Advancing serologic diagnosis: assessing the efficacy of rErpY-like protein in human leptospirosis detection.

Leptospirosis is a globally distributed infectious disease caused by pathogenic spirochetes of the Leptospira genus, often overlooked. It is estimated that the disease affects approximately one million people annually, resulting in more than 58,900 deaths. The gold standard for serodiagnosis of leptospirosis is the Microscopic Agglutination Test (MAT). However, the limitations of this technique necessitate the exploration of alternative diagnostic methods. In this study, we evaluated the ErpY-like recombinant protein (rErpY-like) in the development of a serologic diagnostic assay for human leptospirosis. Eighty-six human sera samples, characterized by MAT, underwent evaluation through indirect IgM-ELISA and IgG-ELISA. The sensitivity and specificity values obtained from IgM-ELISA were 60% and 76%, respectively, while those from IgG-ELISA were 96.4% and 100%, respectively. The use of the rErpY-like protein in both IgM-ELISA and IgG-ELISA proves to be a sensitive and specific method for antibody detection. This could potentially serve as a valuable alternative tool in the diagnosis of human leptospirosis.

An evaluation of techniques to diagnose Dioctophyme renale in dogs.

Dioctophyme renale is a nematode with zoonotic potential that affects the kidneys of carnivorous, wild, and domestic mammals. In this study, we sought to evaluate the indirect ELISA method against routine methods used to diagnose dioctophimosis. Hence, 38 dogs parasitized by D. renale, as confirmed by surgery, were selected. The dogs were evaluated by abdominal ultrasound and urinalysis, and their sera were tested by indirect ELISA using D. renale adult secretion and excretion antigen (DES). Five dogs were followed up with serum collections on day 0 (day of surgery) and 30, 60, and 90 days after surgery to evaluate antibody kinetics. Abdominal ultrasound and indirect ELISA successfully diagnosed 37 dogs parasitized by D. renale, while urinalysis diagnosed 29 animals. The positive animals were parasitized with 1-7 parasites; 17 dogs were infected by male and female parasites, 15 only by female parasites, and six were parasitized only by male parasites. When assessing specificity and sensitivity, all techniques showed 100% specificity and 81.6%, 97.4%, and 97.4% sensitivity for urinalysis, ultrasound, and ELISA, respectively (p < 0.001). The five positive dogs that were followed up after surgery showed a progressive decrease in mean absorbances in indirect ELISA (0.644, 0.516, 0.511, and 0.440, respectively). This study demonstrated that the indirect ELISA using the DE antigen could diagnose dioctophimosis regardless of the number, sex, and location of the parasites, with the potential to be used in epidemiological research and implementing immunological and molecular studies, opening new lines of research on D. renale.

Dioctophyme renale é um nematódeo que possui potencial zoonótico e acomete o rim de mamíferos carnívoros, silvestres e domésticos. Neste estudo busca-se avaliar a técnica de ELISA indireto frente metodologias de rotina utilizadas no diagnóstico da dioctofimose. Para isto, 38 cães participaram do estudo, sendo todos parasitados por D. renale, confirmados por cirurgia. Esses cães foram avaliados por ultrassom abdominal, urinálise e seus soros testado por ELISA indireto utilizando antígeno de excreção e secreção (DES) de adultos de D. renale. Destes, cinco cães foram acompanhados com coletas de soro, no dia zero (dia da cirurgia) e após 30, 60 e 90 dias da cirurgia para avaliar a cinética dos anticorpos. O ultrassom abdominal e ELISA indireto apresentaram sucesso no diagnóstico de 37 cães parasitados por D. renale, enquanto que a urinálise diagnosticou 29 animais. Os animais positivos possuíam de um a sete parasitos, 17 cães apresentaram infecções por macho e fêmeas, 15 somente por fêmeas e seis foram parasitados apenas por machos. Na avaliação da especificidade e sensibilidade, todas as técnicas apresentaram 100% de especificidade e 81,6%, 97,4%, 97,4% de sensibilidade para urinálise, ultrassom e ELISA, respectivamente (p < 0,001). Os cinco cães positivos que foram acompanhados após a cirurgia apresentaram diminuição progressiva nas médias de absorbâncias no ELISA indireto (0,644, 0,516, 0,511 e 0,440, respectivamente). O estudo demonstrou que o ELISA indireto utilizando o antígeno DES poderia diagnosticar dioctofimose, independentemente do número, sexo e localização dos parasitos, com potencial para ser utilizada em estudos epidemiológicos e na implementação de estudos imunológicos e moleculares, abrindo novas linhas de pesquisa sobre Dioctophyme renale.

Subclinical circulation of Chicken infectious anaemia virus- A Sero-molecular study.

Chicken infectious anaemia-an important immunosuppressive viral disease of chicken-gained much attention in the recent past. Based on huge mortality and production loss observed in the fast-growing poultry sector, the present study aimed to find out the current status of the chicken infectious anaemia virus (CIAV), among chicken flocks in the Punjab state of India by sero-molecular study. The sera from the blood samples were tested for anti-CIAV antibodies by indirect ELISA and also compared with haematological parameters. DNA from sero-positive samples underwent PCR amplification, sequencing and phylogenetic analysis of the most conserved genomic region (VP3 gene) to detect viraemia in asymptomatic birds. The serological study using indirect ELISA showed a high sero-positivity of 77.27% in chicken flocks. Additionally, the present study also revealed the high molecular evidence (72.54%) of CIAV in apparently healthy birds. Genetic analysis showed that all CIAVs have conserved VP3 genes without any nucleotide substitutions, indicating presence of CIAV and its subclinical circulation among apparently healthy flocks. The wide distribution of CIAV among birds may be the reason for huge mortality and production loss. Further, it is suggested that studies be conducted to find out the co-involvement of CIAV with other immunosuppressive microbial agents and the immunosuppressive effect of CIAV in apparently healthy birds. Also, its role in vaccine failure and outbreaks of various other avian diseases needs to be explored.

Serological Cross-Reaction between Six Thiadiazine by Indirect ELISA Test and Their Antimicrobial Activity.

Malaria is a parasitic infection caused by a protozoon of the genus Plasmodium, transmitted to humans by female biting mosquitoes of the genus Anopheles. Chloroquine and its derivates have caused the parasite to develop drug resistance in endemic areas. For this reason, new anti-malarial drugs as treatments are crucial. This work aimed to evaluate the humoral response. with hyper-immune sera, of mice immunized with six derivatives of tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) by indirect ELISA test. The cross-reactivity between the compounds as antigens and their microbial activity on Gram-positive and Gram-negative bacteria was evaluated. The results of the humoral evaluation by indirect ELISA show that three bis-THTTs react with almost all of the above. Besides, three compounds used as antigens stimulate the BALB/c mice's immune system. The best combination of two antigens as a combined therapy displays similar absorbances between the antigens in the mixture, showing similar recognition by antibodies and their compounds. In addition, our results showed that different bis-THTT presented antimicrobial activity on Gram-positive bacteria, mainly on Staphylococcus aureus strains, and no inhibitory activity was observed on the Gram-negative bacteria tested.

Determination of Proinflammatory and Antiinflammatory Cytokines by ELISA Technique.

The main objective of this chapter is to determine inflammatory and anti-inflammatory cytokines in patients with or without preeclampsia by means of the enzyme-linked immunosorbent assay (ELISA) technique. In this chapter, 16 cell cultures were obtained from different patients admitted to the hospital with term vaginal delivery or cesarean section. Here, we describe the ability to quantify the presence of cytokines in cell culture supernatants. The supernatants of the cell cultures were collected and concentrated. The concentration of IL-6 and VEGF-R1 was measured by ELISA to determine the prevalence of alterations in the samples studied. We observed that the sensitivity of the kit allowed the detection of several cytokines in a range between 2 and 200 pg/mL. The test was performed using the ELISpot method (5×), which allowed a higher level of precision.

Design and optimization of an IgG human ELISA assay reactive to recombinant RBD SARS-CoV-2 protein.

Serology assays are essential tools to mitigate the effect of COVID-19, help to identify previous SARS-CoV-2 infections or vaccination, and provide data for surveillance and epidemiologic studies. In this study, we report the production and purification process of the receptor-binding domain (RBD) of SARS-CoV-2 in HEK293 cells, which allowed the design, optimization, and validation of an indirect ELISA (iELISA) for the detection of human anti-RBD antibodies. To find the optimal conditions of this iELISA, a multivariate strategy was performed throughout design of experiments (DoE) and response surface methodology (RSM), one of the main tools of quality by design (QbD) approach. The adoption of this strategy helped to reduce the time and cost during the method development stage and to define an optimum condition within the analyzed design region. The assay was then validated, exhibiting a sensitivity of 94.24 (86.01-98.42%; 95% CI) and a specificity of 95.96% (89.98-98.89%; 95% CI). Besides, the degree of agreement between quality results assessed using kappa's value was 0.92. Hence, this iELISA represents a high-throughput technique, simple to perform, reliable, and feasible to be scaled up to satisfy the current demands. Since RBD is proposed as the coating antigen, the intended use of this iELISA is not only the detection of previous exposure to the virus, but also the possibility of detecting protective immunity. KEY POINTS: • RBD was produced in 1-L bioreactor and highly purified. • An iELISA assay was optimized applying QbD concepts. • The validation procedure demonstrated that this iELISA is accurate and precise.

Seroincidencia de anticuerpos IgG del SARS-CoV-2 y factores de riesgo en trabajadores sanitarios asintomáticos del hospital Departamental de Villavicencio

Objetivo: Estimar la seroincidencia acumulada de inmunoglobulinas (Ig) clase G (IgG) anti-SARS-CoV-2 en trabajadores de la salud asintomáticos y su asociación epidemiológica dentro de las áreas funcionales del Hospital Departamental de Villavicencio (HDV). Metodología: Se llevó a cabo un estudio observacional analítico longitudinal de una cohorte de trabajadores, donde cada 21 días, en tres oportunidades, se midieron IgG anti-SARS-CoV-2 en suero sanguíneo, a través de ELISA indirecto, en una muestra representativa aleatoria (n= 105) de trabajadores sanitarios del hospital (N= 756). Como instrumento de recolección de datos se utilizó una encuesta, donde cada trabajador sanitario declaró no haber sido diagnosticado con COVID-19, e igualmente registró la información sobre las variables independientes: sexo, edad, condición laboral, área funcional y comorbilidades. Resultados: La prevalencia inicial para SARS-CoV-2 entre los trabajadores sanitarios asintomáticos del HDV fue de 9,52 % (IC 95 % 5,25-16,65). La seroincidencia acumulada durante 42 días fue de 12,38 % (IC 95 % 7,38-20,04). El riesgo relativo (RR) se utilizó para establecer los factores de riesgo asociados a las variables independientes. El sexo masculino (RR ajustado = 3,34, IC 95 % 1,98-5,86), obesidad (RR ajustado = 10,98, IC 95 % 1,41-85,98) y sexo femenino (RR ajustado = 2,15, IC 95 % 1,12-4,31) en las áreas funcionales de Hospitalización, Medicina Crítica y Urgencias, respectivamente, son factores de riesgo en el HDV. Conclusión: Un total de 13 de 105 trabajadores sanitarios del hospital seroconvirtieron positivamente para SARS-CoV-2 y fueron asintomáticos durante 42 días de seguimiento epidemiológico. Además, existen factores de riesgo importantes en su exposición a este virus en el HDV.

Objective: To estimate the cumulative seroincidence of antisars-CoV-2 immunoglobulin (Ig) class G (IgG) in asymptomatic health care workers and its epidemiological association within the functional areas of the Villavicencio Departmental Hospital (HDV). Methodology: A longitudinal analytical observational study of a cohort of workers was conducted in which anti- SARS-CoV-2 IgG levels in blood serum were measured every 21 days on three occasions using an indirect ELISA in a random representative sample (n = 105) of hospital health workers (N = 756). The data collection tool was a survey in which each healthcare worker indicated that they had not been diagnosed with COVID-19 and provided information on the independent variables: sex, age, job status, functional area, and comorbidities. Results: The baseline prevalence for SARS-CoV-2 among asymptomatic HDV healthcare workers was 9.52% (CI 95% 5.25-16.65). Cumulative seroincidence over 42 days was 12.38% (CI 95% 7.38-20.04). Relative risk (RR) was used to establish the risk factors associated with the independent variables. Male sex (adjusted RR 3.34, CI 95% 1.98-5.86), obesity (adjusted RR 10.98, CI 95% 1.41- 85.98) and female sex (adjusted RR 2.15, CI 95% 1.12-4.31) in the functional areas of Hospitalization, Critical Medicine and Emergency, respectively, are risk factors in the HDV. Conclusion: During 42 days of epidemiological follow-up, 13 out of 105 hospital healthcare workers seroconverted positively for SARS-CoV-2 and remained asymptomatic. Additionally, significant risk factors are associated with their exposure to this virus in the HDV.

Objetivo: Estimar a incidência zero acumulada de imunoglobulinas (Ig) classe G (IgG) anti-SARS-CoV-2 em profissionais de saúde assintomáticos e sua associação epidemiológica dentro das áreas funcionais do Hospital Estadual de Villavicencio (HDV). Metodologia: Foi realizado um estudo observacional analítico longitudinal de uma coorte de profissionais, no qual a cada 21 dias, em três ocasiões mediram-se IgG anti-SARS-CoV-2 em soro sanguíneo, através de ELISA indireto, em uma amostra representativa aleatória (n = 105) de profissionais de saúde do hospital (N =756). Como instrumento de recolecção de dados foi usada uma pesquisa, onde cada profissional de saúde declarou não ter sido diagnosticado com COVID-19, e igualmente registrou a informação sobre as variáveis independentes: sexo, idade, condições de trabalho, área de atuação e comorbidades. Resultados: A prevalência inicial para SARS-CoV-2 entre os profissionais de saúde assintomáticos do HDV foi de 9,52% (IC 95% 5,25-16,65). A incidência zero acumulada durante 42 dias foi de 12,38% (IC 95% 7,38-20,04). O risco relativo (RR) foi utilizado para estabelecer os fatores de risco associados às variáveis independentes. O sexo masculino (RR ajustado 3,34, IC 95% 1,98-5,86), obesidade (RR ajustado 10,98, IC 95% 1,41-85,98) e sexo feminino (RR ajustado 2,15, IC 95% 1,12-4,31) nas áreas funcionais de Internação, Unidade de Terapia Intensiva e Urgências, respectivamente, são fatores de risco no HDV. Conclusão: Um total de 13 de 105 profissionais de saúde do hospital foram detectados positivamente para SARS-CoV-2 e foram assintomáticos durante 42 dias de seguimento epidemiológico. Além disso, existem importantes fatores de risco na sua exposição a este vírus no HDV.

The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high genetic and antigenic polymorphism. The objective of this study was to determine if GP-45 contains conserved B-cell epitopes, and if they would induce neutralizing antibodies. The comparative analysis of nucleotide and amino acids sequences revealed a high percentage of similarity between field isolates. Antibodies against peptides containing conserved B-cell epitopes of GP-45 were generated. Antibodies present in the sera of mice immunized with GP-45 peptides specifically recognize B. bigemina by the IFAT. More than 95% of cattle naturally infected with B. bigemina contained antibodies against conserved GP-45 peptides tested by ELISA. Finally, sera from rabbits immunized with GP-45 peptides were evaluated in vitro neutralization tests and it was shown that they reduced the percentage of parasitemia compared to sera from rabbits immunized with adjuvant. GP-45 from geographically distant isolates of B. bigemina contains conserved B-cell epitopes that induce neutralizing antibodies suggesting that this gene and its product play a critical role in the survival of the parasite under field conditions.

Evaluation of B. melitensis whole-cell lysate antigen-based indirect ELISA for the serodiagnosis of caprine brucellosis.

Different fractions of Brucella (B) abortus or Brucella melitensis have been used as antigens for the detection of anti-Brucella antibodies in goat sera, being their accomplishment cumbersome and time consuming. In an attempt to achieve a simpler enzyme-linked immunosorbent assay (ELISA) antigen preparation method for serodiagnosis of caprine brucellosis, we developed and evaluated a B. melitensis whole-cell lysate antigen-based indirect ELISA (Bm-WCL iELISA). A total of 162 serum samples from female crossbred goats collected from non-vaccinated herds against brucellosis were classified according to the buffered plate antigen (BPA) screening test and the complement fixation (CF) test and used for the indirect ELISA (iELISA) evaluation. The Bm-WCL iELISA showed a high Se and Sp [95.7% (CI 88.1% - 98.8%), and 92.4% (CI 83.4% - 96.7%), respectively] to detect the serological response against Brucella in commercial goat herds, and an almost perfect agreement with combined official tests results (κ = 0.88), when goat sera with concordant results in both official serological tests (BPA and CF; n = 136) were used. However, the agreement dropped to substantial (k > 0.73) when 26 goat serum samples with BPA and CF not concordant results were incorporated for the iELISA performance evaluation and the comparison was made for each test independently. Comparison of the Bm-WCL iELISA results with Brucella abortus sLPS iELISA showed almost perfect agreement (κ > 0.83). Even when a larger number of samples are needed to validate this test, these preliminary results encourage the optimization of the Brucella melitensis whole cell lysate antigen-based iELISA.

Seroprevalence and associated factors of infectious bovine rhinotracheitis and bovine viral diarrhea in dairy cows in the Caparaó region, Espírito Santo, Brazil / Soroprevalência e fatores associados à rinotraqueíte infecciosa bovina e diarreia viral bovina em vacas leiteiras na região do Caparaó, Espírito Santo, Brasil

This study evaluated the seroprevalence and associated factors of Infectious Bovine Rhinotracheitis (IBR) and Bovine Viral Diarrhea (BVD), and to analyze the possible relationship between IBR, BVD, and the occurrence of mastitis. For this purpose, 854 crossbred dairy cows were evaluated from 69 properties allocated in the 12 municipalities that make up the Caparaó region, Espírito Santo (ES), Brazil. The seroprevalence of IBR and BVD was determined using the indirect ELISA test. Associations between variables were estimated using the prevalence ratio (PR) with a 95% confidence interval. The chi-square test was used to verify the significance of the associations (P < 0.05). The average prevalence of IBR and BVD was 48.59% and 26.46%, respectively. Animals seroreactive for IBR were more likely to develop subclinical mastitis (P < 0.01; PR: 1.27), and cows that were seroreactive for BVD were more likely to develop clinical mastitis (P < 0.01; PR: 2.24). Mechanical milking was considered a factor associated with IBR (P < 0.01; PR: 1.36) and BVD (P < 0.01; PR: 1.25). Reproductive management by natural breeding was considered a factor associated with IBR (P < 0.01; PR: 1.22). Animals seroreactive for BVD were more likely to develop reproductive problems (P < 0.01; PR: 1.34). It was concluded that the agents that cause IBR and BVD are widely disseminated in dairy cattle herds in the municipalities of the Caparaó region, ES, Brazil. The presence of IBR and BVD increased the chances of cows developing subclinical mastitis and clinical mastitis, respectively, and the cows that were mechanically milked were more likely to be seroreactive for IBR and BVD.

O objetivo do presente estudo foi avaliar a soroprevalência e os fatores de risco associados à Rinotraqueíte Infecciosa Bovina (IBR) e Diarreia Viral Bovina (BVD), e analisar a possível relação entre IBR, BVD e à ocorrência de mastite. Para tanto, foram avaliadas 854 vacas leiteiras mestiças de 69 propriedades localizadas nos 12 municípios que compõem a região do Caparaó, Espírito Santo (ES), Brasil. A soroprevalência de IBR e BVD foram determinadas pelo teste ELISA indireto. As associações entre variáveis ​​foram estimadas pela razão de prevalência (PR) com intervalo de confiança de 95%. O teste do qui-quadrado foi utilizado para verificar a significância das associações (P < 0,05). A prevalência média de IBR e BVD foi de 48% e 26%, respectivamente. Os animais sororreagentes para IBR foram mais propensos a desenvolver mastite subclínica (P < 0,01; PR: 1,27), e as vacas sororreagentes para BVD foram mais propensas a desenvolver mastite clínica (P < 0,01; PR: 2,24). À ordenha mecânica foi considerada um fator associado a IBR (P < 0,01; PR: 1,36) e BVD (P < 0,01; PR: 1,25). O manejo reprodutivo por monta natural foi considerado um fator associado IBR (P < 0,01; PR: 1,22). Os animais sororreagentes para BVD foram mais propensos a desenvolverem problemas reprodutivos (P < 0,01; PR: 1,34). Concluiu-se que os agentes causadores de IBR e BVD estão amplamente disseminados em rebanhos leiteiros nos municípios da região de Caparaó, ES, Brasil. A presença de IBR e BVD aumentaram as chances das vacas desenvolverem mastite subclínica e mastite clínica, respectivamente, e as vacas que foram ordenhadas mecanicamente apresentaram maior probabilidade de serem sororreagente para IBR e BVD.

Epidemiology of canine visceral leishmaniasis in a vulnerable region in Brazil / Soroepidemiologia da leishmaniose visceral canina em uma região vulnerável do Brasil

Visceral leishmaniasis (VL) is a neglected and endemic zoonosis that occurs throughout Brazil; nevertheless, few studies have focused on the early detection of the disease. The municipality of Ourinhos is a non-receptive, silent and vulnerable area for VL, where the seroprevalence of this disease has so far not been investigated. The present study aimed to determine the seroprevalence of canine VL in Ourinhos-SP, and to identify the presence of risk factors. Blood samples were obtained from 604 dogs during a rabies vaccination campaign together with application of a socioeconomic questionnaire, environmental and animal characteristics and tutor's knowledge about the disease. The samples were subjected to indirect ELISA and new samples were collected from reactive and suspect animals, including whole blood and lymph node aspiration evaluated by parasitological method, complete blood count and PCR. No animal was diagnosed as positive based on the combination of direct and indirect tests and the tutors' answers indicated little knowledge about leishmaniasis, being often confused with other diseases transmitted by arthropods; hence, according to the proposed methods, the presence of canine leishmaniasis in the city of Ourinhos was not confirmed and health education campaigns about the disease should be carried out.(AU)

A leishmaniose visceral (LV) é uma zoonose negligenciada e endêmica presente em todas as regiões do Brasil, mas mesmo assim poucos estudos têm objetivado a detecção inicial da doença. O município de Ourinhos - SP é uma área não receptiva, silenciosa e vulnerável à LV, não havendo até o momento estudos que tenham investigado a soroprevalência no município. Nesse sentido, o presente estudo objetivou determinar a soroprevalência da LV canina em Ourinhos-SP, bem como associar a presença de fatores de risco. Amostras sanguíneas de 604 cães foram obtidas juntamente com a aplicação de questionário socioeconômico, características ambientais e dos animais e conhecimento sobre a doença. As amostras foram submetidas à sorologia por ELISA e novas amostras coletadas de cães reagentes ou suspeitos foram analisadas por método parasitológico direto, hemograma e PCR. Nenhum animal foi considerado positivo na combinação de testes direto e indireto, e as respostas dos tutores indicaram pouco conhecimento sobre a leishmaniose, sendo muitas vezes confundida com outras doenças transmitidas por artrópodes. Dessa forma, de acordo com os métodos propostos, a presença de leishmaniose canina, na cidade de Ourinhos, não foi confirmada. Por isso campanhas de educação em saúde sobre a doença deveriam ser realizadas.(AU)

Seroprevalence and associated factors of infectious bovine rhinotracheitis and bovine viral diarrhea in dairy cows in the Caparaó region, Espírito Santo, Brazil / Soroprevalência e fatores associados à rinotraqueíte infecciosa bovina e diarreia viral bovina em vacas leiteiras na região do Caparaó, Espírito Santo, Brasil

This study evaluated the seroprevalence and associated factors of Infectious Bovine Rhinotracheitis (IBR) and Bovine Viral Diarrhea (BVD), and to analyze the possible relationship between IBR, BVD, and the occurrence of mastitis. For this purpose, 854 crossbred dairy cows were evaluated from 69 properties allocated in the 12 municipalities that make up the Caparaó region, Espírito Santo (ES), Brazil. The seroprevalence of IBR and BVD was determined using the indirect ELISA test. Associations between variables were estimated using the prevalence ratio (PR) with a 95% confidence interval. The chi-square test was used to verify the significance of the associations (P < 0.05). The average prevalence of IBR and BVD was 48.59% and 26.46%, respectively. Animals seroreactive for IBR were more likely to develop subclinical mastitis (P < 0.01; PR: 1.27), and cows that were seroreactive for BVD were more likely to develop clinical mastitis (P < 0.01; PR: 2.24). Mechanical milking was considered a factor associated with IBR (P < 0.01; PR: 1.36) and BVD (P < 0.01; PR: 1.25). Reproductive management by natural breeding was considered a factor associated with IBR (P < 0.01; PR: 1.22). Animals seroreactive for BVD were more likely to develop reproductive problems (P < 0.01; PR: 1.34). It was concluded that the agents that cause IBR and BVD are widely disseminated in dairy cattle herds in the municipalities of the Caparaó region, ES, Brazil. The presence of IBR and BVD increased the chances of cows developing subclinical mastitis and clinical mastitis, respectively, and the cows that were mechanically milked were more likely to be seroreactive for IBR and BVD.(AU)

O objetivo do presente estudo foi avaliar a soroprevalência e os fatores de risco associados à Rinotraqueíte Infecciosa Bovina (IBR) e Diarreia Viral Bovina (BVD), e analisar a possível relação entre IBR, BVD e à ocorrência de mastite. Para tanto, foram avaliadas 854 vacas leiteiras mestiças de 69 propriedades localizadas nos 12 municípios que compõem a região do Caparaó, Espírito Santo (ES), Brasil. A soroprevalência de IBR e BVD foram determinadas pelo teste ELISA indireto. As associações entre variáveis ​​foram estimadas pela razão de prevalência (PR) com intervalo de confiança de 95%. O teste do qui-quadrado foi utilizado para verificar a significância das associações (P < 0,05). A prevalência média de IBR e BVD foi de 48% e 26%, respectivamente. Os animais sororreagentes para IBR foram mais propensos a desenvolver mastite subclínica (P < 0,01; PR: 1,27), e as vacas sororreagentes para BVD foram mais propensas a desenvolver mastite clínica (P < 0,01; PR: 2,24). À ordenha mecânica foi considerada um fator associado a IBR (P < 0,01; PR: 1,36) e BVD (P < 0,01; PR: 1,25). O manejo reprodutivo por monta natural foi considerado um fator associado IBR (P < 0,01; PR: 1,22). Os animais sororreagentes para BVD foram mais propensos a desenvolverem problemas reprodutivos (P < 0,01; PR: 1,34). Concluiu-se que os agentes causadores de IBR e BVD estão amplamente disseminados em rebanhos leiteiros nos municípios da região de Caparaó, ES, Brasil. A presença de IBR e BVD aumentaram as chances das vacas desenvolverem mastite subclínica e mastite clínica, respectivamente, e as vacas que foram ordenhadas mecanicamente apresentaram maior probabilidade de serem sororreagente para IBR e BVD.(AU)

Epidemiology of canine visceral leishmaniasis in a vulnerable region in Brazil / Soroepidemiologia da leishmaniose visceral canina em uma região vulnerável do Brasil

Abstract Visceral leishmaniasis (VL) is a neglected and endemic zoonosis that occurs throughout Brazil; nevertheless, few studies have focused on the early detection of the disease. The municipality of Ourinhos is a non-receptive, silent and vulnerable area for VL, where the seroprevalence of this disease has so far not been investigated. The present study aimed to determine the seroprevalence of canine VL in Ourinhos-SP, and to identify the presence of risk factors. Blood samples were obtained from 604 dogs during a rabies vaccination campaign together with application of a socioeconomic questionnaire, environmental and animal characteristics and tutor's knowledge about the disease. The samples were subjected to indirect ELISA and new samples were collected from reactive and suspect animals, including whole blood and lymph node aspiration evaluated by parasitological method, complete blood count and PCR. No animal was diagnosed as positive based on the combination of direct and indirect tests and the tutors' answers indicated little knowledge about leishmaniasis, being often confused with other diseases transmitted by arthropods; hence, according to the proposed methods, the presence of canine leishmaniasis in the city of Ourinhos was not confirmed and health education campaigns about the disease should be carried out.

Resumo A leishmaniose visceral (LV) é uma zoonose negligenciada e endêmica presente em todas as regiões do Brasil, mas mesmo assim poucos estudos têm objetivado a detecção inicial da doença. O município de Ourinhos - SP é uma área não receptiva, silenciosa e vulnerável à LV, não havendo até o momento estudos que tenham investigado a soroprevalência no município. Nesse sentido, o presente estudo objetivou determinar a soroprevalência da LV canina em Ourinhos-SP, bem como associar a presença de fatores de risco. Amostras sanguíneas de 604 cães foram obtidas juntamente com a aplicação de questionário socioeconômico, características ambientais e dos animais e conhecimento sobre a doença. As amostras foram submetidas à sorologia por ELISA e novas amostras coletadas de cães reagentes ou suspeitos foram analisadas por método parasitológico direto, hemograma e PCR. Nenhum animal foi considerado positivo na combinação de testes direto e indireto, e as respostas dos tutores indicaram pouco conhecimento sobre a leishmaniose, sendo muitas vezes confundida com outras doenças transmitidas por artrópodes. Dessa forma, de acordo com os métodos propostos, a presença de leishmaniose canina, na cidade de Ourinhos, não foi confirmada. Por isso campanhas de educação em saúde sobre a doença deveriam ser realizadas.

Seroprevalence and associated factors of infectious bovine rhinotracheitis and bovine viral diarrhea in dairy cows in the Caparaó region, Espírito Santo, Brazil / Soroprevalência e fatores associados à rinotraqueíte infecciosa bovina e diarreia viral bovina em vacas leiteiras na região do Caparaó, Espírito Santo, Brasil

ABSTRACT: This study evaluated the seroprevalence and associated factors of Infectious Bovine Rhinotracheitis (IBR) and Bovine Viral Diarrhea (BVD), and to analyze the possible relationship between IBR, BVD, and the occurrence of mastitis. For this purpose, 854 crossbred dairy cows were evaluated from 69 properties allocated in the 12 municipalities that make up the Caparaó region, Espírito Santo (ES), Brazil. The seroprevalence of IBR and BVD was determined using the indirect ELISA test. Associations between variables were estimated using the prevalence ratio (PR) with a 95% confidence interval. The chi-square test was used to verify the significance of the associations (P < 0.05). The average prevalence of IBR and BVD was 48.59% and 26.46%, respectively. Animals seroreactive for IBR were more likely to develop subclinical mastitis (P < 0.01; PR: 1.27), and cows that were seroreactive for BVD were more likely to develop clinical mastitis (P < 0.01; PR: 2.24). Mechanical milking was considered a factor associated with IBR (P < 0.01; PR: 1.36) and BVD (P < 0.01; PR: 1.25). Reproductive management by natural breeding was considered a factor associated with IBR (P < 0.01; PR: 1.22). Animals seroreactive for BVD were more likely to develop reproductive problems (P < 0.01; PR: 1.34). It was concluded that the agents that cause IBR and BVD are widely disseminated in dairy cattle herds in the municipalities of the Caparaó region, ES, Brazil. The presence of IBR and BVD increased the chances of cows developing subclinical mastitis and clinical mastitis, respectively, and the cows that were mechanically milked were more likely to be seroreactive for IBR and BVD.

RESUMO: O objetivo do presente estudo foi avaliar a soroprevalência e os fatores de risco associados à Rinotraqueíte Infecciosa Bovina (IBR) e Diarreia Viral Bovina (BVD), e analisar a possível relação entre IBR, BVD e à ocorrência de mastite. Para tanto, foram avaliadas 854 vacas leiteiras mestiças de 69 propriedades localizadas nos 12 municípios que compõem a região do Caparaó, Espírito Santo (ES), Brasil. A soroprevalência de IBR e BVD foram determinadas pelo teste ELISA indireto. As associações entre variáveis ​​foram estimadas pela razão de prevalência (PR) com intervalo de confiança de 95%. O teste do qui-quadrado foi utilizado para verificar a significância das associações (P < 0,05). A prevalência média de IBR e BVD foi de 48% e 26%, respectivamente. Os animais sororreagentes para IBR foram mais propensos a desenvolver mastite subclínica (P < 0,01; PR: 1,27), e as vacas sororreagentes para BVD foram mais propensas a desenvolver mastite clínica (P < 0,01; PR: 2,24). À ordenha mecânica foi considerada um fator associado a IBR (P < 0,01; PR: 1,36) e BVD (P < 0,01; PR: 1,25). O manejo reprodutivo por monta natural foi considerado um fator associado IBR (P < 0,01; PR: 1,22). Os animais sororreagentes para BVD foram mais propensos a desenvolverem problemas reprodutivos (P < 0,01; PR: 1,34). Concluiu-se que os agentes causadores de IBR e BVD estão amplamente disseminados em rebanhos leiteiros nos municípios da região de Caparaó, ES, Brasil. A presença de IBR e BVD aumentaram as chances das vacas desenvolverem mastite subclínica e mastite clínica, respectivamente, e as vacas que foram ordenhadas mecanicamente apresentaram maior probabilidade de serem sororreagente para IBR e BVD.

Factors associated with the seroprevalence of caseous lymphadenitis in sheep from Northeastern Brazil.

Caseous lymphadenitis (CL) is one of the main infectious diseases in sheep, responsible for great economic losses, mainly in Northeast region of Brazil, which has the largest sheep flock in the country. Thus, the aim of this survey was to determine the factors associated with the seroprevalence of this disease in sheep from Northeastern Brazil using a planned sample of flocks and animals. Samples were collected from 2312 adult and young sheep of both sex from 196 farms, located in 51 municipalities in five Northeastern Brazilian states (Ceará, Paraíba, Piauí, Rio Grande do Norte, and Sergipe). Serological diagnosis was performed using the indirect ELISA technique. Factors associated with seropositivity were identified by variables extracted from epidemiological questionnaires administered to breeders, using univariable and multivariable analyses. Factors associated with prevalence were purebred sheep (Prevalence ratio - PR = 1.189; P = 0.017), ram acquisition from animal expositions (PR = 1.192; P = 0.020), offspring is separated from ewes (PR = 1.132; P = 0.048), water supplied to sheep from ponds (PR = 1.365; P = 0.002), and delayed disposal of infected animals (PR = 1.263; P = 0.027). Flock-level and animal-level prevalences were 93.88 % (95 % CI = 89.60-96.46%) and 37.46 % (95 % CI = 35.51-39.45%), respectively. In view of the high prevalence found here and based on the associated factors, the implementation of an efficient CL control programme in Northeastern Brazil is recommended.

Improved serodiagnosis of histoplasmosis by use of deglycosylated extracellular released antigens of Histoplasma capsulatum.

The diagnosis of histoplasmosis depends on various approaches: direct clinical examination, fungus isolation from cultures of clinical samples, histopathological evaluation, and serological testing. In serodiagnostic assays, the Histoplasma capsulatum H and M antigenic glycoproteins have been extensively used. However, both antigens showed limitations attributed mainly to their cross-reactivity with glycoproteins from other pathogenic fungi, which compromises specificity, and generates false positives, misdiagnosis, and therapeutic failure. In this work, we deglycosylated extracellular released antigens from the Venezuelan 7090 H. capsulatum clinical isolate, using chemical and enzymatic methods and evaluated their effectiveness by indirect enzyme-linked immunosorbent assay (ELISA) with sera from patients with either histoplasmosis or PCM. Prior to deglycosylation, the extracellular released antigen showed 62% of sensitivity 66% of specificity and 68% of cross-reactivity with paracoccidioidomicosis sera. The chemically deglycosylated extracellular released antigen, for 8 or 18  h showed 72 and 52% sensitivity with 98% and 92% specificity, respectively. Moreover, cross-reactivity with Paracoccidioides decreased to 4 and 16%, following deglycosylation for 8 or 18 h, respectively. The enzymatically treated antigen showed 52% of sensitivity, 92% of specificity and 8% cross-reactivity against Paracoccidioides. Deglycosylation of the H. capsulatum antigen improves its specificity and decreases its cross-reactivity against Paracoccidioides when using indirect ELISA for serodiagnosis. Therefore, it is recommended to deglycosylate the fungal extracellular released antigen for clinical serodiagnosis, and to monitor humoral immune responses during therapy of patients with the different clinical forms of histoplasmosis.

Seroepidemiology of ovine toxoplasmosis and neosporosis in breeding rams from Rio Grande do Sul, Brazil.

This study determined the prevalence of ovine toxoplasmosis and neosporosis and the risk factors associated with the development of these diseases in breeding rams from the State of Rio Grande do Sul (RS), Southern Brazil. Serum samples (n = 1,800) from breeding rams maintained on 705 sheep farms from seven mesoregions were evaluated serologically to detect anti-IgG Toxoplasma gondii by indirect ELISA and anti-IgG Neospora caninum by the indirect immunofluorescence assay (IFA). The prevalence of T. gondii was 33.05% (595/1,800); seropositivity to N. caninum was 18.44% (332/1,800). Additionally, there was simultaneous seropositivity (8.94%;161/1,800) to N. caninum and T. gondii. The variables size of the property (<500 ha) (Prevalence Ratio, PR = 1.36); breeding system (semi-intensive/intensive) (PR = 1.23); and natural mounting without control (PR = 1.50) were considered as risk factors for the occurrence of T. gondii. Size of the property (<500 ha) (PR = 1.58) and natural mounting without control (PR = 2.32) were risk factors associated with the prevalence of N. caninum in rams. Additionally, separation of ewes prior to parturition was considered as a protective factor for the occurrence of T. gondii (PR = 0.82) and N. caninum (PR = 0.74). These results demonstrated that these two parasitic disease agents are endemic in rams throughout all regions of RS.

Frequency of bovine cysticercosis in the state of Rondônia, Brazil.

Bovine cysticercosis remains as one of the most important cause of carcasses and viscera condemnation in Brazilian slaughterhouses. However, the efficiency of post-mortem inspection for the diagnosis of this zoonotic disease is relatively low, and few available studies were performed through serological exams. This study evaluated the frequency of bovine cysticercosis in cattle herds located in different farms of the state of Rondônia, Brazil. Among the 987 animals slaughtered from 33 farms, 21 animals (Frequency: 2.13%; 95C.I. 1.23-3.03) were considered as positive through indirect ELISA and confirmed by Immunoblot tests and the cysticercosis was detected in 12 farms (36.36% - C.I. 95% 19.95-52.78). The disease was detected in the municipalities Vale do Paraíso (12.50%), Theobroma (8.11%), Guaporé (7.27%), Rolim de Moura (5.71%), Presidente Médici (5.0%), Ouro Preto do Oeste (4.69%), Nova União (1.77%), Nova Brasilândia d'Oeste (1.14%) and Ministro Andreazza (1.01%). Therefore, prophylactic measures should be taken to improve beef production, control bovine cysticercosis and reduce costs to public health in this Brazilian state.

Diagnóstico serológico de Neospora caninum en vacas del municipio de Tuta, Boyacá / Serological diagnosis of Neospora caninum in cows of Tuta, Boyacá

RESUMEN Neospora caninum es un parásito protozoario del filo Apicomplexa que ha sido reconocido como causante de abortos y fallas reproductiva en el ganado de todo el mundo. Aunque en Colombia existen algunos estudios sobre la seroprevalencia de esta enfermedad, la información sigue siendo limitada. Objetivo: establecer la seroprevalencia de N. caninum en vacas lecheras del municipio de Tuta (Boyacá, Colombia). Materiales y Métodos: se muestrearon 375 animales. Las muestras se procesaron bajo la técnica de ELISA indirecta y se realizó un análisis estadístico con la prueba de chi-cuadrado de asociación-independencia para determinar si existía relación entre la presencia de anticuerpos contra N. caninum y las diferentes variables reproductivas. Resultados: el 52% de los individuos fueron positivos a anticuerpos contra N. caninum y la única variable reproductiva que presentó relación estadística con la presencia del protozoo fue la repetición de celo; por otra parte, no existió relación entre edad y raza de los bovinos y la presencia de N. caninum. Conclusión: la seroprevalencia es elevada si se compara con los datos reportados con anterioridad en otras regiones del país; no obstante, estos resultados no demostraron la presencia de enfermedad en los animales analizados, pero sí la evidencia antigénica, lo que sugiere que en algún momento de la vida se infectaron con el agente y promovieron la formación de anticuerpos específicos.

ABSTRACT Neospora caninum is a protozoan parasite of the phylum Apicomplexa, and has been recognized as a major cause of abortion and reproductive failure in cattle in the world, Although in Colombia there are some studies on the seroprevalence of this disease, even so the information remains limited. Objective: the aim of this study was to establish the seroprevalence of N. caninum in dairy cows of Tuta, (Boyacá, Colombia). Materials and methods: 375 animals were sampled. The samples were processed under indirect ELISA technique, a statistical analysis was performed with the chi-square association independence test to determine if there was a relationship between the presence of antibodies against N. caninum and the different reproductive variables. Results: it was observed that 52% of the individuals were positive for antibodies against N. caninum. the only reproductive variable that presented a statistical relationship with the presence of the protozoan was repetition of estrus, there was no relationship between age and breed of the cattle and the presentation of N.caninum. Conclusions: the seroprevalence is high if they if you keep in mind the seroprevalence data previously reported in other regions of the country, however, these results do not demonstrate the presence of disease in the animals analyzed, but the antigenic evidence, which implies that at some point life they became infected with the agent and promoted the formation of specific antibodies.

The use of ErpY-like recombinant protein from Leptospira interrogans in the development of an immunodiagnostic test for swine leptospirosis.

Swine leptospirosis poses a major problem in the agricultural sector. The gold standard for serodiagnosis of leptospirosis is Microscopic Agglutination Test (MAT). However, the limitations of this technique make the search for alternative diagnostic methods inevitable. In the present study, ErpY-like recombinant protein (rErpY-like), produced in Escherichia coli and used as antigen in indirect enzyme-linked immunosorbent assay (ELISA), was evaluated for its efficacy as a novel diagnostic tool for swine leptospirosis. For the study, 72 samples of swine sera characterized by microscopic agglutination test (MAT), were evaluated by indirect ELISA. The sensitivity, specificity and accuracy values obtained from the analysis were 96.8%, 100%, and 99%, respectively, thereby suggesting that rErpY-like ELISA being a sensitive and specific method for antibodies detection in swine populations could be used as an alternative for diagnosis of swine leptospirosis.