Comparative analysis of a novel N-butanol-prepared antigen vs thermo-resistant and sonicated antigens for human leptospirosis detection.

The diagnosis of human leptospirosis is mainly based on serological assays. Since the extraction by N-butanol has only been studied as an antigen for the diagnosis of cattle leptospirosis, this study aimed to investigate the feasibility of the N-butanol preparation for the diagnosis of human leptospirosis and compare it with sonicated and thermo-resistant antigens in IgM dot-blot test. Paired serum samples from 147 laboratory-confirmed leptospirosis cases were tested. The control group consisted of 148 serum samples from healthy individuals and nonleptospirosis cases. N-butanol antigens from serovar Copenhageni (ButC3) and serovar Patoc (ButP3) showed reactivity with antileptospiral antibodies from patients with confirmed leptospirosis. In the acute phase, sensitivities of IgM dot-blot assay with ButC3 and ButP3 antigens were 47.6% and 51.0%, respectively. In the convalescent phase, sensitivities were 95.9% (ButC3) and 93.2% (ButP3), and no significant differences were observed among the IgM dot-blot tests with other antigens. The specificity of the IgM dot-blot test with ButC3 antigen was good (92.6%), but with ButP3 (83.1%), it was significantly lower than with the other tests. The IgM dot-blot test described in this study is simple to perform and presents reliable visual results. Antigens prepared by N-butanol proved to be valuable diagnostic markers of leptospirosis.

Evaluation of the Histoplasma capsulatum 100-kilodalton antigen dot blot for the rapid diagnosis of progressive histoplasmosis in HIV/AIDS patients.

Among people living with HIV (PLHIV), progressive disseminated histoplasmosis (PDH) represents an important cause of mortality. Since antigen detection allows a rapid diagnosis and the instauration of a specific treatment this study aimed to evaluate the analytical performance of the Hcp100 dot blot, an in-house assay that detects the Histoplasma capsulatum 100-kilodalton antigen in urine and compare it with 2 commercially available assays the Histoplasma Urine Antigen Lateral Flow Assay (MVD-LFA) (MiraVista® Diagnostics) and the Clarus Histoplasma Galactomannan EIA (Clarus HGM) (IMMY). Urine specimens from 23 PLHIV with PDH, 13 patients with other infectious diseases, and 20 healthy individuals were tested. The Hcp100 dot blot showed higher sensitivity (87.0%), specificity (97.0%) and accuracy (92.9%) than the MVD-LFA (73.9%, 78.8%, and 76.8%, respectively) and the Clarus HGM (78.3%, 90.9%, and 85.7%, respectively). The Hcp100 dot blot had high analytical performance and would be a valuable screening tool for diagnosing PDH among PLHIV.

Development of an instrument-free and low-cost ELISA dot-blot test to detect antibodies against SARS-CoV-2.

Most laboratory tests to detect the presence of anti-SARS-CoV-2 antibodies use enzyme-linked immunosorbent assays (ELISA) or chemiluminescence immunoassays (CLIA); however, equipment for these immunoassays is unavailable in many areas of low- and middle-income countries. Rapid lateral flow immunoassay (LFIA) tests are an equipment-free option, but their high price may make them less suitable for conducting seroprevalence surveys. Here, we describe a simple dual antigen ELISA dot-blot test to detect anti-SARS-CoV-2 IgG antibodies with high sensitivity (94-98%) and specificity (92-100%), compared to commercially available ELISA and CLIA options. Additionally, this ELISA dot-blot test can be completed in one hour using minimal laboratory equipment. Importantly, this immunoassay is significantly more affordable than most LFIA tests available on the global market. The dot-blot strips may be stored for up to 7 days under freezing conditions. This ELISA dot-blot test is a cost-effective option for conducting seroprevalence screenings in areas lacking ELISA or CLIA facilities, compared to LFIA tests.

Role of recombinant Histoplasma capsulatum 100-kilodalton antigen in the diagnosis of histoplasmosis among HIV/AIDS patients: Antigenuria and antibodies detection.

BACKGROUND: Diagnosing progressive disseminated histoplasmosis (PDH) is still challenging in many countries where this disease is highly endemic. Definitive diagnosis is established by culture and/or by cytology/histopathology but both procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has a low sensitivity in immunocompromised individuals. Commercially available antigen detection assays have high sensitivity in PDH cases; however, they are expensive and only performed in few laboratories. AIMS: To describe the potential use of a novel ELISA for antibodies testing and a dot blot assay for antigen testing for diagnosing PDH using the recombinant 100 kDa protein of Histoplasma capsulatum (Hcp100) and their polyclonal antibodies as novel reagents, respectively. METHODS: Serum and urine samples from a cohort of patients with HIV/AIDS and proven PDH were studied for the detection of anti-Hcp100 antibodies by ELISA and Hcp100 antigen by dot blot, respectively. Sensitivity, specificity and cross-reactions with other diseases were estimated for each assay and compared with those obtained using histoplasmin (HMN) as a reagent for antibodies detection by ELISA and immunodiffusion, and using a commercial antigenuria test. RESULTS: Antibodies detection by the Hcp100 ELISA demonstrated 78.6% sensitivity and 88.4% specificity, versus 85.7% sensitivity and 81.0% specificity for the HMN ELISA and 26.1% sensitivity and 100% specificity for the immunodiffusion assay. Antigen detection by the Hcp100 dot blot demonstrated 89.3% sensitivity and 97.0% specificity versus 82.1% sensitivity and 90.9% specificity for the commercial test. CONCLUSION: The immunoassays described herein based on Hcp100 would be a valuable screening tool for diagnosing PDH.

Development and evaluation of polyclonal antibody-based antigen detection ELISA and dot blot assays as a less costly diagnostic alternative for pathogenic Leptospira infection.

Leptospirosis is an infectious, zoonotic disease of worldwide distribution, the cause of which is infection by pathogenic Leptospira. In Chile, dairy cattle are recognized a significant source in the maintenance and transmission of this infection, which causes economic losses and represents an infection threat to workers in the dairy industry. The infection is underestimated in cattle, due to the lack of clinical, pathognomonic signs, as well as the low efficiency of current diagnostic techniques. In this study, we developed antigen ELISA and dot blot assays, based on polyclonal antibodies, to detect pathogenic Leptospira in the urine samples of dairy cattle. The proposed tests showed an acceptable diagnostic accuracy, based on an analytical sensitivity of 1·104 Leptospira per mL for ELISA, and 3.2·103 for dot blot. These results corresponded with those obtained by qPCR, and the use of urine samples allowed us to propose new diagnostic alternatives for pathogenic Leptospira infection at a low cost, which can provide information on active infection status, which is a key element in control programs both at individual and herd level.

Identificação de peptídeos potenciais candidatos para desenvolvimento de vacina contra Leptospirose

Leptospirosis is caused by bacteria of the genus Leptospira. More than 500,000 severe cases are reported annually, with a mortality rate of over 10%. Infection in humans usually occurs through direct or indirect contact with the urine of carrier animals. Reservoir animals do not present clinical signs of the acute disease, but renal colonization by Leptospira is frequent. Leptospirosis affects populations in both urban and rural areas, especially in tropical and subtropical climates areas. Conditions such as heavy rainfall and areas without sanitary services, favor the incidence and spread of leptospirosis. Humans are accidental hosts and may have asymptomatic manifestations or severe disease leading to death. The Leptospira genus harbors a wide variety of serovars, hindering the development of vaccine and early diagnosis, and consequently the control of the disease. The aim of this study was to identify Leptospira protein peptides conserved with immunogenic potential and capacity for recognition by antibodies present in human sera from diagnosed patients with leptospirosis. For this, five peptides were designed and selected in silico and analyzed by Dot-blot technique. The results demonstrated the five peptides were recognized by antibodies from human serum. The Pep 9 was recognized by two sera samples. In this study, the immunoinformatics tools were important to help in the peptides screening, and optimizing time and reducing financial expenses. The Dot-blot technique indicate to be effective for testing the recognition of epitopes that may induce an immune system response. The identification of peptides that are recognized by antibodies is a promising approach for the development of vaccines and diagnostic method for leptospirosis.

A leptospirose é causada por bactérias do gênero Leptospira. Anualmente são relatados mais de 500.000 casos graves, apresentando uma taxa de mortalidade superior a 10%. A infecção em humanos geralmente ocorre por contato direto ou indireto com a urina de animais portadores. Os humanos são hospedeiros acidentais e podem ter manifestações assintomáticas até condições graves evoluindo para óbito. Condições climáticas como chuvas intensas e condições sociais como áreas sem serviços sanitários, favorecem a incidência e disseminação da leptospirose. O gênero Leptospira alberga grande variedade de sorovares dificultando o desenvolvimento de uma vacina eficiente e diagnóstico precoce, e consequentemente o controle da doença. O presente estudo visa desenhar e identificar peptídeos de proteínas de Leptospira com potencial imunogênico e com capacidade de reconhecimento por anticorpos presentes em soros de pacientes diagnosticados com leptospirose. Para isso, cinco peptídeos foram desenhados e analisados in silício e por técnica de Dot-blot. Os resultados demonstraram a capacidade de reconhecimento dos cinco peptídeos testados frente às amostras de soro humano. Nesse estudo o uso da imunoinformática foi importante para auxiliar na escolha dos peptídeos, otimizando o tempo e diminuindo gastos financeiros. A técnica Dot-blot se mostrou eficaz para analisar o reconhecimento dos peptídeos de proteínas de Leptospira por anticorpos presentes nas amostras de soros analisadas. A identificação de peptídeos que sejam reconhecidos por anticorpos, é uma abordagem promissora para o desenvolvimento de vacinas e método de diagnostico para a leptospirose.

Multiparametric assay of screening for the diagnosis of mycoses of interest in Public Health: standardization of methodology / Ensaio multiparamétrico de triagem para diagnóstico de micoses de interesse em Saúde Pública: padronização da metodologia

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).

Análise e identificação de peptídeos como possíveis candidatos para o desenvolvimento de vacinas e métodos diagnóstico da leptospirose

Leptospirosis is caused by bacteria of the genus Leptospira and is responsible for about 60 thousand deaths annually worldwide. The infection occurs through direct or indirect contact with contaminated animals' urine because the bacteria colonize the renal tubes e are excreted in the urine, which can cause renal insufficiency. The bacteria can also spread into the lungs and liver, causing fever, lungs hemorrhage, and hepatic insufficiency with lethality in about 15% of cases. Nowadays, there are about 13 pathogenic species of Leptospira with more than 200 serovars described in the literature. This diversity hinders vaccine and diagnostic methods development, which could cover such variability. Thus, the study aims to check peptides' recognition capacity from Leptospira proteins by hamsters’ serum previously immunized with an anti-Leptospirosis’ vaccine with reduced LPS. To do so, seven peptides were designed and analyzed in silico, synthesized, and evaluated by the Dot Blot technique. The selected peptides were applied into a 0,45 μm nitrocellulose membrane and incubated with the serum of immunized animals to verify possible recognitions. Next, the membranes were incubated with a secondary antibody conjugated with peroxidase. The membranes were revealed using ECL kit and the chemiluminescent signal was detected by the Cambridge Uvitec. Preliminary results showed specific recognition capability of three of the seven designed peptides from Leptospira proteins. The remaining peptides did not show a detectable signal or showed possible non-specific interactions. Therefore, we concluded that the Dot Blot technique could be a simple and effective technique used to analyze and identification of epitopes and proteins able to induce antibodies in immunizations or natural infections. Besides that, the identification of peptides able to recognize specifically anti-Leptospira antibodies is a promising strategy for diagnostic test development and identification of possible candidates for vaccines development.

A leptospirose é causada por bactérias do gênero Leptospira, sendo responsável por aproximadamente 60 mil mortes anualmente ao redor do mundo. O contágio se dá pelo contato direto ou indireto com a urina de animais contaminados, pois as bactérias colonizam os túbulos renais e são excretadas na urina podendo causar insuficiência renal. As bactérias também se disseminam nos pulmões e no fígado, podendo causar febre, hemorragias pulmonares e insuficiência hepática com letalidade em torno de 15% dos casos. Atualmente, cerca de 13 espécies patogênicas de Leptospira são descritas na literatura com mais de 200 sorovares. Essa diversidade dificulta o desenvolvimento de vacinas e métodos de diagnósticos que consigam cobrir tamanha variedade. Dessa forma, o estudo almeja averiguar a capacidade de reconhecimento de peptídeos oriundos de proteínas de Leptospira por soro de hamsters, previamente imunizados com vacinas anti-leptospirose com LPS reduzido. Para isso, sete peptídeos foram desenhados e analisados in silico, sintetizados e avaliados por técnica de Dot Blot. Os peptídeos selecionados foram aplicados em membranas de nitrocelulose 0,45 μm e incubados com o soro de animais imunizados, para verificar possíveis reconhecimentos. Em seguida, as membranas foram incubadas com o anticorpo secundário conjugado a peroxidase. As membranas foram reveladas com o kit ECL e o sinal detectado por quimioluminescência utilizando o aparelho Uvitec Cambridge. Os resultados preliminares demonstraram capacidade de reconhecimento especifico de três dos sete peptídeos desenhados a partir de proteínas de Leptospira. Os demais peptídeos não apresentaram sinal detectável ou evidenciaram possíveis interações inespecíficas. Com isso, concluímos que a técnica de Dot Blot pode ser uma ferramenta simples e eficaz utilizada para a análise e identificação de epítopos e peptídeos de proteínas capazes de induzir anticorpos em imunizações ou infecções naturais. Além disso, identificar peptídeos capazes de reconhecer especificamente anticorpos anti-Leptospira é uma estratégia promissora para o desenvolvimento de testes de diagnóstico e identificação de possíveis candidatos para o desenvolvimento de vacinas.

Análise e identificação de peptídeos como possíveis candidatos para o desenvolvimento de vacinas e métodos diagnóstico da leptospirose

Leptospirosis is caused by bacteria of the genus Leptospira and is responsible for about 60 thousand deaths annually worldwide. The infection occurs through direct or indirect contact with contaminated animals' urine because the bacteria colonize the renal tubes e are excreted in the urine, which can cause renal insufficiency. The bacteria can also spread into the lungs and liver, causing fever, lungs hemorrhage, and hepatic insufficiency with lethality in about 15% of cases. Nowadays, there are about 13 pathogenic species of Leptospira with more than 200 serovars described in the literature. This diversity hinders vaccine and diagnostic methods development, which could cover such variability. Thus, the study aims to check peptides' recognition capacity from Leptospira proteins by hamsters’ serum previously immunized with an anti-Leptospirosis’ vaccine with reduced LPS. To do so, seven peptides were designed and analyzed in silico, synthesized, and evaluated by the Dot Blot technique. The selected peptides were applied into a 0,45 μm nitrocellulose membrane and incubated with the serum of immunized animals to verify possible recognitions. Next, the membranes were incubated with a secondary antibody conjugated with peroxidase. The membranes were revealed using ECL kit and the chemiluminescent signal was detected by the Cambridge Uvitec. Preliminary results showed specific recognition capability of three of the seven designed peptides from Leptospira proteins. The remaining peptides did not show a detectable signal or showed possible non-specific interactions. Therefore, we concluded that the Dot Blot technique could be a simple and effective technique used to analyze and identification of epitopes and proteins able to induce antibodies in immunizations or natural infections. Besides that, the identification of peptides able to recognize specifically anti-Leptospira antibodies is a promising strategy for diagnostic test development and identification of possible candidates for vaccines development.

A leptospirose é causada por bactérias do gênero Leptospira, sendo responsável por aproximadamente 60 mil mortes anualmente ao redor do mundo. O contágio se dá pelo contato direto ou indireto com a urina de animais contaminados, pois as bactérias colonizam os túbulos renais e são excretadas na urina podendo causar insuficiência renal. As bactérias também se disseminam nos pulmões e no fígado, podendo causar febre, hemorragias pulmonares e insuficiência hepática com letalidade em torno de 15% dos casos. Atualmente, cerca de 13 espécies patogênicas de Leptospira são descritas na literatura com mais de 200 sorovares. Essa diversidade dificulta o desenvolvimento de vacinas e métodos de diagnósticos que consigam cobrir tamanha variedade. Dessa forma, o estudo almeja averiguar a capacidade de reconhecimento de peptídeos oriundos de proteínas de Leptospira por soro de hamsters, previamente imunizados com vacinas anti-leptospirose com LPS reduzido. Para isso, sete peptídeos foram desenhados e analisados in silico, sintetizados e avaliados por técnica de Dot Blot. Os peptídeos selecionados foram aplicados em membranas de nitrocelulose 0,45 μm e incubados com o soro de animais imunizados, para verificar possíveis reconhecimentos. Em seguida, as membranas foram incubadas com o anticorpo secundário conjugado a peroxidase. As membranas foram reveladas com o kit ECL e o sinal detectado por quimioluminescência utilizando o aparelho Uvitec Cambridge. Os resultados preliminares demonstraram capacidade de reconhecimento especifico de três dos sete peptídeos desenhados a partir de proteínas de Leptospira. Os demais peptídeos não apresentaram sinal detectável ou evidenciaram possíveis interações inespecíficas. Com isso, concluimos que a técnica de Dot Blot pode ser uma ferramenta simples e eficaz utilizada para a análise e identificação de epítopos e peptídeos de proteínas capazes de induzir anticorpos em imunizações ou infecções naturais. Além disso, identificar peptídeos capazes de reconhecer especificamente anticorpos anti-Leptospira é uma estratégia promissora para o desenvolvimento de testes de diagnóstico e identificação de possíveis candidatos para o desenvolvimento de vacinas.

Dot blot platform as a novel diagnostic kit: rapid, accurate, and on-site detection of Schistosoma mansoni in urine samples of hard to detect individuals.

Rapid diagnostics provide actionable information for patient care at the time and site of an encounter with the health care system. The mainstay of infectious diseases care is early detection (case finding) and treatment completion, but for many, it is hard to identify positive individuals, as is the case of infection with low burden in schistosomiasis, a parasitic disease common in the tropics and subtropics. We developed a new, accurate, and fast Dot blot methodology (iDot) to indirectly detect Schistosoma mansoni in individuals with very low parasite burden using urine samples. Accuracy of 0.74 was obtained with a significant difference between negative and positive patients and a substantial agreement was found when iDot was compared with five available methods. Our analysis also revealed the superiority of iDot in detecting negative individuals from non-endemic sites, thus, presenting the lowest rate of false positives. This new method called iDot is convenient and suitable for qualitative and quantitative detection of schistosomiasis in individuals with low parasite burden.

Quantification of matrix metalloproteinases MMP-8 and MMP-9 in gingival overgrowth.

BACKGROUND: Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in extracellular matrix remodeling of all body tissues, including oral tissues such as gingival tissue. Expression levels of MMPs are widely studied as important biomarkers for explaining the biochemical mechanisms and evolution of many oral diseases. OBJECTIVE: Demonstrate the sensitivity, reproducibility, repeatability, and robustness of the dot blot assay for the relative quantification of MMP-8 and MMP-9 expression levels in patients with GO associated with orthodontic treatment. METHODS: A validated dot blot assay was used to compare the relative expression levels of MMP-8 and MMP-9 in gingival samples. Methodological variability, reproducibility, sensitivity and robustness were determined with the use of control samples from healthy donors (G1). Next, expression levels were measured in gingival tissue from patients with mild and moderate gingival overgrowth associated with orthodontic treatment (G3 and G4) and patients without gingival overgrowth but with a history of using orthodontic appliances (G2). RESULTS: Dot blot assay demonstrated that MMP-8 and MMP-9 expression levels were higher in patients with gingival overgrowth and distinguished those with moderate clinical grade (G4) from those with mild overgrowth (G3). In addition, patients with a history of orthodontic treatment showed similar expression levels to the control group two years after removing orthodontic appliances. CONCLUSIONS: With the assay used, we were able to detect differences in MMP-8 and MMP-9 expression in patients with different levels of severity of gingival overgrowth. Dot blot could be used to measure MMPs during the onset and progression of gingival overgrowth.

Nano dot blot: An alternative technique for protein identification and quantification in a high throughput format.

BACKGROUND: Dot blot technique has been used in a similar way to western blotting, with the major difference being the lack of protein separation with electrophoresis. Protein samples are spotted over a membrane paper, the identification and quantification of a protein is achieved by immunodetection procedures such as colorimetry, fluorescence or chemiluminescence. This technique is widely accepted, but it uses large amounts of sample and antibodies to reveal the presence of the target protein. Significant milestones have been reached to achieve better results with the use of less sample and reagents; however, the ninety-six-well format is still in use. NEW METHOD: In this work, we propose an innovation to this technique, reducing the amount of sample and antibodies to identify a specific protein when compared to the regular dot blot method. Procedure consists of using a sample volume of approximately 200 nanoliters deposited with a multineedle device developed by our group. RESULTS: Five samples of standard protein or antigen can be spotted in a Cartesian format to identify and quantify the protein involved in physiological or pathological conditions. In addition, at least five replicates of sample or antigen are used to enable better statistics to calculate the concentration of every standard and the protein present in a sample. CONCLUSIONS: Hundreds of samples can be deposited in a few minutes and analyzed in a single experimental session. To validate this method, which we called nano dot blot, six proteins involved in the inflammation process were tested in acute and chronic rat models of seizures.

Application of a dot blot hybridization assay for genotyping Streptococcus uberis from Brazilian dairy herds.

Streptococcus uberis is a major cause of environmental mastitis in many regions, and it is associated with clinical and subclinical infections. Although the main source of infection is the environment, reports of strains with a contagious profile have been described. Dot blot hybridization analysis allows the rapid identification of S. uberis population structures within and between herds, and it helps to identify strain diversity as well as possible clonal lineages that directly affect the control of bovine mastitis caused by this pathogen. The aim of this study was to evaluate the diversity of S. uberis isolates obtained from clinical (n = 22) and subclinical (n = 22) cases of mastitis in dairy herds (n = 13) in Brazil over a period of 12 mo. We submitted 44 S. uberis isolates to dot blot hybridization followed by automatic data analysis. We identified 8 different hybridization patterns using genetic markers associated with virulence factors and taxonomy, indicating diversity of S. uberis within the population and suggesting environmental transmission. However, the evidence of identical dot blot patterns in different mammary quarters from the same animal also suggested local contagious transmission. Of the virulence genes evaluated, we found a high prevalence of the genes sua, pauA, and gapC, highlighting the importance of these virulence factors for the adhesion, invasion, and multiplication of S. uberis in subclinical and clinical intramammary infections.

Comparison of different analytical methods to evaluate the heat shock protein (HSP) response in fruits. Application to tomatoes subjected to stress treatments.

Heat shock proteins (HSP) are synthesized in living tissues exposed to transient increase in temperature and play a central role in the protective response against heat and other stresses. In fruits, this response to heat treatment provides resistance to a physiological alteration known as chilling injury. Despite the physiological importance of this group of proteins, publications comparing different methodological alternatives for their analysis are rather scarce. In the present paper, we conducted a comparative study using different electrophoretic and immunological techniques to evaluate the HSP response in fruits. Proteins were extracted from tomato fruit exposed to an HSP-inducing temperature (38 °C) for different times (0, 3, 20, and 27 h). Different alternatives of analysis (SDS-PAGE, SDS-PAGE followed by IEF, Western blot, and dot blot) were performed, and their potential application discussed. The study was complemented with a practical application, in which tomatoes were subjected to heat and anaerobic treatments and then stored in a chill-inducing temperature. This application evidences the relevance of knowing the level of proteins attained by stress treatments which correlates with the acquired tolerance.

Performance of the Dot-blot test method for detecting antibodies to Sarcocystis spp. in cattle / Desempenho do teste Dot-blot para detecção de anticorpos para Sarcocystis spp. em bovinos

Serological techniques can detect antibodies against Sarcocystis spp., Neospora caninum and Toxoplasma gondii antigens in single or mixed infections. Immunofluorescent antibody tests (IFAT) is considered the gold standard technique for Sarcocystosis diagnostic in cattle serum and a positive IFAT result reflects Sarcocystis spp. infection. Therefore, the aims of the present study were to compare IFAT and Dot-blot for sarcocystosis diagnostic in experimentally infected mice and to investigate serological cross-reactions with N. caninum and T. gondii in these methods. Mice (Mus musculus) were inoculated intraperitoneally with bradizoites of Sarcocystis spp. or tachyzoites of N. caninum or T. gondii. Serum samples were obtained and analyzed by IFAT and Dot-blot for the three protozoa. Serum from N. caninum and T. gondii experimentally infected mice were tested by IFAT and reacted only to N. caninum or T. gondii antigens, respectively. Specific antibodies against Sarcocystis spp. were present in all animals experimentally infected with this protozoan, with IFAT titers from 10 to 800. Serum samples from mice experimentally infected with Sarcocystis spp., N. caninum and T. gondii and tested by Dot-blot demonstrated no cross reaction between protozoa. A Dot-blot using Sarcocystis spp. antigen appears to be a good alternative to IFAT in the serological diagnosis of Sarcocystosis.(AU)

As técnicas sorológicas podem detectar anticorpos contra os antígenos de Sarcocystis spp., Neospora caninum e Toxoplasma gondii em infecções únicas ou mistas. O teste de anticorpos imunofluorescentes (IFAT) é considerado a técnica padrão-ouro para o diagnóstico de sarcocistose no soro de bovinos e um resultado positivo de IFAT reflete Sarcocystis spp. infecção. Portanto, os objetivos do presente estudo foram comparar IFAT e Dot-blot para diagnóstico de sarcocistose em camundongos infectados experimentalmente e investigar reações cruzadas sorológicas com N. caninum e T. gondii nesses métodos. Os camundongos (Mus musculus) foram inoculados intraperitonealmente com bradizoítos de Sarcocystis spp. ou taquizoítos de N. caninum ou T. gondii. As amostras de soro foram obtidas e analisadas por IFAT e Dot-blot para os três protozoários. O soro de N. caninum e T. gondii infectados experimentalmente foram testados por IFAT e reagiram apenas aos antígenos de N. caninum ou T. gondii, respectivamente. Anticorpos específicos contra Sarcocystis spp. estavam presentes em todos os animais experimentalmente infectados com este protozoário, com títulos de IFAT de 10 a 800. Amostras de soro de camundongos infectados experimentalmente com Sarcocystis spp., N. caninum e T. gondii e testadas por Dot-blot não demonstraram reação cruzada entre protozoários. Um Dot-blot usando Sarcocystis spp. O antígeno parece ser uma boa alternativa ao IFAT no diagnóstico sorológico da sarcocistose.(AU)

Performance of the Dot-blot test method for detecting antibodies to Sarcocystis spp. in cattle / Desempenho do teste Dot-blot para detecção de anticorpos para Sarcocystis spp. em bovinos

Serological techniques can detect antibodies against Sarcocystis spp., Neospora caninum and Toxoplasma gondii antigens in single or mixed infections. Immunofluorescent antibody tests (IFAT) is considered the gold standard technique for Sarcocystosis diagnostic in cattle serum and a positive IFAT result reflects Sarcocystis spp. infection. Therefore, the aims of the present study were to compare IFAT and Dot-blot for sarcocystosis diagnostic in experimentally infected mice and to investigate serological cross-reactions with N. caninum and T. gondii in these methods. Mice (Mus musculus) were inoculated intraperitoneally with bradizoites of Sarcocystis spp. or tachyzoites of N. caninum or T. gondii. Serum samples were obtained and analyzed by IFAT and Dot-blot for the three protozoa. Serum from N. caninum and T. gondii experimentally infected mice were tested by IFAT and reacted only to N. caninum or T. gondii antigens, respectively. Specific antibodies against Sarcocystis spp. were present in all animals experimentally infected with this protozoan, with IFAT titers from 10 to 800. Serum samples from mice experimentally infected with Sarcocystis spp., N. caninum and T. gondii and tested by Dot-blot demonstrated no cross reaction between protozoa. A Dot-blot using Sarcocystis spp. antigen appears to be a good alternative to IFAT in the serological diagnosis of Sarcocystosis.(AU)

As técnicas sorológicas podem detectar anticorpos contra os antígenos de Sarcocystis spp., Neospora caninum e Toxoplasma gondii em infecções únicas ou mistas. O teste de anticorpos imunofluorescentes (IFAT) é considerado a técnica padrão-ouro para o diagnóstico de sarcocistose no soro de bovinos e um resultado positivo de IFAT reflete Sarcocystis spp. infecção. Portanto, os objetivos do presente estudo foram comparar IFAT e Dot-blot para diagnóstico de sarcocistose em camundongos infectados experimentalmente e investigar reações cruzadas sorológicas com N. caninum e T. gondii nesses métodos. Os camundongos (Mus musculus) foram inoculados intraperitonealmente com bradizoítos de Sarcocystis spp. ou taquizoítos de N. caninum ou T. gondii. As amostras de soro foram obtidas e analisadas por IFAT e Dot-blot para os três protozoários. O soro de N. caninum e T. gondii infectados experimentalmente foram testados por IFAT e reagiram apenas aos antígenos de N. caninum ou T. gondii, respectivamente. Anticorpos específicos contra Sarcocystis spp. estavam presentes em todos os animais experimentalmente infectados com este protozoário, com títulos de IFAT de 10 a 800. Amostras de soro de camundongos infectados experimentalmente com Sarcocystis spp., N. caninum e T. gondii e testadas por Dot-blot não demonstraram reação cruzada entre protozoários. Um Dot-blot usando Sarcocystis spp. O antígeno parece ser uma boa alternativa ao IFAT no diagnóstico sorológico da sarcocistose.(AU)

Egg Albumin as a Protein Marker to Study Dispersal of Noctuidae in the Agroecosystem.

Knowledge of dispersal and spatial dynamics of pest populations is fundamental for implementation of integrated pest management and integrated resistance management. This study evaluated 1) the effectiveness of egg white albumin protein to mark larvae and adults of two polyphagous and highly mobile pests, Spodoptera frugiperda (J.E. Smith) (fall armyworm) and Helicoverpa zea (Boddie) (corn earworm) (Lepidoptera: Noctuidae), and 2) the sensitivity of polyvinylidene difluoride membrane (dot blot) in detecting albumin on marked insects. Laboratory and field experiments tested egg albumin as a protein marker, which was detected using two enzyme-linked immunosorbent assay (ELISA), microplate, and dot blot. In the laboratory, 100% of the moths sprayed with 20% egg white solution acquired the albumin marker, which was detected through the last time point tested (5 d) after application. Egg albumin was not effective at long-term marking of larvae, detected only prior the molting to the next instar. Albumin application in field cages resulted in a high percentage of moths detected as marked at 24 h and 5 d for both species. Egg albumin applied in the open field resulted in 15% of the recaptured corn earworm moths marked with most of them collected 150 m from the application area, although some were captured as far as 1,600 m within approximately 6 d after adult emergence. The results indicated egg albumin is a suitable marker to study the dispersion of fall armyworm and corn earworm in the agroecosystem and dot blot was as effective to detect egg albumin as was indirect ELISA.

Detection of miRNAs by Tissue Printing and Dot Blot Hybridization.

Tissue printing and dot blot are simple techniques to detect miRNA expression and localization, allowing a better understanding of the function of a miRNA. In this work, we describe a tissue printing and a dot blot hybridization protocol for miRNA detection and localization in plant tissues, which opens the possibility of analyzing spatiotemporal expression patterns of miRNAs.

Validación de una técnica de Dot blot para la detección de Cercospora kikuchii en plantas de soja / Validation of the technique based on Dot blot for the detection of Cercospora kikuchii in soybean plants

Cercospora kikuchii, fitopatógeno usual en plantas de soja, ocasiona deterioro en la scosechas. Su identificación precoz y correcta evitaría el uso indebido de plaguicidas y permitiría iniciar un tratamiento adecuado. Una técnica rápida, económica y de fácil ejecución es el Dot blot, capaz de reconocer la presencia de una proteína específica del género conocida como CFP (Cercosporin Facilitator Protein). El objetivo de este trabajo fue validar dicha técnica para garantizar la fiabilidad del resultado. Para ello, se procesaron 29 plantas de soja infectadas y 31 plantas sanas teniendo en cuenta una confianza deseada del 95% y un error permitido del 5%. La técnica presentó una sensibilidad diagnóstica del 93,3% y una especificidad diagnóstica del 96,7%. La eficacia fue del 95% y los valores predictivos positivo y negativo del 96,6 y el 93,5%, respectivamente. Estos resultados la postulan como una herramienta útil para detectar precozmente c. kikuchii en plantas de soja.

Cercospora kikuchii is a common pathogen in soybean plants that causes crop spoilage. Its early and precise identification would prevent the misuse of pesticides and allow the initiation of an appropriate treatment. A quick, economical and easy-to-execute technique is the Dot blot, capable of recognizing the presence of a genus-specific protein called CFP (Cercosporin Facilitator Protein). The objective was to validate this technique to guarantee the reliability of the results. For that purpose, 29 infected soybean plants and 31 healthy plants were processed, taking into account a 95% desired confidence level and a permissible error of 5%. The technique provided a diagnostic sensitivity of 93.3% and a diagnostic specificity of 96.7%. The efficiency was 95% and positive and negative predictive values were 96.6% and 93.5%, respectively. These results postulate it as a useful resource for the early detection of C. kikuchii in soybean plants.

Evaluation of monoclonal antibodies that detect conserved proteins from Respiratory Syncytial Virus, Metapneumovirus and Adenovirus in human samples.

Human Respiratory Syncytial Virus (hRSV), human Metapneumovirus (hMPV) and Adenovirus (ADV), are three of the most prevalent viruses responsible for pneumonia and bronchiolitis in children and elderly worldwide, accounting for a high number of hospitalizations annually. Diagnosis of these viruses is required to take clinical actions that allow an appropriate patient management. Thereby, new strategies to design fast diagnostic methods are highly required. In the present work, six monoclonal antibodies (mAbs, two for each virus) specific for conserved proteins from hRSV, hMPV and ADV were generated and evaluated through different immunological techniques, based on detection of purified protein, viral particles and human samples. In vitro evaluation of these antibodies showed higher specificity and sensitivity than commercial antibodies tested in this study. These antibodies were used to design a sandwich ELISA tests that allowed the detection of hRSV, hMPV, and ADV in human nasopharyngeal swabs. We observed that hRSV and ADV were detected with sensitivity and specificity equivalent to a current Direct Fluorescence Assay (DFA) methodology. However, hMPV was detected with more sensitivity than DFA. Our data suggest that these new mAbs can efficiently identify infected samples and discriminate from patients infected with other respiratory pathogens.