between microbes and cancer has been reported repeatedly; however, it is not clear if molecular tumour properties are connected to specific microbial colonisation patterns. This is due mainly to the current technical and analytical strategy limitations to characterise tumour-associated bacteria. METHODS: Here, we propose an approach to detect bacterial signals in human RNA sequencing data and associate them with the clinical and molecular properties of the tumours. The method was tested on public datasets from The Cancer Genome Atlas, and its accuracy was assessed on a new cohort of colorectal cancer patients. RESULTS: Our analysis shows that intratumoural microbiome composition is correlated with survival, anatomic location, microsatellite instability, consensus molecular subtype and immune cell infiltration in colon tumours. In particular, we find Faecalibacterium prausnitzii, Coprococcus comes, Bacteroides spp., Fusobacterium spp. and Clostridium spp. to be strongly associated with tumour properties. CONCLUSIONS: We implemented an approach to concurrently analyse clinical and molecular properties of the tumour as well as the composition of the associated microbiome. Our results may improve patient stratification and pave the path for mechanistic studies on microbiota-tumour crosstalk.

GZMKhigh CD8+ T effector memory cells are associated with CD15high neutrophil abundance in non-metastatic colorectal tumors and predict poor clinical outcome.

CD8+ T cells are a major prognostic determinant in solid tumors, including colorectal cancer (CRC). However, understanding how the interplay between different immune cells impacts on clinical outcome is still in its infancy. Here, we describe that the interaction of tumor infiltrating neutrophils expressing high levels of CD15 with CD8+ T effector memory cells (TEM) correlates with tumor progression. Mechanistically, stromal cell-derived factor-1 (CXCL12/SDF-1) promotes the retention of neutrophils within tumors, increasing the crosstalk with CD8+ T cells. As a consequence of the contact-mediated interaction with neutrophils, CD8+ T cells are skewed to produce high levels of GZMK, which in turn decreases E-cadherin on the intestinal epithelium and favors tumor progression. Overall, our results highlight the emergence of GZMKhigh CD8+ TEM in non-metastatic CRC tumors as a hallmark driven by the interaction with neutrophils, which could implement current patient stratification and be targeted by novel therapeutics.

Acute phase proteins and markers of oxidative status in water buffalos during the transition from late pregnancy to early lactation.

The transition period, from pregnancy to lactation, implies comprehensive metabolic and endocrine changes including a systemic inflammatory reaction and oxidative stress around calving in dairy cows. The aim of the present study was a longitudinal characterization of the serum concentration of acute phase proteins (APP), i.e., haptoglobin (Hp), serum amyloid A (SAA) and acidic glycoprotein (AGP), as well as of markers for oxidative stress in another large dairy animal, i.e. water buffalo, during the transition from late pregnancy to early lactation. As indicators of oxidative status, derivatives of reactive oxygen metabolites (dROM), ferric reducing ability (FRAP), thiobarbituric acid reactive substances (TBARS), and advanced oxidation protein products (AOPP) were determined in serum. Indicators for metabolic stress included nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB) and adiponectin. Bovine specific ELISA methods for Hp and adiponectin were adapted and validated for their application to water buffalo samples. Blood samples were collected weekly from 11 pluriparous water buffalo cows (lactation number 4.6 ± 1.6; daily milk yield 9.0 ± 1.9 kg; means ± SD) from 6 weeks (wk) ante partum (ap) until 8 wk post partum (pp). The maximum concentrations of Hp were observed in wk 1 pp, followed by a decrease towards values lower than before calving starting from wk 3 pp. The concentrations of SAA also peaked in wk 1 pp and then returned to basal values. The AGP serum concentrations increased suddenly from the first to the second wk pp and remained elevated for all the observation period. Indicators of oxidative status which changed in concentration during the transition period were dROM, AOPP and the oxidative stress index (OSi) (dROM/FRAP ratio). Briefly, dROM and AOPP values were lower pp as compared to ap, and OSi was largely following the pattern of dROM due to the constant FRAP values. The TBARS values did not change during the observation period. From the metabolic indicators, adiponectin was not changing with time, whereas greater NEFA and BHB values were observed ap than pp. The time course of NEFA and of some indicators for oxidative status (dROM, OSi and AOPP) point to greater metabolic load in late pregnancy as compared with the first wk of lactation - contrary to the common situation in dairy cows. Both BHB and NEFA values remained below the thresholds applied for dairy cows to define subclinical or clinical ketosis, thus indicating that the buffaloes included in this study were not under metabolic stress. The increase in concentration of the APP around calving supports the concept that an inflammatory reaction is a physiological epiphenomenon of the onset of lactation in water buffalos that is independent of metabolic stress.

In-vitro effect of heat stress on bovine monocytes lifespan and polarization.

Heat stress (HS) has a negative impact on dairy cows' health, milk production, reproductive performance and immune defenses. Cellular and molecular responses to high temperatures in bovine polymorphonuclear cells and peripheral blood mononuclear cells (PBMCs) have been investigated so far. On the contrary, the effects of high temperatures on isolated monocytes remain almost undisclosed. The aim of this study was to unravel the in vitro effects of high temperatures, simulating a severe HS related body hyperthermia, on bovine lifespan and M1/M2 polarisation. The PBMCs were isolated from whole blood of 9 healthy dairy cattle. Monocytes were sorted by magnetic activated cell sorting and cultured over night at 39 °C (normothermia) or 41 °C (HS). Apoptotic rate and viability were assessed and mRNA abundance for heat shock proteins (HSPs), heat transcription factors (HSFs) and genes involved in monocyte/macrophage polarization (STAT1, STAT2, STAT3, STAT6, IL1ß, TGF1ß, IL-10, COX2) were quantified by qPCR. We found that apoptosis increased in monocytes exposed to 41 °C, as compared to control, while viability conversely decreased. HS increased the abundance of HSF1 and HSP70. The concomitant decrease of STAT1 and STAT2 and the increase of STAT6 genes abundance at 41 °C suggest, at transcriptional factors level, a polarization of monocytes from a classical activated M1 to a non-classically activated M2 monocytes. In conclusion, the exposure of bovine monocytes to high temperatures affects their lifespan as well as the abundance of genes involved in HS response and in monocyte/macrophages polarization phenotype, confirming that bovine immune response may be significantly affected by hyperthermia.

Cerumen microbial community shifts between healthy and otitis affected dogs.

Otitis externa is a common multifactorial disease in dogs, characterized by broad and complex modifications of the ear microbiota. The goal of our study was to describe the ear cerumen microbiota of healthy dogs, within the same animal and between different animals, and to compare the cerumen microbiota of otitis affected dogs with that of healthy animals. The present study included 26 healthy dogs, 16 animals affected by bilateral otitis externa and 4 animals affected by monolateral otitis externa. For each animal cerumen samples from the right and left ear were separately collected with sterile swabs, and processed for DNA extraction and PCR amplification of the 16S rRNA gene. Amplicon libraries were sequenced using an Ion Torrent Personal Genome Machine (PGM), and taxonomical assignment and clustering were performed using QIIME 2 software. Our results indicate that the bacterial community of the cerumen in healthy dogs was characterized by extensive variability, with the most abundant phyla represented by Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Fusobacteria. The analysis of both alpha and beta diversity between pairs of left and right ear samples from the same dog within the group of affected animals displayed higher differences than between paired samples across healthy dogs. Moreover we observed reduced bacterial richness in the affected group as compared with controls and increased variability in population structure within otitis affected animals, often associated with the proliferation of a single bacterial taxon over the others. Moreover, Staphylococcus and Pseudomonas resulted to be the bacterial genera responsible for most distances between the two groups, in association with differences in the bacterial community structure. The cerumen microbiota in healthy dogs exhibits a complex bacterial population which undergoes significant modifications in otitis affected animals.

Characterization of skin surface and dermal microbiota in dogs with mast cell tumor.

The skin microbiota interacts with the host immune response to maintain the homeostasis. Changes in the skin microbiota are linked to the onset and the progression of several diseases, including tumors. We characterized the skin surface and dermal microbiota of 11 dogs affected by spontaneous mast cell tumor (MCT), using skin contralateral sites as intra-animal healthy controls. The microbial profile differed between healthy and tumor skin surfaces and dermis, demonstrating that the change in microbiota composition is related to the presence of MCT. The number of observed taxa between MCT and healthy skin surfaces was detected, showing a decrease in number and heterogeneity of taxa over the skin surface of MCT, at both inter- and intra-individual level. Preliminary data on bacterial population of MCT dermis, obtained only on three dogs, demonstrated an intra-individual reduction of taxa number when compared to the skin surface. Taxonomy reveals an increase of Firmicutes phylum and Corynebacteriaceae family in MCT skin surface when compared to the healthy contralateral. In conclusion, we demonstrate that microbial population of skin surface and dermis is related to mast cell tumor. Our study provides the basis for future investigations aiming to better define the interaction between mast cell tumors, microbiota and host immune response.

Characterization of circulating miRNA signature in water buffaloes (Bubalus bubalis) during Brucella abortus infection and evaluation as potential biomarkers for non-invasive diagnosis in vaginal fluid.

Brucellosis is an infectious disease caused by bacteria from the Brucella genus that can be transmitted to humans through contact with infected animals or contaminated animal products. Brucellosis also causes financial losses in animal production. Ruminants are highly susceptible to brucellosis, and the causative agent water buffaloes (Bubalus bubalis) is Brucella abortus. Circulating microRNAs (miRNAs) are cropping up as promising biomarkers for several infectious diseases. The goals of this study were to characterize the serum miRNA signature associated with brucellosis in water buffaloes and investigate the miRNAs' potential use as biomarkers in vaginal fluids. Next Generation Sequencing was used to assess miRNA expression profiles in Brucella-positive and Brucella-negative blood sera; dysregulated miRNAs in blood serum and vaginal fluids were validated using RT-qPCR. ROC curves were generated to evaluate the diagnostic value of miRNAs for Brucella. GO and KEGG pathway enrichment analyses were exploited to investigate the biological functions of dysregulated miRNAs. The results showed that 20 miRNAs were modulated, of which, 12 were upregulated and 8 were downregulated. These findings were corroborated by RT-qPCR, and ROC curves indicated that the miRNAs can serve as potential biomarkers for Brucella. GO and KEGG pathway analyses pointed out that some of these miRNAs are related to immune response and apoptosis. These results provided an overview of miRNA expression profiles and highlighted potential biomarkers for Brucella infection in water buffaloes. We also demonstrated the potential of vaginal fluids in studies involving microRNA detection. Further functional and mechanistic studies of these miRNAs may improve our understanding of the biological processes involved in Brucella infection and host immune response.

Impact of intramammary inoculation of inactivated Lactobacillus rhamnosus and antibiotics on the milk microbiota of water buffalo with subclinical mastitis.

Water buffalo mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies to avoid or reduce antibiotics' based therapies, in particular during subclinical mastitis. The use of Generally Regarded As Safe bacteria (GRAS) such as Lactobacillus rhamnosus to restore the unbalance in mammary gland microbiota could provide potential corrective measures. The aim of this study was to investigate the changes in milk microbiota after the intramammary treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters naturally affected by subclinical mastitis as compared to antibiotic therapy.A number of 43 quarters affected by subclinical mastitis with no signs of clinical inflammation and aerobic culture positive for pathogens were included in the study. The experimental design was as follows: 11 quarters were treated with antibiotics, 15 with inactivated cultures of Lactobacillus rhmnosus and 17 with PBS as negative control, by means of intrammary injection. Samples were collected at eight time points, pre- (T-29, T-21, T-15, T-7, T0 days) and post- treatment (T1, T2, and T6 days). Microbiological culture and Somatic Cell Count (SCC) were perfomed on all the samples, and microbiota was determined on milk samples collected at T0 and T6 by amplifying the V4 region of 16S rRNA gene by PCR and sequencing using next generation sequencing technique. Treatment with Lactobacillus rhamnosus elicited a strong chemotactic response, as determined by a significant increase of leukocytes in milk, but did not change the microbiological culture results of the treated quarters. For what concerns the analysis of the microbiota, the treatment with Lactobacillus rhamnosus induced the modification in relative abundance of some genera such as Pseudomonas and 5-7N15. As expected, antibiotic treatment caused major changes in microbiota structure with an increase of Methylobacterium relative abundance. No changes were detected after PBS treatment. In conclusion, the present findings demonstrated that the in vivo intrammmary treatment with Lactobacillus rhamnosus has a transient pro-inflammatory activity by increasing SCC and is capable to modify the microbiota of milk after six days from inoculation, albeit slightly, even when the bacterial cultures were heat inactivated. Further studies are necessary to assess the potential use of this GRAS as supportive therapy against mastitis.

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon.

Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Conclusions: Both full-length 16S rRNA and the 16S-ITS-23S of the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the 16S-ITS-23S of the rrn operon would be the best choice.

The microbiota of water buffalo milk during mastitis.

The aim of this study was to define the microbiota of water buffalo milk during sub-clinical and clinical mastitis, as compared to healthy status, by using high-throughput sequencing of the 16S rRNA gene. A total of 137 quarter samples were included in the experimental design: 27 samples derived from healthy, culture negative quarters, with a Somatic Cell Count (SCC) of less than 200,000 cells/ml; 27 samples from quarters with clinical mastitis; 83 samples were collected from quarters with subclinical mastitis, with a SCC number greater of 200,000 cells/ml and/or culture positive for udder pathogens, without clinical signs of mastitis. Bacterial DNA was purified and the 16S rRNA genes were individually amplified and sequenced. Significant differences were found in milk samples from healthy quarters and those with sub-clinical and clinical mastitis. The microbiota diversity of milk from healthy quarters was richer as compared to samples with sub-clinical mastitis, whose microbiota diversity was in turn richer as compared to those from clinical mastitis. The core microbiota of water buffalo milk, defined as the asset of microorganisms shared by all healthy milk samples, includes 15 genera, namely Micrococcus, Propionibacterium, 5-7N15, Solibacillus, Staphylococcus, Aerococcus, Facklamia, Trichococcus, Turicibacter, 02d06, SMB53, Clostridium, Acinetobacter, Psychrobacter and Pseudomonas. Only two genera (Acinetobacter and Pseudomonas) were present in all the samples from sub-clinical mastitis, and no genus was shared across all in clinical mastitis milk samples. The presence of mastitis was found to be related to the change in the relative abundance of genera, such as Psychrobacter, whose relative abundance decreased from 16.26% in the milk samples from healthy quarters to 3.2% in clinical mastitis. Other genera, such as SMB53 and Solibacillus, were decreased as well. Discriminant analysis presents the evidence that the microbial community of healthy and clinical mastitis could be discriminated on the background of their microbiota profiles.