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Platelet lysate (PL) is investigated as a potential replacement for fetal bovine serum (FBS) in cell culture. However, there is limited research on its impact on the immune profile of equine mesenchymal stromal cells (eMSCs). This study aimed to evaluate the effects of different PL formulations on the proliferative capacity, multipotentiality, and immune profile of equine adipose tissue-derived MSCs (eAD-MSCs). In vitro growth kinetics and trilineage differentiation of eAD-MSCs (n = 7) were assessed under three culture conditions: medium-concentration PL (MPL), high-concentration PL (HPL), and FBS as a control. The immune profile was evaluated by studying the expression of immunogenic receptors such as MHC I, MHC II, and immunomodulatory molecules IL-6, IL-10, and TNF-α, determined by gene expression, surface marker expression, and cytokine quantification. Both PL formulations, pooled from 5 donors, exhibited 3.3 and 6.5-fold higher platelet counts than baseline plasma for MPL and HPL, respectively. Higher concentrations of TGF-ß and PDGF were found in both PL formulations compared to baseline. Furthermore, MPL and HPL subcultures demonstrated proliferative, clonogenic, and multipotent capacities similar to FBS. The immune profile of PL-cultured cells exhibited gene expression levels related to immunogenicity and immunomodulation similar to the reference condition, and the surface antigen presence of MHC II was also similar. However, HPL media exhibited higher IL-6, IL-10, and TNF-α concentrations in the culture supernatant. In conclusion, both PL media contained higher concentrations of growth factors compared to FBS, supporting the in vitro culture of eAD-MSCs with proliferative, clonogenic, and multipotent capacity similar to the reference medium. Nonetheless, PL usage led to a variation in the immunomodulatory cytokine microenvironment, with higher concentrations of IL-6, IL-10, and TNF-α in HPL media compared to MPL and FBS.
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Bovine respiratory disease (BRD) is a common global health problem in dairy cattle. The definitive diagnosis of BRD is complex because its etiology involves several predisposing and determining factors. This report describes the etiology of a BRD outbreak in a dairy herd in the mesoregion of Central Eastern Paraná, which simultaneously affected young (calves and heifers) and adult (cows) Holstein-Friesian cattle. Nine biological samples, consisting of five lung samples from two cows and three suckling calves, and four nasal swab samples from heifers, were used for etiological diagnosis. The nucleic acids extracted from lung fragments and nasal swabs were subjected to PCR and RT-PCR assays for partial amplification of the genes of five viruses [bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoAHV1), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (BPIV-3), and bovine coronavirus (BCoV)] and four bacteria (Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) involved in the etiology of BRD. All nine biological samples from the animals with BRD tested negative for BoAHV1, BRSV, BPIV-3, BCoV, and H. somni. Therefore, the involvement of these microorganisms in the etiology of BRD outbreak can be ruled out. It was possible to identify the presence of BVDV and M. bovis in singular and mixed infections of the lower respiratory tract in cattle. BVDV was also identified in two nasal swabs: one as a single etiological agent and the other in association with two bacteria (P. multocida and M. haemolytica). The phylogenetic analysis conducted in the nucleotide sequence of the 5'UTR region and Npro gene of the BVDV amplicons demonstrated that the BVDV field strains of this BRD outbreak belong to subgenotype 2b. To the best of our knowledge, this is the first report of BVDV-2b involvement in the etiology of BRD in Brazil. Finally, it is necessary to highlight that the cattle were obtained from an open dairy herd with biannual vaccinations for BVDV-1a and - 2a.
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Bovine leukemia virus (BLV) establishes a lifelong persistent infection in dairy cattle. White blood cell count (WBC) is correlated with proviral load in the blood and milk of BLV-infected cattle, and testing WBC can be used to assess both BLV infectiousness levels and risk of BLV transmission from different types of infected animals. The objective of the study was to compare effective transmission rates (ß) and the basic reproduction ratio (R o) among two types of BLV-infected dairy cows in Chile: those affected with persistent lymphocytosis (PL) vs. aleukemic (AL).The estimated (ß) coefficient was higher in PL cattle [1.1; 95% Confidence interval (CI) (-1.6, 3.8)], compared to AL cattle (-3.1; 95% CI = -3.7, -2.5). In addition, the R o was higher in PL cattle (60.4; 95% CI = 3.5; 820.6), compared to AL cattle (1.5; 95% CI = 0.7, 3.1). The ratio between PL/AL expected rate of cases was 73.9. The estimated effective transmission rate and the Ro were higher in PL cattle compared to AL cattle. The WBC test is a convenient alternative that can be considered for risk identification and risk management of BLV infection in dairy herds; particularly in livestock regions where laboratory capacity is limited (e.g., use of PCR or gene sequencing techniques) and/or molecular tests are not cost-effective. Therefore, when prevalence of infection is high, the removal of PL cattle should be engaged to control BLV within-herds.
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The translation of silver-based nanotechnology 'from bench to bedside' requires a deep understanding of the molecular aspects of its biological action, which remains controversial at low concentrations and non-spherical morphologies. Here, we present a hemocompatibility approach based on the effect of the distinctive electronic charge distribution in silver nanoparticles (nanosilver) on blood components. According to spectroscopic, volumetric, microscopic, dynamic light scattering measurements, pro-coagulant activity tests, and cellular inspection, we determine that at extremely low nanosilver concentrations (0.125-2.5µg ml-1), there is a relevant interaction effect on the serum albumin and red blood cells (RBCs). This explanation has its origin in the surface charge distribution of nanosilver particles and their electron-mediated energy transfer mechanism. Prism-shaped nanoparticles, with anisotropic charge distributions, act at the surface level, generating a compaction of the native protein molecule. In contrast, the spherical nanosilver particle, by exhibiting isotropic surface charge, generates a polar environment comparable to the solvent. Both morphologies induce aggregation at NPs/bovine serum albumin ≈ 0.044 molar ratio values without altering the coagulation cascade tests; however, the spherical-shaped nanosilver exerts a negative impact on RBCs. Overall, our results suggest that the electron distributions of nanosilver particles, even at extremely low concentrations, are a critical factor influencing the molecular structure of blood proteins' and RBCs' membranes. Isotropic forms of nanosilver should be considered with caution, as they are not always the least harmful.
Assuntos
Eritrócitos , Nanopartículas Metálicas , Soroalbumina Bovina , Prata , Prata/química , Nanopartículas Metálicas/química , Eritrócitos/metabolismo , Eritrócitos/química , Humanos , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Propriedades de Superfície , Animais , Bovinos , Coagulação Sanguínea/efeitos dos fármacos , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/química , Teste de MateriaisRESUMO
After ejaculation, mammalian sperm undergo a series of molecular events conducive to the acquisition of fertilizing competence. These events are collectively known as capacitation and involve acrosomal responsiveness and a vigorous sperm motility called hyperactivation. When mimicked in the laboratory, capacitating bovine sperm medium contains bicarbonate, calcium, albumin and heparin, among other components. In this study, we aimed at establishing a new capacitation protocol for bovine sperm, using calcium ionophore. Similar to our findings using mouse sperm, bovine sperm treated with Ca2+ ionophore A23187 were quickly immobilized. However, these sperm initiated capacitation after ionophore removal in fresh medium without heparin, and independent of the Protein Kinase A. When A23187-treated sperm were used on in vitro fertilization (IVF) procedures without heparin, eggs showed cleavage rates similar to standardized IVF protocols using heparin containg synthetic oviduct fluid (IVF-SOF). However, when A23187 pre-treated sperm were further used for inseminating eggs in complete IVF-SOF-heparin, a significantly higher percentage of embryo development was observed, suggesting a synergism between two different signaling pathways during bovine sperm capacitation. These results have the potential to improve current protocols for bovine IVF that could also be applied in other species of commercial interest.
Assuntos
Calcimicina , Ionóforos de Cálcio , Criopreservação , Fertilização in vitro , Preservação do Sêmen , Capacitação Espermática , Espermatozoides , Animais , Bovinos , Masculino , Ionóforos de Cálcio/farmacologia , Criopreservação/veterinária , Criopreservação/métodos , Fertilização in vitro/veterinária , Fertilização in vitro/métodos , Calcimicina/farmacologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Capacitação Espermática/efeitos dos fármacos , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Feminino , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacosRESUMO
The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70 kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.
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Apoptose , Proteínas Quinases Dependentes de AMP Cíclico , Células Epiteliais , Ciclo Estral , Transdução de Sinais , Animais , Feminino , Células Epiteliais/metabolismo , Bovinos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclo Estral/fisiologia , Ciclo Estral/metabolismo , Oviductos/metabolismo , Oviductos/citologia , Tubas Uterinas/metabolismo , Tubas Uterinas/citologia , FosforilaçãoRESUMO
Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.
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Lactoferrina , Mannheimia haemolytica , Metaloproteases , Proteólise , Lactoferrina/metabolismo , Lactoferrina/farmacologia , Metaloproteases/metabolismo , Metaloproteases/antagonistas & inibidores , Animais , Apoproteínas/metabolismo , Apoproteínas/química , Simulação de Acoplamento Molecular , Ovinos , Bovinos , Colagenases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Zinco/metabolismoRESUMO
OBJECTIVE: To evaluate the histomorphometric and computerized microtomographic (Micro-CT) analysis of the regenerated bone tissue from maxillary sinus augmentation surgery, with and without using the collagen membrane on the external osteotomy window. MATERIALS AND METHODS: Twelve patients were selected for this prospective, controlled, and randomized study. The patients were submitted to bilateral maxillary sinus surgery in a split-mouth design. On the test side, the maxillary sinus augmentation procedure included using Geistlich Bio-Oss® and a Geistlich Bio-Gide® collagen membrane covering the lateral osteotomy window. On the control side, only Geistlich Bio-Oss® was used without the presence of the membrane. After 6 months, the surgeries for implant installation were performed. In this surgical phase, specimens of the regenerated tissue were collected for histological and Micro-CT analysis. RESULTS: In the histomorphometric evaluation, the mean (±SD) percentages of newly formed bone were 43.9% (±11.5) and 40.8% (±8.9) in the test and control groups, respectively. The corresponding values of the Micro-CT analysis were 36.6% (±3.4) and 37.2% (±4.7) in the test and control groups, respectively. There was no statistically significant difference between the test and control groups in the two methods. In addition, there was no statistically significant difference between the mean percentage of biomaterial remaining between the test and control groups. However, the mean percentage of newly formed bone was significantly higher and the mean percentage of remaining biomaterial was significantly lower in the histomorphometric analysis compared to the values obtained through microtomography. CONCLUSION: The additional use of collagen membranes in maxillary sinus surgery does not offer advantages in newly formed bone.
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Regeneração Óssea , Colágeno , Levantamento do Assoalho do Seio Maxilar , Microtomografia por Raio-X , Humanos , Levantamento do Assoalho do Seio Maxilar/métodos , Masculino , Feminino , Estudos Prospectivos , Pessoa de Meia-Idade , Minerais , Membranas Artificiais , Substitutos Ósseos/uso terapêutico , Adulto , Seio Maxilar/cirurgia , Seio Maxilar/diagnóstico por imagem , Seio Maxilar/patologia , Implantação Dentária Endóssea/métodosRESUMO
Herpesviruses are significant pathogens of ruminants. In water buffaloes (Bubalus bubalis), however, herpesviruses have not been thoroughly studied. Although bubaline alphaherpesvirus 1 (BuAHV1) and bovine alphaherpesvirus 1 (BoAHV1) have already been recovered from water buffaloes, to date, no reports on the occurrence of bovine alphaherpesvirus 5 (BoAHV5) in these animals have been published. Therefore, the aim of this study was to search for BuAHV1, BoAHV1, and BoAHV5 in palatine tonsils of apparently healthy water buffaloes from the Pará state, Northern Brazil. Tissue samples of tonsils (n = 293) were screened by a nested PCR (nPCR) targeting a region of UL44 (gC coding gene), followed by sequencing, to detect and differentiate between the viral types. Viral genome segments were detected in 18 out of 293 (6.1%) of the palatine tonsil samples. Two animals carried genomes of BoAHV1 only, eleven animals carried BoAHV5 genomes only, and four animals carried BuAHV1 only. Another animal had both BoAHV1 and BoAHV5 genomes in its tonsils. No infectious virus could be recovered from any of the samples. The BuAHV1 sequences identified here were more closely related to BuAHV1 genomes identified in India. Phylogenetic analyses suggested a closer relationship between the recovered BoAHV5 and BuAHV1 genomes. Therefore, evidence is provided here to confirm that not only BoAHV1 and BuAHV1, but also BoAHV5, can infect water buffaloes. This report highlights (i) the first detection of BoAHV5 in water buffaloes and (ii) the occurrence of coinfections with BoAHV1 and BoAHV5 in that species. Such findings and the similarity of BoAHV5 to Indian herpesvirus genomes suggest that the origin of type 5 may be linked to recombinations between bovine and bubaline herpesviruses within bubalines, since the scenario for generation of recombinants in buffaloes is potentially present.
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Alphaherpesvirinae , Búfalos , Infecções por Herpesviridae , Tonsila Palatina , Animais , Bovinos , Alphaherpesvirinae/genética , Alphaherpesvirinae/isolamento & purificação , Alphaherpesvirinae/classificação , Brasil , DNA Viral/genética , Genoma Viral , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Tonsila Palatina/virologia , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
The reproductive efficiency and milk yield of cows are crucial factors in a dairy farm's profitability. However, abortions can have a negative impact on these factors. While the morbidity of abortion has been estimated in many countries, information on the burden on dairy cattle in tropical conditions is limited, and Costa Rica is a good example. This study aims to assess the incidence and recurrence of bovine abortion in dairy cattle from Costa Rica. The study analysed the morbidity of abortion in Costa Rican dairy herds between 2010 and 2022. The incidence rate (IR) and the recurrence rate (ReR) were calculated per 100 cow-months at risk using data from the Veterinary Automated Management and Production Control Programme (VAMPP). The dataset comprised 1032,457 lactations from 330,265 cows in 1134 specialized dairy herds. Abortions were classified either as early foetal mortality (EFM) or late foetal mortality (LFM). Rates were estimated based on cow breed, lactation number, and ecological zone to which the farm belongs. The IR of general abortion, EFM, and LFM cases were 0.98, 0.41, and 0.57 per 100 cow-months at risk, respectively. No statistically significant differences were found in the IR between cow breed, lactation number, and ecological zone, nor for the trend of abortions over calving years. The first ReR (for cows that had one previous abortion during the lactation) was 0.95, and the second ReR (for cows that had two previous abortions during the lactation) was 1.41 per 100 cow-months at risk. These results suggest that bovine abortions are an important ongoing problem in dairy farms in Costa Rica with potentially detrimental effects on the reproductive and productive performance of cows and may be representative of other specialized tropical dairy systems in Latin America.