Effect of the Season on Blood Changes of Oxidative Stress Index in the Italian Mediterranean Buffalo (Bubalis bubalis).

Studies in cattle have shown that high temperatures increase the production of reactive oxygen species (ROS) causing an imbalance between ROS and the ability of antioxidant systems to detoxify and remove the reactive intermediates. As such studies remain limited in buffalo, the effect of temperature on oxidative stress was investigated through the oxidative stress index (OSi). Blood samples were collected from 40 buffaloes over 12 time points distributed over two years (2021, 2022). Samples were taken monthly during the hot and cold seasons. Plasma free oxygen radicals were determined using the d-ROMs test (Diacron, Italy), modified for a microplate procedure, and the results were expressed in arbitrary Carratelli Units (U.CARR). Plasma antioxidants were determined by using the BAP test (Diacron) in a dedicated spectrophotometer (Carpe Diem Free, Diacron). The OSi parameter was calculated as d-ROMs/BAP × 100. Temperature and humidity were recorded daily during the trial to calculate the Temperature Humidity Index (THI). For statistical analysis, year and season and their interactions were included in the model. The results of this study showed for the first time the effect of season on the oxidative stress in buffalo. The minimum and maximum THI values for the hot and cold season recorded during the experimental period were 79.27 ± 2.20 and 63.42 ± 3.20, respectively. Levels of d-ROMs and BAP were affected by the seasons (133.0 vs. 145.1 U.CARR, p = 0.0189, and 2489.19 vs. 2392.43 mml/L, p = 0.033, in the hot and cold season, respectively). A significant year × season interaction was found both for d-ROMs and BAP (p = 0.06 and p < 0.0001, respectively). Moreover, OSi was affected by season, showing a growing trend from hot to cold season (5.35 vs. 6.17, p < 0.0001), but, interestingly, it was unaffected by annual variation. Therefore, Osi could be considered a better and independent marker of oxidative status in buffalo, with respect to the evaluation of single determinations of d-ROMs and BAP. Lastly, there were no differences in the plasma 25OHD levels between seasons; concentrations were 12.24 and 10.26 ng/mL in the hot and cold season, respectively.

Candidate genes associated with heat stress and breeding strategies to relieve its effects in dairy cattle: a deeper insight into the genetic architecture and immune response to heat stress.

The need for food products of animal origin is increasing worldwide. Satisfying these needs in a way that has minimal impact on the environment requires cutting-edge technologies and techniques to enhance the genetic quality of cattle. Heat stress (HS), in particular, is affecting dairy cattle with increasing frequency and severity. As future climatic challenges become more evident, identifying dairy cows that are more tolerant to HS will be important for breeding dairy herds that are better adapted to future environmental conditions and for supporting the sustainability of dairy farming. While research into the genetics of HS in the context of the effect of global warming on dairy cattle is gaining momentum, the specific genomic regions involved in heat tolerance are still not well documented. Advances in omics information, QTL mapping, transcriptome profiling and genome-wide association studies (GWAS) have identified genomic regions and variants associated with tolerance to HS. Such studies could provide deeper insights into the genetic basis for response to HS and make an important contribution to future breeding for heat tolerance, which will help to offset the adverse effects of HS in dairy cattle. Overall, there is a great interest in identifying candidate genes and the proportion of genetic variation associated with heat tolerance in dairy cattle, and this area of research is currently very active worldwide. This review provides comprehensive information pertaining to some of the notable recent studies on the genetic architecture of HS in dairy cattle, with particular emphasis on the identified candidate genes associated with heat tolerance in dairy cattle. Since effective breeding programs require optimal knowledge of the impaired immunity and associated health complications caused by HS, the underlying mechanisms by which HS modulates the immune response and renders animals susceptible to various health disorders are explained. In addition, future breeding strategies to relieve HS in dairy cattle and improve their welfare while maintaining milk production are discussed.

Evaluation of Hematological Profiles and Monocyte Subpopulations in Water Buffalo Calves after Immunization with Two Different IBR Marker Vaccines and Subsequent Infection with Bubaline alphaherpesvirus-1.

Bubaline alphaherpesvirus-1 (BuAHV-1) and Bovine alphaherpesvirus-1 (BoAHV-1) are respiratory viruses that can cause an infection known as "Infectious Bovine Rhinotracheitis" (IBR) in both water buffalo and bovine species. As the main disease control strategy, vaccination can protect animals from clinical disease through the development of specific humoral and cell-mediated immune responses. In the present study, the time-related circulatory kinetics of hematological profile and bubaline monocyte subsets have been investigated in vaccinated buffalo calves after challenge infections with BuAHV-1. Thirteen buffalo calves were selected and grouped into the VAX-1 group, which received an IBR-live-attenuated gE-/tk-deleted marker vaccine; the VAX-2 group, which received an IBR-inactivated gE-deleted marker vaccine; the CNT group, which remained an unvaccinated control. Fifty-five days after the first vaccination, the animals were infected with 5 × 105.00 TCID50/mL of wild-type BuAHV-1 strain via the intranasal route. Whole blood samples were collected at 0, 2, 4, 7, 10, 15, 30, and 63 days post-challenge (PCDs) for the analysis of hematological profiles and the enumeration of monocyte subsets via flow cytometry. The analysis of leukocyte compositions revealed that neutrophils were the main leukocyte population, with a relative increase during the acute infection. On the other hand, a general decrease in the proportion of lymphocytes was observed early in the post-infection, both for the VAX-1 and VAX-2 groups, while in the CNT group, the decrease was observed later at +30 and +63 PCDs. An overall infection-induced increase in blood total monocytes was observed in all groups. The rise was especially marked in the animals vaccinated with an IBR-live-attenuated gE-/tK-deleted marker vaccine (VAX-1 group). A multicolor flow cytometry panel was used to identify the bubaline monocyte subpopulations (classical = cM; intermediate = intM; and non-classical = ncM) and to investigate their variations during BuAHV-1 infection. Our results showed an early increase in cMs followed by a second wave of intMs. This increase was observed mainly after stimulation with live-attenuated viruses in the VAX-1 group compared with the animals vaccinated with the inactivated vaccine or the non-vaccinated animal group. In summary, the present study characterized, for the first time, the hematological profile and distribution of blood monocyte subsets in vaccinated and non-vaccinated water buffalo in response to experimental infection with BuAHV-1. Although not experimentally proven, our results support the hypothesis of a linear developmental relationship between monocyte subsets.

Flow cytometric detection of IFN-γ production and Caspase-3 activation in CD4+ T lymphocytes to discriminate between healthy and Mycobacterium bovis naturally infected water buffaloes.

Tuberculosis has a negative economic impact on buffalo farming, and it poses a potential threat to human health. Interferon-gamma (IFN-γ) plays a central role in protection against mycobacterial diseases, illustrating the importance of T-cell mediated immune responses in tuberculosis infection. Recently, the expression of Caspase-3, a critical executor of apoptosis, in M. tuberculosis-specific IFN-γ+CD4+ T cells was used as a new marker to distinguish active from latent tuberculosis infection in humans. The aims of this work were to develop a whole blood flow cytometric assay to detect the production of IFN-γ and the activation of Caspase-3 by CD4+ T lymphocytes from water buffalo and to evaluate whether these parameters can discriminate between healthy and M. bovis naturally infected buffaloes. A total of 35 Italian Mediterranean buffaloes were grouped in two groups: uninfected and M. bovis infected (based on the results of antemortem diagnostic tests: single intradermal tuberculin (SIT) and ELISA IFN-γ tests). Whole blood was incubated for 6 h with tubercular antigens: PPD-B, PPD-A, ESAT-6/CFP-10 and a new mix of precocious secreted antigens (PA). Our results showed a significant increase in the percentage of IFN-γ+CD4+ T cells in infected compared to the uninfected animals after each stimulus. Improved sensitivity of the assay was obtained by including the stimulation with the new mix of PA. Interestingly, we observed a concomitant decrease in percentage of Caspase-3+CD4+ T cells in M. bovis infected animals compared to the control healthy ones, regardless of the stimulus used. Overall, these results showed that M. bovis infection activates CD4+ T lymphocytes to produce IFN-γ and at the same time causes a concomitant decrease of Caspase-3 activation in CD4+ T cells. This study for the first time in water buffalo describes the development of a whole blood flow cytometric assay for the detection of IFN-γ producing CD4+ T cells and proposes the expression of active Caspase-3 as an additional bovine TB biomarker. Although further studies are needed to better understand the mechanisms of Caspase-3-mediated cell death during tuberculosis, our data can help to better understand the cellular immune response to M. bovis infection in buffalo species.

In-depth immunophenotyping reveals significant alteration of lymphocytes in buffalo with brucellosis.

Water buffalo (Bubalus bubalis) has a prominent position in the livestock industry worldwide but still suffers from limited knowledge on the mechanisms regulating the immune against infections, including brucellosis (BRC), one of the most significant neglected zoonotic diseases of livestock. Seventy-three buffalo were recruited for the study. Thirty-five were naturally infected with Brucella spp. The aims of the study were to (i) verify the cross-reactivity of 16 monoclonal antibodies (mAbs) developed against human, bovine, and ovine antigens; (ii) evaluate lymphocyte subset alterations in BRC positive buffalo; (iii) evaluate the use of the canonical discriminant analysis (CDA), with flow cytometric data, to discriminate BRC positive from negative animals. A new set of eight mAbs (anti CD3e, CD16, CD18, CD45R0, CD79a; CD172a) were shown to cross-react with water buffalo orthologous molecules. BRC positive animals presented a significant (p < 0.0001) decrease in the percentage of PBMC (29.5 vs. 40.3), total, T and B lymphocytes (23.0 vs. 35.5, 19.2 vs. 28.9, 2.6 vs. 5.7, respectively). In contrast, they showed an increase in percentage of granulocytes (65.2 vs. 55.1; p < 0.0001) and B lymphocytes CD21neg (22.9 vs. 16.1; p = 0.0067), a higher T/B lymphocyte ratio (10.3 vs. 6.4; p = 0.0011) and CD3+ /CD21+ (14.7 vs. 8.3; p = 0.0005) ratio. The CDA, applied to 33 different flow cytometric traits, allowed the discrimination of all BRC positive from negative buffalo. Although this is a preliminary study, our results show that flow cytometry can be used in a wide range of applications in livestock diseases, including in support of uncertain BRC diagnoses.

BCR::ABL1 levels at first month after TKI discontinuation predict subsequent maintenance of treatment-free remission: A study from the "GRUPPO TRIVENETO LMC".

We analyzed BCR::ABL1 expression at stop and in the first month after discontinuation in 168 chronic myeloid leukemia patients who stopped imatinib or 2nd generation tyrosine kinase inhibitors (2G-TKIs) while in sustained deep molecular response. Patients were divided among those who maintained response (group 1, n = 123) and those who lost major molecular response (group 2, n = 45). Mean BCR::ABL1 RNA levels 1 month after discontinuation were higher in group 2 than in group 1 (p = 0.0005) and the difference was more evident 2 months after stop (p < 0.0001). The same trend was found both for imatinib and 2G-TKIs. A receiver operating characteristic (ROC) analysis to determine a threshold value of BCR::ABL1 at 1 month after discontinuation identified a cut-off value of 0.0051%, with 92.2% specificity, 31.7% sensitivity and a likelihood ratio of 4.087.

Hyperthermia-induced changes in leukocyte survival and phagocytosis: a comparative study in bovine and buffalo leukocytes.

Heat stress negatively affects health, welfare, and livestock productivity by impairing immune function, increasing disease incidence. In recent years, there has been increasing interest in understanding the immune system of water buffalo due to the growing economic impact of this species for the high quality and nutritional value of buffalo milk. While there are common responses across bovine and buffalo species, there are also some species-specific variations in the physiological responses to heat stress, mainly attributed to differences in metabolism and heat dissipation efficiency. At cellular level, the exposure to thermal stress induces several anomalies in cell functions. However, there is limited knowledge about the differential response of bovine and buffalo leucocytes to early and late exposure to different degrees of thermal exposure. The aim of this study was to compare the in vitro effect of hyperthermia on apoptosis and phagocytosis in leukocytes from bovine and buffalo species. For this, whole blood samples of six bovines and nine buffaloes were incubated at 39°C (mimicking normothermia condition) or 41°C (mimicking heat stress condition) for 1, 2, and 4 h. Two flow cytometric assays were then performed to evaluate apoptosis and determine functional capacity of phagocytic cells (neutrophils and monocytes). The results showed that the viability of bovine and buffalo leukocytes was differently affected by temperature and time of in vitro exposure. A higher percentage of apoptotic leukocytes was observed in bovines than in buffaloes at 39°C (3.19 vs. 1.51, p < 0.05) and 41°C (4.01 vs. 1.69, p < 0.05) and for all incubation time points (p < 0.05). In contrast, no difference was observed in the fraction of necrotic leukocytes between the two species. In both species, lymphocytes showed the highest sensitivity to hyperthermia, showing an increased apoptosis rates along with increased incubation time. In bovine, apoptotic lymphocytes increased from 5.79 to 12.7% at 39°C (p < 0.05), in buffalo, this population increased from 1.50 to 3.57% at 39°C and from 2.90 to 4.99% at 41°C (p < 0.05). Although no significant differences were found between the two species regarding the percentage of phagocytic neutrophils, lower phagocytosis capacity values (MFI, mean fluorescence intensity) were found in bovines compared with buffaloes at 41°C (27960.72 vs. 53676.45, p > 0.05). However, for monocytes, the differences between species were significant for both phagocytosis activity and capacity with lower percentages of bovine phagocytic monocytes after 2 h at 39°C and after 1 h at 41°C. The bovine monocytes showed lower MFI values for all temperature and time variations than buffaloes (37538.91 vs. 90445.47 at 39°C and 33752.91 vs. 70278.79 at 41°C, p < 0.05). In conclusion, the current study represents the first report on the comparative analysis of the effect of in vitro heat stress on bovine and buffalo leukocyte populations, highlighting that the leukocytes of buffalo exhibit relatively higher thermal adaptation than bovine cells.

Combined effects of CXCL8 (IL-8) and CXCR2 (IL-8R) gene polymorphisms on deregressed MACE EBV indexes of milk-related traits in Simmental bulls.

CXCL8 (also known as IL-8) is a member of the CXC subfamily of chemokines that binds two of the seven transmembrane G-protein-coupled receptors (GPCRs), CXCR1 and CXCR2, to mediate and regulate leucocyte accumulation and activation at sites of inflammation. They are known to play a critical role in both disease susceptibility and infection outcome. The aim of this study was to investigate the entire sequences of CXCL8 and CXCR2 genes in thirty-one Simmental sires to evaluate the effects of genomic variants on the indexes of the bulls for milk, fat and protein yields, and for somatic cell score (SCS). Five new single nucleotide polymorphisms (SNPs) were found in CXCR2 gene. The analysis of association indicated that one SNP in CXCL8 and two in CXCR2 influenced the considered traits. To evaluate the existence of functional haplotypic effects, combinations among the three genomic variants (SNP 1 in CXCL8, SNP 6 and SNP 7 in CXCR2) were investigated. Four different haplotypic alleles were identified in the experimental population, one of which at a high frequency (61%). Bulls with Hap 4 (G-C-G at SNP 1, SNP 6, and SNP 7 respectively) had more favourable indexes for SCS (P < 0.05). These results suggest that the SNPs in CXCL8 and CXCR2 may be potential genetic markers to improve udder health in the Simmental breed.

Interferon Tau (IFNt) and Interferon-Stimulated Genes (ISGs) Expression in Peripheral Blood Leukocytes and Correlation with Circulating Pregnancy-Associated Glycoproteins (PAGs) during Peri-Implantation and Early Pregnancy in Buffalo Cows.

The objective of this study was to analyze interferon-stimulated genes (ISGs) and interferon tau (IFNt) gene expression in peripheral blood leukocytes during the peri-implantation period and until 40 days of pregnancy in buffalo cows. Relationships were also examined between the expression of ISGs and IFNt and pregnancy-associated glycoproteins (PAGs) peripheral plasma concentration. Buffalo cows were synchronized and artificially inseminated (d 0). Blood samples were collected on days 0, 18, 28 and 40 after artificial insemination (AI) for peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) isolation and PAGs radioimmunoassay analysis. The study was carried out on 21 buffalo cows divided ex post into Pregnant (n = 12) and Non-pregnant (n = 9) groups. Steady state levels of OAS1, MX2, ISG15 and IFNt mRNA were measured by RT-qPCR and their estimated marginal means (p < 0.01 for all) were higher in pregnant than non-pregnant buffaloes, both in PBMCs and PMNs. In PBMCs, pairwise comparisons showed that OAS1 and MX2 expressions differed between pregnant and non-pregnant buffaloes on all the days of observation (p < 0.001), while significant differences in ISG15 and IFNt started from day 28 post-AI (p < 0.05). In PMNs, ISG15 expression differed between groups only at days 18 and 28 (p < 0.001), while comparisons were always significant for IFNt (p < 0.05). The expression of all genes, except ISG15 as determined in PMNs, was positively associated with PAGs plasma concentrations (p < 0.05). This work showed a significant increase in ISGs and IFNt expressions in PBMCs and PMNs in buffalo during the peri-implantation period and early pregnancy, and their correlation with PAGs plasma concentration.

Comprehensive phenotyping of peripheral blood monocytes in healthy bovine.

Monocytes are bone marrow derived innate myeloid cells that circulate in the blood and play important roles in infection and inflammation. As part of the mononuclear phagocytic system, monocytes provide innate effector functions, support the adaptive immune response, and play a role in the maintenance of tissue homeostasis. In addition to their role in sensing pathogen-associated molecular patterns using several pattern recognition receptors, monocytes are characterized by their ability to ingest and kill microbes, to produce cytokines and chemokines, and to present antigens to T cells. For a long time, monocytes have been considered as a homogenous cell population, characterized by the expression of CD14, the receptor of lipopolysaccharide. Studies in several species have shown that the monocyte population consists of phenotypically and functionally different cell subsets. In this review, we report a comprehensive phenotyping of monocyte subsets in cattle. In addition, the most characterizing cell markers and gating strategies for detailed immunophenotyping of bovine monocyte subsets are discussed.

Novel SNPs and haplotypes identified in the CD4 gene and their influence on deregressed MACE EBV indexes of milk-related traits in Simmental breed.

Cluster of differentiation 4 (CD4) is the accessory protein non-covalently bound to the T cell receptor that recognizes an invariant region of MHC class II on antigen presenting cells. Its cytoplasmic tail, physically associated with a protein tyrosine kinase, is important in the activation of helper/inducer T lymphocytes. In Bos taurus, CD4 gene is located on chromosome 5 from which two isoforms are transcribed, with a different number of amino acids due to splicing of exon 7 and variation in the reading frame. The aim of this study was to investigate the sequence of the entire CD4 gene in Simmental sires to evaluate the effects of genomic variants on the indexes of the bulls for milk, fat and protein yields, as well as somatic cell score. The associations among genomic variants and indexes were analysed using the Allele and GLM procedures of SAS 9.4. The analysis indicated that only four of the thirty-one identified SNPs influenced the considered traits. Identified variants insist on coding zones and intronic sequences, where we revealed the presence of sites for transcription factors. To evaluate the existence of haplotypic effects, combinations among the four genomic variants (SNP 3, SNP 8, SNP 11 and SNP 19) were investigated. Six different haplotypic alleles were identified, but only four of them were frequent enough to allow for an evaluation of any haplotypic effect (at least six copies in the examined sample): Hap1, Hap2, Hap3 and Hap6. The analysis of associations between the selected haplotypes in the CD4 gene with milk related indexes showed that bulls with Hap2 (T-A-C-C) had better indexes for milk and protein yields (P < 0.05), whereas the presence of the Hap1 haplotype (A-G-A-T) caused a significant decrease of the index for protein yield (P < 0.05). Frequencies of the two alleles Hap1 and Hap2 (9 and 36% respectively) make them of interest for their possible inclusion in breeding schemes and support the hypothesis of considering this gene as a candidate for the improvement of milk-related traits in the Simmental breed.

Comparison of metabolic, oxidative and inflammatory status of Simmental × Holstein crossbred with parental breeds during the peripartal and early lactation periods.

The aim of the research reported in this paper was to evaluate plasma concentrations of energy, oxidative and inflammatory biomarkers of Simmental (sire) × Holstein (dam) crossbred cows, in comparison with the two parental breeds during the peripartal and early lactation periods and to estimate the effects of heterosis for these traits. Thirty-three animals, managed under the same conditions, 8 Simmental (SI), 9 Holstein (HO) and 16 crossbred (CR) cows were enrolled in this study. Glucose, non-esterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), total protein, albumin, creatinine and urea were determined in blood sampled at six different time points (30 ± 3 and 15 ± 3 d before the expected calving date, at calving and 15, 30 and 60 d after calving). Furthermore, derived reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP), interleukin-6 (IL-6), haptoglobin (Hp) and serum amyloid A protein (SAA) were determined to evaluate inflammatory and oxidative status. Results showed that the CR group had significantly lower average values of glucose and NEFA when compared to HO group; signifcantly lower values of urea than SI group and significantly higher values of creatinine than HO. Furthermore, CR cows showed the lowest average value of d-ROMs with respect to SI and HO parental breeds. Finally, the average value of haptoglobin was significantly lower in CR and HO groups, when compared to SI group. As for the heterosis we found the highest (positive) percentage for CK (98%) and BAP (47%) and the lowest (negative) percentage for OSi (-75%) and d-ROMs (-39%). A negative percentage was also found for the glucose (-11%) and NEFA (-20%) toward the Simmental parental breed. Our results suggest a different response among the three genetic groups during the peripartal and early lactation periods. In particular, CR and SI cows seem more adaptable regarding energy metabolism and oxidative status. Heterosis led to a positive effect on those parameters in Simmental (sire) × Holstein (dam) crossbred cows F1 population (50% Simmental and 50% Holstein).

Effect of IL8 haplotype on immunological traits in periparturient dairy cows.

Interleukin 8 (IL8) is a major mediator of the innate immune response. Polymorphisms in this gene are associated with susceptibility to inflammatory disease in humans. Two major promoter polymorphic haplotypes (IL8-h1 and IL8-h2) segregating in cattle populations have shown a significant effect on the immune response profile in calves but their implications for transition cow immunity have not been established. The aims of this study were to assess functional relevance of the IL8 haplotypes on the immunological traits of periparturient cows (n = 32) belonging to three genetic groups: Holstein (HO), Simmental (SI) and their crosses (CR) and to evaluate the frequency of IL8 haplotypes in the HO (dairy) and SI (dual purpose) pure breeds. IL8 haplotypes showed a significant effect on circulating number of both T helper lymphocytes (P = 0.0133) and T cytotoxic lymphocytes (P = 0.0024). Differences in percentage of CD14+ monocytes and T lymphocyte subsets were found between haplotype groups at different time points. Plasma concentrations of Serum Amyloid A (SAA) and Haptoglobin (Hp) were enhanced at calving in IL8-h2 (P = 0.0019, P = 0.0029) and IL8-het (P = 0.050 and P = 0.052) respectively, compared with IL8-h1 cows. In contrast, significantly lower levels of reactive oxygen metabolites (d-ROMs) activation were identified in IL8-h2 and IL8-het cows after calving compared with IL8-h1 cows. Furthermore, genotyping results showed that SI cows have a high frequency of the homozygous IL8-h2 haplotype compared to the HO cows (87.5 % vs 40 %) which reflects the different selective pressure between the two pure breeds. In conclusion, our preliminary data suggests that IL8 promoter haplotype is associated with significant and dynamic changes in immunological traits during peripartum and early lactation period. Future work will focus on a more comprehensive assessment of immune changes in additional cows.

Increased PARylation impacts the DNA methylation process in type 2 diabetes mellitus.

BACKGROUND: Epigenetic modifications, such as DNA methylation, can influence the genetic susceptibility to type 2 diabetes mellitus (T2DM) and the progression of the disease. Our previous studies demonstrated that the regulation of the DNA methylation pattern involves the poly(ADP-ribosyl)ation (PARylation) process, a post-translational modification of proteins catalysed by the poly(ADP-ribose) polymerase (PARP) enzymes. Experimental data showed that the hyperactivation of PARylation is associated with impaired glucose metabolism and the development of T2DM. Aims of this case-control study were to investigate the association between PARylation and global and site-specific DNA methylation in T2DM and to evaluate metabolic correlates. RESULTS: Data were collected from 61 subjects affected by T2DM and 48 healthy individuals, recruited as controls. Global levels of poly(ADP-ribose) (PAR, a surrogate of PARP activity), cytosine methylation (5-methylcytosine, 5mC) and de-methylation intermediates 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were determined in peripheral blood cells by ELISA-based methodologies. Site-specific DNA methylation profiling of SOCS3, SREBF1 and TXNIP candidate genes was performed by mass spectrometry-based bisulfite sequencing, methyl-sensitive endonucleases digestion and by DNA immuno-precipitation. T2DM subjects presented higher PAR levels than controls. In T2DM individuals, increased PAR levels were significantly associated with higher HbA1c levels and the accumulation of the de-methylation intermediates 5hmC and 5fC in the genome. In addition, T2DM patients with higher PAR levels showed reduced methylation with increased 5hmC and 5fC levels in specific SOCS3 sites, up-regulated SOCS3 expression compared to both T2DM subjects with low PAR levels and controls. CONCLUSIONS: This study demonstrates the activation of PARylation processes in patients with T2DM, particularly in those with poor glycaemic control. PARylation is linked to dysregulation of DNA methylation pattern via activation of the DNA de-methylation cascade and may be at the basis of the differential gene expression observed in presence of diabetes.

New Insights into the Significance of PARP-1 Activation: Flow Cytometric Detection of Poly(ADP-Ribose) as a Marker of Bovine Intramammary Infection.

Bovine intramammary infections are common diseases affecting dairy cattle worldwide and represent a major focus of veterinary research due to financial losses and food safety concerns. The identification of new biomarkers of intramammary infection, useful for monitoring the health of dairy cows and wellness verification, represents a key advancement having potential beneficial effects on public health. In vitro experiments using bovine peripheral blood mononuclear cells (PBMC), stimulated with the bacterial endotoxin lipopolysaccharide (LPS) enabled a flow cytometric assay in order to evaluate in vivo poly-ADP-ribose (PAR) levels. Results showed a significant increase of PAR after 1 h of treatment, which is consistent with the involvement of PARP activity in the inflammatory response. This study investigated PARP-1 activation in leukocyte subpopulations from bovine milk samples during udder infection. A flow cytometric assay was, therefore, performed to evaluate the PAR content in milk leukocyte subsets of cows with and without intramammary infection (IMI). Results showed that milk lymphocytes and macrophages isolated from cows with IMI had a significant increase of PAR content compared to uninfected samples. These results suggest mastitis as a new model for the study of the role of PARP in zoonotic inflammatory diseases, opening a new perspective to the "One Health" approach.

Evaluation of Safety and Efficacy of an Inactivated Marker Vaccine against Bovine alphaherpesvirus 1 (BoHV-1) in Water Buffalo (Bubalus bubalis).

Recent studies have explored the seropositivity of Bovine alphaherpesvirus 1 (BoHV-1) in water buffaloes, suggesting the urgency for developing strategies to eradicate the virus involving both cattle and water buffaloes. However, in Europe, the glycoprotein E (gE) deleted marker vaccines against BoHV-1 are commercially available only for the cattle industry. This study, for the first time, evaluated the safety and efficacy of a commercial inactivated gE-deleted marker vaccine in water buffalo. Five animals devoid of BoHV-1-neutralizing antibodies were vaccinated via intramuscular route. Five additional animals served as an unvaccinated control group. Sixty days after the first immunization, all animals were experimentally infected with a virulent BoHV-1via intranasal route. A detectable BoHV-1-humoral immune response was observed in the vaccinated group on post-vaccination day 30, whereas the antibodies appeared on post-challenge day 10 in the control group. Moreover, the vaccinated animals neither show viral shedding nor clinical signs compared to the control upon challenge. However, post-challenge, the BoHV-1-specific humoral and cell-mediated immune responses were significantly more increased in vaccinated animals than the control animals. Overall, the present study provides evidence of both the safety and efficacy of an inactivated gE-deleted marker vaccine against BoHV-1 in water buffaloes.

Assessment of Multicolor Flow Cytometry Panels to Study Leukocyte Subset Alterations in Water Buffalo (Bubalus bubalis) During BVDV Acute Infection.

The identification of cross-reactive monoclonal antibodies (mAbs) that recognize orthologous leukocyte differentiation molecules (LDM) in buffaloes has overcome a major impediment limiting research on the immune response to pathogens and development of vaccines. As reported, two pilot trials were conducted to accomplish two objectives: (1) demonstrate that multiparameter flow cytometry can be conducted equally well in buffalo with mAbs directly and indirectly labeled with fluorochromes in research and (2) flow cytometry can be used to compare and extend studies on diseases of economic importance to buffalo using bovine viral diarrhea virus (BVDV) as a model pathogen. Pregnant buffalo cows were infected with BVDV-1 at 81 (trial 1) and 203 (trial 2) days post artificial insemination and flow cytometric evaluations were performed at 0, 3, 4, and 14 days after infection (dpi). Fluorochrome conjugated mAbs were used in trial 1, and fluorochrome conjugated goat isotype specific anti-mouse antibodies were used to label mAbs in trial 2. Flow cytometric analysis revealed a transient lymphopenia occurs during the 1st days following infection similar to lymphopenia reported in cattle. In particular, significant differences were observed between pre- and post-infection absolute values of T lymphocytes (-56%, P < 0.01). CD21+ B lymphocytes (-65%, P = 0.04), and Natural Killer cells (-72%, P < 0.001). No significant differences were observed in monocytes and neutrophil absolute values, or the CD4:CD8 ratio. Animal health status was followed until 15 days after calving. No clinical signs of infection were observed during the evaluation period, however, animals in trial 1 developed complications later the infection. One cow aborted at 57 days post-infection, the second cow developed a prolapse a day after calving and died. These two animals also showed a more pronounced lymphopenia in comparison with animals infected at 203 days of pregnancy (e.g., -77 vs. -22% T lymphocytes at 3 dpi, respectively). The pilot studies have demonstrated that it is possible to use multicolour multiparameter flow cytometry to study the immune response to pathogens affecting the health of buffalo.

Simmental × Holstein crossbred: comparison of immunological traits with parental breeds during peripartum and early lactation period.

The experiment described in this research communication aimed to compare the immunological traits of Simmental (sire) × Holstein (dam) crossbred cows with the two parental breeds in the peripartum and early lactation period and to estimate the effects of heterosis for these traits. Flow cytometric evaluation of leukocyte subpopulations was assessed in 16 Crossbred (CR), 8 Holstein (HO) and 8 Simmental (SI) cows. Estimated average values of innate and adaptive immune cells showed statistically significant differences between the crossbred cows and parental breeds. Interestingly, the most relevant differences between the three groups related to adaptive immune cells. In particular, the CR cows showed a lower percentage of CD3+ T lymphocytes compared with the SI group (P < 0.0001) and the highest proportions of CD21+ B lymphocytes among the three groups (P < 0.0001). Furthermore, we found the highest positive value of heterosis for the CD21+ B lymphocytes (7.0) and the lowest negative value for CD3+ T lymphocytes (-4.8) in F1 derived population. It seems reasonable to believe that these differences could affect immune function of crossbred cows.