Diagnostic potential of anti-rNcp-43 polyclonal antibodies for the detection of Neospora caninum.

Neosporosis is a disease caused by the apicomplexan parasite Neospora caninum, which is closely related to Toxoplasma gondii. N. caninum infection represents an important cause of reproductive failure in sheep, goats, horses, and cattle worldwide. The diagnosis of neosporosis is based on the detection of pathogen-specific antibodies in animal sera or the presence of tissue cysts. However, morphological similarities and serological cross-reactivity between N. caninum and T. gondii can result in the misdiagnosis. In this study, the N. caninum tachyzoite surface protein Ncp-43 was expressed in a recombinant form to elicit polyclonal antibodies (pAb) response. The pAb was purified and conjugated to horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC) to detect the recombinant and native Ncp-43 proteins, respectively. The pAb and pAb/HRP were able to recognize rNcp-43 by dot blot and ELISA, and pAb/FITC immunolabeled the apical complex of tachyzoites. A blocking enzyme-linked immunosorbent assay (b-ELISA) was performed to evaluate pAb/HRP as a diagnostic tool. The mean percent inhibition for the positive and negative serum samples from cattle with neosporosis was significantly different (P < 0.0001). These results suggest that the pAb may bind to the same epitopes of Ncp-43 as anti-N. caninum antibodies in the positive samples tested. The b-ELISA using the pAb/HRP can facilitate diagnostic testing for neosporosis, since fewer steps are involved, and cross-reactivity with secondary antibodies is avoided. In summary, this report describes the production of antibodies against N. caninum, and evaluates the potential of these tools for the development of new diagnostic tests for neosporosis.

Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method.

Diagnosis of leptospirosis by PCR is hampered due to the presence of substances on biological fluids. Here, we report an immunomagnetic separation step prior to PCR which improved the detection of Leptospira spp. in blood and urine samples from dogs. It resulted in a significant improvement on sensitivity for diagnosis of canine leptospirosis.

Infection with Leptospira kirschneri Serovar Mozdok: First Report from the Southern Hemisphere.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira spp. In this study, we characterized two Leptospira kirschneri serogroup Pomona serovar Mozdok isolates, one obtained from a dog and the other from a patient with severe leptospirosis, 4 years later. Histopathological analysis showed that both isolates caused severe tissue damage when used to infect hamsters. While L. kirschneri serogroup Pomona serovar Mozdok is endemic in animals in Europe, there is only one report of human leptospirosis in the literature. Although strains belonging to L. kirschneri serogroup Pomona have been identified in cases of human leptospirosis in Europe, serovar Mozdok has not yet been implicated. The 4-year interval between isolations and the fact that this is the first report of serovar Mozdok as the causative agent of human leptospirosis in the southern hemisphere, demonstrates its epidemiological importance to public health. Moreover, the presence of serovar Mozdok in Brazil has the potential to affect vaccine and diagnostic test development.

Immunological and molecular characterization of Leptospira interrogans isolated from a bovine foetus.

Cattle are commonly infected with pathogenic leptospires, and similarly to rodents, they excrete the bacteria in their urine and can transmit the pathogen from animal to animal or animal to human. Thus, surveillance and monitoring systems for detection of new Leptospira serovars are important for the control of leptospirosis. Here, we report the isolation of a spirochete from a stillborn bovine foetus and its characterization by immunological and molecular techniques. A variable number tandem repeat profile using seven discriminatory primers identified the spirochete as belonging to species Leptospira interrogans serogroup Australis serovar Muenchen. A phenotypic analysis using monoclonal antibodies (mAbs) against leptospiral membrane-associated proteins confirmed the expression of important virulence and pathogenicity factors (LipL32 and LigBrep). Out of 120 reference sera tested, 22 positive (36.66%) and 9 negative (15%) also reacted with the new isolate. Furthermore, the serovar Muenchen isolate was virulent in hamster model. The animal inoculated developed acute lethal infection characterized by hepatic, pulmonary and renal lesions. Local isolates exhibited unique characteristics that differed from those of reference strains; therefore, isolation of leptospires is useful in the surveillance of local pathogenic serovars. In conclusion, the data obtained from this study can contribute to the epidemiological understanding and control of leptospirosis in southern Brazil.

The Mycoplasma hyopneumoniae recombinant heat shock protein P42 induces an immune response in pigs under field conditions.

Enzootic pneumonia (EP), resulting from Mycoplasma hyopneumoniae infection is one of the most prevalent diseases in pigs and is a major cause of economic losses to the swine industry worldwide. EP is often controlled by vaccination with inactivated, adjuvanted whole-cell bacterin. However, these bacterins provide only partial protection and do not prevent M. hyopneumoniae colonization. Attempts to develop vaccines that are more efficient have made use of the recombinant DNA technology. The objective of this study was to assess the potential of recombinant M. hyopneumoniae heat shock protein P42 in vaccine preparations against EP, using piglets housed under field conditions in a M. hyopneumoniae-positive farm. The cellular and humoral immune responses were elicited after a single intramuscular inoculation of rP42 in an oil-based adjuvant, or in conjunction with whole-cell vaccine preparation. The production of INF-γ and IL-10 cytokines was quantified in the supernatant of the cultured mononuclear cells. The rP42 emulsified in oil-based adjuvant was able to trigger a strong humoral immune response. Further, it induced a cellular immune response, accompanied by the production of antibodies that reacted with the native M. hyopneumoniae protein. The rP42 mediated induction of cellular and humoral immune response in the host suggests that rP42 emulsified in an oil-based adjuvant holds promise as an effective recombinant subunit vaccine against EP.

Development of an indirect ELISA-NcSRS2 for detection of Neospora caninum antibodies in cattle.

Neosporosis is of alarming economic concern in the cattle industry. The effectiveness of diagnostic tests for detecting specific antibodies against Neospora caninum is hampered by potential cross-reaction with other coccidia. Use of a single specific antigen might improve test specificity. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the truncated protein NcSRS2 expressed in Escherichia coli. The ELISA results were compared with those of the indirect fluorescence antibody test (IFAT). Receiver Operating Characteristic (ROC) and Tests in the Absence of a Gold Standard (TAGS) analysis revealed an assay having 96% specificity and 95% sensitivity when applied to 145 positive and 352 negative sera from two distinct cattle populations. Using OD ≤ 0.095 as the cut-off point, the assay's negative and positive predictive values ranged from 98.8% to 50.8% and from 58.8% to 99.1%, respectively, depending on neosporosis prevalence in a given area. The novel ELISA-NcSRS2 format described in the present report constitutes a specific and sensitive method for detecting N. caninum in cattle.

Production of leptospiral LipL32 antigen in Pichia pastoris and its use in an enzyme-linked immunosorbent assay

The production of recombinant LipL32 protein using Escherichia coli has been used extensively for the development of vaccines and diagnostic tests for leptospirosis. However, E. coli has demonstrated limitations, including low yield and lack of post-translational modifications. In this study, rLipL32 was produced in eukaryotic expression system (Pichia pastoris) and evaluated the antigen by enzyme-linked immunosorbent assay (ELISA). The yield obtained from the culture supernatant reached 270 mg/L and ELISA showed an accuracy of 95.34%. In summary, the production of rLipL32 using P. pastoris did not impair the antigenic characteristics of this antigen and ensured its use for detecting the leptospiral antibodies in swine sera.

Efeitos da suplementação carboidratada e de diferentes tipos de treinamento físico sobre as concentrações de células sanguíneas / Effects of carbohydrate supplementation and different types of exercise training on the blood cells concentrations

INTRODUÇÃO: A participação dos atletas em sessões de exercício intenso e prolongado pode fazer declinar o número circulante e a capacidade funcional dos leucócitos. Por outro lado, o consumo de uma solução carboidratada pode atenuar os efeitos imunossupressivos do exercício. OBJETIVO: Verificar os efeitos do exercício aeróbio e anaeróbio, além da suplementação carboidratada sobre as concentrações sanguíneas da contagem total e diferencial de leucócitos, hemoglobina e glicose sérica de ratos Wistar. MÉTODOS: Sessenta e nove Ratos machos Wistar (60 dias) foram divididos em seis grupos: sedentários não suplementados (n = 12) e suplementados (n = 12); treinados em estado estável máximo de lactato (EEML) não suplementados (n = 11) e suplementados (n = 11); treinados em alta intensidade não suplementados (n = 12) e suplementados (n = 11). O protocolo de treinamento consistiu de oito semanas de natação em padrão contínuo em EEML (60 min.dia-1) ou intermitente (dois períodos de 30 minutos, com intervalo de 10 minutos), com sobrecargas correspondentes a 5% e 10% do peso corporal, respectivamente. Durante 37 dias os animais foram suplementados com uma dose diária de 0,48 g.kg-1 de maltodextrina dissolvida em água ou receberam água pura. RESULTADOS: Não houve efeito da suplementação carboidratada e dos dois tipos de treinamento nas concentrações de leucócitos sanguíneos. O exercício anaeróbio (p = 0,04) e o uso da maltodextrina (p = 0,003) proporcionaram elevações nas concentrações de hemoglobinas sanguíneas, enquanto o exercício aeróbio ocasionou aumento na concentração da glicose sérica (p < 0,02). CONCLUSÃO: Os diferentes tipos de exercícios não estiveram envolvidos com leucopenia, hipoglicemia ou anemia que poderiam levar a fadiga muscular precoce e queda do desempenho.

INTRODUCTION: The participation of athletes in sessions of intense and prolonged exercise can lower the number and functional capacity of circulating leukocytes. On the other hand, the intake of a carbohydrate solution can attenuated the immunosuppressive effects of exercise. OBJECTIVE: To evaluate the effects of aerobic and anaerobic exercises, and carbohydrate supplementation on blood concentrations of total and differential counts leukocytes, hemoglobin and serum glucose in Wistar rats. METHODS: 69 Male Wistar rats (60 days) were divided into six groups: sedentary non-supplemented (n = 12) and supplemented (n = 12); trained in Maximum Lactate Steady State (EEML) not supplemented (n = 11) and supplemented (n = 11) trained at high intensity non-supplemented (n = 12) and supplemented (n = 11). The training protocol consisted of eight weeks of continuous swimming pattern EEML (60min.day-1) or intermittent (two periods of 30 minutes, for exercise with 10 minutes rest), with overloads corresponding to 5% and 10% of body weight, respectively. For 37 days the animals were supplemented with a daily dose of 0.48 g.kg-1 maltodextrin dissolved in water or pure water. RESULTS: There was no effect of carbohydrate supplementation and the two types of training on blood concentrations of leukocytes. The anaerobic exercise (p = 0.04) and the use of maltodextrin (p = 0.003) resulted in increases in blood hemoglobin concentrations, while aerobic exercise caused an increase in the concentration of serum glucose (p < 0.02). CONCLUSION: The different types of exercises were not involved with leukopenia, anemia, or hypoglycemia that could lead to early muscle fatigue and decreased performance.

Perfil lipídico e glicêmico de ratos treinados em exercício aeróbio ou anaeróbio e suplementados com maltodextrina / Lipids and glucose levels of trained rats in aerobic or anaerobic conditions and supplemented with maltodextrin / Los lípidos y los niveles de glucosa de las ratas entrenadas en condiciones aeróbicas o anaeróbicas y se complementa con maltodextrina

OBJETIVO: verificar as alterações no perfil lipídico e glicêmico de ratos treinados, suplementados com maltodextrina. MATERIAIS E MÉTODOS: o protocolo de treinamento consistiu de 8 semanas de natação em padrão contínuo (60min.dia-1) ou intermitente (2 períodos de 30min, com intervalo de 10min), com sobrecargas correspondentes a 5% e 10% do peso corporal, respectivamente. Durante 37 dias os animais foram suplementados com uma dose diária de 0,48g.kg-1 de maltodextrina dissolvida em água ou receberam água pura. RESULTADOS: o exercício aeróbio ocasionou aumento significativo no nível glicêmico. Os exercícios e a maltodextrina não causaram alterações no perfil lipídico. CONCLUSÃO: o exercício aeróbio proporcionou elevação no nível glicêmico, não causando hipoglicemia.

The objective was to verify changes in lipids and glucose levels of trained rats supplemented with maltodextrin. MATERIALS AND METHODS: The training protocol consisted of 8 weeks of continuous swimming pattern (60min.dia-1) or intermittent (2 periods of 30 minutes, with an interval of 10 minutes), with overloads corresponding to 5% and 10% of body weight, respectively. For 37 days the animals were supplemented with a daily dose of 0.48 g.kg-1 maltodextrin dissolved in water or pure water. RESULTS: Aerobic exercise caused a significant increase in blood glucose levels. The exercises and maltodextrin did not cause changes in lipid profile. CONCLUSION: Aerobic exercise gave rise in blood glucose levels, without causing hypoglycemia.

El objetivo fue comprobar los cambios en los lípidos y los niveles de glucosa de las ratas entrenadas complementados con maltodextrina. MATERIAL Y MÉTODOS: El protocolo consistió en 8 semanas de patrón de natación continua (60min.dia-1) o intermitente (2 períodos de 30 min, con un intervalo de 10 min), con sobrecargas que corresponden al 5% y el 10% del peso corporal. Durante 37 días los animales fueron suplementados con una dosis diaria de 0,48 g.kg-1 maltodextrina disuelta en agua o agua pura. RESULTADOS: El ejercicio aeróbico provocó un aumento significativo en los niveles de glucosa en la sangre. Los ejercicios y la maltodextrina no causó cambios en el perfil lipídico. CONCLUSIONES: El ejercicio aeróbico dio lugar en los niveles de glucosa en la sangre, sin causar hipoglucemia.

Efeitos do treinamento moderado contínuo sobre parâmetros imunológico e metabólico de ratos suplementados com maltodextrina / Effects of continuous moderate training on immune and metabolic parameters of rats supplemented with maltodextrin

O objetivo deste estudo foi verificar os efeitos do exercício moderado contínuo sobre a contagem total e diferencial de leucócitos, as concentrações de glicose sérica e os teores lipídicos de ratos suplementados e não suplementados com solução carboidratada. Para tanto, 35 ratos Wistar, machos foram distribuídos em quatro grupos: sedentários não suplementado (n = 10) e suplementado (n = 8); treinados em exercício aeróbio moderado contínuo não suplementado (n = 9) e suplementado (n = 8). O período de treinamento foi de seis semanas de natação em padrão contínuo com sobrecarga correspondente a 3% do peso corporal. Durante cinco dias os animais foram suplementados com uma dose diária de 0,48 g.kg-1 de maltodextrina dissolvida em água ou receberam água pura. O exercício moderado causou uma diminuição significativa na glicemia (p < 0,001) e no número de linfócitos sanguíneos (p < 0,01), entretanto, a maltodextrina proporcionou um aumento significativo nos linfócitos dos animais treinados (p < 0,03). Não houve efeito do treinamento e da maltodextrina no perfil lipídico. Conclui-se que com seis semanas de treinamento foi possível causar queda no número de linfócitos e concentração de glicose sérica, mas com cinco dias de suplementação o declínio na contagem de linfócitos foi atenuado sem, no entanto, causar elevações no perfil lipídico.

The objective of this study was to determine the effects of continuous moderate exercise on the differential and total count leukocyte, serum glucose concentration and lipid levels of rats supplemented and not supplemented with carbohydrate solution. To this purpose, thirty-five male Wistar rats were divided into four groups: sedentary non-supplemented (n = 10) and supplemented (n = 8), trained in continuous moderate aerobic exercise non-supplemented (n = 9) and supplemented (n = 8). The training of continuous swimming was developed during six week with 3% body weight overload. For five days the animals were supplemented with 0.48 g.kg-1 maltodextrin daily dose dissolved in water or pure water. Moderate exercise caused a significant decrease in blood glucose (p < 0.001) and in the number of blood lymphocytes (p < 0.01). Additionally, the maltodextrin significantly increased the number of lymphocytes of trained animals (p < 0.03). There was no effect of training and maltodextrin on the lipid profile. The results allowed to conclude that with six weeks of training it was possible to cause a drop in the number of lymphocytes and in the concentration of serum glucose, but with five days of supplementation the decline in the lymphocyte count was attenuated without, however, cause elevations in the lipid profile.

Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method

Diagnosis of leptospirosis by PCR is hampered due to the presence of substances on biological fluids. Here, we report an immunomagnetic separation step prior to PCR which improved the detection of Leptospira spp. in blood and urine samples from dogs. It resulted in a significant improvement on sensitivity for diagnosis of canine leptospirosis.