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The application and evaluation of highly efficient chromatographic techniques with tandem mass spectrometry for the detection and quantitation of 108 pesticides and metabolites, some considered persistent organic pollutants, was performed in muscle samples obtained from 25 birds of prey belonging to the families Accipitridae, Falconidae, and Strigidae presented dead in 2013 to Grupo de Rehabilitación de la Fauna Autóctona y su Hábitat, in Madrid, Spain. Pesticides with prohibited use were detected at high concentrations in the muscle samples analyzed. Based on its high sensitivity to detect pesticides in muscle, the described chromatographic techniques with tandem mass spectrometry should be considered an alternative testing methodology to those commonly used for routine application in ecotoxicological forensic research.

Infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) are both important viruses seriously affecting poultry industry worldwide. In this study, reverse-transcription LAMP (RT-LAMP) was combined with lateral flow dipstick (LFD) forming a novel detection tool which could simultaneously detect IBV and NDV visually. Primers targeted the 5'-untranslated region (5'-UTR) of IBV genome and the conserved region of NDV large polymerase gene (LP). The specificity and sensitivity of this multiple reverse transcription-LAMP-LFD (mRT-LAMP-LFD) assay were compared with those of conventional RT-PCR, nested RT-PCR (nRT-PCR), quantification RT-PCR (qRT-PCR), and RT-LAMP monitored by electrophoresis. No non-specific amplifications were observed when the assays were tested with unrelated viruses. According to the sensitivity study, when detecting IBV or NDV alone, the lowest detection limits of mRT-LAMP-LFD were 100.8 IBV RNA copies/reaction and 100.7 NDV RNA copies/reaction. Furthermore, when detecting IBV and NDV simultaneously, the lowest detection limit was the same as that of the single detection assays. In the clinical sample study, mRT-LAMP-LFD performed the best among these assays. When tested with IBV or NDV single infected samples, the mean detection rates were 98.65% and 97.25%, respectively. In the IBV and NDV co-infected sample study, the mean detection rates of IBV and NDV were both 95%. This study showed that mRT-LAMP-LFD was a promising qualitative detection tool suitable for field single or multiple IBV and NDV detection., (© 2019 Poultry Science Association Inc.)

Fowl adenovirus serotype-4 (FAdV-4) has been recognized as a predominant threat to the broilers aged from three to five weeks. Hydropericardium syndrome (HPS) is one of its major clinical diseases by FAdV-4 resulting in heavy economic losses. In this study, a loop-mediated isothermal amplification coupling with a lateral flow dipstick (LAMP-LFD) was developed for rapid and specific detection of fowl adenovirus serotype-4. The optimized LAMP-LFD can be completed in 60 min at 65 °C. The minimum detection limits of PCR, real-time PCR, nested PCR and LAMP-LFD are 1 × 10 4 copies/μl, 1 × 10 2 copies/μl, 10 copies/μl and 10 copies/μl respectively. Moreover, the specificity of the LAMP-LFD assay is satisfactory and does not produce cross reactions with other species. In field samples, 150 samples were assayed by PCR and LAMP-LFD. They agreed on the diagnosis "positive" in 13% of clinical samples, and they agreed on the diagnosis "negative" in 85% of clinical samples. Their probability of agreement is p 0  = 147/150 = 13% + 85% = 98%. LAMP-LFD can potentially be modified and applied as a diagnostic tool for FAdV-4 infection especially in resource-limited areas, such as small breeding farms and basic veterinary labs to offer an affordable diagnostic., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Visceral leishmaniasis is a major public health concern in Brazil and the domestic dog is the main source of infection. In this study, we aimed to evaluate the accuracy and reliability of a rapid chromatographic immunoassay based on a dual-path platform for the diagnosis of canine visceral leishmaniasis (CVL)., Methods: Sampling consisted of 428 domestic dogs selected from two neighborhoods in the municipality of Fortaleza, Ceara state, Brazil. The reference standard was composed of three parasitological tests and was applied samples from 333 dogs. The rapid test was used to analyse whole blood and serum samples., Results: Accuracy of the rapid test in whole blood samples through visual reading (n=305), serum samples through electronic reading (n=333) and serum samples through visual reading (n=333), yielded sensitivities of 87.5% (21/24; 95% CI: 66.5 to 96.7), 88% (22/25; 95% CI: 67.5 to 96.8) and 88% (22/25; 95% CI: 67.5 to 96.8), and specificities of 73.3% (206/281; 95% CI: 67.7 to 78.4), 68.2% (210/308; 95% CI: 62.2 to 74.3) and 69.2% (213/308; 95% CI: 63.7 to 74.3), respectively. Agreement between the visual and electronic readings in 428 serum samples were classified as almost perfect (Kappa Index=0.88; 95% CI: 0.83 to 0.93). The positive predictive value of the test using whole blood samples was 21.9% for the 7.9% prevalence detected by the reference standard in the study sample. A sensitivity analysis of the positive predictive value revealed that it remained below 50% in scenarios with a prevalence of up to 20%., Conclusions: The similarity of the accuracy values of the rapid test using whole blood or serum samples, together with its reliable performance in sera through visual and electronic reading, suggests that it may contribute as a screening test for routine use under field-conditions. However, future studies need to improve the accuracy of the test so that it can be successfully implemented in public health programs., (© The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Canine visceral leishmaniasis (CVL) is the major source of human visceral leishmaniasis (VL) and is transmitted from dogs to sand flies to humans. To control the spread of this disease, early and accurate detection of infected dogs is critical but challenging. Here we demonstrate the potential of the Dual-Path Platform (DPP(®)) CVL rapid test for detecting K26/K39-reactive antibodies in sera from clinically symptomatic (n=60) and asymptomatic (n=60) Leishmania infantum-infected dogs. For the specificity evaluation, assays were performed using known negative diagnostic serum samples (n=59) and cross-reaction control sera (n=11) from animals born in a VL-free area of Brazil. The diagnostic kit displayed high specificity (96%) but low sensitivity (47%) in identifying parasite-positive dogs without signs of CVL. However, the test sensitivity was significantly higher (98%) in diseased cases, indicating that this convenient test may be useful to identify the most infectious dogs. Efforts should be pursued to obtain a more sensitive DPP-multiplexed test parameter (i.e. based on simultaneous yet separate antibody detection of carefully selected multiple antigens of diagnostic utility) for effective serodiagnosis of early-infected dogs, as this will likely allow more efficient canine removal regimens than those used in practice by public health services., (Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

Cryptosporidium is an important protozoan parasite that causes diarrhea in neonates and young bovines. The objective of the present study was to determine the frequency of Cryptosporidium infection in animals of dairy farms of the Metropolitan Region (Santiago), Chile. Fecal samples of 205 newborn calves with diarrhea were studied and used for comparing the efficiency of two microscopic staining methods for diagnosis of the parasite, the auramine (AU) and a modified Ziehl-Neelsen (ZN) procedure. Out of the 205 fecal samples, we detected oocysts in 115 (56.1%) with AU and 102 (49.8%) with ZN. Comparison of results obtained with the two microscopic techniques showed significant difference (p<0.05), AU being more sensitive. On the other hand, concordance between the two methods was almost perfect (kappa value of 0.83). The results with these two operator dependent methods were confirmed using an operator independent immunochromatographic (IC) method. The IC method also enabled us to determine the identity of the parasite species as that of Cryptosporidium parvum. Identification of the parasite species was further corroborated by performing a Cryptosporidium species-specific polymerase chain reaction (PCR) test on few samples taken at random. Overall, the results showed a high number of infected animals suggesting the parasite C. parvum as a major parasitic disease agent of neonatal calves with diarrhea in dairy farms of the Metropolitan Region (Santiago) of Chile., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In the current study, a rapid chromatographic immunoassay submitted for registration in Europe was used to analyze PrP(sc) in 13 different areas of brain from 10 confirmed classical scrapie cases. The levels of PrP(sc) in the different areas of brain were plotted to draw a brain PrP(sc) distribution curve. This curve was compared with the brain PrP(sc) distribution curve obtained from immunoblotting and immunohistochemistry tests on the same samples. The distribution of PrP(sc) in different areas of the brain was similar, irrespective of the test applied, indicating that any of the 3 tests could be used for the characterization of classical cases of scrapie.

An immunochromatographic test (ICT) using recombinant BgSA1 (rBgSA1) for the detection of antibodies against Babesia gibsoni was developed and evaluated. Only the serum samples collected from dogs infected with B. gibsoni were positive in the ICT, but the serum samples from dogs infected with closely related parasites and from healthy dogs were negative. The specific antibodies could be detected in a dog experimentally infected with B. gibsoni at both the acute and chronic infection stages by the ICT. To evaluate the clinical application of the ICT, a total of 94 serum samples collected from domestic dogs in Japan were tested with the ICT and the previously established enzyme-linked immunosorbent assay (ELISA) with rBgSA1. Twenty-one of the tested samples (22.3%) were positive in both the ICT and the ELISA. The concordance between the ELISA and the ICT was found to be 95.8%. These results suggested that the ICT using rBgSA1 is rapid, simple, accurate, and suitable for the diagnosis of B. gibsoni infection of dogs in the field.

A simple yellow head virus (YHV) "strip test" was developed using monoclonal antibody Y19 (against the p20 structural protein) conjugated with colloidal gold as the detector antibody. Rabbit anti-recombinant p20 (rp20) protein antibody was used as a capture antibody at the test line (T) and goat anti-mouse IgG antibody (GAM) was used as the capture antibody at the control line (C). The ready-to-use strip was housed in a plastic case for convenient application and stored in the desiccated plastic bag. A sample volume of 100 microl of either haemolymph or gill or appendage homogenates in application buffer was applied to the sample chamber at one end of the strip and allowed to flow by chromatography through the nitrocellulose membrane to the other end. In test samples containing YHV, the virus would bind to colloidal gold conjugated monoclonal antibody and the resulting complex would be captured by the rabbit anti-rp20 antibody at the test line to give a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moved across the test line to be captured by the GAM to form a band at the control line (C). In the sample without YHV or below the limit of detection for the kit, only the control line was demonstrated. This method was about 500 times less sensitive than that of one-step RT-PCR, but slightly more sensitive than dot blotting. Therefore, it could be used for primary screening of individual shrimp or pooled shrimp samples to confirm high levels of YHV infection or YHV disease outbreaks. This kit can be used to detect gill associated virus (GAV) infection as well since the monoclonal antibody used in this kit cross-reacted well with GAV. The beneficial features of this kit are that simple, convenient, and rapid results that can be obtained without the requirement of sophisticated tools or special skills.

Background: Reliable analytical methods are required to monitor neomycin residue levels in the livestock products. In particular, a more simple and rapid detection method is required in the veterinary fields., Methods: Competitive direct ELISA and immunochromatographic assay were developed using monoclonal antibody to detect neomycin in the animal plasma and milk., Results: No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA methods, indicating that the antibody is highly specific for neomycin. Based on the standard curves, the detection limits were determined to be 6.85 ng/ml in PBS, 3.61 ng/ml in plasma, and 2.73 ng/ml in milk, respectively. Recoveries of neomycin from spiked plasma and milk at levels of 50-200 ng/ml ranged from 87% to 108%. Concentration of intramuscularly injected neomycin was successfully monitored in the rabbit plasma through competitive direct ELISA. Immunochromatographic method was also developed using colloidal gold-conjugated monoclonal antibody. Through this method, the detection limits were estimated to be about 10 ng/ml of neomycin in PBS, plasma, and milk., Conclusions: Immunochromatographic assay developed in this study is suitable for the simple screening of neomycin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA.

The serological diagnosis of canine visceral leishmaniasis (CVL) remains problematic because there areno reliable commercially available tests. Most laboratories use domestically prepared tests such as the enzyme-linked immunosorbent assay (ELISA) or the indirect immunofluorescent antibody test (IFAT). We evaluated rapid immunochromatographic (RICH) test kits for the diagnosis of CVL. The tests were assembled with either Leishmania chagasi recombinant antigens K39 or K26 and with either gold-labelled Staphylococcus aureus protein A or Streptococcus pyogenes protein G. Fifty sera from dogs with CVL, 14 sera from dogs with Chagas disease, and 50 sera from normal healthy dogs were tested. The results show that the RICH test using recombinant antigen K39 has a sensitivity of 96% and 100% specificity for the diagnosis of CVL. No significant differences were observed in the tests assembled with either protein A or protein G. The RICH tests using recombinant antigen K26 were equally specific but less sensitive than those using K39. However, the 2 antigens complemented each other and increased the overall sensitivity of the test. Because of its simplicity and performance the RICH test is a quick and reliable alternative for the diagnosis of CVL either in conventional laboratories or for remote areas where laboratories are not readily accessible for conventional assays.