Classical BSE dismissed as the cause of CWD in Norwegian red deer despite strain similarities between both prion agents.

The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrPSc immunohistochemistry staining in the brainstem and the absence of detectable PrPSc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.

Development and validation of a chemiluminescent western blot assay for glanders (Burkholderia mallei) serodetection.

Glanders, caused by Burkholderia mallei, is a zoonotic disease of equids. Serologic testing for glanders is required by disease-free countries before international movement of equids. The World Organisation for Animal Health Terrestrial Manual recommends the complement fixation test (CFT) for clearance of individual animals for movement, but the CFT is prone to false-positive results. A colorimetric western blot (WB) assay was developed and validated to resolve false-positive CFT results; however, that assay is relatively time-consuming, and the interpretation is subjective. We present here a procedurally similar chemiluminescent WB assay that performs comparably to the validated colorimetric WB assay and offers noticeable benefits of decreased time-to-result and greater ease of interpretation.

Recombinantly expressed virus-like particles (VLPs) of canine circovirus for development of an indirect ELISA.

Canine circovirus (CanineCV) is an emerging pathogen in domestic dogs, detected in multiple countries in association with varying clinical and pathological presentations including diarrhoea, vasculitis, granulomatous inflammation, and respiratory signs. Understanding the pathology of CanineCV is confounded by the fact that it has been detected in asymptomatic dogs as well as in diseased dogs concurrently infected with known pathogens. Recombinantly expressed self-assembling Virus-like particles (VLPs) lack viral genomic material but imitate the capsid surface conformations of wild type virion, allowing arrays of biological applications including subunit vaccine development and immunodiagnostics. In this study, full length CanineCV capsid gene was expressed in Escherichia coli followed by two-step purification process to yield soluble capsid protein in high concentration. Transmission electron microscopy (TEM) confirmed the capsid antigen self-assembled into 17-20 nm VLPs in glutathione S-transferase (GST) buffer, later utilised to develop an indirect enzyme-linked immunosorbent assay (iELISA). The respective sensitivity and specificity of the proposed iELISA were 94.10% and 88.40% compared with those obtained from Western blot. The mean OD450 value for western blot positive samples was 1.22 (range 0.12-3.39) and negative samples was 0.21 (range 0.07-0.41). An optimal OD450 cut-off of 0.35 was determined by ROC curve analysis. Median inter-assay and intra-assay validation revealed that the iELISA test results were reproducible with coefficients of variation 7.70 (range 5.6-11.9) and 4.21 (range 1.2-7.4). Our results demonstrated that VLP-based iELISA is a highly sensitive method for serological diagnosis of CanineCV infections in dogs, suitable for large-scale epidemiological studies.

Development and standardization of a western blotting test for detection of antibodies against B. abortus / Desenvolvimento e padronização do teste Western blotting para detecção de anticorpos contra B. abortus

Brucellosis is an infectious disease caused by bacteria of the genus Brucella spp. with diagnosis based on use of serological techniques. The present study aimed to develop and standardize a western blotting (WB) test for detection of antibodies against B. abortus. Samples from two groups of cattle were analyzed: group I: 60 serum samples from true positive and true negative vaccinated animals (30 positive samples from infected animals according to rose bengal test (RBT), 2-mercaptoethanol, serum agglutination test (SAT) and complement fixation test (CFT) and 30 RBT negatives samples); group II: 383 field samples (90 positive and 293 CFT negative sera). The most reactive band in the western blotting, which properly identified and separated infected from non - infected had a molecular weight of ≤ 20kDa. The sensitivity, specificity and accuracy of the WB compared to RBT was 93%, 99%, 98%, respectively and k= 0.938. When compared to CFT, the sensitivity, specificity and accuracy of the WB was 97%, 98% and 97%, respectively and k= 0.929. The WB developed and standardized in the present study is a serological test with potential use as a confirmatory test for the diagnosis of bovine brucellosis.(AU)

A brucelose é uma doença infectocontagiosa, causada por bactérias do gênero Brucella spp., com diagnóstico baseado no emprego de técnicas sorológicas. Objetivou-se neste estudo desenvolver e padronizar um teste Western blotting (WB) para detecção de anticorpos contra B. abortus. Foram analisados dois grupos de amostras bovinas: grupo I, com 60 amostras de animais verdadeiros positivos e verdadeiros negativos vacinados (30 amostras positivas de animais infectados e positivos nos testes de antígeno acidificado tamponado (AAT), 2 - mercaptoetanol (2 - ME), soroaglutinação lenta em tubos (SAT) e fixação do complemento e de 30 amostras negativas no AAT); grupo II, com 383 amostras de campo, sendo 90 soropositivas e 293 soronegativas no TFC. O resultado da análise do WB revelou peso molecular ≤20kDa como sendo a área mais reativa e característica para identificação e separação dos animais infectados dos não infectados. A sensibilidade, a especificidade e a acurácia do WB, quando este foi comparado com o AAT, foram, respectivamente, 93%, 99% e 98%, e k= 0,938. Quando comparadas com a TFC, a sensibilidade, a especificidade e a acurácia foram 97%, 98% e 97%, respectivamente, e k= 0,929. O WB padronizado neste estudo mostrou-se um teste sorológico com potencial uso como teste confirmatório no diagnóstico da brucelose bovina.(AU)

Expressão da enzima indoleamina-2, 3-dioxigenase em truta arco-íris (Oncorhynchus mykiss) / Expression of the enzyme indoleamine-2, 3-dioxygenase in Rainbow Trout (Oncorhynchus mykiss

A indoleamina 2,3-dioxigenase (IDO) é uma enzima que cataboliza o aminoácido triptofano, levando à inibição da proliferação de linfócitos T, seja pela exaustão desse aminoácido no ambiente, ou pela indução via catabólitos induzindo-os a apoptose. Em mamíferos, esta enzima atua em diversas condições do organismo como a gestação, infecções, inflamações crônicas, transplantes e tumores, atuando na regulação imunológica. Estudos recentes identificaram a presença de moléculas homólogas a IDO em espécies filogeneticamente inferiores, cuja função parece estar restrita ao metabolismo do triptofano como fonte de energia. Este estudo teve por objetivo averiguar a expressão da IDO em células sanguíneas e órgãos hematopoiéticos de truta arco-íris pela imuno-histoquímica, buscando evidências de que a mesma poderia, nesta espécie, estar relacionada ao sistema imune. A expressão de IDO foi observada nos órgãos hematopoiéticos estudados incluindo o rim cefálico que apresentou marcação em células interrenais e leucócitos; baço, na qual a marcação restringiu à alguns leucócitos; no fígado a marcação ficou limitada à apenas algumas células dentro dos vasos sanguíneos e nas extensões sanguíneas pode-se visualizar a marcação de alguns leucócitos como os monócitos, linfócitos e neutrófilos. A predominância da marcação da IDO nesses tecidos pode constituir uma evidência de que a IDO identificada na O. mykiss esteja relacionada ao sistema imunológico nessa espécie...

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catabolizes the amino acid tryptophan, leading to inhibition of T lymphocyte proliferation, whether by depletion of this amino acid in the environment, or by induction via the catabolites inducing apoptosis. In mammals, this enzyme acts on various conditions of the body such as pregnancy, infections, chronic inflammation, transplantation and tumors, acting in immune regulation. Recent studies have identified the presence of homologous molecules IDO lower phylogenetically related species, whose function appears to be confined to the tryptophan metabolism as an energy source. This study aimed to investigate the expression of IDO in blood cells and hematopoietic organs of rainbow trout by immunohistochemistry, seeking evidence that it could, this species is related to the immune system. The expression of IDO was observed in hematopoietic organs studied including head kidney that show labeling in interrenal cells and leukocytes; spleen, in which the marking restricted to a few leukocytes in the liver;, labeling was restricted to only certain cells within the blood vessels and the blood extensions can view the marking of some leukocytes including monocytes, lymphocytes and neutrophils. The predominance of IDO marking these tissues may constitute evidence that IDO identified in O. mykiss is related to the immune system in this species...

Use of serological diagnostic techniques in the control and eradication of caprine arthritis encephalitis: an update / Uso de técnicas sorológicas de diagnóstico no controle e erradicação da artrite encefalite caprina: atualização

Caprine arthritis encephalitis (CAE) is a chronic disease caused by a small ruminant lentivirus (SRLV), which causes significant losses in goat breeding. The actual state of animal infection with SRLV is difficult to determine due to a complex pathogenesis of the virus, including factors such as delayed or intermittent seroconversion in serological tests. Several serological techniques are available for disease diagnosis, such as screening or confirmation tests, which are different in sensitivity and specificity. Regarding the choice of the test to be applied, availability of commercial immunoreagents, team training, antigen used, and cost of techniques must be considered. This review presents the serological methods available for use in different stages of CAE control and eradication programs, and management measures to be adopted in conjunction with serological diagnosis of the disease...

A artrite encefalite caprina (CAE) é uma enfermidade crônica causada por um lentivírus de pequenos ruminantes (LVPR), que ocasiona perdas significativas na caprinocultura. O estado real da infecção animal pelo LVPR é de difícil determinação em virtude da complexa patogenia do vírus, incluindo fatores como soroconversão tardia ou intermitente em testes sorológicos. Para o diagnóstico da enfermidade, diversas técnicas sorológicas estão disponíveis, como testes de triagem ou confirmatórios, com variações na sensibilidade e especificidade. Para escolha do teste a ser usado, a disponibilidade de imunorreagentes comerciais, o treinamento da equipe, o antígeno utilizado, e o custo das técnicas devem ser considerados. Esta revisão apresenta os métodos sorológicos disponíveis para uso em diferentes fases dos programas de controle e erradicação da CAE e as medidas de manejo que devem ser adotadas em conjunto com o diagnóstico sorológico da enfermidade...

Diagnostic methods to cutaneous leishmaniasis detection in domestic dogs and cats

Abstract: Cutaneous leishmaniasis is caused by different species of Leishmania. In domestic animals such as dogs and cats, the diagnostic consists of clinical, epidemiological and serological tests, which changes among countries all around the world. Because of this diversity in the methods selected, we propose this systematic literature review to identify the methods of laboratory diagnosis used to detect cutaneous leishmaniasis in domestic dogs and cats in the Americas. Articles published in the last 5 years were searched in PubMed, ISI Web of Science, LILACS and Scielo, and we selected 10 papers about cutaneous leishmaniasis in dogs and cats in the Americas. In Brazil, often the indirect immunofluorescence and enzyme immunoassay (ELISA) have been applied. Other countries like United States and Mexico have been using antigenic fractions for antibodies detections by Western blot. ELISA and Western blot showed a higher sensitivity and efficacy in the detection of leishmaniasis. Analysis of sensibility and specificity of the methods was rarely used. Although confirmatory to leishmaniasis, direct methods for parasites detection and polymerase chain reaction showed low positivity in disease detection. We suggested that more than one method should be used for the detection of feline and canine leishmaniasis. Serological methods such as Western blot and enzyme immunoassay have a high efficacy in the diagnosis of this disease.

Padronização do Elisa indireto e Western Blot para diagnóstico da artrite-encefalite caprina / Standardization of indirect Elisa and Western Blot for diagnosis of Caprine Arthritis-Encephalitis

A artrite-encefalite caprina (CAE) é diagnosticada rotineiramente pela técnica de imunodifusão em gel de agarose (IDGA), que é considerada pouco sensível. Objetivou-se com este estudo padronizar testes de Elisa-i e Western Blot (WB) para diagnóstico precoce de anticorpos em caprinos contra CAEV e comparar os resultados obtidos nesses testes com a prova de IDGA. Para a padronização dos testes Elisa-i e WB, utilizaram-se diferentes concentrações e diluições de antígeno, soros e conjugado. No Elisa-i, adotaram-se microplacas rígidas com 96 poços, sendo a combinação de concentração de 0,5µg/poço de antígeno e diluições de 1:100 de soro e 1:1500 de conjugado a que apresentou melhor resultado. No WB foram utilizadas membranas de nitrocelulose, definindo-se as diluições de 1:50 de soro e 1:15000 de conjugado. Para avaliar o desempenho das técnicas, 222 amostras de soro caprino foram testadas e os dados obtidos foram comparados com o IDGA. A sensibilidade e a especificidade do Elisa-i/IDGA, WB/IDGA e WB/Elisa-i foram de 70% e 91%, 100% e 72,6%, 84,6% e 76,5%, concomitantemente. O índice Kappa desses testes foi de 0,35, 0,2 e 0,36, respectivamente. As técnicas de Elisa-i e WB apresentaram-se mais sensíveis que a IDGA, podendo ser utilizadas como ferramentas para o diagnóstico precoce da CAE...

Caprine arthritis-encephalitis (CAE) is routinely diagnosed with the Agarose Gel Immunodiffusion (AGID) technique, which is considered to have low sensitivity. The objective of this study was to standardize testing i-Elisa and Western Blot for early detection of antibodies against CAEV in goats and compare the results obtained in these tests with proof of AGID. For standardization of i-Elisa and WB, different concentrations and dilutions of antigen, sera and conjugate were used. In the i-Elisa, rigid microplate with 96 wells was adopted, and the combination that showed the best result was a concentration of 0.5µg/ well of antigen and dilutions of the serum of 1:100 and conjugate of 1:1500. In the WB nitrocellulose membranes were used, and the dilutions of the serum were defined at 1:50 and conjugate at 1:15000. To evaluate the performance of the techniques, 222 goat serum samples were tested and the data were compared with the AGID. The sensitivity and specificity of Elisa-i/IDGA, WB/AGID and WB/Elisa-i were 70% and 91%, 100% and 72.6%, 84.6% and 76.5%, concomitantly. The Kappa index of these tests was 0.35, 0.2 and 0.36, respectively. The i-Elisa and WB techniques were more sensitive than the AGID and can be used as tools for early diagnosis of CAE...

Stanniocalcin-1 protects bovine intestinal epithelial cells from oxidative stress-induced damage

Chronic enteritis can produce an excess of reactive oxygen species resulting in cellular damage. Stanniocalcin-1(STC-1) reportedly possesses anti-oxidative activity, the aim of this study was to define more clearly the direct contribution of STC-1 to anti-oxidative stress in cattle. In this study, primary intestinal epithelial cells (IECs) were exposed to hydrogen peroxide (H2O2) for different time intervals to mimic chronic enteritis-induced cellular damage. Prior to treatment with 200 microM H2O2, the cells were transfected with a recombinant plasmid for 48 h to over-express STC-1. Acridine orange/ethidium bromide (AO/EB) double staining and trypan blue exclusion assays were then performed to measure cell viability and apoptosis of the cells, respectively. The expression of STC-1 and apoptosis-related proteins in the cells was monitored by real-time PCR and Western blotting. The results indicated that both STC-1 mRNA and protein expression levels positively correlated with the duration of H2O2 treatment. H2O2 damaged the bovine IECs in a time-dependent manner, and this effect was attenuated by STC-1 over-expression. Furthermore, over-expression of STC-1 up-regulated Bcl-2 protein expression and slightly down-regulated caspase-3 production in the damaged cells. Findings from this study suggested that STC-1 plays a protective role in intestinal cells through an antioxidant mechanism.

Expression of E-cadherin in pig kidney

E-cadherin is a cell adhesion molecule that plays an important role in maintaining renal epithelial polarity and integrity. The purpose of this study was to determine the exact cellular localization of E-cadherin in pig kidney. Kidney tissues from pigs were processed for light and electron microscopy immunocytochemistry, and immunoblot analysis. E-cadhedrin bands of the same size were detected by immunoblot of samples from rat and pig kidneys. In pig kidney, strong E-cadherin expression was observed in the basolateral plasma membrane of the tubular epithelial cells. E-cadherin immunolabeling was not detected in glomeruli or blood vessels of pig kidney. Double-labeling results demonstrated that E-cadherin was expressed in the calbindin D28k-positive distal convoluted tubule and H(+)-ATPase-positive collecting duct, but not in the aquaporin 1-positive, N-cadherin-positive proximal tubule. In contrast to rat, E-cadherin immunoreactivity was not expressed at detectable levels in the Tamm-Horsfall protein-positive thick ascending limb of pig kidney. Immunoelectron microscopy confirmed that E-cadherin was localized in both the lateral membranes and basal infoldings of the collecting duct. These results suggest that E-cadherin may be a critical adhesion molecule in the distal convoluted tubule and collecting duct cells of pig kidney.

Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease.

Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice

The outer membrane proteins (OMPs) of Brucella (B.) abortus have been extensively studied, but their immunogenicity and protective ability against B. abortus infection are still unclear. In the present study, B. abortus Omp28, a group 3 antigen, was amplified by PCR and cloned into a maltose fusion protein expression system. Recombinant Omp28 (rOmp28) was expressed in Escherichia coli and was then purified. Immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive mouse serum. Furthermore, humoral- or cell-mediated immune responses measured by the production of IgG1 or IgG2a in rOmp28-immunized mice and the ability of rOmp28 immunization to protect against B. abortus infection were evaluated in a mouse model. In the immunogenicity analysis, the mean titers of IgG1 and IgG2a produced by rOmp28-immunized mice were 20-fold higher than those of PBS-treated mice throughout the entire experimental period. Furthermore, spleen proliferation and bacterial burden in the spleen of rOmp28-immunized mice were approximately 1.5-fold lower than those of PBS-treated mice when challenged with virulent B. abortus. These findings suggest that rOmp28 from B. abortus is a good candidate for manufacturing an effective subunit vaccine against B. abortus infection in animals.

Biomarkers for identifying the early phases of osteoarthritis secondary to medial patellar luxation in dogs

The levels of tartrate resistant acid phosphatase (TRAP), matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in synovial fluid (SF) and serum in cases of canine osteoarthritis (OA) were measured. OA was induced by a surgically-created medial patellar luxation in the left stifle of 24 dogs. SF and blood samples were collected at 1.5- and 3-month intervals, respectively. Every 3 months, one dog was euthanatized to collect tissue samples from both stifles. TRAP levels in SF and serum were measured using a spectrophotometer, and TRAP-positive cells in joint tissues were identified by enzyme histochemistry. MMP-2 and TIMP-2 in SF and serum were detected by Western blotting and ELISA, respectively. TRAP in SF from the stifles and serum was significantly increased (p < 0.05) after 3 months. TIMP-2 in SF and serum was significantly decreased (p < 0.05), whereas MMP-2 in SF was significantly increased (p < 0.05) during the progression of OA. Histochemistry revealed an increased number of TRAP-positive cells in tissues from OA-affected joints. Assays measuring TRAP, MMP-2, and TIMP-2 in SF and serum, and methods that detect increased numbers of TRAP-positive cells in the joint tissues can play an important role in identifying the early phases of degenerative changes in canine joint components.

CMTM3 is reduced in prostate cancer and inhibits migration, invasion and growth of LNCaP cells

Purpose. A novel tumor suppressor gene CKLF-like MARVEL transmembrane domain-containing member 3 (CMTM3) is reduced or undetectable in many kinds of cancers and relates tumor malignant features. We detected its role in prostate cancer for possibility of target therapy as accumulating evidence has shown that CMTM3 is a promising tumor suppressor gene (TSG) for gene therapy. Methods. The expression of CMTM3 detected in prostate tissue microarray, specimens and cell lines were evaluated by immunohistochemistry and semi-quantitative PCR and Western blot, respectively. After being transfected with CMTM3 adenovirus or vector (mock), the proliferation and migration and invasion of LNCaP cells were detected by transwell assay and matrigel assay, respectively. Furthermore, the effects of CMTM3 on tumor growth were performed in nude mice xenograft in vivo. Results. We found CMTM3 was reduced in PCa tissues and cells compared with BPH tissues, and its expression in PCa tissues was related to the Gleason score. Moreover, after being transfected with adenovirus, ectopic expression of CMTM3 in LNCaP cells led to significant inhibition of cell proliferation and migration and invasion compared with the control (P < 0.05), which may be attributed to decreased Erk1/2 activity as p-Erk1/2 was remarkably reduced when CMTM3 was overexpressed. Finally, restoration of CMTM3 significantly suppressed xenograft tumor growth in vivo (P < 0.01) (AU)

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Diagnostico de la Leishmaniasis visceral canina mediante las técnicas western blot e inmunofluorescencia indirecta / Diagnóstico de leishmaniose visceral canina através da técnicade western blot e imunofluorescencia / Canine visceral leishmaniasis diagnosis by means of western blot and indirect immunofluorescence test

Objetivo: un estudio epidemiológico transversal fue desarrollado para establecer la seroprevalencia de la Leishmaniasis Visceral (LV) en caninos localizados en la zona endémica del departamento del Tolima por medio de las pruebas de Inmunofluorescencia Indirecta (IFI) y Western Blot (WB). Materiales y métodos: se incluyeron en el estudio 211 caninos mestizos procedentes de seis municipios de la zona endémica de leishmaniasis visceral del departamento del Tolima. Se utilizó como antígeno la cepa colombiana de Leishmania infantum MHOM/COL/CL044B para IFI y WB. Resultados: se estableció una seroprevalencia de la Leishmaniasis Visceral Canina en el grupo de animales evaluados del 41,7 y22, 2% por medio de IFI y WB, respectivamente. La concordancia entre las dos técnicas fue negativa (k= -0,102; p = 0,084). Se detectaron 12 fracciones antigénicas en la prueba WB entre los 13 y 91 kDa, de las cuales la de 14 kDa se considera valiosa en el diagnóstico precoz de la infección, para evaluar la resolución de la enfermedad y la evolución del tratamiento. Se sugiere desarrollar un constructo de pruebas diagnósticas directas e indirectas que permitan confirmar el estado real de la infección de los caninos, para orientar eficientemente los programas de control de la enfermedad en zonas endémicas de leishmaniasis visceral.

Objective: a cross-sectional epidemiological study was carried out in order to establish the prevalence of canine visceral leishmaniasis in the endemic area of Tolima department by means of Indirect Immunofluorescence (IFAT) and Western Blotting (WB) tests. Materials and Methods: the study included 211 mongrel dogs from six municipalities of the endemic area of visceral leishmaniasis (VL) of the Tolima department. The Colombian Leishmania infantum MHOM/CO/CL044B strain was used as antigen for IFI and WB. Results: The seroprevalence in the group tested was 41.7% and 22.2% by IFAT and WB, respectively. The agreement between the two techniques was negative (k = -0.102; p = 0.084). 12 antigenic fractions were detected between 13 and 91 kDa, of which 14 kDa is considered valuable in early diagnosis of the infection, in order to evaluate the resolution of the disease and treatment progress. The article suggests developing a construct of direct and indirect diagnostic tests for confirming the real state of the animals’ infection to guide disease control programs in endemic areas of visceral leishmaniasis.

Objetivo: uma pesquisa epidemiológica transversal foi desenvolvida para estabelecer a soroprevalência da Leishmaniose Visceral (LV) canina localizados na zona endêmica do Estado do Tolima por médio das provas de Imunofluorescência indireta (IFI) e Western Blot (WB). Materiais e Métodos se incluíram na pesquisa 211 canina mestiça procedente de seis prefeituras da zona endêmica de Leishmaniose visceral do Estado de Tolima. Utilizou-se como antígeno a cepa Colombiana de Leishmania infantum MHOM/COL/CL044B para IFI e WB...

Development and characterization of stable cell lines constitutively expressing the porcine reproductive and respiratory syndrome virus nucleocapsid protein

Despite global efforts to control porcine reproductive and respiratory syndrome virus (PRRSV) infection, the virus continues to cause economic problems in the swine industry worldwide. In this study, we attempted to generate and characterize a panel of stable BHK cell lines that constitutively express the nucleocapsid (N) protein of type 1 or type 2 PRRSV. The established BHK cell lines were found to react well with N-specific antibodies as well as the hyperimmune serum of pigs raised against each genotype of PRRSV. Taken together, the data implicate a potential usefulness for the newly generated stable cell lines as a diagnostic reagent for PRRSV serology.

Transcriptional and translational expression of calbindin-D9k in the duodenum, kidney and uterus of a female canine model

Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein expressed in tissues in the intestine, uterus, placenta, kidney, pituitary gland and bone. Its exact function is unknown, but it is considered to regulate intracytoplasmic concentration and transport of free ions (Ca2+). CaBP-9k protein is involved in intestinal calcium absorption in the intestine and in the regulation of myometrial activity by intracellular calcium in the uterus. Renal CaBP-9k protein is expressed at the site of calcium re-absorption in the kidney and expressed in distal convoluted tubules, where it is thought to facilitate calcium re-absorption. Expression of the CaBP-9k gene has been explored in most mammalians except in a canine model. Presently, we elucidated the expression of CaBP-9k mRNA and protein in the duodenum, kidney and uterus in a canine model involving two adult (2.5-year-old) female beagles. To collect tissues, the dogs were euthanized and then the abdominal cavity was exposed by midline incision. The proximal duodenum, cortex of kidney and uterine horn were collected. Expression of CaBP-9k mRNA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. CaBP-9k protein expression and localization were ascertained by Western blot analysis and immunohistochemistry, respectively. CaBP-9k mRNA was detected in the duodenum, but not in the kidney and uterus. Its protein was expressed only in the enterocytes of the duodenum. Taken together, the results indicate that CaBP-9k mRNA and protein are highly expressed in the enterocytes of the duodenum of a canine model, consistent with findings in other mammalian species.

Estudio comparativo de pruebas serológicas para el diagnóstico de la leishmaniasis visceral canina: nota técnica / Comparative study of serological test for the diagnosis of canine visceral leishmaniasis: technical note

El objetivo del presente estudio consistió en comparar la concordancia entre los resultados obtenidos por las técnicas IFI, ELISA y Western Blot en 72 sueros caninos procedentes de siete municipios de la zona endémica de leishmaniasis visceral zoonótica (LVZ) del departamento del Tolima (Colombia). Se utilizó como antígeno la cepa colombiana de Leishmania infantum MHOM/CO/CL044B para IFI, ELISA y WB, y el antígeno rK39 para una prueba de ELISA disponible comercialmente. Se encontró que la concordancia entre las diferentes técnicas comparadas fue menor del 16 por ciento (k<16 por ciento), lo que sugiere que las pruebas no son consistentes y por lo tanto, no son aceptables como método de diagnóstico en el presente estudio. La baja asociación de las pruebas serológicas utilizadas en el diagnóstico de L. infantum sugiere que es necesario desarrollar estudios que permitan establecer un algoritmo de pruebas diagnósticas en el país para confirmar el estado real de la infección en los animales y de esta forma orientar eficientemente los recursos de salud pública destinados para el control de la enfermedad.

The goal of present study was to compare the agreement between the results obtained by ELISA, IFI and WB tests in 72 canine serums from the South of the Tolima Department (a visceral leishmaniasis endemic area). The Colombian Leishmania infantum MHOM/CO/CL044B strain was used as antigen for ELISA, IFI and WB test, and the rK39 antigen for the commercial ELISA test. The agreement among the compared techniques was smaller than 16 percent (k<16 percent), suggesting that the tests are not consistent and therefore not acceptable as a diagnostic tool in this study. The low association between the serological tests used in diagnosing Leishmania infantum suggested the need for further studies aimed at establishing an algorithm for diagnostic tests in Colombia for confirming the real state of the animals´ infection and thereby efficiently orientating public health resources allocated for controlling visceral leishmaniasis.

The sero-prevalence of Toxoplasma gondii in British marine mammals

Serum samples from 101 stranded or bycatch cetaceans from British waters were screened for Toxoplasma gondii-specific antibodies using the Sabin Feldman Dye Test. Relatively high seropositivity was recorded in short-beaked Delphinus delphis and this study presents the first documented case of Toxoplasma in a humpback whale Megaptera novaeangliae.

Detección de la subunidad NMDAR-1 del receptor N-metil-D-asparato (NMDAR) en el hipotálamo del ovino mediante el análisis de Western blot / Detection of the N-methyl-D-asparate receptor subunit-1 (NMDAR-1) in the hypothalamus of the sheep bye Western blot analysis

Los aminoácidos excitatorios (AAEs), L-glutamato y aspartato, son considerados las principales sustancias endógenas neuroactivas involucradas en la transmisión excitatoria. El receptorN-metíl-D-aspartato (NMDAR) es uno de los principales receptores de los AAEs. Este receptor estimula la secreción de la hormona luteinizante (LH) al facilitar la secreción de la hormona liberadora de las gonadotrofinas (GnRH). En este estudio se plantearon dos objetivos: Estandarizar la técnica de Western blot para identificar la subunidad-1 del NMDAR (NMDAR-1) en hipotálamo del ovino; y determinar si existe diferencia en la distribución de la NMDAR-1 en los diferentes cuadrantes hipotalámicos (cuadrantes dorsal-rostral, ventral-rostral, dorsal-caudal, y ventral-caudal) del ovino. Se utilizaron 100 µg de proteína total para identificar la NMDAR-1 en el hipotálamo ovino. Se evidenció un efecto del cuadrante o porción del hipotálamo ovino sobre la expresión proteica de la NMDAR-1. En particular, el cuadrante dorsal-rostral (31,5567 densidad óptica ± 13,0831;P<0,05) expresó más cantidad del receptor que el cuadrante ventral caudal (17,1517 densidad óptica ± 13,0831; P<0,05) y que el cuadrante ventral-rostral (16,4683 densidad óptica ±13,0831; P<0,05). Estos resultados demuestran que el receptor NMDA se expresa más en la región rostral del hipotálamo del ovino, área en la cual se encuentran distribuidos los cuerpos celulares y axones de las neuronas productoras de GnRH. Estos resultados sugieren la participación de la vía glutamatérgica en la regulación de la reproducción de los ovinos.

The excitatory amino acids (EAAs), L-glutamate and aspartate, are considered the major endogenous neuroactive substances involved in excitatory neurotransmission. The N-methyl-D-aspartate receptor (NMDAR) is one of the main excitatory amino acid receptors. Exogenous activation of the NMDAR results in the release of gonadotrophin-releasing hormone (GnRH) from the hypothalamus and subsequent release of luteinizing hormone (LH) from the anterior pituitary. The present study had two aims: to standardize the optimal amount of total protein required to identify the NMDAR subtype-1 (NMDAR-1) in the hypothalamus of the sheep; to determine whether a difference exists in the distribution of NMDAR-1 in different hypothalamic regions (dorsal-rostral, ventral-rostral, dorsal-caudal, and ventral-caudal quadrant) in male sheep. The optimal amount of total hypothalamic protein required to visualize the NMDAR-1 was 100 µg. There was an effect of quadrant on the expression of NMDAR-1 in the hypothalamus of the intact male sheep. In particular, dorsal-rostral quadrant (31.5567 optical density ± 13.0831) expressed more receptor than ventral-caudal quadrant (17.1517 optical density ± 13.0831; P<0.05) and ventral-rostral quadrant (16.4683 optical density ± 13.0831; P<0.05). These data suggest that NMDAR is more expressed in the rostral area of the hypothalamus, where the cell bodies and axons of the GnRH neurons are localized. Thus, these data allow suggest that glutamatergic pathway potentially is involved in modulating the reproductive activity in the ovine.