A Bayesian approach to estimate the accuracy of in-house ELISA assay to measure rabies antibodies from compulsory vaccinated dogs and cattle

Rabies is a vaccine-preventable disease that causes acute encephalitis in mammals, and it is still a significant public health problem in numerous countries. Infected dogs represent the main vectors involved in human rabies. Additionally, cattle rearing close to geographic areas where vampire bats are found presents an important connection with rural epidemiology. We applied two in-house enzyme-linked immunosorbent assay (ELISA) methodologies, considered alternatives to measure antibodies from vaccinated dogs and cattle, without employing the gold standard approach. The ELISA assays were performed on individual serum samples taken from domestic adult dogs and cows compulsory vaccinated against rabies (147 urban dogs and 64 cows; n = 211). The sandwich and liquid-phase competitive ELISA (scELISA and lpcELISA), considered in-house assays, were performed according to previous works. The only statistical methodology that allows this study is the Bayesian approach, developed to replace the conventional Hui-Walter paradigm. For conditional independent Bayesian model (one population, two tests and no gold standard) the prior information for sensitivity and specificity of each test, mode, prevalence and transformed (α, β) were submitted to Bayesian inference. The in-house lpcELISA revealed 16 - out of 261 serum samples - negative results, whereas in scELISA all results were positive. The Bayesian approach showed that prior information was specified for all parameters; posterior medians were Se scELISA 89%, Sp scELISA 88%, Sp lpcELISA 95% Se lpcELISA 98%, and prevalence (pi) of 99%, without the use of gold standard analysis to measure specific anti-rabies antibodies.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Experimental infectious bursal disease in the ostrich (Struthio camelus)

Clinically severe disease was produced in ostriches aged 4 weeks by oral infection with avirulent strain of infectious bursal disease virus (vIBDV), namely strain Faragher 52/70. Four days after infection the birds were humanely killed and tissue samples, including thymus, bursa of Fabricius (BF), brain and kidney were collected for examination. Histopathologically, the thymus and BF showed severe lymphoid depletion and necrosis, while immunolabelling with a polyclonal antibody demonstrated abundant viral antigen. (C) 2007 Elsevier Ltd. All rights reserved

Pathology and Tissue Distribution of Turkey Coronavirus in Experimentally Infected Chicks and Turkey Poults

Twenty 1-day-old specific pathogen free chicks and 20 1-day-old commercially derived turkey poults were inoculated with a Brazilian strain of turkey coronavirus (TCoV) to study the pathogenicity and virus distribution up to 14 days post-moculanon by histopathology, immunohistochemistry, reverse transcriptase polymerase chain reaction and sequencing. At 2-14 dpi, TCoV antigens were detected in the paranasal sinus and lachrymal accessory gland (Harderian gland) of infected chicks and in the ileum, ileocaecal junction and caecum of infected poults Lymphocytic inflammation was present in these tissues TCoV was re-isolated from pooled tissue suspensions of nasal concha, Harderian gland and paranasal sinus from chicks, as well as from the ileum, ileocaecal junction and caecum of poults, after three consecutive passages in 28-day-old embryonated turkey eggs Viral RNA corresponding to the spike gene region (1178-2073 genome position) was amplified from the upper respiratory tract of chickens and from the intestinal tract of poults and phylogenetic analysis confirmed the identity as TCoV This is the first description of TCoV antigens and mRNA in upper respiratory tissues in experimentally infected chickens (C) 2010 Elsevier Ltd All rights reservedFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs

Aiming to improve the diagnosis of canine leishmaniasis (CanL) in an endemic area of the Northwest region of São Paulo State, Brazil, the efficacy of parasitological, immunological and molecular diagnostic methods were studied. Dogs with and without clinical sips of the disease and positive for Leishmania, by direct parasite identification on lymph node smears and/or specific antibody detection by ELISA, were selected for the study. According to the clinical signs, 89 dogs attending the Veterinary Hospital of UNESP in Aracatuba (SP, Brazil) were divided into three groups: symptomatic (36%), oligosymptomatic (22%) and asymptomatic (22%). Twenty-six dogs from an area non-endemic for CanL were used as negative controls (20%). Fine-needle aspiration biopsies (FNA) of popliteal lymph nodes were collected and Diff-Quick (R)-stained for optical microscopy. Direct immumofluorescence, immunocytochemistry and parasite DNA amplification by PCR were also performed. After euthanasia, fragments of popliteal lymph nodes, spleen, bone marrow and liver were collected and processed for HE and immunohistochemistry. Parasite detection by both HE and immunohistochemistry was specifically more effective in lymph nodes, when compared with the other organs. Immunolabeling provided higher sensitivity for parasite detection in the tissues. In the symptomatic group, assay sensitivity was 75.61% for direct parasite search on Diff-Quick (R)-stained FNAs, 92.68% for direct immunofluorescence, 92.68% for immunocytochemistry and 100% for PCR; the corresponding values in the other clinical groups were: 32, 60, 76 and 96% (oligosymptomatic), and 39.13, 73.91, 100 and 95.65% (asymptomatic). Results of the control animals from the CanL non-endemic area were all negative, indicating that the methods used were 100% specific. (C) 2006 Elsevier B.V. All rights reserved

Validation of an immunohistochemistry assay to detect turkey coronavirus: A rapid and simple screening tool for limited resource settings

The objective of the present study was to develop and apply the direct immunohistochemistry (D-IHC) assay to search for turkey coronavirus (TCoV) antigens in formalin-fixed embedded-paraffin tissues by the use of biotin-labeled polyclonal antibody. Twenty-eight-day-old embryonated turkey eggs (n = 50) were inoculated with TCoV-purified virus, and 3 d after inoculation, sections from ileum, ileum-cecal junction, and ceca were harvested, fixed in neutral formalin, and embedded in paraffin blocks and used as positive control. In addition, a total of 100 field samples from ileum, ileum-cecal junction, and ceca, collected from 30 to 45-d-old turkeys poults experiencing an outbreak of acute enteritis, were used to search for TCoV by the same D-IHC. All results were compared with those obtained by conventional RT-PCR and indirect fluorescent antibody assay (IFA) for all tested samples. Turkey coronavirus was detected in experimentally infected embryo tissues and also in field samples in 100% of ileum-cecal junction and ceca by the 3 detection procedures. With IFA as a reference assay, sensitivity and specificity of D-IHC were 98 and 58%, whereas sensitivity and specificity of reverse transcription-PCR were 96 and 66%, calculated from the total of tested samples from experimental infection. Each of the examined procedures was highly specific (D-IHC, 93%; RT-PCR, 90%), sensitive (D-IHC, 85%; RT-PCR, 86%), and agreement of both D-IHC and RT-PCR was 99 and 100%, respectively, compared with IFA results obtained from all the field samples. These findings demonstrated the utility of D-IHC for direct detection of TCoV from field samples and considering the sensitivity and specificity found here, can be used as an alternative technique