RESUMO
Fasciolosis is an important economic disease of livestock. There is a global interest in the development of protective vaccines since the current anthelmintic therapy is no longer sustainable. A better knowledge of the host-parasite interaction is needed to design effective vaccines. To date, few studies have evaluated host-parasite interaction by comparing infected and reinfected animals. The present study evaluates the microscopical hepatic lesions in sheep infected and reinfected with Fasciola hepatica during the acute and chronic stages of infection. The histopathological study revealed the presence of necrotizing foci (NF1) associated with larvae migration during the early stages of infection in the primoinfected (PI) and reinfected (RI) groups. In the late stages of infection of the PI group and at the early and late stages of infection in the RI groups, extensive necrotizing/hemorrhagic foci (NF2) were found in the vicinity of enlarged bile ducts, some containing adult flukes, suggesting parasites may have caused NF2 while feeding. The immunohistochemical study revealed an increase in Foxp3+ T cells in both PI and RI groups with respect to the UC group and in the infiltrates adjacent to NF1 in the RI groups with respect to the PI group, suggesting the F. hepatica induce Foxp3 T cell expansion to facilitate parasite survival. In addition, in both the PI and RI groups, and during acute and chronic stages of the infection, a poor expression of iNOS was found accompanied by a strong expression of CD163, suggesting a marked M2 activation of macrophages in the hepatic lesions, which may be related with healing processes, and it also may facilitate parasite survival. The main differences between PI and RI animals were the more severe infiltration of eosinophils and Foxp3+ T cells, whereas RI did not modify M2 activation of macrophages which occurs since the early stages of primoinfection.
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Real-life pollution usually involves simultaneous co-exposure to different chemicals. Metals and drugs are frequently and abundantly released into the environment, where they interact and bioaccumulate. Few studies analyze potential interactions between metals and pharmaceuticals in these mixtures, although their joint effects cannot be inferred from their individual properties. We have previously demonstrated that the mixture (PC) of the metals Cd and Hg, the metalloid As and the pharmaceuticals diclofenac (DCF) and flumequine (FLQ) impairs hepatic proteostasis. To gain a deeper vision of how PC affects mouse liver homeostasis, we evaluated here the effects of PC exposure upon some biochemical and morphometric parameters, and on the transcriptional profiles of selected group of genes. We found that exposure to PC caused oxidative damage that exceeded the antioxidant capacity of cells. The excessive oxidative stress response resulted in an overabundance of reducing equivalents, which hindered the metabolism and transport of metabolites, including cholesterol and bile acids, between organs. These processes have been linked to metabolic and inflammatory disorders, cancer, and neurodegenerative diseases. Therefore, our findings suggest that unintended exposure to mixtures of environmental pollutants may underlie the etiology of many human diseases. Fortunately, we also found that a diet enriched with selenium mitigated the harmful effects of this combination of toxicants.
Assuntos
Fígado , Estresse Oxidativo , Selênio , Animais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Selênio/farmacologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Arsênio/toxicidade , Dieta , Diclofenaco/toxicidadeRESUMO
Gene expression for Th1/Th2 cytokines (IL-4 and IFN-É£), regulatory cytokines (TGF-ß and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-É£ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-É£ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-ß in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.
Assuntos
Citocinas , Fasciola hepatica , Fasciolíase , Fatores de Transcrição Forkhead , Doenças dos Ovinos , Animais , Fasciolíase/veterinária , Fasciolíase/prevenção & controle , Fasciolíase/imunologia , Fasciola hepatica/imunologia , Ovinos , Fatores de Transcrição Forkhead/metabolismo , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Citocinas/metabolismo , Fígado/parasitologia , Fígado/imunologia , Vacinas/imunologia , Vacinas/administração & dosagem , Células Th1/imunologia , Linfonodos/imunologia , Feminino , Células Th2/imunologiaRESUMO
Fasciola hepatica is distributed worldwide, causing substantial economic losses in the animal husbandry industry. Human fasciolosis is an emerging zoonosis in Andean America, Asia, and Africa. The control of the disease, both in humans and animals, is based on using anthelmintic drugs, which has resulted in increased resistance to the most effective anthelmintics, such as triclabendazole, in many countries. This, together with the concerns about drug residues in food and the environment, has increased the interest in preventive measures such as a vaccine to help control the disease in endemic areas. Despite important efforts over the past two decades and the work carried out with numerous vaccine candidates, none of them has demonstrated consistent and reproducible protection in target species. This is at least in part due to the high immunomodulation capacity of the parasite, making ineffective the host response in susceptible species such as ruminants. It is widely accepted that a deeper knowledge of the host-parasite interactions is needed for a more rational design of vaccine candidates. In recent years, the use of emerging technologies has notably increased the amount of data about these interactions. In the present study, current knowledge of host-parasite interactions and their implication in Fasciola hepatica vaccine development is reviewed.
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This study performs an analysis that will enable the evaluation of the quality, durability, and structure of repaired cartilaginous extracellular matrix tissue using an autologous-based particulated autograft cartilage and platelet-rich plasma treatment (PACI + PRP). A single-blind controlled experiment was conducted on 28 sheep to evaluate the efficacy of the PACI + PRP treatment for cartilage defects. Full-thickness 8 mm diameter defects were created in the weight-bearing area of both knees. The right knees received PACI + PRP. The left knees were treated with Ringer's lactate solution (RLS) or hyaluronic acid (HA) injections. Sheep were euthanized at 9- or 18-months post-surgery. An extensive immunohistochemical analysis was performed to assess collagen types (I, II, III, V, VI, IX, X, XI) and aggrecan positivity. A semiquantitative scoring system provided a detailed evaluation of immunostaining. Collagens and aggrecan scores in the PACI + PRP groups were similar to healthy cartilage. Significant differences were found in collagens associated with matrix maturity (II and V), degradation (IX), structure and mechanics (VI), and hypertrophy (X) between healthy cartilage and RLS- or HA-repaired cartilage. The PACI + PRP treatment advanced the repair cartilage process in chondral defects with mature hyaline cartilage and enhanced the structural and mechanical qualities with better consistent cartilage, less susceptible to degradation and without hypertrophic formation over time.
Assuntos
Doenças das Cartilagens , Cartilagem Articular , Plasma Rico em Plaquetas , Animais , Ovinos , Cartilagem Articular/cirurgia , Agrecanas/genética , Agrecanas/metabolismo , Método Simples-Cego , Doenças das Cartilagens/metabolismo , Plasma Rico em Plaquetas/metabolismo , Ácido Hialurônico/metabolismo , Colágeno/metabolismoRESUMO
Potential endocrine-disrupting properties of cyanotoxins, such as microcystin-LR (MC-LR) and cylindrospermopsin (CYN) are of concern due to their increasing occurrence, the scarcity of reports on the topic (particularly for CYN) and the impact of human's health at different levels. Thus, this work performed for the first time the uterotrophic bioassay in rats, following the Organization for Economic Cooperation and Development (OECD) Test Guideline 440, to explore the oestrogenic properties of CYN and MC-LR (75, 150, 300 µg/kg b.w./day) in ovariectomized (OVX) rats. Results revealed neither changes in the wet and blotted uterus weights nor in the morphometric study of uteri. Moreover, among the steroid hormones analysed in serum, the most remarkable effect was the dose-dependent increase in progesterone (P) levels in rats exposed to MC-LR. Additionally, a histopathology study of thyroids and serum levels of thyroids hormones were determined. Tissue affectation (follicular hypertrophy, exfoliated epithelium, hyperplasia) was observed, as well as increased T3 and T4 levels in rats exposed to both toxins. Taken together, these results point out that CYN and MC-LR are not oestrogenic compounds at the conditions tested in the uterotrophic assay in OVX rats, but, however, thyroid disruption effects cannot be discarded.
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Toxinas Bacterianas , Glândula Tireoide , Humanos , Animais , Ratos , Organização para a Cooperação e Desenvolvimento Econômico , Estrogênios/toxicidade , Toxinas Bacterianas/toxicidade , Toxinas de Cianobactérias , Microcistinas/toxicidade , Microcistinas/análiseRESUMO
The expression of proinflammatory (IL-1ß, IFN-γ, TNF-α) and regulatory (IL-10, TGF-ß, IL-4) cytokines, as well as the transcription factor FoxP3, was quantified in the liver and hepatic lymph node (HLN) of sheep primoinfected and reinfected with Fasciola hepatica at early (4, 8 and 16 days post-infection [dpi]) and late (100 dpi) stages. The liver exerted a Th2 immune response at very early stages after the primoinfection with F. hepatica that induced the downregulation of IFN-γ, followed by a Th1/Th2/Treg response although the late stages were characterised by the expression of Th1/Th2 immune mediators. Contrarily, in reinfected sheep a robust mixed Th1/Th2/Treg immune response was found at very early stages meanwhile at late stages we observed a Th2/Treg immune response overcoming the expression of Th1 immune mediators. However, the HLN displayed a completely different Th1/Th2/Treg expression profile compared to the liver. Primoinfections with F. hepatica in HLN induced a mixed Th1/Th2/Treg environment from early stages, establishing a Th2 immune response at a late stage. However, the reinfected sheep exerted a Th2 immune response at early stages led by the IL-4 expression in opposition to the Th1/Th2/Treg found in the liver, meanwhile at late stages the HLN of reinfected sheep exerted a mixed Th1/Th2/Treg immune response. This is the first work publishing the expression of immune mediators in the liver and HLN from reinfected sheep with F. hepatica. The study of the immune responses exerted by the natural host in the target organs directly implied in the development of F. hepatica are crucial to better understand the immunopathogenesis of the fasciolosis being a key factor to develop effective vaccines.
Assuntos
Fasciola hepatica , Fasciolíase , Doenças dos Ovinos , Ovinos , Animais , Fasciola hepatica/fisiologia , Interleucina-4 , Reinfecção/patologia , Reinfecção/veterinária , Linfócitos T Reguladores , Fasciolíase/veterinária , Fígado/patologia , Fatores de Transcrição , Imunidade , Linfonodos , Doenças dos Ovinos/patologiaRESUMO
PURPOSE: Articular cartilage is vulnerable to multiple types of damage and it has limited reparative and regenerative capacities due to its absence of vascularity. Although a large number of therapeutic strategies exist to treat chondral defects, they have some limitations, such as fibrocartilage formation. Therefore, the goal of the present study was to evaluate the chondrogenic regenerative properties of an autologous-made matrix of particulated cartilage and platelet-rich plasma (PACI + PRP) implantation for the treatment of full-thickness chondral defects in sheep. METHODS: A full-thickness 8 mm diameter cartilage defect was created in the weight-bearing area of the medial femoral condyle in both knees of 16 sheep. The right knees of all animals were treated with particulated autograft cartilage implantation and platelet-rich plasma, while the left knees were injected with Ringer's lactate solution or hyaluronic acid. The sheep were killed 9 or 18 months after surgery. Macroscopic evaluations were performed using three different scoring systems, and histopathological evaluations were performed using a modified scoring system based on different scoring systems. RESULTS: The PACI + PRP groups showed statistically significant differences in the percentage of defect repair and chondrocytes in the newly formed cartilage tissue at 18 months compared to 9 months. CONCLUSIONS: The results suggest that macroscopic appearance, histological structure and chondrocyte repair were improved when using PACI + PRP treatment for chondral defects, producing an outcome similar to the surrounding healthy cartilage. PACI + PRP is a totally autologous, easy, and unexpensive treatment that can be performed in one-step procedure and is useful as a therapeutic option for knee chondral defects.
Assuntos
Doenças das Cartilagens , Cartilagem Articular , Plasma Rico em Plaquetas , Animais , Ovinos , Articulação do Joelho/cirurgia , Cartilagem Articular/patologia , Doenças das Cartilagens/patologia , Condrócitos/transplanteRESUMO
Fasciola hepatica is a trematode parasite that infects animals and humans causing fasciolosis, a worldwide-distributed disease responsible for important economic losses and health problems. This disease is of growing public health concern since parasite isolates resistant to the current treatment (triclabendazole) have increasingly been described. F. hepatica infects its vertebrate host after ingestion of the encysted parasite (metacercariae), which are found in the water or attached to plants. Upon ingestion, newly excysted juveniles of F. hepatica (FhNEJ) emerge in the intestinal lumen and cross the intestinal barrier, reach the peritoneum and migrate to the biliary ducts, where adult worms fully develop. Despite the efforts made to develop new therapeutic and preventive tools, to date, protection against F. hepatica obtained in different animal models is far from optimal. Early events of host-FhNEJ interactions are of paramount importance for the infection progress in fasciolosis, especially those occurring at the host-parasite interface. Nevertheless, studies of FhNEJ responses to the changing host environment encountered during migration across host tissues are still scarce. Here, we set-up an ex vivo model coupled with quantitative SWATH-MS proteomics to study early host-parasite interaction events in fasciolosis. After comparing tegument and somatic fractions from control parasites and FhNEJ that managed to cross a mouse intestinal section ex vivo, a set of parasite proteins whose expression was statistically different were found. These included upregulation of cathepsins L3 and L4, proteolytic inhibitor Fh serpin 2, and a number of molecules linked with nutrient uptake and metabolism, including histone H4, H2A and H2B, low density lipoprotein receptor, tetraspanin, fatty acid binding protein a and glutathione-S-transferase. Downregulated proteins in FhNEJ after gut passage were more numerous than the upregulated ones, and included the heath shock proteins HSP90 and alpha crystallin, amongst others. This study brings new insights into early host-parasite interactions in fasciolosis and sheds light on the proteomic changes in FhNEJ triggered upon excystment and intestinal wall crossing, which could serve to define new targets for the prevention and treatment of this widespread parasitic disease.
Assuntos
Fasciola hepatica , Fasciolíase , alfa-Cristalinas , Animais , Catepsinas , Fasciola hepatica/metabolismo , Fasciolíase/parasitologia , Proteínas de Ligação a Ácido Graxo , Glutationa/metabolismo , Proteínas de Helminto/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Proteômica , Receptores de LDL/metabolismo , Transferases/metabolismo , Triclabendazol , alfa-Cristalinas/metabolismoRESUMO
Cylindrospermopsin (CYN) is a ubiquitous cyanotoxin showing increasing incidence worldwide. CYN has been classified as a cytotoxin and, among its toxic effects, its immunotoxicity is scarcely studied. This work investigates for the first time the influence of oral CYN exposure (18.75; 37.5 and 75 µg/kg b.w./day, for 28 days) on the mRNA expression of selected interleukin (IL) genes (IL-1ß, IL-2, IL-6, Tumor Necrosis Factor alpha (TNF-α), Interferon gamma (IFN-γ)) in the thymus and the spleen of male and female rats, by quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, their serum levels were also measured by a multiplex-bead-based immunoassay, and a histopathological study was performed. CYN produced immunomodulation mainly in the thymus of rats exposed to 75 µg CYN/kg b.w./day in both sexes. However, in the spleen only IL-1ß and IL-2 (males), and TNF-α and IFN-γ (females) expression was modified after CYN exposure. Only female rats exposed to 18.75 µg CYN/kg b.w./day showed a significant decrease in TNF-α serum levels. There were no significant differences in the weight or histopathology in the organs studied. Further research is needed to obtain a deeper view of the molecular mechanisms involved in CYN immunotoxicity and its consequences on long-term exposures.
Assuntos
Toxinas de Cianobactérias/metabolismo , Toxinas de Cianobactérias/toxicidade , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Administração Oral , Animais , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Feminino , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Ratos , Baço/efeitos dos fármacos , Baço/metabolismo , Timo/efeitos dos fármacos , Timo/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
The organosulfur compounds (OSC) extracted from Allium spp. exhibit antibacterial, antifungal, and antioxidant properties. The agri-food industry is taking advantage of these properties by using them as natural feed and food additives. In the present work, an acute and a subchronic 90-days toxicity studies have been conducted for the first time to assess the safety of the OSC propyl-propane-thiosulfinate (PTS). Both studies were carried out following the Organization for Economic Co-operation and Development test guidelines (425 and 408, respectively). The acute study provided a maximum tolerated dose (MTD) of 175 mg/kg and the subchronic study established the Non Observed Adverse Effect Level (NOAEL) ≥ 55 mg/kg body weight (b.w.)/day in both sexes. In addition, the subchronic study performed on rats exposed to 14, 28 and 55 mg/kg b.w./day PTS, revealed no changes in any of the hematological parameters measured as well as no differences in body weight and water/food consumption. However, biochemical parameters were altered in some groups, although they were not biologically significant (Ca2+ in female rats, and the thyroids hormones T3 and T4 in rat males). Furthermore, the histopathological assessment evidenced no abnormality on the gastrointestinal, respiratory, lymphoid, urinary, circulatory, nervous, musculoskeletal, and reproductive systems.
Assuntos
Allium/química , Extratos Vegetais/farmacologia , Ácidos Tiossulfônicos/toxicidade , Animais , Relação Dose-Resposta a Droga , Feminino , Dose Letal Mediana , Masculino , Extratos Vegetais/química , Ratos , Testes de ToxicidadeRESUMO
Several collagen subtypes have been identified in hyaline articular cartilage. The main and most abundant collagens are type II, IX and XI collagens. The minor and less abundant collagens are type III, IV, V, VI, X, XII, XIV, XVI, XXII, and XXVII collagens. All these collagens have been found to play a key role in healthy cartilage, regardless of whether they are more or less abundant. Additionally, an exhaustive evaluation of collagen fibrils in a repaired cartilage tissue after a chondral lesion is necessary to determine the quality of the repaired tissue and even whether or not this repaired tissue is considered hyaline cartilage. Therefore, this review aims to describe in depth all the collagen types found in the normal articular cartilage structure, and based on this, establish the parameters that allow one to consider a repaired cartilage tissue as a hyaline cartilage.
Assuntos
Doenças das Cartilagens/metabolismo , Cartilagem Articular/metabolismo , Colágeno/metabolismo , Cartilagem Hialina/metabolismo , Animais , Doenças das Cartilagens/patologia , Cartilagem Articular/patologia , Humanos , Cartilagem Hialina/patologiaRESUMO
Infection with the zoonotic trematode Fasciola hepatica, common in many regions with a temperate climate, leads to delayed growth and loss of productivity in cattle, while infection in sheep can have more severe effects, potentially leading to death. Previous transcriptomic analyses revealed upregulation of TGFB1, cell death and Toll-like receptor signalling, T-cell activation, and inhibition of nitric oxide production in macrophages in response to infection. However, the differences between ovine and bovine responses have not yet been explored. The objective of this study was to further investigate the transcriptomic response of ovine peripheral blood mononuclear cells (PBMC) to F. hepatica infection, and to elucidate the differences between ovine and bovine PBMC responses. Sixteen male Merino sheep were randomly assigned to infected or control groups (n = 8 per group) and orally infected with 120 F. hepatica metacercariae. Transcriptomic data was generated from PBMC at 0, 2 and 16 weeks post-infection (wpi), and analysed for differentially expressed (DE) genes between infected and control animals at each time point (analysis 1), and for each group relative to time 0 (analysis 2). Analysis 2 was then compared to a similar study performed previously on bovine PBMC. A total of 453 DE genes were found at 2 wpi, and 2 DE genes at 16 wpi (FDR < 0.1, analysis 1). Significantly overrepresented biological pathways at 2 wpi included role of PKR in interferon induction and anti-viral response, death receptor signalling and RIG-I-like receptor signalling, which suggested that an activation of innate response to intracellular nucleic acids and inhibition of cellular apoptosis were taking place. Comparison of analysis 2 with the previous bovine transcriptomic study revealed that anti-inflammatory response pathways which were significantly overrepresented in the acute phase in cattle, including IL-10 signalling, Th2 pathway, and Th1 and Th2 activation were upregulated only in the chronic phase in sheep. We propose that the earlier activation of anti-inflammatory responses in cattle, as compared with sheep, may be related to the general absence of acute clinical signs in cattle. These findings offer scope for "smart vaccination" strategies for this important livestock parasite.
Assuntos
Doenças dos Bovinos/genética , Fasciolíase/veterinária , Leucócitos Mononucleares/metabolismo , Doenças dos Ovinos/genética , Transcriptoma , Animais , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Fasciola hepatica/imunologia , Fasciolíase/genética , Fasciolíase/imunologia , Fasciolíase/parasitologia , Perfilação da Expressão Gênica/veterinária , Redes Reguladoras de Genes , Interações Hospedeiro-Parasita , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Masculino , Fenótipo , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Transdução de Sinais , Especificidade da Espécie , Fatores de TempoRESUMO
Fasciolosis is an important economic disease of livestock. There is a global interest in the development of protective vaccines since current anthelmintic therapy is no longer sustainable. A better knowledge of the host-parasite interaction is needed for the design of effective vaccines. The present study evaluates the microscopical hepatic lesions in sheep immunized with a partially protective vaccine (VAC1), a non-protective vaccine (VAC2), and an infected control group (IC). The nature of granulomatous inflammation associated with degeneration of adult flukes found in the VAC1 group was characterized by immunohistochemistry. Hepatic lesions (fibrous perihepatitis, chronic tracts, bile duct hyperplasia, infiltration of eosinophils and lymphocytes and plasma cells) were significantly less severe in the VAC1 group than in the IC group. Dead adult flukes within bile ducts were observed only in the VAC1 group and were surrounded by a severe granulomatous inflammation composed by macrophages and multinucleate giant cells with a high expression of lysozyme, CD163 and S100 markers, and a low expression of CD68. Numerous CD3+ T lymphocytes and scarce infiltrate of FoxP3+ Treg and CD208+ dendritic cells were present. This is the first report describing degenerated flukes associated to a severe granulomatous inflammation in bile ducts in a F. hepatica vaccine trial.
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Propyl-propanethiosulfinate (PTS) is a component of Allium essential oils. This organosulfur molecule can be used as a feed additive to decrease the appearance of bacterial resistances caused by the residues of antibiotics. In previous in vitro genotoxicity studies, contradictory results were reported for PTS. In this work, the in vivo genotoxicity of PTS in male and female rats was assessed for the first time, following OECD (Organisation for Economic Co-operation and Development) guidelines. After oral administration (doses: 5.5, 17.4, and 55.0 mg/kg PTS body weight), a combination of the micronucleus (MN) assay (OECD 474) in bone marrow and the standard and enzyme-modified comet assay (OECD 489) was performed. After necropsy, histopathological studies were also carried out. The results did not show the in vivo genotoxicity of PTS at any doses assayed, revealed by the absence of increased MN, and DNA strand breaks or oxidative DNA damage in the standard and enzyme-modified comet assays. The histopathological study revealed that only the highest dose tested (55.0 mg/kg) in the liver and all dose groups in the stomach presented minimal pathological lesions in the organs studied. Consequently, the present work confirms that PTS is not genotoxic at the doses assayed, and it is a promising natural alternative to synthetic preservatives and antibiotics in animal feed.
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Fasciola hepatica is a trematode parasite responsible for major economic losses in livestock production, and is also a food-borne zoonotic agent in developing rural regions. For years, the immunoregulatory mechanisms employed by the parasite have hampered efforts to develop a successful vaccine candidate. Given that a comprehensive understanding of the immune response to infection is needed, we investigated the gene expression changes in ovine hepatic lymph nodes after experimental infection with F. hepatica. Lymph nodes from uninfected and infected animals were processed for RNA sequencing (RNA-seq) at 16 weeks post-infection. Comparison of groups revealed 5,132 differentially-expressed genes (DEGs). An inhibition of pro-inflammatory pathways, which has previously been described during fasciolosis, was evident in our data. However, other signals previously identified in ruminant peripheral blood mononuclear cells (PBMC) or liver tissue, such as activation of TGF-ß or apoptosis-related pathways were not detected. We found inhibition of some key immunological pathways, including natural killer (NK) cell activity and IgE-mediated signaling. These may point to additional some as yet unrecognized mechanisms employed by the parasite to evade the host immune response. Understanding these, and leveraging information from this and other omics studies, will be important for the development of future vaccine prototypes against this parasite.
Assuntos
Fasciola hepatica/patogenicidade , Fasciolíase/parasitologia , Perfilação da Expressão Gênica , Imunoglobulina E/metabolismo , Células Matadoras Naturais/metabolismo , Fígado/parasitologia , Linfonodos/parasitologia , Transcriptoma , Animais , Modelos Animais de Doenças , Fasciola hepatica/imunologia , Fasciolíase/genética , Fasciolíase/imunologia , Fasciolíase/metabolismo , Interações Hospedeiro-Parasita , Células Matadoras Naturais/imunologia , Fígado/imunologia , Fígado/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Masculino , Carneiro Doméstico , Transdução de SinaisRESUMO
Cylindrospermopsin (CYN) is a toxin with a world-wide increasing occurrence. It can induce toxic effects both in humans and the environment, and toxicity studies are needed to complete its toxicological profile. In this sense, in vivo oral toxicity studies with pure CYN are scarce. The aim of this work was to perform a repeated dose 28-day oral study in rats following the OECD guideline 407 to provide information on health hazard likely to arise from this kind of exposure. Male and female Sprague-Dawley rats were dosed with 18.75, 37.5 and 75 µg CYN/kg b.w./day. After the study period, no clinical signs or mortality and no significant differences in final body weight, body weight gain and total feed intake in both sexes were observed. Only in females some biochemical parameters (triglycerides (TRIG) levels and aspartate aminotransferase (AST) activity) as well as changes in the weight of organs (absolute liver weight values, relative kidney/body weight ratios or relative liver weight/brain weight ratios) were altered, but without toxicological relevance. Histopathological analysis revealed a very mild affectation of liver and kidney in rats. These results suggest the need to perform longer oral toxicity studies to define the potential consequences of long term CYN exposure.
Assuntos
Alcaloides/toxicidade , Administração Oral , Alcaloides/administração & dosagem , Animais , Peso Corporal/efeitos dos fármacos , Testes de Química Clínica , Toxinas de Cianobactérias , Relação Dose-Resposta a Droga , Comportamento de Ingestão de Líquido/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Feminino , Testes Hematológicos , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
In this work we report the protection found in a vaccination trial performed in sheep with two different vaccines composed each one by a cocktail of antigens (rCL1, rPrx, rHDM and rLAP) formulated in two different adjuvants (Montanide ISA 61 VG (G1) and Alhydrogel®(G2)). The parameters of protection tested were fluke burden, faecal egg count and evaluation of hepatic lesions. In vaccinated group 1 we found a significant decrease in fluke burden in comparison to both unimmunised and infected control group (37.2%; p = 0.002) and to vaccinated group 2 (Alhydrogel®) (27.08%; p = 0.016). The lower fluke burden found in G1 was accompanied by a decrease in egg output of 28.71% in comparison with the infected control group. Additionally, gross hepatic lesions found in vaccine 1 group showed a significant decrease (p = 0.03) in comparison with unimmunised-infected group. The serological study showed the highest level for both IgG1 and IgG2 in animals from group 1. All these data support the hypothesis of protection found in vaccine 1 group.
Assuntos
Fasciola hepatica/imunologia , Fasciolíase/veterinária , Doenças dos Ovinos/prevenção & controle , Vacinação/veterinária , Vacinas Combinadas/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Fasciolíase/imunologia , Fasciolíase/prevenção & controle , Ovinos , Doenças dos Ovinos/imunologia , Carneiro DomésticoRESUMO
BACKGROUND: Fasciolosis is one of the most important parasitic diseases of livestock. The need for better control strategies gave rise to the identification of various vaccine candidates. The recombinant form of a member of the cysteine protease family, cathepsin L1 of Fasciola hepatica (FhCL1) has been a vaccine target for the past few decades since it has been shown to behave as an immunodominant antigen. However, when FhCL1 was used as vaccine, it has been observed to elicit significant protection in some trials, whereas no protection was provided in others. METHODS: In order to improve vaccine development strategy, we conducted a linear B-cell epitope mapping of FhCL1 in sheep vaccinated with FhCL1, FhHDM, FhLAP and FhPrx plus Montanide and with significant reduction of the fluke burden, sheep vaccinated with FhCL1, FhHDM, FhLAP and FhPrx plus aluminium hydroxide and with non-significant reduction of the fluke burden, and in unvaccinated-infected sheep. RESULTS: Our study showed that the pattern and dynamic of peptide recognition varied noticeably between both vaccinated groups, and that the regions 55-63 and 77-84, which are within the propeptide, and regions 102-114 and 265-273 of FhCL1 were specifically recognised only by vaccinated sheep with significant reduction of the fluke burden. In addition, these animals also showed significant production of specific IgG2, whereas none was observed in vaccinated-Aluminium hydroxide and in infected control animals. CONCLUSIONS: We have identified 42 residues of FhCL1 that contributed to protective immunity against infection with F. hepatica in sheep. Our results provide indications in relation to key aspects of the immune response. Given the variable outcomes of vaccination trials conducted in ruminants to date, this study adds new insights to improve strategies of vaccine development.
Assuntos
Catepsinas/imunologia , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B , Fasciola hepatica/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Catepsina L , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Ensaio de Imunoadsorção Enzimática/veterinária , Epitopos de Linfócito B/imunologia , Fasciolíase/imunologia , Fasciolíase/veterinária , Gado/imunologia , Gado/parasitologia , Modelos Moleculares , Conformação Molecular , Peptídeos/imunologia , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Vacinas/imunologiaRESUMO
Fasciola hepatica has been shown to have a high capacity for immunomodulation of the host response, making the development of protective vaccines extremely difficult. One of these immunomodulation mechanisms is the impairment of dendritic cells (DC) maturation and, therefore, suppression of antigenic presentation. The aim of this study was to evaluate the pathological changes as well as the characterization of two antigen presenting cells, DC (CD1b, CD83 and MHC-II positive) and follicular dendritic cells (FDC) (CNA.42, S100 and CD83 positive) by immunohistochemistry in the hepatic lymph nodes (HLN) and livers of sheep during the early stages of infection with F. hepatica [9 and 18 days post-infection (dpi)], compared with an uninfected group (UC) as a control. The results revealed a marked hyperplasia of HLN germinal centres at 9 and, in particular, 18 dpi, with respect to the UC group, with coincidental increased expression of CNA.42 in FDC of lymphoid follicles and CD1b in the DC of paracortical areas at 18 dpi. However, the expression of MHC-II and CD83 decreased at 9 and, particularly, at 18 dpi in HLN compared with that in the UC group. Since both markers are related to active presentation of antigens by DC and FDC, the results of the present study suggest that, despite the marked hyperplasia of HLN and increase in DC and FDC numbers during early stages of infection, the DC and FDC antigenic presentation capacity, as suggested by the expression of the markers MHC-II and CD83, is suppressed by the parasite. This suppression was not observed in the liver, probably because of the low number of DC. This is the first study of the immunophenotype of DCs and FDC in sheep infected with F. hepatica.