RESUMEN
The aim of this study was to develop and validate a SYBR Green qPCR assay to detect and quantify a fragment of the 18S rRNA gene of Rangelia vitalii in canine blood. Repeatability of the qPCR was determined by the intra- and inter-assay variations. The qPCR showed efficiency of E=101.30 (r(2)=0.996), detecting as few as one copy of plasmid containing the target DNA. Specificity of the assay was performed using DNA samples of Babesia canis, B. gibsoni, Ehrlichia canis, E. ewingii and Leishmania sp. No cross-reactivity was observed. Field samples consisting of blood from 265 dogs from Porto Alegre, Brazil were also tested. A total of 24 (9.05%) samples were positive for R. vitalii. Amplicons of 50% of positive samples were confirmed to be R. vitalii by Sanger sequencing. The positive samples had an average of 3.5×10(5) organisms/mL of blood (range: 1.27×10(3)-1.88×10(6)) based on the plasmid-generated standard curve. In conclusion, the SYBR Green qPCR assay developed herein is sensitive and specific and can be used as a diagnostic tool for detection and quantification of R. vitalii in canine blood samples.
Asunto(s)
Enfermedades de los Perros/diagnóstico , Piroplasmida/genética , Infecciones Protozoarias en Animales/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Medicina Veterinaria/métodos , Animales , Brasil , Enfermedades de los Perros/sangre , Enfermedades de los Perros/parasitología , Perros , Infecciones Protozoarias en Animales/sangre , Infecciones Protozoarias en Animales/parasitología , ARN Ribosómico 18S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Sensibilidad y EspecificidadRESUMEN
The aim of this study was to characterize the response of acute phase proteins (APP) in rabbits experimentally infected with Trypanosoma evansi (T. evansi), and to relate the findings with serum immunoglobulins levels, in order to verify the relation between APP and the immune response of rabbits. A total of 12 animals were used in this experiment and divided into 2 groups, control and infected, of six rabbits each. The experimental period was 118 days, and blood was collected on days 0, 5, 20, 35, 65, 95 and 118 post-infection (PI). The infection with T. evansi stimulated APP and immunoglobulins production, once the infected animals showed an increase in C-reactive protein, haptoglobin, alpha 2-macroglobulin and IgM levels. The elevation in IgM levels observed in this study, when related to the increase in C-reactive protein and haptoglobin levels, suggests the involvement of these proteins in host defense against flagellated protozoa, with possible participation in the control of the parasitemia in rabbits infected with T. evansi.
Asunto(s)
Proteínas de Fase Aguda/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Parasitemia/veterinaria , Conejos/parasitología , Tripanosomiasis/veterinaria , Animales , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Parasitemia/inmunología , Parasitemia/parasitología , Conejos/inmunología , Estadísticas no Paramétricas , Trypanosoma/inmunología , Tripanosomiasis/inmunología , Tripanosomiasis/parasitologíaRESUMEN
The aim of this study was to evaluate biochemical parameters of iron metabolism in rats experimentally infected with Trypanosoma evansi. To this end, 20 rats (Wistar) were intraperitoneally inoculated with blood containing trypomastigotes 10(6) (Group T) and 12 animals were used as negative control (Group C) and received saline (0.2 mL) through same route. Blood samples were collected by cardiac puncture on day 5 (C5, T5) and 30 (C30, T30) post-inoculation (pi) to perform complete blood count and determination of serum iron, transferrin, ferritin, total and latent iron fixation capacity, transferrin saturation and prohepcidin concentration. Also, bone marrow samples were collected, to perform Pearls staining reaction. Levels of iron, total and latent iron binding capacity and prohepcidin concentration were lower (P<0.05) in infected rats (T5 and T30 groups) compared to controls. On the other hand, levels of transferrin and ferritin were higher when compared to controls (P<0.05). The transferrin saturation increased on day 5 pi, but decreased on day 30 pi. The Pearls reaction showed a higher accumulation of iron in the bone marrow of infected animals in day 5 pi (P<0.01). Infection with T. evansi in rats caused anemia and changes in iron metabolism associated to the peaks of parasitemia. These results suggest that changes in iron metabolism may be related to the host immune response to infection and anemic status of infected animals.
Asunto(s)
Hierro/metabolismo , Tripanosomiasis/metabolismo , Anemia Ferropénica/inmunología , Anemia Ferropénica/parasitología , Animales , Péptidos Catiónicos Antimicrobianos/sangre , Médula Ósea/metabolismo , Perros , Recuento de Eritrocitos , Índices de Eritrocitos , Ferritinas/metabolismo , Hematócrito , Hemoglobinas/análisis , Hemosiderina/metabolismo , Hepcidinas , Sistema Inmunológico/metabolismo , Hierro/sangre , Masculino , Parasitemia/inmunología , Parasitemia/parasitología , Precursores de Proteínas/sangre , Ratas , Ratas Wistar , Transferrina/metabolismo , Trypanosoma/crecimiento & desarrollo , Tripanosomiasis/sangre , Tripanosomiasis/complicaciones , Tripanosomiasis/inmunologíaRESUMEN
Background: The cholinergic system is involved in many biological functions in mammals and is associated with pathogenesis of infectious diseases, as has participation in transmission of nerve impulses in cholinergic synapses, haematopoiesis, regulation of inflammatory markers, production and coordination of movement, and memory. Rangelia vitalii is a parasite endemic to south of Brazil. This parasite multiplies in the blood and can be visualized in plasma in its free form and/or within leukocytes and erythrocytes, causing various pathologies. Therefore, the purpose of this study was to investigate the activity of cholinergic system enzymes in dogs experimentally infected with R. vitalii. Materials, Methods & Results: Twelve dogs were used, divided into two groups: control group (n = 5), consisting of healthy animals, and infected group with R. vitalii (n = 7). Fresh blood samples of these infected animals were inoculated in seven dogs (2 mL/dog through the jugular vein). Blood samples were collected on days 0, 10 and 20 post-infection (PI). Butyrylcholinesterase (BChE) activity was measured in serum and acetylcholinesterase (AChE) in lymphocytes and whole blood. Boold samples were diluted 1:50 (v/v) in lysis solution (0.1 mmol/L potassium/sodium phosphate buffer containing 0.03% Triton X-100) and frozen (-20 ºC by 7 days) to determine AChE activity in whole blood. Lymphocytes were also obtained from whole blood with EDTA by gradient separation using Ficoll-Histopaque™ plus to AChE activity this cell. After analysis of the samples, was observed that the dogs infected with R. vitalii presented a signifi cant (P < 0.01) increase in AChE activity in whole blood on days 10 and 20 PI. However, the infected group showed a reduced activity in AChE in lymphocytes (P < 0.01) and BChE in serum (P < 0.05) on day 20 PI. Discussion: According to the literature, infected dogs R. vitalii develop regenerative anemia evidenced by an increase in the erytroid precursors in bone marrow associated with alterations of leucogram as leukopenia, neutropenia, eosinopenia, lymphocytosis and monocytosis. Furthermore, it was observed severe thrombocytopenia, with alteration in platelet aggregation and activity of enzymes involved in the control of ATP, ADP and adenosine levels on platelets, thereby influencing hemostasis and contributing to the typical bleeding disease. AChE activity in whole blood was increased in dogs parasitized by R. vitalii observed in this study. This increase may be a compensatory effect to severe anemia caused by the parasite infection, because this enzyme is involved in the maturation of erythrocytes and in the regulation of hematopoiesis. In the present study, we found that the reduction in AChE activity in lymphocytes is associated to lymphocytosis; and it is known that ACh is produced within lymphocytes and has the ability to negatively modulate the immune response, acting directly on the inhibition of inflammatory mediators. Therefore, the decrease of AChE activity may have an anti-inflammatory action in order to have more free ACh to bind lymphocytes and inhibit inflammation. The enzyme BChE can also act as an inflammatory marker in various diseases, similar to AChE, because the enzyme can hydrolyze acetylcholine when AChE is inhibited. In conclusion, our results indicate that canine rangeliosis alters the activity of cholinesterase's, which may be involved in the pathogenesis of the disease, as well as various pathological conditions.
Asunto(s)
Animales , Femenino , Perros , Infecciones Protozoarias en Animales/inducido químicamente , Babesiosis/sangre , Colinesterasas/análisis , Receptores Colinérgicos/análisis , Enfermedades de los Perros/sangreRESUMEN
This study aimed to test an alternative protocol with human plasma to control Trypanosoma evansi infection in mice. Plasma from an apparently 27-year-old healthy male, blood type A+, was used in the study. A concentration of 100 mg.dL(-1) apolipoprotein L1 (APOL1) was detected in the plasma. Forty mice were divided into four groups with 10 animals each. Group A comprised uninfected animals. Mice from groups B, C and D were inoculated with a T. evansi isolate. Group B was used as a positive control. At three days post-infection (DPI), the mice were administered intraperitoneally with human plasma. A single dose of 0.2 mL plasma was given to those in group C. The mice from group D were administered five doses of 0.2 mL plasma with a 24 hours interval between the doses. Group B showed high increasing parasitemia that led to their death within 5 DPI. Both treatments eliminated parasites from the blood and increased the longevity of animals. An efficacy of 50 (group C) and 80% (group D) of human plasma trypanocidal activity was found using PCR. This therapeutic success was likely achieved in the group D due to their higher levels of APOL1 compared with group C.
Asunto(s)
Fenómenos Fisiológicos Sanguíneos , Plasma , Trypanosoma , Adulto , Animales , Humanos , Masculino , RatonesRESUMEN
This study aimed to test an alternative protocol with human plasma to control Trypanosoma evansi infection in mice. Plasma from an apparently 27-year-old healthy male, blood type A+, was used in the study. A concentration of 100 mg.dL-1 apolipoprotein L1 (APOL1) was detected in the plasma. Forty mice were divided into four groups with 10 animals each. Group A comprised uninfected animals. Mice from groups B, C and D were inoculated with a T. evansi isolate. Group B was used as a positive control. At three days post-infection (DPI), the mice were administered intraperitoneally with human plasma. A single dose of 0.2 mL plasma was given to those in group C. The mice from group D were administered five doses of 0.2 mL plasma with a 24 hours interval between the doses. Group B showed high increasing parasitemia that led to their death within 5 DPI. Both treatments eliminated parasites from the blood and increased the longevity of animals. An efficacy of 50 (group C) and 80% (group D) of human plasma trypanocidal activity was found using PCR. This therapeutic success was likely achieved in the group D due to their higher levels of APOL1 compared with group C.
Este estudo teve como objetivo testar um protocolo alternativo com plasma humano para controlar a infecção por Trypanosoma evansi em camundongos. O plasma foi oriundo de um homem aparentemente saudável, com idade entre 27 anos e tipo de sangue A+. Foi detectada uma concentração de 100 mg.dL -1 de apolipoproteína L1 (APOL1) no plasma. Quarenta camundongos foram divididos em quatro grupos, contendo dez animais cada. Grupo A, composto de animais não infectados. Os roedores dos grupos B, C e D foram inoculados intraperitonealmente com um isolado de T. evansi. O Grupo B foi usado como um controle positivo. Três dias pós-infecção (DPI), os camundongos foram tratados com plasma humano. Uma dose única de 0,2 mL de plasma foi administrada nos roedores do grupo C. Os ratos do grupo D receberam cinco doses de 0,2 mL de plasma em intervalos de 24 horas. Os ratos do grupo B apresentaram parasitemia crescente, o que ocasionou a morte dos animais em 5 DPI. Ambos os tratamentos foram capazes de eliminar o parasito do sangue e aumentar a longevidade dos animais. O método da PCR detectou uma eficácia de 50% (grupo C) e 80% (grupo D) no tratamento com plasma humano. Este sucesso terapêutico obtido nos animais do grupo D provavelmente foi por receber maiores níveis de APOL1, comparado ao grupo C.
Asunto(s)
Adulto , Animales , Humanos , Masculino , Ratones , Fenómenos Fisiológicos Sanguíneos , Plasma , TrypanosomaRESUMEN
This study aimed to test an alternative protocol with human plasma to control Trypanosoma evansi infection in mice. Plasma from an apparently 27-year-old healthy male, blood type A+, was used in the study. A concentration of 100 mg.dL-1 apolipoprotein L1 (APOL1) was detected in the plasma. Forty mice were divided into four groups with 10 animals each. Group A comprised uninfected animals. Mice from groups B, C and D were inoculated with a T. evansi isolate. Group B was used as a positive control. At three days post-infection (DPI), the mice were administered intraperitoneally with human plasma. A single dose of 0.2 mL plasma was given to those in group C. The mice from group D were administered five doses of 0.2 mL plasma with a 24 hours interval between the doses. Group B showed high increasing parasitemia that led to their death within 5 DPI. Both treatments eliminated parasites from the blood and increased the longevity of animals. An efficacy of 50 (group C) and 80% (group D) of human plasma trypanocidal activity was found using PCR. This therapeutic success was likely achieved in the group D due to their higher levels of APOL1 compared with group C.(AU)
Este estudo teve como objetivo testar um protocolo alternativo com plasma humano para controlar a infecção por Trypanosoma evansi em camundongos. O plasma foi oriundo de um homem aparentemente saudável, com idade entre 27 anos e tipo de sangue A+. Foi detectada uma concentração de 100 mg.dL -1 de apolipoproteína L1 (APOL1) no plasma. Quarenta camundongos foram divididos em quatro grupos, contendo dez animais cada. Grupo A, composto de animais não infectados. Os roedores dos grupos B, C e D foram inoculados intraperitonealmente com um isolado de T. evansi. O Grupo B foi usado como um controle positivo. Três dias pós-infecção (DPI), os camundongos foram tratados com plasma humano. Uma dose única de 0,2 mL de plasma foi administrada nos roedores do grupo C. Os ratos do grupo D receberam cinco doses de 0,2 mL de plasma em intervalos de 24 horas. Os ratos do grupo B apresentaram parasitemia crescente, o que ocasionou a morte dos animais em 5 DPI. Ambos os tratamentos foram capazes de eliminar o parasito do sangue e aumentar a longevidade dos animais. O método da PCR detectou uma eficácia de 50% (grupo C) e 80% (grupo D) no tratamento com plasma humano. Este sucesso terapêutico obtido nos animais do grupo D provavelmente foi por receber maiores níveis de APOL1, comparado ao grupo C.(AU)
Asunto(s)
Humanos , Animales , Masculino , Adulto , Ratones , Fenómenos Fisiológicos Sanguíneos , Plasma , TrypanosomaRESUMEN
Background: In Brazil, the official buffalo herd has about three million animals, distributed over many states of the country. Many times, abnormalities found in proteinogram are not related to illness conditions, but with physiological organic and individuals conditions, that are relatively constant. The interpretation of biochemical constituents depends on the disponibility of knowledge of the variation that exists among different species of animals. Factors like age, stage of development, breed, hormones, pregnancy, nutrition, stress and loss of fluid are directly related to changes in proteinogram. Proteinogram is an important auxiliary exam, helpfull to clinical biochemistry, and represents one of the most reliable methods for identification of blood proteins. The aim of this research was to evaluate the proteinogram of buffaloes of different ages reared in extensive system in Rio Grande do Sul State, Brazil. Materials, Methods & Results: Forty-five buffaloes were separated into three groups: group 1 (n = 15), six-months old animals; group 2 (n = 16), twelve-month old animals and group 3 (n = 14), twenty-four months old animals. The total serum protein were determined by the biuret method, using commercial reagent Labtest® and the analysis were realized in semi-automatic spectrophotometer BioPlus-Bio-2000®, according to fabricant instructions. The fractionation of serum proteins were determined by electrophoresis on cellulose acetate strips celugell Labex®. The samples were layered on the strip and, after the closing of the horizontal plane, it was applied a constant voltage of 220 volts for 15 min. Significant differences were verified among groups in the following protein fractions: total serum protein, betaglobulin, gammaglobulin and albumin:globulin ratio. In correlation tests, there was a positive correlation between total proteins and gamaglobulins, albumin:globulin ratio and albumin and negative correlation between the albumin:globulin ratio and gamaglobulins. Discussion: With age, there is a significant increase in total serum protein, as can be seen in our work. Thus, it is possible to justify the increase in total serum proteins with the increase of gamaglobulins. In a study realized, who evaluated pregnant sheeps, observed that the total serum protein concentration diminishes with the proximity of the parturition, indicating the importance in evaluating the total serum proteins in different moments of life in different species. With age, the total serum protein tend to increase, resulting in decrease in albumin, with progressive increase in globulins. However, in this study, it was not noticed any significant statistic difference in albumin among the groups. Proteins that compose the alfaglobulin, are acute phase proteins and their concentrations increase rapidly in response to antigenic and traumatic stimuli in bovine. However, in our study, no statistic difference was observed. The proteins that compose the betaglobulin fraction are: lipoprotein, transferrin, ferritin, hemopexin, complement C3, protein C-reative, complement C4, plasminogen and fibrinogen. Some authors observed a decrease in fraction beta at the end of gestation in sheeps, other did not observed any variation in this fraction in neonatal bovine. The proteins that compose the gamaglobulin fraction are immunoglobulins IgG, IgA, IgE and IgM. In general, due to the age, there is an increase in gamaglobulin concentration, because of a higher exposure of organism to pathogens including bacteria, viruses, fungi and parasites. Similar data were observed in this study. The results indicate that there are some variations according to the analyzed age. Then, some abnormalities found in the protein profile may be due to physiological variations.(AU)
Asunto(s)
Animales , Búfalos/fisiología , alfa-Globulinas/fisiología , gammaglobulinas/fisiología , Albúminas/fisiología , Envejecimiento/sangreRESUMEN
Background: In Brazil, the official buffalo herd has about three million animals, distributed over many states of the country. Many times, abnormalities found in proteinogram are not related to illness conditions, but with physiological organic and individuals conditions, that are relatively constant. The interpretation of biochemical constituents depends on the disponibility of knowledge of the variation that exists among different species of animals. Factors like age, stage of development, breed, hormones, pregnancy, nutrition, stress and loss of fluid are directly related to changes in proteinogram. Proteinogram is an important auxiliary exam, helpfull to clinical biochemistry, and represents one of the most reliable methods for identification of blood proteins. The aim of this research was to evaluate the proteinogram of buffaloes of different ages reared in extensive system in Rio Grande do Sul State, Brazil. Materials, Methods & Results: Forty-five buffaloes were separated into three groups: group 1 (n = 15), six-months old animals; group 2 (n = 16), twelve-month old animals and group 3 (n = 14), twenty-four months old animals. The total serum protein were determined by the biuret method, using commercial reagent Labtest® and the analysis were realized in semi-automatic spectrophotometer BioPlus-Bio-2000®, according to fabricant instructions. The fractionation of serum proteins were determined by electrophoresis on cellulose acetate strips celugell Labex®. The samples were layered on the strip and, after the closing of the horizontal plane, it was applied a constant voltage of 220 volts for 15 min. Significant differences were verified among groups in the following protein fractions: total serum protein, betaglobulin, gammaglobulin and albumin:globulin ratio. In correlation tests, there was a positive correlation between total proteins and gamaglobulins, albumin:globulin ratio and albumin and negative correlation between the albumin:globulin ratio and gamaglobulins. Discussion: With age, there is a significant increase in total serum protein, as can be seen in our work. Thus, it is possible to justify the increase in total serum proteins with the increase of gamaglobulins. In a study realized, who evaluated pregnant sheeps, observed that the total serum protein concentration diminishes with the proximity of the parturition, indicating the importance in evaluating the total serum proteins in different moments of life in different species. With age, the total serum protein tend to increase, resulting in decrease in albumin, with progressive increase in globulins. However, in this study, it was not noticed any significant statistic difference in albumin among the groups. Proteins that compose the alfaglobulin, are acute phase proteins and their concentrations increase rapidly in response to antigenic and traumatic stimuli in bovine. However, in our study, no statistic difference was observed. The proteins that compose the betaglobulin fraction are: lipoprotein, transferrin, ferritin, hemopexin, complement C3, protein C-reative, complement C4, plasminogen and fibrinogen. Some authors observed a decrease in fraction beta at the end of gestation in sheeps, other did not observed any variation in this fraction in neonatal bovine. The proteins that compose the gamaglobulin fraction are immunoglobulins IgG, IgA, IgE and IgM. In general, due to the age, there is an increase in gamaglobulin concentration, because of a higher exposure of organism to pathogens including bacteria, viruses, fungi and parasites. Similar data were observed in this study. The results indicate that there are some variations according to the analyzed age. Then, some abnormalities found in the protein profile may be due to physiological variations.
Asunto(s)
Animales , Albúminas/fisiología , Búfalos/fisiología , Envejecimiento/sangre , alfa-Globulinas/fisiología , gammaglobulinas/fisiologíaRESUMEN
This study aimed to assess the plasma lipid peroxidation and the susceptibility of erythrocytes to in vitro peroxidation as indicators of oxidative damage in erythrocytes and their roles in the pathogenesis of anemia during the early acute phase of Trypanosoma evansi infection in rats. Fifty male Wistar rats were randomly distributed into seven groups: three trypanosome-infected groups (T(2), T(4) and T(6); n=10 animals per group) and four uninfected controls (C(0), C(2), C(4) and C(6); n=5 animals per group). Animals from trypanosome-infected groups were inoculated intraperitoneally with 10(6) trypanosomes. Blood samples were collected by cardiac puncture before infection (day 0; group C(0)) or on the 2nd (C(2) and T(2)), 4th (C(4) and T(4)) and 6th (C(6) and T(6)) day post-infection (dpi). Samples were analyzed for red blood cell (RBC) count, hemoglobin (Hb) concentration, packed cell volume (PCV), plasma malondialdehyde (MDA) and in vitro peroxidation of erythrocytes. The mean values of the hematological indices gradually decreased in the infected rats compared with the control. MDA was significantly increased (P<0.001) on the 6th dpi in infected versus control animals and was negatively correlated with PCV (P<0.001; R(2)=0.372). The values for erythrocyte in vitro peroxidation were higher for groups T(4) and T(6) than for the control rats (P<0.01). A positive correlation between erythrocyte peroxidation and MDA (P<0.001; R(2)=0.414) was observed. The results of this study indicate that T. evansi infection in rats is associated with oxidative stress, indicated by lipid peroxidation and oxidative damage in erythrocyte membranes, as demonstrated by in vitro peroxidation. This may be one of the causes of anemia in acute trypanosomosis.