Padronização de um teste ELISA indireto para diagnóstico da cinomose canina / Standardization of an indirect ELISA test for canine diagnosis diagnosis

A cinomose é uma enfermidade causada pelo vírus Canine Distemper Virus (CDV). Essa doença afeta principalmente cães, mas também acomete outras espécies domésticas e selvagens. A imunidade do animal está relacionada ao grau que a esse patógeno vai atingir o organismo do indivíduo. Ela afeta a respiração do animal, pode causar vômito, diarreia, convulsões, podendo levar o animal à óbito. O objetivo do presente trabalho foi padronizar um teste ELISA indireto com antígeno de superfície para o diagnostico cinomose utilizando amostras de soro canino. Para padronização da técnica, fez-se necessário o estudo da diluição do antígeno para identificar a melhor concentração para sensibilização da placa. O teste foi aplicado primeiramente com diferentes diluições do antígeno para detecção do melhor desempenho do antígeno. Feito isso, foi testado em um banco de soro de 45 animais comprovadamente positivos no teste ELISA comercial e em soro de 45 animais comprovadamente negativos no teste ELISA comercial, posteriormente foi calculado o ponto de corte, especificidade e sensibilidade do teste. O teste ELISA indireto se mostrou com excelência como um teste de diagnóstico para a cinomose canina, obtendo-se ponto de corte de densidade óptica de 0,229, sensibilidade de 95,5% e especificidade de 84,4%.

Distemper is a disease or the disease by the CDV virus, Distemper Virus. This disease mainly affects dogs, but also affects other domestic and wild species. The animal's immunity is related to the degree to which it will reach the individual's organism. It affects the animal's breathing, can cause vomiting, diarrhea, convulsions, and can lead to death. The aim of the present work test was to standardize an indirect ELISA for distemper diagnosis in experiments using a surface antigen. For the study of technical identification, it was necessary to specify the antigen for the best concentration of plaque sensitization. The test was initially applied with different dilutions of the antigen to detect the best performance of the antigen. This was tested in a serum bank of 45 animals proven positive in the commercial ELISA test and in the serum of 45 animals proven negative in the commercial ELISA test, later it was tested on the cut-off point, specificity and sensitivity of the test. The indirect ELISA test proved to be excellent as a diagnostic test for canine distemper, with an optical density cut-off of 0.229, sensitivity of 95.5% and specificity of 84.4% being obtained.

Review of methods for the detection of Lawsonia intracellularis infection in pigs.

Lawsonia intracellularis is an obligate intracellular bacterium associated with enteric disease in pigs. Clinical signs include weight loss, diarrhea, and, in some cases, sudden death. The hallmark lesion is the thickening of the intestinal mucosa caused by increased epithelial cell replication, known as proliferative enteropathy. The immune response to L. intracellularis is not well defined, and detection of the infection, especially in the early stages, is still a significant challenge. We review here the main approaches used to identify this important but poorly understood pathogen. Detection of L. intracellularis infection as the cause of clinical disease is confounded by the high prevalence of the pathogen in many countries and that several other pathogens can produce similar clinical signs. A single L. intracellularis-specific ELISA and several amplification assays are available commercially to aid detection and surveillance, although histopathology remains the primary way to reach a conclusive diagnosis. There are major gaps in our understanding of L. intracellularis pathogenesis, especially how the host responds to infection and the factors that drive infection toward different clinical outcomes. Knowledge of pathogenesis will increase the predictive value of antemortem tests to guide appropriate interventions, including identification and treatment of subclinically affected pigs in the early stages of disease, given that this important manifestation reduces pig productivity and contributes to the economic burden of L. intracellularis worldwide.

Recombinase-aided amplification-lateral flow dipstick assay-a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus.

The purpose of this study was to explore a specific, simple, and sensitive method for diagnosis of avian infectious laryngotracheitis virus. Recombinase-aided amplification (RAA) and lateral flow dipstick (LFD) were combined for labeling the optimized RAA probe with 6-carboxyfluorescein (FAM) and the 5'-end of the downstream primer with biotin, respectively. By optimizing the reaction time, temperature, and primer concentration of RAA, a RAA-LFD assay, which could be used for detection of infectious laryngotracheitis, was established. After the specificity and sensitivity test, the target gene fragments could be amplified by RAA-LFD assay in 20 min under isothermal conditions (37°C), and the amplification products could be visually observed and determined by LFD within 3 min. There was no cross-reaction with nucleic acids of other avian pathogens, the lowest detectable limit of RAA-LFD was 102 copies/µL, and the sensitivity of this method was 100 times higher than that of conventional PCR with the lowest detectable limit of 104 copies/µL. The results showed that RAA-LFD assay was highly sensitive, easy to use, and more suitable for clinical detection.

[Comparison between oral fluid samples and pooled serum samples for the detection of antibodies against Porcine Reproductive and Respiratory Syndrome Virus in weaning pig herds]. / Vergleich von Speichelproben und ­gepoolten Serumproben für den ­Nachweis von Antikörpern gegen das Porzine Reproduktive und Respiratorische ­Syndrom Virus in ­Aufzuchtbeständen.

INTRODUCTION: Monitoring of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in pig farms is performed usually by testing for antibodies against PRRSV in serum samples. A new method is the detection of PRRSV antibodies in porcine saliva. In this study serum samples and saliva were collected in nine farms suspicious for PRRSV and tested for the presence of PRRSV antibodies. In total 220 serum and 41 saliva samples were taken from pigs at the age of 8 weeks (± 1 week). One saliva and one pooled serum sample (1:5) were tested from each pen. In total 11 (Cut-off 0.4/0.3) or 14 (Cut-off 0.2) serum samples and 23 saliva out of 41 pens were positive for PRRSV antibodies. Cohen`s Kappa testing showed a moderate agreement (κ = 0.446). Saliva samples compared to pooled serum samples were very sensitive, the specificity was 60 and 67, respectively.

INTRODUCTION: La surveillance du virus du syndrome reproducteur et respiratoire porcin (PRRSV) dans les élevages de porcs est généralement effectuée en recherchant des anticorps contre le PRRSV dans des échantillons de sérum. Une nouvelle méthode est la détection des anticorps anti-­PRRSV dans la salive porcine. Dans cette étude, des échantillons de sérum et de salive ont été prélevés dans neuf exploitations suspectes de PRRSV et testés quant à la présence d'anticorps PRRSV. Au total, 220 échantillons de sérum et 41 échantillons de salive ont été prélevés sur des porcs à l'âge de 8 semaines (± 1 semaine). De chaque boxe, un échantillon de salive et un échantillon de sérums regroupé (1: 5) ont été testés. Au total, 11 échantillons de sérum (seuil 0,4/0,3) ou 14 (seuil 0,2) et 23 de salive sur 41 boxes étaient positifs quant aux anticorps anti-PRRSV. Le test Kappa de Cohen a montré une corrélation modérée (κ = 0,446). Les échantillons de salive étaient très sensibles par rapport aux échantillons de sérum regroupés, la spécificité n'était toutefois que de 60 respectivement 67.

Noninvasive strategies for surveillance of swine viral diseases: a review.

In view of the intensive development of the swine industry, monitoring and surveillance of infectious diseases require low-cost, effective, and representative population sampling methods. We present herein the state of knowledge, to date, in the use of alternative strategies in the monitoring of swine health. Blood sampling, the most commonly used method in veterinary medicine to obtain samples for monitoring swine health, is labor-intensive and expensive, which has resulted in a search for alternative sampling strategies. Oral fluid (OF) is a good alternative to serum for pooled sample analysis, especially for low-prevalence pathogens. Detection of viral nucleic acids or antiviral antibodies in OF is used to detect numerous viruses in the swine population. Meat juice is used as an alternative to serum in serologic testing. Processing fluid obtained during processing of piglets (castration and tail-docking) may also be used to detect viruses. These matrices are simple, safe, cost-effective, and allow testing of many individuals at the same time. The latest methods, such as snout swabs and udder skin wipes, are also promising. These alternative samples are easy to acquire, and do not affect animal welfare negatively.

Bovine Respiratory Disease Diagnosis: What Progress Has Been Made in Clinical Diagnosis?

Bovine respiratory disease (BRD) complex is a worldwide health problem in cattle and is a major reason for antimicrobial use in young cattle. Several challenges may explain why it is difficult to make progress in the management of this disease. This article defines the limitation of BRD complex nomenclature, which may not easily distinguish upper versus lower respiratory tract infection and infectious bronchopneumonia versus other types of respiratory diseases. It then discusses the obstacles to clinical diagnosis and reviews the current knowledge of readily available diagnostic test to reach a diagnosis of infectious bronchopneumonia.

Determining the predictive capability of a Clinical Assessment Scoring Chart to differentiate severity of the clinical consequences of neonatal calf diarrhea relative to gold-standard blood gas analysis.

Neonatal calf diarrhea (NCD) is a major problem to calf health worldwide, in terms of both morbidity and mortality. A five-point ordinal scale clinical assessment scoring (CAS) chart was utilized to assess calves suffering from NCD-related clinical abnormalities (acidosis and dehydration) on commercial farms. The objective of this research was to determine the predictive capability of this CAS chart against gold standard blood gas parameters, designed to assist farmers in the accurate assessment of the clinical consequences of NCD. A total of 443 diarrheic and non-diarrheic calves were enrolled in the study. The CAS chart rated a calf's health from no clinical signs to varying degrees of clinical severity on a 0 (clinically normal) to 4 (grave) scale, based on clinical indicators including calf demeanour, ear position, mobility, suckle reflex, desire-to-feed, and enophthalmos. Blood gas analysis was conducted for individual calves, consisting of pH, base excess, Na+, K+, Ca2+, Cl-, glucose, total hemoglobin, bicarbonate, anion gap, and strong ion difference. Statistical evaluation was performed by comparison of the CAS score with blood gas profiles using ordinal logistic regression and a non-parametric estimation of the Receiver Operating Characteristics (ROC). The ROC analysis indicated that the CAS chart had acceptable specificity (>95%) with low sensitivity (<60%) in differentiating clinically normal from acidotic/dehydrated cases. Assessment of individual severity classes indicated that the chart can predict and differentiate both clinically normal and advanced cases from the other severity classes (peak estimations >80%) but had reduced accuracy in differentiating mild and moderate cases (peak estimations >50%). The chart, as presented, provides a simple tool to differentiate clinically normal from calves suffering the consequences of diarrhea, but fails to accurately differentiate severity for NCD related acidosis and dehydration. Further efforts are required to enhance the sensitivity and differential diagnostic value of this type of chart.

Diagnostic Methods for Detecting Internal Parasites of Livestock.

Internal parasites are a major concern in livestock production because they can impact the health and well-being of animals clinically and subclinically, and ultimately cause significant production loss. Among these internal parasites are nematodes, tapeworms, flukes, and coccidian protozoans. This review focuses on the diagnostic tests that are routinely performed by veterinarians and diagnostic laboratories, but also highlights recently developed tools that may improve diagnostic capabilities, including molecular and immunodiagnostic tests. Overall, diagnostic tests for parasites of livestock are an integral part of health management practices, and for assessing individual animal and herd health.

Superglue slide impression (SSI) method: a novel diagnostic application for canine demodicosis.

The conventional gold standard diagnostic method for canine demodicosis, the deep skin scrapings (DSS), is traumatic to the animal and appears aggressive in the eyes of the owner. A less invasive, sensitive, easy-to-perform and field-oriented diagnostic method for the rapid diagnosis of canine demodicosis is warranted. The present study aimed to develop a rapid less invasive diagnostic method using superglue (cyanoacrylate adhesive) slide impression (SSI). Ninety-seven client-owned dogs presented with clinical symptoms and signs suggestive of demodicosis were examined using SSI for detection of Demodex mites. A clean microscope slide was taken and a drop of superglue was placed on the slide. Immediately, the superglue-bearing slide surface was applied to the previously squeezed selected skin lesion with gentle pressure for 30 s. The slide was removed from the skin lesion and a drop of immersion oil was placed over the SSI. Another clean cover slide was applied and examined under the microscope at low-power magnification (× 10 lens). Of the 97 dogs, 90 dogs (92.8%) were detected positive for demodectic mites using the SSI method, whereas 86 (88.7%) dogs were found positive using the DSS technique. The SSI method was found to be equally sensitive to the DSS method. In summary, the SSI method is a new quick, sensitive, easy-to-perform, owner- and animal-friendly, less traumatic and field-oriented diagnostic application for demodicosis in dogs. It can be used for harvesting the live demodectic mites and monitoring miticidal therapies.

Evaluation of DPP® and SNAP® Rapid Tests for diagnosis of Leishmania infantum canine infections.

INTRODUCTION: Visceral leishmaniasis is a disease that affects humans, wildlife, and domestic species. Since dogs play a key role in urban Leishmania spp. transmission, the Brazilian government maintains the Monitoring and Control Program of Visceral Leishmaniasis (VLMCP) in endemic regions, which promotes awareness campaigns aiming to enhance the control of the infection. The VLMCP recommends the Dual Path Platform (DPP®) canine visceral leishmaniasis test (Bio-Manguinhos, Brazil) for screening and enzyme-linked immunosorbent assay to confirm the infection. The DPP® test is produced and distributed by the Health Ministry to the Municipal Health Centers responsible for the local VLMCP. The test is not available to all the clinics, forcing some veterinarians to use other rapid tests for screening and diagnosis of this disease in their daily routine. METHODS: The present study was conducted to compare the performance of the DPP® and SNAP® tests using sera from the dogs with confirmed infections of L. infantum and from the dogs with no previous testing, residing in areas with a low Leishmania infection. RESULTS: There was 97.0% agreement between the two tests. Sensitivity and specificity of the SNAP® test were 96.3% and 100%, respectively. Agreement between both the antibody tests and the parasitological detection methods was 96.8%. The DPP® test had 95.8% sensitivity and 100% specificity. CONCLUSIONS: The SNAP® and the DPP® tests were virtually equivalent in terms of detection of canine antibodies against L. infantum, and both the tests demonstrated high and similar levels of sensitivity and specificity.

Evaluation of DPP® and SNAP® Rapid Tests for diagnosis of Leishmania infantum canine infections

Abstract INTRODUCTION: Visceral leishmaniasis is a disease that affects humans, wildlife, and domestic species. Since dogs play a key role in urban Leishmania spp. transmission, the Brazilian government maintains the Monitoring and Control Program of Visceral Leishmaniasis (VLMCP) in endemic regions, which promotes awareness campaigns aiming to enhance the control of the infection. The VLMCP recommends the Dual Path Platform (DPP®) canine visceral leishmaniasis test (Bio-Manguinhos, Brazil) for screening and enzyme-linked immunosorbent assay to confirm the infection. The DPP® test is produced and distributed by the Health Ministry to the Municipal Health Centers responsible for the local VLMCP. The test is not available to all the clinics, forcing some veterinarians to use other rapid tests for screening and diagnosis of this disease in their daily routine. METHODS: The present study was conducted to compare the performance of the DPP® and SNAP® tests using sera from the dogs with confirmed infections of L. infantum and from the dogs with no previous testing, residing in areas with a low Leishmania infection. RESULTS: There was 97.0% agreement between the two tests. Sensitivity and specificity of the SNAP® test were 96.3% and 100%, respectively. Agreement between both the antibody tests and the parasitological detection methods was 96.8%. The DPP® test had 95.8% sensitivity and 100% specificity. CONCLUSIONS: The SNAP® and the DPP® tests were virtually equivalent in terms of detection of canine antibodies against L. infantum, and both the tests demonstrated high and similar levels of sensitivity and specificity.

Brucellosis in the Addis Ababa dairy cattle: the myths and the realities.

BACKGROUND: Bovine brucellosis is considered as an important disease among livestock and people in sub-Saharan African countries including Ethiopia. A cross-sectional study was conducted from November 2016 to May 2017 to estimate the prevalence and associated risk factors, and to assess knowledge-attitude and practices (KAP) of farm workers about bovine brucellosis in Addis Ababa dairy farms. RESULTS: A total of 1550 cattle from 127 dairy farms were serially tested using the Rose Bengal Plate Test (RBPT), Competitive Enzyme-Linked Immunosorbant Assay (c-ELISA) and Complement Fixation Test (CFT). Forty-three (2.77%) of the collected sera were positive by the RBPT and only one of these was positive by c-ELISA (0.06%) and none was positive by CFT. The knowledge of farm workers towards the disease was very low and risk factors associated with Brucella infection were apparent in the study area. CONCLUSION: Seropositivity for Brucella spp. was found in only a very small percentage by c-ELISA test, although risk factors for transmitting Brucella infection were present. The results suggest that bovine brucellosis is currently not a generalized problem in dairy cattle of Addis Ababa. Since this favorable disease situation is not the result of informed policy, there is no guarantee that it will continue unchanged. Setting clear policy in control of the disease and implementing "One Health" are the most constructive approaches recommended.

Evaluation of nonwoven fabrics for nasal wipe sampling for influenza A virus in swine.

Influenza A virus (IAV) is a zoonotic pathogen threatening animal and public health; therefore, detection and monitoring of IAV in animal populations are critical components of a surveillance program. Swine are important hosts of IAV, wherein the virus can undergo rapid evolution. Several methods (i.e., nasal swabs, nasal wipes, and oral fluids) have been used to collect samples from swine for IAV surveillance. We utilized nasal wipes made from cotton gauze and multiple, polyester or mixed polyester fabrics to compare performance in the molecular detection and isolation of IAV. In vitro experiments revealed that no polyester or mixed polyester fabric was superior to cotton gauze for molecular IAV detection; however, 3 polyester or mixed polyester fabrics yielded significantly more viable IAV than cotton. In a field trial, both cotton gauze and the polyester or mixed polyester fabric yielded similar proportions of IAV isolates from swine. The results indicate that cotton gauze remains a practical and useful material for swine nasal wipes.

Comparison of three diagnostic methods for Salmonella enterica serovars detection in chicken rinse / Comparação de três métodos diagnósticos para detecção de Salmonella enterica em lavados de carcaça de frango

Salmonella detection is a key point in food safety testing, because of the frequent association of this pathogen with food poisoning in humans. The standard bacteriological tests currently used for Salmonella-detection are time-consuming; therefore, there is a need to develop alternative methods to accelerate the detection. In order to accelerate Salmonella diagnosis, we used the immunomagnetic separation assay associated with bacteriophage P22 for the rapid detection of the following Salmonella serovars in chicken rinses of drumsticks, artificially contaminated with 5, 10, and 100 CFU/25mL of bacteria: Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg), Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium). The efficiency of the technique, represented by the time required for detection of positive and negative samples, was compared with that of the standard diagnostic tests used for this pathogen, the bacteriological assay and the polymerase chain reaction (PCR)-based test. This study confirmed the ability of the bacteriophage-associated immunomagnetic separation assay to identify 99.6% of Salmonella-positive samples of the three serovars tested. In contrast, the bacteriological assay and PCR-based test detected 95.1% and 98.5% of the Salmonella-positive samples respectively.(AU)

A detecção de Salmonella é um ponto crucial para a segurança alimentar, devido a frequente associação deste patógeno com infecções alimentares em humanos. O método padrão para detecção de Salmonella é o bacteriológico, mas o tempo requerido para o processamento das amostras e o diagnóstico final é longo, por isso existe a necessidade de desenvolvimento de métodos alternativos que visem acelerar esta etapa. Para isto utilizamos a separação imunomagnética associada ao bacteriófago P22 como técnica de detecção rápida para os seguintes sorovares de Salmonella: Salmonella enterica subsp. enterica sorovar Heidelberg (S. Heidelberg), Salmonella enterica subsp. enterica sorovar Enteritidis (S. Enteritidis) e Salmonella enterica subsp. enterica sorovar Typhimurium (S. Typhimurium), os quais foram inoculados artificialmente em lavados de sobre-coxas de frango nas seguintes concentrações: 5, 10 e 100 UFC/25mL. A eficiência da técnica, representada pelo tempo requerido para detecção de amostras positivas ou negativas, foi comparado com os testes rotineiramente utilizados para detecção de Salmonella, o exame bacteriológico e a reação em cadeia da polimerase (PCR). Este estudo confirmou a capacidade do teste de separação imunomagnética associado a bacteriófago, o qual identificou 99,6% das amostras positivas para Salmonella, dos três sorovares testados. Já o bacteriológico e PCR identificaram respectivamente 95,1% e 98,5% das amostras positivas.(AU)

Assessment of Chronic Wasting Disease Prion Shedding in Deer Saliva with Occupancy Modeling.

The detection of prions is difficult due to the peculiarity of the pathogen, which is a misfolded form of a normal protein. The specificity and sensitivity of detection methods are imperfect in complex samples, including in excreta. Here, we combined optimized prion amplification procedures with a statistical method that accounts for false-positive and false-negative errors to test deer saliva for chronic wasting disease (CWD) prions. This approach enabled us to discriminate the shedding of prions in saliva and the detection of prions in saliva-a distinction crucial to understanding the role of prion shedding in disease transmission and for diagnosis. We found that assay sensitivity and specificity were indeed imperfect, and we were able to draw several conclusions pertinent to CWD biology from our analyses: (i) the shedding of prions in saliva increases with time postinoculation, but is common throughout the preclinical phase of disease; (ii) the shedding propensity is influenced neither by sex nor by prion protein genotype at codon 96; and (iii) the source of prion-containing inoculum used to infect deer affects the likelihood of prion shedding in saliva; oral inoculation of deer with CWD-positive saliva resulted in 2.77 times the likelihood of prion shedding in saliva compared to that from inoculation with CWD-positive brain. These results are pertinent to horizontal CWD transmission in wild cervids. Moreover, the approach described is applicable to other diagnostic assays with imperfect detection.

Identifying free-text features to improve automated classification of structured histopathology reports for feline small intestinal disease.

The histologic evaluation of gastrointestinal (GI) biopsies is the standard for diagnosis of a variety of GI diseases (e.g., inflammatory bowel disease [IBD] and alimentary lymphoma [ALA]). The World Small Animal Veterinary Association (WSAVA) Gastrointestinal International Standardization Group proposed a reporting standard for GI biopsies consisting of a defined set of microscopic features. We compared the machine classification accuracy of free-text microscopic findings with those represented in the WSAVA format with a diagnosis of IBD and ALA. Unstructured free-text duodenal biopsy pathology reports from cats ( n = 60) with a diagnosis of IBD ( n = 20), ALA ( n = 20), or normal ( n = 20) were identified. Biopsy samples from these cases were then scored following the WSAVA guidelines to create a set of structured reports. Three supervised machine-learning algorithms were trained using the structured and then the unstructured reports. Diagnosis classification accuracy for the 3 algorithms was compared using the structured and unstructured reports. Using naive Bayes and neural networks, unstructured information-based models achieved higher diagnostic accuracy (0.90 and 0.88, respectively) compared to the structured information-based models (0.74 and 0.72, respectively). Results suggest that discriminating diagnostic information was lost using current WSAVA microscopic guideline features. Addition of free-text features (number of plasma cells) increased WSAVA auto-classification performance. The methodologies reported in our study represent a way of identifying candidate microscopic features for use in structured histopathology reports.