Resumo
A cinomose é uma enfermidade causada pelo vírus Canine Distemper Virus (CDV). Essa doença afeta principalmente cães, mas também acomete outras espécies domésticas e selvagens. A imunidade do animal está relacionada ao grau que a esse patógeno vai atingir o organismo do indivíduo. Ela afeta a respiração do animal, pode causar vômito, diarreia, convulsões, podendo levar o animal à óbito. O objetivo do presente trabalho foi padronizar um teste ELISA indireto com antígeno de superfície para o diagnostico cinomose utilizando amostras de soro canino. Para padronização da técnica, fez-se necessário o estudo da diluição do antígeno para identificar a melhor concentração para sensibilização da placa. O teste foi aplicado primeiramente com diferentes diluições do antígeno para detecção do melhor desempenho do antígeno. Feito isso, foi testado em um banco de soro de 45 animais comprovadamente positivos no teste ELISA comercial e em soro de 45 animais comprovadamente negativos no teste ELISA comercial, posteriormente foi calculado o ponto de corte, especificidade e sensibilidade do teste. O teste ELISA indireto se mostrou com excelência como um teste de diagnóstico para a cinomose canina, obtendo-se ponto de corte de densidade óptica de 0,229, sensibilidade de 95,5% e especificidade de 84,4%.
Distemper is a disease or the disease by the CDV virus, Distemper Virus. This disease mainly affects dogs, but also affects other domestic and wild species. The animal's immunity is related to the degree to which it will reach the individual's organism. It affects the animal's breathing, can cause vomiting, diarrhea, convulsions, and can lead to death. The aim of the present work test was to standardize an indirect ELISA for distemper diagnosis in experiments using a surface antigen. For the study of technical identification, it was necessary to specify the antigen for the best concentration of plaque sensitization. The test was initially applied with different dilutions of the antigen to detect the best performance of the antigen. This was tested in a serum bank of 45 animals proven positive in the commercial ELISA test and in the serum of 45 animals proven negative in the commercial ELISA test, later it was tested on the cut-off point, specificity and sensitivity of the test. The indirect ELISA test proved to be excellent as a diagnostic test for canine distemper, with an optical density cut-off of 0.229, sensitivity of 95.5% and specificity of 84.4% being obtained.
Assuntos
Animais , Cães , Testes Imunológicos/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Técnicas e Procedimentos Diagnósticos/veterinária , Cinomose/diagnóstico , Vírus da Cinomose Canina , Cães/imunologia , Antígenos Virais/análiseResumo
The standardization and validation of a multiplex assay requires the combination of important parameters such as sensitivity and specificity, acceptable levels of performance, robustness, and reproducibility. We standardized a multiparametric Dot-blot aimed at the serological screening of paracoccidioidomycosis, histoplasmosis, and aspergillosis. A total of 148 serum were evaluated: 10 from healthy subjects, 36 from patients with paracoccidioidomycosis, 62 from patients with histoplasmosis, and 40 from patients with aspergillosis. It was found that the multiparametric Dot-blot showed a high percentage of cross-reactivity. However, when evaluated individually, in the serological screening of histoplasmosis, a good performance was observed when compared to the double immunodiffusion assay, considered the gold standard test, with 100% co-positivity and 83.3% co-negativity. The performance of serological screening for aspergillosis was not satisfactory when compared to double immunodiffusion, showing 71.4% co-positivity and 100% co-negativity. The evaluation of the stability of nitrocellulose membranes showed that membranes sensitized with H. capsulatum antigen remained stable for 90 days and those sensitized with A. fumigatus antigen for 30 days. We conclude that the use of crude antigens was not suitable for the standardization of the multiparametric Dot-blot assay, due to the high cross-reactivity, and that further tests should be performed with purified proteins (AU).
A padronização e validação de um ensaio multiplex requer a combinação de parâmetros importantes, como sensibilidade e especificidade, níveis aceitáveis de desempenho, robustez e reprodutibilidade. Este trabalho padronizou um Dot-blot multiparamétrico visando a triagem sorológica da paracoccidioidomicose, histoplasmose e aspergilose. Foram avaliadas 148 amostras de soro: 10 de indivíduos saudáveis, 36 de pacientes com paracoccidioidomicose, 62 de pacientes com histoplasmose e 40 de pacientes com aspergilose. Verificou-se que o Dot-blot multiparamétrico apresentou elevado percentual de reatividade cruzada. Entretanto, quando avaliado individualmente, na triagem sorológica da histoplasmose observou-se bom desempenho quando comparado ao ensaio de imunodifusão dupla, considerado o teste padrão ouro, com 100% de co-positividade e 83,3% de co-negatividade. O desempenho da triagem sorológica da aspergilose não foi satisfatório quando comparado a imunodifusão dupla, apresentando 71,4% de co-positividade e 100% de co-negatividade. A avaliação da estabilidade das membranas de nitrocelulose mostrou que membranas sensibilizadas com antígeno de H. capsulatum permaneceram estáveis por 90 dias e as sensibilizadas com antígeno de A. fumigatus, por 30 dias. Concluímos que o uso de antígenos brutos não foi adequado para a padronização do ensaio de Dot-blot multiparamétrico, devido ao alto índice de reatividade cruzada, e que novos testes devem ser realizados com proteínas purificadas (AU).
Assuntos
Paracoccidioidomicose , Aspergilose , Padrões de Referência , Testes Imunológicos , Saúde Pública , Metodologia como Assunto , Histoplasmose , Micoses/diagnósticoResumo
Background: Pseudopterygium, also known as aberrant conjunctival growth, is poorly described in the literature, althoughit is known that this abnormality is uncommon and affects dwarf rabbits and their crossbreeds. The etiology of this diseaseis unknown, but there are hypotheses that the conjunctival growth cause may have its origins in immunological factors,inflammation, traumatic conditions, or cartilage dysplasias. Thus, this study reports the treatment efficacy applied in arabbit, through the continuous use of tracolimus eye drops, after surgical procedure of conjunctival fold resection, as away of controlling the pseudopterygium in rabbits.Case: This case report discusses the positive results from the surgical and therapeutic conduct of a clinical case attended bythe Ophthalmology and Microsurgical Veterinary Service at the Hospital Veterinário Universitário (HVU) of the UFSM. Thepatient was a male rabbit, sterilized, approximately 2-year-old, crossed with a dwarf rabbit. The owners main complaintwas the change in the aspect of the left eye, with progressive worsening in the previous four weeks. In the ophthalmologicalexamination, the animal did not present impaired vision or discomfort, however, a vascularized pink membrane was noted,which consisted of a fold of the bulbar conjunctiva, that grew centripetally and covered 90% of the cornea in 360 degrees.The diagnosis was confirmed through visual inspection and the patients history. The eye alteration had a characteristicaspect, described as proliferation of the bulbar conjunctiva over the cornea, in a centripetal manner and without signs ofinflammation. In addition, other ophthalmological alterations were ruled out during the patients physical and specificexamination. The patient was referred for anesthetic evaluation and, in addition, pre-surgical blood tests were performed,which were normal, according to the expected ranges for the species...
Assuntos
Animais , Coelhos , Proteínas de Ligação a Tacrolimo , Proteínas de Ligação a Tacrolimo/administração & dosagem , Proteínas de Ligação a Tacrolimo/uso terapêutico , Túnica Conjuntiva/anormalidades , Túnica Conjuntiva/crescimento & desenvolvimento , Fatores Imunológicos , Imunomodulação , Pterígio/veterináriaResumo
Background: Pseudopterygium, also known as aberrant conjunctival growth, is poorly described in the literature, althoughit is known that this abnormality is uncommon and affects dwarf rabbits and their crossbreeds. The etiology of this diseaseis unknown, but there are hypotheses that the conjunctival growth cause may have its origins in immunological factors,inflammation, traumatic conditions, or cartilage dysplasias. Thus, this study reports the treatment efficacy applied in arabbit, through the continuous use of tracolimus eye drops, after surgical procedure of conjunctival fold resection, as away of controlling the pseudopterygium in rabbits.Case: This case report discusses the positive results from the surgical and therapeutic conduct of a clinical case attended bythe Ophthalmology and Microsurgical Veterinary Service at the Hospital Veterinário Universitário (HVU) of the UFSM. Thepatient was a male rabbit, sterilized, approximately 2-year-old, crossed with a dwarf rabbit. The owners main complaintwas the change in the aspect of the left eye, with progressive worsening in the previous four weeks. In the ophthalmologicalexamination, the animal did not present impaired vision or discomfort, however, a vascularized pink membrane was noted,which consisted of a fold of the bulbar conjunctiva, that grew centripetally and covered 90% of the cornea in 360 degrees.The diagnosis was confirmed through visual inspection and the patients history. The eye alteration had a characteristicaspect, described as proliferation of the bulbar conjunctiva over the cornea, in a centripetal manner and without signs ofinflammation. In addition, other ophthalmological alterations were ruled out during the patients physical and specificexamination. The patient was referred for anesthetic evaluation and, in addition, pre-surgical blood tests were performed,which were normal, according to the expected ranges for the species...(AU)
Assuntos
Animais , Coelhos , Túnica Conjuntiva/anormalidades , Túnica Conjuntiva/crescimento & desenvolvimento , Proteínas de Ligação a Tacrolimo , Proteínas de Ligação a Tacrolimo/administração & dosagem , Proteínas de Ligação a Tacrolimo/uso terapêutico , Pterígio/veterinária , Fatores Imunológicos , ImunomodulaçãoResumo
The protozoan Neospora caninum is known worldwide as one of the main causes of abortion in cattle. During infection, rhoptry proteins present in the apical complex of the parasite play important roles in adhesion and parasitophorous vacuole formation. The use of N. caninum ROP2 in experimental vaccines has shown promising protective results. In our study we performed cloning and expression in Escherichia coli of an antigenic portion of N. caninum ROP2. The recombinant protein (rROP2) was obtained in insoluble form, and the purified protein showed a size of approximately 18kDa. Even being a small truncate NcROP2 region, it was possible to conserve the antigenic epitopes which were recognized by bovine serum naturally infected with N. caninum. Vaccination with rROP2 on aluminum hydroxide adjuvant induced high levels of rROP2-specific IgG antibodies capable of recognizing native protein in tachyzoite lysates. In conclusion, our approaches were effective in obtaining the rROP2 protein, which induced specific mouse immune response and was also recognized by sera from N. caninum naturally infected cattle. These results suggest that it is a promising antigen for the development of neosporosis subunit vaccines as well as a suitable antigen for use in immunodiagnosis.(AU)
O protozoário Neospora caninum é conhecido mundialmente como uma das principais causas de aborto em bovinos. Durante a infecção, as proteínas rhoptry presentes no complexo apical do parasita desempenham papel importante na adesão e formação de vacúolos parasitóforos. O uso de ROP2 de N. caninum em vacinas experimentais tem mostrado resultados de proteção promissores. Em nosso estudo, realizamos a clonagem e expressão em Escherichia coli de uma porção antigênica de N. caninum ROP2. A proteína recombinante (rROP2) foi obtida na forma insolúvel, e a proteína purificada apresentou tamanho aproximado de 18kDa. Mesmo sendo uma pequena região truncada de NcROP2, foi possível conservar os epítopos antigênicos que foram reconhecidos pelo soro de bovinos naturalmente infectados com N. caninum. A vacinação com rROP2 adsorvida no adjuvante de hidróxido de alumínio induziu altos níveis de anticorpos IgG anti-rROP2, capazes de reconhecer a proteína nativa em lisados de taquizoítos. Em conclusão, nossas abordagens foram eficazes na obtenção da proteína rROP2, que induziu resposta imune específica em camundongos e também foi reconhecida por soros de bovinos naturalmente infectados com N. caninum. Estes resultados sugerem que rROP2 é um antígeno promissor para o desenvolvimento de vacinas de subunidades de neosporose, bem como um antígeno adequado para uso em imunodiagnóstico.(AU)
Assuntos
Testes Imunológicos , Imunoglobulina G , Vacinas , Neospora , Clonagem de OrganismosResumo
The protozoan Neospora caninum is known worldwide as one of the main causes of abortion in cattle. During infection, rhoptry proteins present in the apical complex of the parasite play important roles in adhesion and parasitophorous vacuole formation. The use of N. caninum ROP2 in experimental vaccines has shown promising protective results. In our study we performed cloning and expression in Escherichia coli of an antigenic portion of N. caninum ROP2. The recombinant protein (rROP2) was obtained in insoluble form, and the purified protein showed a size of approximately 18kDa. Even being a small truncate NcROP2 region, it was possible to conserve the antigenic epitopes which were recognized by bovine serum naturally infected with N. caninum. Vaccination with rROP2 on aluminum hydroxide adjuvant induced high levels of rROP2-specific IgG antibodies capable of recognizing native protein in tachyzoite lysates. In conclusion, our approaches were effective in obtaining the rROP2 protein, which induced specific mouse immune response and was also recognized by sera from N. caninum naturally infected cattle. These results suggest that it is a promising antigen for the development of neosporosis subunit vaccines as well as a suitable antigen for use in immunodiagnosis.(AU)
O protozoário Neospora caninum é conhecido mundialmente como uma das principais causas de aborto em bovinos. Durante a infecção, as proteínas rhoptry presentes no complexo apical do parasita desempenham papel importante na adesão e formação de vacúolos parasitóforos. O uso de ROP2 de N. caninum em vacinas experimentais tem mostrado resultados de proteção promissores. Em nosso estudo, realizamos a clonagem e expressão em Escherichia coli de uma porção antigênica de N. caninum ROP2. A proteína recombinante (rROP2) foi obtida na forma insolúvel, e a proteína purificada apresentou tamanho aproximado de 18kDa. Mesmo sendo uma pequena região truncada de NcROP2, foi possível conservar os epítopos antigênicos que foram reconhecidos pelo soro de bovinos naturalmente infectados com N. caninum. A vacinação com rROP2 adsorvida no adjuvante de hidróxido de alumínio induziu altos níveis de anticorpos IgG anti-rROP2, capazes de reconhecer a proteína nativa em lisados de taquizoítos. Em conclusão, nossas abordagens foram eficazes na obtenção da proteína rROP2, que induziu resposta imune específica em camundongos e também foi reconhecida por soros de bovinos naturalmente infectados com N. caninum. Estes resultados sugerem que rROP2 é um antígeno promissor para o desenvolvimento de vacinas de subunidades de neosporose, bem como um antígeno adequado para uso em imunodiagnóstico.(AU)
Assuntos
Testes Imunológicos , Imunoglobulina G , Vacinas , Neospora , Clonagem de OrganismosResumo
This study evaluated the physicochemical and morphological properties of a marine sponge protein extract (PE) using scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDS), analysis of mass loss and pH and in vitro and in vivo. Scanning electron microscopy showed that PE fibers present a granular aspect and irregular structure and the element carbon followed by oxygen was detected in the EDS analysis. Moreover, a 29% of mass loss was observed after 14 days and the pH slightly modified after 14 days. Cell viability of fibroblast cells (L929) of control and PE at a concentration of 25% demonstrated higher values compared to the groups. Osteoblast cell viability of PE at 25 and 50% was significantly higher. Comet assay on day 1 showed higher values for PE at 25%. In addition, in vivo experiments demonstrated that in the treated animals, the bone defects were filled with biomaterial particles, granulation tissue and some areas of newly formed bone. Furthermore, similar immunoexpression of Runx-2 and Cox-2 was observed. Taken together, all results suggest that PE is biocompatible, present non-citotoxicity in the in vitro studies (at the lower concentration) and in the in vivo studies and it can be considered as an alternative source of collagen for tissue engineering proposals.
Assuntos
Poríferos/química , Testes Imunológicos de Citotoxicidade , Testes de Mutagenicidade , Técnicas In VitroResumo
This study evaluated the physicochemical and morphological properties of a marine sponge protein extract (PE) using scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDS), analysis of mass loss and pH and in vitro and in vivo. Scanning electron microscopy showed that PE fibers present a granular aspect and irregular structure and the element carbon followed by oxygen was detected in the EDS analysis. Moreover, a 29% of mass loss was observed after 14 days and the pH slightly modified after 14 days. Cell viability of fibroblast cells (L929) of control and PE at a concentration of 25% demonstrated higher values compared to the groups. Osteoblast cell viability of PE at 25 and 50% was significantly higher. Comet assay on day 1 showed higher values for PE at 25%. In addition, in vivo experiments demonstrated that in the treated animals, the bone defects were filled with biomaterial particles, granulation tissue and some areas of newly formed bone. Furthermore, similar immunoexpression of Runx-2 and Cox-2 was observed. Taken together, all results suggest that PE is biocompatible, present non-citotoxicity in the in vitro studies (at the lower concentration) and in the in vivo studies and it can be considered as an alternative source of collagen for tissue engineering proposals.(AU)
Assuntos
Testes de Mutagenicidade , Testes Imunológicos de Citotoxicidade , Técnicas In Vitro , Poríferos/químicaResumo
The selective activity of an antineoplastic drug is related to its ability to promote cytotoxic action on tumor cells and preserve the integrity of non-neoplastic cells. Beta-lapachone is extracted from the sawdust of Ipe wood, a thick bark tree from the Ipe wood found in the Brazilian Cerrado biome. This study aimed to evaluate the cytotoxic action of beta-lapachone in an endothelial cell line. The EA.hy926 cells were seeded in two groups, G1 and G2, cultured and exposed to beta-lapachone at concentrations of 0.0, 0.01, 0.03, 0.1, 0.3, 1 and 3 & 956;M for 24 hours. G1 remained under normal cultivation conditions and G2 was subjected to oxidative stress through an ischemia and reperfusion assay, in a deoxygenated sealed chamber. The cytotoxicity assay was performed using the tetrazolium reduction method. In G1, the cytotoxicity ranged from 0.0 to 10.0%; and in G2 between 0.0 and 6.3%. No statistically significant difference was observed between the obtained values. Moreover, we found no cytotoxic action of beta-lapachone on endothelial cells, and the results point out that the drug might have preserved the cells integrity against oxidative stress under the conditions of this experiment. This promising result suggests the possibility of beta-lapachone as a chemotherapy drug with selective activity.
Assuntos
Células Endoteliais/fisiologia , Células Endoteliais/química , Naftoquinonas , Testes Imunológicos de CitotoxicidadeResumo
The selective activity of an antineoplastic drug is related to its ability to promote cytotoxic action on tumor cells and preserve the integrity of non-neoplastic cells. Beta-lapachone is extracted from the sawdust of Ipe wood, a thick bark tree from the Ipe wood found in the Brazilian Cerrado biome. This study aimed to evaluate the cytotoxic action of beta-lapachone in an endothelial cell line. The EA.hy926 cells were seeded in two groups, G1 and G2, cultured and exposed to beta-lapachone at concentrations of 0.0, 0.01, 0.03, 0.1, 0.3, 1 and 3 & 956;M for 24 hours. G1 remained under normal cultivation conditions and G2 was subjected to oxidative stress through an ischemia and reperfusion assay, in a deoxygenated sealed chamber. The cytotoxicity assay was performed using the tetrazolium reduction method. In G1, the cytotoxicity ranged from 0.0 to 10.0%; and in G2 between 0.0 and 6.3%. No statistically significant difference was observed between the obtained values. Moreover, we found no cytotoxic action of beta-lapachone on endothelial cells, and the results point out that the drug might have preserved the cells integrity against oxidative stress under the conditions of this experiment. This promising result suggests the possibility of beta-lapachone as a chemotherapy drug with selective activity.(AU)
Assuntos
Testes Imunológicos de Citotoxicidade , Naftoquinonas , Células Endoteliais/química , Células Endoteliais/fisiologiaResumo
Background: Small ruminant lentivirus (SRLV) belong to genus Lentivirus, family Retroviridae. These viruses causecaprine arthritis encephalitis (CAE) and maedi visna (MV), infectious diseases that cause economic, production, and reproductive losses. There are no effective treatments or vaccines for these diseases. Thus, early detection via serology hasgreat importance for control of SRLV. Therefore, the objective of this review is to demonstrate the potential of the westernblot (WB) test as an immunodiagnostic test for SRLV.Review: In general, immunodiagnosis of SRLV is performed via agar gel immunodiffusion (AGID) and indirect enzymelinked immunosorbent assay (ELISA), which can detect antibodies in several different biological samples but is used preferably with serum and blood plasma. However, WB has demonstrated efficacy in the early diagnosis of immunoglobulinsagainst SRLV, presenting higher sensitivity and specificity than the serological tests usually used, because this techniquecan detect antibodies at a dilution as much as 256 times greater than that of AGID and 32 times greater than that of ELISA.SRLV infection and consequent immunological activation result in the induction of cellular and humoral responses. Additionally, around the third week, production of antibodies directed mainly toward viral capsid proteins (p25 and p28)occurs. After the fifth week, production of immunoglobulins directed toward other viral proteins occurs. Because of thepersistence of SRLV infection, serology is considered to be the most practical means to diagnosis. Each serological testhas a percentage specificity and distinct sensitivity, as well as advantages and disadvantages in its applicability. It shouldbe noted that there is no gold standard test for diagnosis of SRLV infection. Moreover, SRLV are characterized by escapemechanisms such as genetic diversity, mutagenic potential, viral intermittence...
Assuntos
Animais , Infecções por Lentivirus/diagnóstico , Infecções por Lentivirus/veterinária , Ruminantes/virologia , Western Blotting/veterinária , Imunoglobulinas , Testes Sorológicos/veterináriaResumo
Background: Small ruminant lentivirus (SRLV) belong to genus Lentivirus, family Retroviridae. These viruses causecaprine arthritis encephalitis (CAE) and maedi visna (MV), infectious diseases that cause economic, production, and reproductive losses. There are no effective treatments or vaccines for these diseases. Thus, early detection via serology hasgreat importance for control of SRLV. Therefore, the objective of this review is to demonstrate the potential of the westernblot (WB) test as an immunodiagnostic test for SRLV.Review: In general, immunodiagnosis of SRLV is performed via agar gel immunodiffusion (AGID) and indirect enzymelinked immunosorbent assay (ELISA), which can detect antibodies in several different biological samples but is used preferably with serum and blood plasma. However, WB has demonstrated efficacy in the early diagnosis of immunoglobulinsagainst SRLV, presenting higher sensitivity and specificity than the serological tests usually used, because this techniquecan detect antibodies at a dilution as much as 256 times greater than that of AGID and 32 times greater than that of ELISA.SRLV infection and consequent immunological activation result in the induction of cellular and humoral responses. Additionally, around the third week, production of antibodies directed mainly toward viral capsid proteins (p25 and p28)occurs. After the fifth week, production of immunoglobulins directed toward other viral proteins occurs. Because of thepersistence of SRLV infection, serology is considered to be the most practical means to diagnosis. Each serological testhas a percentage specificity and distinct sensitivity, as well as advantages and disadvantages in its applicability. It shouldbe noted that there is no gold standard test for diagnosis of SRLV infection. Moreover, SRLV are characterized by escapemechanisms such as genetic diversity, mutagenic potential, viral intermittence...(AU)
Assuntos
Animais , Infecções por Lentivirus/veterinária , Infecções por Lentivirus/diagnóstico , Western Blotting/veterinária , Ruminantes/virologia , Imunoglobulinas , Testes Sorológicos/veterináriaResumo
Canine visceral leishmaniasis (CVL) is a zoonotic disease of high lethality caused by Leishmania infantum in the Americas. In the infected dog, the amastigotes are scarce in blood, especially in the late phase of the disease. This study aimed to report a rare case of L. infantum amastigotes found in neutrophils from peripheral blood of a naturally infected dog in terminal phase of CVL, also describing its clinical status before and after treatment with miltefosine 2%. The dog, which presented as polysymptomatic and with classical signs and symptoms of CVL was submitted to the following tests: Dual Path Platform (DPP) rapid test, ELISA and parasitological examination of peripheral blood. Hematological and biochemical parameters were obtained before and after treatment. All diagnostic tests were positive for CVL. The identification of L. infantum amastigotes inside neutrophils from peripheral blood was confirmed through microscopy, and the species was confirmed by molecular analysis. At the end of the treatment, peripheral parasitemia was not detected, and improvements were observed in clinical and laboratorial parameters. Finally, this atypical finding can be used as example to raise discussions about the real immunological role of neutrophils in late phases of CVL and its clinical/therapeutic implications.(AU)
A leishmaniose visceral canina (LVC) é uma doença zoonótica de alta letalidade causada por Leishmania infantum nas Américas. No cão infectado, as formas amastigotas são escassas no sangue, principalmente na fase tardia da doença. Este estudo teve como objetivo relatar um caso raro de amastigotas de L. infantum encontradas em neutrófilos do sangue periférico de um cão naturalmente infectado e terminal da LVC, descrevendo também seu estado clínico antes e após o tratamento com miltefosina a 2%. O cão, que se apresentou como polissintomático e com sinais e sintomas clássicos da LVC foi submetido aos seguintes testes: teste rápido Dual Path Platform (DPP), ELISA e exame parasitológico de sangue periférico. Os parâmetros hematológicos e bioquímicos foram obtidos antes e após o tratamento. Todos os testes diagnósticos foram positivos para LVC. A identificação de formas amastigotas de L. infantum, dentro de neutrófilos do sangue periférico foi confirmada por microscopia, e a espécie foi confirmada por análise molecular. Ao final do tratamento, não foi detectada parasitemia periférica, observando-se melhora dos parâmetros clínicos e laboratoriais. Por fim, esse achado atípico pode ser usado como exemplo para levantar discussões sobre o real papel imunológico dos neutrófilos nas fases tardias da LVC e suas implicações clínicas/terapêuticas.(AU)
Assuntos
Animais , Cães , Cães/parasitologia , Leishmaniose/diagnóstico , Leishmania infantum , Cães/sangue , NeutrófilosResumo
Pyometra has several immunological and molecular changes that are responsible for uterine inflammation and the disease may or may not have infections. This study aimed to isolate and identify bacteria in the uterine content of bitches with pyometra, to analyze the susceptibility profile to antibiotics, detect ß-lactamase enzyme production by phenotypic tests, and resistance genes to ß-lactams. Eighteen samples of uterine content were collected by aspiration puncture. The samples were inoculated in bacteriological media and identified by biochemical tests. Subsequently, antibiogram tests, screening for detection of ß-lactamases, and Real-Time PCR for detection of resistance genes was performed. Escherichia coli, Klebsiella spp., Enterobacter aerogenes, Citrobacter spp., Staphylococcus spp., and Streptococcus spp. were identified in the analyzed samples of uterine content. In the antibiogram test, 90.5% of the isolates showed resistance to at least one antibiotic, and of these, 36.8% were considered MDR, with three Staphylococcus spp., three E. coli, and one Klebsiellaspp. Concerning bacterial resistance to the groups of antibiotics tested, 38.1% of the isolates were resistant to at least one type of ß-lactam, 33.3% to tetracycline, 19.0% to aminoglycosides, and 14.3% to fluoroquinolones, macrolides, and trimethoprim-sulfamethoxazole. In the phenotypic test to detect ß-lactamase production, E. coli samples were negative and Klebsiella spp. was positive for the production of AmpC, which presented the blaCMY, blaSPM, and blaSIM genes. Bacteria that are resistant to antibiotics represent a great challenge and laboratory support is therefore essential, without which therapeutic success decreases and death may be inevitable.(AU)
A piometra apresenta diversas alterações imunológicas e moleculares que são responsáveis pela inflamação uterina, e a doença pode ser infecciosa ou não. O objetivo deste estudo foi isolar e identificar bactérias no conteúdo uterino de cadelas com piometra, analisar o perfil de suscetibilidade aos antibióticos, detectar a produção de enzimas ß-lactamase por testes fenotípicos e genes de resistência aos ß-lactâmicos. Dezoito amostras de conteúdo uterino foram coletadas por punção aspirativa. As amostras foram inoculadas em meio bacteriológico e identificadas por testes bioquímicos. Posteriormente, foram realizados testes de antibiograma, triagem para detecção de ß-lactamases e PCR em tempo real para detecção de genes de resistência. Escherichia coli, Klebsiella spp., Enterobacter aerogenes, Citrobacter spp., Staphylococcus spp. e Streptococcus spp. foram identificados nas amostras de conteúdo uterino analisadas. No teste de antibiograma, 90,5% dos isolados apresentaram resistência a pelo menos um antibiótico, e destes, 36,8% foram considerados MR, sendo três Staphylococcus spp., três E. coli e uma Klebsiella spp. Sobre a resistência bacteriana aos grupos de antibióticos testados, 38,1% dos isolados foram resistentes a pelo menos um tipo de ß-lactâmico, 33,3% à tetraciclina, 19,0% aos aminoglicosídeos e 14,3% às fluorquinolonas, macrolídeos e trimetoprim-sulfametoxazol. No teste fenotípico para detecção da produção de ß-lactamase, as amostras de E. coli foram negativas, e Klebsiella spp. foi positiva para a produção de AmpC, que apresentou os genes blaCMY, blaSPM e blaSIM. As bactérias resistentes aos antibióticos representam um grande desafio e, portanto, o suporte laboratorial é essencial, sem o qual o sucesso terapêutico diminui e a morte pode ser inevitável.(AU)
Assuntos
Animais , Feminino , Cães , Cães/genética , Cães/microbiologia , Piometra/genética , Genes , Antibacterianos/isolamento & purificaçãoResumo
Pyometra has several immunological and molecular changes that are responsible for uterine inflammation and the disease may or may not have infections. This study aimed to isolate and identify bacteria in the uterine content of bitches with pyometra, to analyze the susceptibility profile to antibiotics, detect ß-lactamase enzyme production by phenotypic tests, and resistance genes to ß-lactams. Eighteen samples of uterine content were collected by aspiration puncture. The samples were inoculated in bacteriological media and identified by biochemical tests. Subsequently, antibiogram tests, screening for detection of ß-lactamases, and Real-Time PCR for detection of resistance genes was performed. Escherichia coli, Klebsiella spp., Enterobacter aerogenes, Citrobacter spp., Staphylococcus spp., and Streptococcus spp. were identified in the analyzed samples of uterine content. In the antibiogram test, 90.5% of the isolates showed resistance to at least one antibiotic, and of these, 36.8% were considered MDR, with three Staphylococcus spp., three E. coli, and one Klebsiellaspp. Concerning bacterial resistance to the groups of antibiotics tested, 38.1% of the isolates were resistant to at least one type of ß-lactam, 33.3% to tetracycline, 19.0% to aminoglycosides, and 14.3% to fluoroquinolones, macrolides, and trimethoprim-sulfamethoxazole. In the phenotypic test to detect ß-lactamase production, E. coli samples were negative and Klebsiella spp. was positive for the production of AmpC, which presented the blaCMY, blaSPM, and blaSIM genes. Bacteria that are resistant to antibiotics represent a great challenge and laboratory support is therefore essential, without which therapeutic success decreases and death may be inevitable.(AU)
A piometra apresenta diversas alterações imunológicas e moleculares que são responsáveis pela inflamação uterina, e a doença pode ser infecciosa ou não. O objetivo deste estudo foi isolar e identificar bactérias no conteúdo uterino de cadelas com piometra, analisar o perfil de suscetibilidade aos antibióticos, detectar a produção de enzimas ß-lactamase por testes fenotípicos e genes de resistência aos ß-lactâmicos. Dezoito amostras de conteúdo uterino foram coletadas por punção aspirativa. As amostras foram inoculadas em meio bacteriológico e identificadas por testes bioquímicos. Posteriormente, foram realizados testes de antibiograma, triagem para detecção de ß-lactamases e PCR em tempo real para detecção de genes de resistência. Escherichia coli, Klebsiella spp., Enterobacter aerogenes, Citrobacter spp., Staphylococcus spp. e Streptococcus spp. foram identificados nas amostras de conteúdo uterino analisadas. No teste de antibiograma, 90,5% dos isolados apresentaram resistência a pelo menos um antibiótico, e destes, 36,8% foram considerados MR, sendo três Staphylococcus spp., três E. coli e uma Klebsiella spp. Sobre a resistência bacteriana aos grupos de antibióticos testados, 38,1% dos isolados foram resistentes a pelo menos um tipo de ß-lactâmico, 33,3% à tetraciclina, 19,0% aos aminoglicosídeos e 14,3% às fluorquinolonas, macrolídeos e trimetoprim-sulfametoxazol. No teste fenotípico para detecção da produção de ß-lactamase, as amostras de E. coli foram negativas, e Klebsiella spp. foi positiva para a produção de AmpC, que apresentou os genes blaCMY, blaSPM e blaSIM. As bactérias resistentes aos antibióticos representam um grande desafio e, portanto, o suporte laboratorial é essencial, sem o qual o sucesso terapêutico diminui e a morte pode ser inevitável.(AU)
Assuntos
Animais , Feminino , Cães , Cães/genética , Cães/microbiologia , Piometra/genética , Genes , Antibacterianos/isolamento & purificaçãoResumo
Abstract Canine visceral leishmaniasis (CVL) is a zoonotic disease of high lethality caused by Leishmania infantum in the Americas. In the infected dog, the amastigotes are scarce in blood, especially in the late phase of the disease. This study aimed to report a rare case of L. infantum amastigotes found in neutrophils from peripheral blood of a naturally infected dog in terminal phase of CVL, also describing its clinical status before and after treatment with miltefosine 2%. The dog, which presented as polysymptomatic and with classical signs and symptoms of CVL was submitted to the following tests: Dual Path Platform (DPP) rapid test, ELISA and parasitological examination of peripheral blood. Hematological and biochemical parameters were obtained before and after treatment. All diagnostic tests were positive for CVL. The identification of L. infantum amastigotes inside neutrophils from peripheral blood was confirmed through microscopy, and the species was confirmed by molecular analysis. At the end of the treatment, peripheral parasitemia was not detected, and improvements were observed in clinical and laboratorial parameters. Finally, this atypical finding can be used as example to raise discussions about the real immunological role of neutrophils in late phases of CVL and its clinical/therapeutic implications.
Resumo A leishmaniose visceral canina (LVC) é uma doença zoonótica de alta letalidade causada por Leishmania infantum nas Américas. No cão infectado, as formas amastigotas são escassas no sangue, principalmente na fase tardia da doença. Este estudo teve como objetivo relatar um caso raro de amastigotas de L. infantum encontradas em neutrófilos do sangue periférico de um cão naturalmente infectado e terminal da LVC, descrevendo também seu estado clínico antes e após o tratamento com miltefosina a 2%. O cão, que se apresentou como polissintomático e com sinais e sintomas clássicos da LVC foi submetido aos seguintes testes: teste rápido Dual Path Platform (DPP), ELISA e exame parasitológico de sangue periférico. Os parâmetros hematológicos e bioquímicos foram obtidos antes e após o tratamento. Todos os testes diagnósticos foram positivos para LVC. A identificação de formas amastigotas de L. infantum, dentro de neutrófilos do sangue periférico foi confirmada por microscopia, e a espécie foi confirmada por análise molecular. Ao final do tratamento, não foi detectada parasitemia periférica, observando-se melhora dos parâmetros clínicos e laboratoriais. Por fim, esse achado atípico pode ser usado como exemplo para levantar discussões sobre o real papel imunológico dos neutrófilos nas fases tardias da LVC e suas implicações clínicas/terapêuticas.
Assuntos
Animais , Cães , Leishmania infantum , Doenças do Cão/diagnóstico , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , NeutrófilosResumo
Bovine viral diarrhoea (BVD) is an infectious and contagious disease affecting cattle that is responsible for causing a wide range of clinical manifestations ranging from inapparent or subclinical infections to an acute and sometimes fatal disease known as mucosal disease. The agent that causes BVD is an RNA virus of the genus Pestivirus and family Flaviridae. BVD is transmitted in two different ways: horizontal, by secretions, and vertically in pregnant cows, where the cow transmits the virus to the foetus. The clinical signs depend on the affected animal, its immunological capacity, and in the case of pregnant females, the gestation phase. A gestational infection can result in several changes, such as congenital anomalies, abortion, or even the birth of so-called persistently infected (PI) animals, which are difficult to detect and have a very important epidemiological role within the herd. The southwest region of Paraná has the largest dairy basin in the state of Paraná and is predominantly composed of family farmers, many of whom adopt measures that increase the health risk of their herd. The objective of this study was to delineate the serological prevalence of non-vaccinated dairy cattle in the municipality of Royalty-PR in relation to the BVD-1 virus, as well as to evaluate the odds ratios, relative risk and risk attributable to the independent variables of breed, age and the community under study. To that end, 317 blood serum samples from non-vaccinated cows from 18 different farms, with various breeds and ages, were evaluated by virus neutralization for the detection of antibodies specific to BVD-1. The results showed that 17.03% of the animals (54/317) had positive BVD-1 serology, and 82.33% (15/18) of the farms had at least one positive animal. Animals of the Jersey breed and the Barra do Sarandi Line community had the highest epidemiological risks, with a strong association with BVD-1 infection.
A Diarreia Viral Bovina (BVD) é uma doença infectocontagiosa que acomete bovinos, responsável por causar uma ampla gama de manifestações clínicas, que variam desde infecções inaparentes ou subclínicas até uma doença aguda e, por vezes, fatal conhecida como Doença das Mucosas. O agente causal da BVD é um RNA vírus do gênero Pestivirus, e da família Flaviridae. A BVD é transmitida de duas formas distintas: horizontal, por secreções, e de forma vertical em vacas prenhes, ocorrendo transmissão da vaca para o feto. Os sinais clínicos dependem do animal acometido, de sua capacidade imunológica, e no caso de fêmeas prenhes, da fase de gestação que a mesma se encontra. Uma infecção no período gestacional pode resultar em várias alterações como: anomalias congênitas, aborto, ou até mesmo no nascimento dos chamados animais persistentemente infectados (PI), que são fontes de difícil detecção e que tem papel epidemiológico muito importante dentro do rebanho bovino. A região Sudoeste do Paraná possui a maior bacia leiteira do estado do Paraná, sendo composta, predominantemente, por pequenos produtores, sendo que muitos destes, adotam medidas que aumentam o risco sanitário do seu rebanho. Objetivou-se com este estudo, delinear a prevalência sorológica de bovinos leiteiros não vacinados do município de Realeza-PR, perante o vírus da BVD-1, bem como, avaliar odds ratio, risco relativo e atribuído as variáveis independentes: raça, idade e comunidade em estudo. Para tal, 317 amostras de soro sanguíneo provenientes de fêmeas não vacinadas, de 18 diferentes propriedades, raças e idades foram avaliadas por meio de vírus neutralização para a detecção de anticorpos específicos a BVD-1. Os resultados demonstram que 17, 03% dos animais (54/317) foram positivos a sorologia de BVD-1, sendo que em 82,33 % (15/18) das propriedades havia ao menos um animal positivo. Animais da raça Jersey e da comunidade de linha Barra do Sarandi...
Assuntos
Animais , Bovinos , Doenças dos Bovinos , Testes Sorológicos/estatística & dados numéricos , Testes Sorológicos/veterinária , Vacinação/veterinária , Vírus da Diarreia Viral Bovina Tipo 1Resumo
Bovine viral diarrhoea (BVD) is an infectious and contagious disease affecting cattle that is responsible for causing a wide range of clinical manifestations ranging from inapparent or subclinical infections to an acute and sometimes fatal disease known as mucosal disease. The agent that causes BVD is an RNA virus of the genus Pestivirus and family Flaviridae. BVD is transmitted in two different ways: horizontal, by secretions, and vertically in pregnant cows, where the cow transmits the virus to the foetus. The clinical signs depend on the affected animal, its immunological capacity, and in the case of pregnant females, the gestation phase. A gestational infection can result in several changes, such as congenital anomalies, abortion, or even the birth of so-called persistently infected (PI) animals, which are difficult to detect and have a very important epidemiological role within the herd. The southwest region of Paraná has the largest dairy basin in the state of Paraná and is predominantly composed of family farmers, many of whom adopt measures that increase the health risk of their herd. The objective of this study was to delineate the serological prevalence of non-vaccinated dairy cattle in the municipality of Royalty-PR in relation to the BVD-1 virus, as well as to evaluate the odds ratios, relative risk and risk attributable to the independent variables of breed, age and the community under study. To that end, 317 blood serum samples from non-vaccinated cows from 18 different farms, with various breeds and ages, were evaluated by virus neutralization for the detection of antibodies specific to BVD-1. The results showed that 17.03% of the animals (54/317) had positive BVD-1 serology, and 82.33% (15/18) of the farms had at least one positive animal. Animals of the Jersey breed and the Barra do Sarandi Line community had the highest epidemiological risks, with a strong association with BVD-1 infection.(AU)
A Diarreia Viral Bovina (BVD) é uma doença infectocontagiosa que acomete bovinos, responsável por causar uma ampla gama de manifestações clínicas, que variam desde infecções inaparentes ou subclínicas até uma doença aguda e, por vezes, fatal conhecida como Doença das Mucosas. O agente causal da BVD é um RNA vírus do gênero Pestivirus, e da família Flaviridae. A BVD é transmitida de duas formas distintas: horizontal, por secreções, e de forma vertical em vacas prenhes, ocorrendo transmissão da vaca para o feto. Os sinais clínicos dependem do animal acometido, de sua capacidade imunológica, e no caso de fêmeas prenhes, da fase de gestação que a mesma se encontra. Uma infecção no período gestacional pode resultar em várias alterações como: anomalias congênitas, aborto, ou até mesmo no nascimento dos chamados animais persistentemente infectados (PI), que são fontes de difícil detecção e que tem papel epidemiológico muito importante dentro do rebanho bovino. A região Sudoeste do Paraná possui a maior bacia leiteira do estado do Paraná, sendo composta, predominantemente, por pequenos produtores, sendo que muitos destes, adotam medidas que aumentam o risco sanitário do seu rebanho. Objetivou-se com este estudo, delinear a prevalência sorológica de bovinos leiteiros não vacinados do município de Realeza-PR, perante o vírus da BVD-1, bem como, avaliar odds ratio, risco relativo e atribuído as variáveis independentes: raça, idade e comunidade em estudo. Para tal, 317 amostras de soro sanguíneo provenientes de fêmeas não vacinadas, de 18 diferentes propriedades, raças e idades foram avaliadas por meio de vírus neutralização para a detecção de anticorpos específicos a BVD-1. Os resultados demonstram que 17, 03% dos animais (54/317) foram positivos a sorologia de BVD-1, sendo que em 82,33 % (15/18) das propriedades havia ao menos um animal positivo. Animais da raça Jersey e da comunidade de linha Barra do Sarandi...(AU)
Assuntos
Animais , Bovinos , Testes Sorológicos/estatística & dados numéricos , Testes Sorológicos/veterinária , Vírus da Diarreia Viral Bovina Tipo 1 , Doenças dos Bovinos , Vacinação/veterináriaResumo
O objetivo do estudo foi produzir anticorpos policlonais para vírus bovino e testar a reatividade em imunoensaios como imunofluorescência, imunoperoxidase e slot blot. Como imunógeno utilizou-se cepas e/ou isolados dos herpesvírus bovino tipo 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus vaccina (VACV) amplificados em cultivo celular. Os coelhos foram imunizados em intervalos regulares e o soro hiperimune produzido foi utilizado como anticorpo primário nos imunoensaios. A diluição de trabalho para cada antissoro foi determinada por diluição seriada limitante e variaram entre 1:800 e 1:51.200. As maiores diluições foram observadas para imunoperoxidase, quando comparadas com a imunofluorescência e slot blot. Diluições menores que 1:800 foram associadas com aumento de reações inespecíficas. Os antissoros anti-BoHV-1, -BoHV-5, -BVDV e -BRSV reagiram frente a isolados heterólogos em ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os antissoros produzidos possuem elevadas concentrações de anticorpos específicos para os vírus e é uma alternativa para a utilização em imunoensaios.
The objective of the study was to produce polyclonal antibodies to bovine viruses and to test the reactivity in immunoassays such as immunofluorescence, immunoperoxidase and slot blot. Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) blue tongue virus (BTV) and vaccinia virus (VACV) were amplified in cell culture and used to immunize rabbits. Animals were immunized at regular intervals and the hyper immune serum produced was used as primary antibody in the immunoassays. The working dilution for each antiserum was determined by limiting serial dilution and varied from 1: 800 to 1: 51,200. The highest dilutions were observed for immunoperoxidase, when compared with immunofluorescence and slot blot. Dilutions lower than 1:800 were associated with the presence of nonspecific reactions. Anti-BoHV-1, -BoHV-5, -BVDV and -BRSV antisera reacted against heterologous isolates in immunofluorescence and immunoperoxidase assays. It is concluded that the produced polyclonal antibodies have high concentrations of virus-specific antibodies and are an alternative for use in immunoassays.
Assuntos
Animais , Coelhos , Imunoensaio/veterinária , Soros Imunes/análise , Testes Imunológicos/métodos , Testes Imunológicos/veterináriaResumo
O objetivo do estudo foi produzir anticorpos policlonais para vírus bovino e testar a reatividade em imunoensaios como imunofluorescência, imunoperoxidase e slot blot. Como imunógeno utilizou-se cepas e/ou isolados dos herpesvírus bovino tipo 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus vaccina (VACV) amplificados em cultivo celular. Os coelhos foram imunizados em intervalos regulares e o soro hiperimune produzido foi utilizado como anticorpo primário nos imunoensaios. A diluição de trabalho para cada antissoro foi determinada por diluição seriada limitante e variaram entre 1:800 e 1:51.200. As maiores diluições foram observadas para imunoperoxidase, quando comparadas com a imunofluorescência e slot blot. Diluições menores que 1:800 foram associadas com aumento de reações inespecíficas. Os antissoros anti-BoHV-1, -BoHV-5, -BVDV e -BRSV reagiram frente a isolados heterólogos em ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os antissoros produzidos possuem elevadas concentrações de anticorpos específicos para os vírus e é uma alternativa para a utilização em imunoensaios.(AU)
The objective of the study was to produce polyclonal antibodies to bovine viruses and to test the reactivity in immunoassays such as immunofluorescence, immunoperoxidase and slot blot. Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) blue tongue virus (BTV) and vaccinia virus (VACV) were amplified in cell culture and used to immunize rabbits. Animals were immunized at regular intervals and the hyper immune serum produced was used as primary antibody in the immunoassays. The working dilution for each antiserum was determined by limiting serial dilution and varied from 1: 800 to 1: 51,200. The highest dilutions were observed for immunoperoxidase, when compared with immunofluorescence and slot blot. Dilutions lower than 1:800 were associated with the presence of nonspecific reactions. Anti-BoHV-1, -BoHV-5, -BVDV and -BRSV antisera reacted against heterologous isolates in immunofluorescence and immunoperoxidase assays. It is concluded that the produced polyclonal antibodies have high concentrations of virus-specific antibodies and are an alternative for use in immunoassays.(AU)