Application and Evaluation of Novel Chromatographic Techniques to Detect and Quantitate 108 Pesticides and Metabolites in Muscle Samples From Wild Birds of Prey.

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.

Simultaneous and visual detection of infectious bronchitis virus and Newcastle disease virus by multiple LAMP and lateral flow dipstick.

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.

A loop-mediated isothermal amplification coupling with a lateral flow dipstick for rapid and specific detection of fowl adenovirus serotype-4.

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 × 104 copies/µl, 1 × 102 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 p0 = 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.

Meat characteristics of cattle fed diets containing whole cottonseed / Características da carne de bovinos alimentados com dietas contendo caroço de algodão

Sensory and instrumental analyses evaluated the meat and fat characteristics of feedlot-finished steers fed a diet containing cottonseed. Global impression, texture, meat color, and fat color were assessed. Thirty Nellore bulls with an average age of 30 ± 6 months and initial body weight of 382.7 ± 28.4kg were kept in feedlot stalls and fed the following cottonseed levels: 0; 2.22 %; 4.44 %; 6.66 %; 8.88 %; 11.11 % of the dietary dry matter. The cottonseed used in this experiment had an average free gossypol content of 4.5g/kg of cottonseed. The overall impression of the samples, assessed by the triangle test for difference, did not differ for more than 62 % of the panelists (P>0.01). The shear strength of roasted meat varied from 6.00 to 6.54kg. According to texture profile analysis (TPA), the hardness, springiness, and chewiness of roast meat ranged from 24.15 to 28.01 N, 0.52 to 0.56, 8.42 to 11.01 N, respectively; of raw meat, 9.51 to 13.86 N, 0.26 to 0.29, and 1.38 to 1.81 N, respectively. The different treatments did not affect meat texture, meat color, or fat color (P>0.05). Meat color, luminosity, and red intensity ranged from 37.71 to 42.85, 20.68 to 25.25, and 6.74 to 8.61, respectively; fat color, luminosity, and yellow intensity ranged from 62.26 to 63.78, 11.13 to 11.62, and 10.53 to 10.86, respectively. Cottonseed intake of up to 1.13kg/animal/day, equivalent to a free gossypol intake of 5.05g/animal/day, in place of soybean meal and ground corn, did not significantly change the global sensory impression, texture, and color of the meat and fat.(AU)

Avaliou-se, por meio de análise sensorial e instrumental, o efeito da adição de caroço de algodão à dieta de bovinos confinados sobre as características da carne e da gordura. Foram avaliadas a impressão global, a textura, a cor da carne e a cor da gordura. Trinta touros da raça Nelore, com médias de idade e peso vivo inicial de 30 ± 6 meses e 382,7 ± 28,4kg, foram confinados e receberam dietas com os seguintes teores de caroço de algodão: 0; 2,22%; 4,44%; 6,66%; 8,88%; 11,11% na matéria seca da dieta. O caroço de algodão utilizado neste experimento apresentou conteúdo médio de 4,5g de gossipol livre/kg de caroço de algodão. A impressão global da carne, avaliada por meio de teste triangular de diferença, mostrou que mais de 62% dos provadores não perceberam diferença significativa (P>0,01) entre as amostras. A textura da carne assada avaliada por meio do teste de força de cisalhamento variou de 6,00 a 6,54kg. Na análise do perfil de textura (TPA) da carne assada, a dureza, a elasticidade e a mastigabilidade variaram, respectivamente, de 24,15 a 28,01N, de 0,52 a 0,56N e de 8,42 a 11,01N. Na TPA da carne crua, variaram, respectivamente, de 9,51 a 13,86N, de 0,26 a 0,29N e de 1,38 a 1,81N. Na avaliação da cor da carne, a luminosidade, a intensidade de vermelho e a intensidade de amarelo da cor da carne variaram, respectivamente, de 37,71 a 42,85, de 20,68 a 25,25 e de 6,74 a 8,61. E para a cor da gordura, variaram, respectivamente, de 62,26 a 63,78, de 11,13 a 11,62 e de 10,53 a 10,86. A textura, a cor da carne e a cor da gordura não apresentaram diferenças significativas (P>0,05) entre os diferentes tratamentos. O consumo de caroço de algodão em até 1,13kg/animal/dia, que resultou no consumo de 5,05g de gossipol livre/animal/dia, em substituição ao farelo de soja e ao grão de milho triturado, não causou alterações significativas nas características da carne quanto à impressão sensorial global, à textura, à cor da carne e à cor da gordura.(AU)

Accuracy and reproducibility of a rapid chromatographic immunoassay for the diagnosis of canine visceral leishmaniasis in Brazil.

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.

Enhanced intestinal epithelial barrier health status on European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides.

The study assesses the effects of dietary mannan oligosaccharides (MOS) in European sea bass (Dicentrarchus labrax) posterior intestinal lipid class composition and its possible relation to the potential prostaglandins production and Gut Associated Lymphoid Tissue (GALT) stimulation. Fish were fed 4 g kg(-1) MOS (Bio-Mos(®) Aquagrade, Alltech, Inc., USA) for eight weeks. Fish fed MOS presented higher (P ≤ 0.05) weight gain, total length, and specific and relative growth rates than fish fed the control diet. Stimulated posterior gut of fish fed MOS showed higher (P ≤ 0.05) prostaglandins production than fish fed the control diet. Lipid class analyses of posterior gut revealed a reduction (P ≤ 0.05) in the neutral lipid fraction in fish fed MOS compared to fish fed the control diet, particularly due to a reduction (P ≤ 0.05) in triacylglycerols content. The polar lipid fraction increased (P ≤ 0.05) in fish fed MOS compared to fish fed the control diet, mainly due to an increase (P ≤ 0.05) in phosphatidylethanolamine and phosphatidylcoline contents. Light microscopy of posterior gut revealed increased number or goblet cells as well as higher level of infiltrated eosinophilic granulocytes for fish fed MOS. Transmission electron microscopy qualitative observations revealed a better preserved cytoarchitecture of the intestinal epithelial barrier in the posterior gut of fish fed MOS. Posterior gut of fish fed MOS presented more densely packed non-damaged enterocytes, better preserved tight junctions structure, healthier and more organized microvilli, and a higher presence of infiltrated lymphocytes and granulocytes compared fish fed the control diet. The present study indicates that dietary MOS enhances European sea bass posterior gut epithelial defense by increasing membrane polar lipids content in relation to a stimulation of the eicosanoid cascade and GALT, promoting posterior gut health status.

Characterization of vitellogenin and its derived yolk proteins in cloudy catshark (Scyliorhinus torazame).

Elasmobranchs (sharks and rays) exhibit unique reproductive characteristics and, in contrast to the situation in teleosts, very little is known about the identity, structure and physical characteristics of their egg yolk proteins. The aims of this study were to (1) detect and purify the vitellogenin (Vtg; egg yolk precursor) and yolk proteins (YPs) of the cloudy catshark (Scyliorhinus torazame), (2) examine the relationships between Vtg and YPs and (3) characterize and classify the deduced primary structure of the Vtg transcript (vtg). The apparent molecular weights of purified Vtg and putative Vtg-related YPs (lipovitellin: Lv, phosvitin: Pv) were determined by gel filtration and were ~560, >669 and ~58 kDa, respectively. Following SDS-PAGE, these purified products (i.e., Vtg, Lv and Pv) appeared as bands of ~210, ~110 and ~22 kDa, respectively. On Western blots, antisera against purified Vtg, Lv and Pv recognized the ~210 kDa Vtg band. Catshark Pv, in contrast to teleost Pvs, had a very low serine content. The catshark Vtg cDNA sequence (vtg) appeared to contain an open-reading frame consisting of domains encoding Lv, Pv and ß'-component (ß'-c). A phylogenetic analysis, with a consideration of genome duplication events, placed catshark vtg into the 'vtgAB type.' It is concluded that at least a single major type of Vtg protein, which is transcribed and translated from catshark vtgAB gene, is the precursor of three egg yolk proteins (Lv, Pv and ß'-c) in catshark.

[Detection of processed animal protein: European experience and perspectives]. / Détection des protéines animales transformées: expérience et perspectives européennes.

European Commission Regulation (EC) No. 152/2009 imposes optical microscopy as the reference method for official controls to detect traces of animal protein in animal feed. Since 1 July 2004, the one-solvent technique has been the only authorised variant of optical microscopy. Its detection limit is 0.1% of meat-and-bone meal. Other techniques--using molecular biology (polymerase chain reaction, immunology), microscopy or near-infrared imaging--have been developed in the past ten years to supplement the official method, which has certain limitations. This paper compares and discusses the different techniques, highlighting the strengths of each technique in order to propose a feasible control scheme to improve the sensitivity and specificity of the technique for the detection of processed animal protein in livestock feed.

Evaluation of a novel chromatographic immunoassay based on Dual-Path Platform technology (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis.

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.

Cryptosporidium parvum in diarrheic calves detected by microscopy and identified by immunochromatographic and molecular methods.

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.

Survey of antibodies to Trypanosoma cruzi and Leishmania spp. in gray and red fox populations from North Carolina and Virginia.

American trypanosomiasis and leishmaniasis are caused by related hemoflagellate parasites, Trypanosoma cruzi and Leishmania spp., which share several common host species. Both zoonotic protozoans are endemic in the United States. Canines, including domestic and wild canids, are reservoir hosts for human infections with T. cruzi and Leishmania spp. The present study examined the seroprevalence of T. cruzi and Leishmania spp. in wild canids from North Carolina and Virginia. Wild canine species tested in this work included 49 gray foxes (Urocyon cinereoargenteus) and 5 red foxes (Vulpes vulpes). Overall, sera samples from 54 foxes (North Carolina  =  43; Virginia  =  11) were tested by immunochromatographic strip assays (ICT). Antibodies to T. cruzi were found in 4 (9%) gray foxes from North Carolina and 2 (18%) gray foxes from Virginia. Antibodies to Leishmania spp. were detected in 1 (2%) gray fox from North Carolina. Our results indicate that wild canids are exposed more frequently to T. cruzi in North Carolina than Leishmania spp. and only T. cruzi in Virginia.

Toxoplasma gondii and Trypanosoma cruzi antibodies in dogs from Virginia.

Toxoplasma gondii and Trypanosoma cruzi are zoonotic protozoan parasites that cause disseminated infections in many vertebrate species. The present study determined the seroprevalence of T. gondii and Tr. cruzi in a population of dogs from Virginia. Serum samples were tested from 90 domestic dogs collected from animal shelters in Virginia. Using an indirect immunofluorescent antibody test, sera were examined at a 1 : 50 dilution and antibodies to T. gondii were found in 19 dogs (21%). Antibodies to Tr. cruzi were determined by qualitative immunochromatographic dipstick assay. One (1%) of the 90 dogs had Tr. cruzi antibodies and it was also seropositive for T. gondii. Our findings indicate that dogs are frequently exposed to T. gondii in Virginia, but that antibodies to Tr. cruzi are rare in the same geographical region.

Rapid chromatographic immunoassay study of brain PrPsc distribution in classical scrapie.

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.

Chromatographic analysis of lipid fractions in healthy dogs and dogs with obesity or hyperadrenocorticism.

Obesity and endogenous hyperadrenocorticism (HAC) are common clinical conditions in veterinary practice, and both conditions have clinical and laboratory similarities, such as weight gain and dyslipidemia. The objective of the present study was to characterize and compare the lipid profiles and plasma lipoprotein fractions in healthy dogs (n = 10), in obese dogs (n = 10), and in dogs with HAC (n = 6). All of the dogs were client owned. The lipoproteins were separated by fast protein liquid chromatography, and the plasma concentrations of total cholesterol and total triacylglycerol (TAG) were determined by enzymatic methods. When compared with the healthy and obese groups, dogs with HAC had a significant increase (P < 0.01) in the total concentrations of TAGs and cholesterol (CHOL), with higher distribution in the very low-density lipoprotein (VLDL)-CHOL fractions. In addition, the distributions of the high-density lipoprotein (HDL)-CHOL and HDL-TAG fractions were significantly lower (P < 0.01) in dogs with HAC than in healthy dogs. Considering the animals in this study, it was determined that the dogs with HAC differed significantly from the healthy and obese dogs regarding the metabolism of CHOL and TAG, as well as their VLDL and HDL fractions. Similar laboratory findings could allow veterinarians to distinguish obese dogs from those with HAC. In addition, dogs with HAC may be at higher risk for developing metabolic and atherosclerotic complications.

Effects of matrix filtration of low-quality boar semen doses on sperm quality.

The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 +/- 0.5 cm), CM Sephadex (length 5 +/- 0.5 cm), glass wool (length 2 +/- 0.5 cm) or glass bead (length 10 +/- 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca((R)) 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l-lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l-lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction.

Evaluation of the Chagas Stat-Pak assay for detection of Trypanosoma cruzi antibodies in wildlife reservoirs.

An immunochromatographic assay (Chagas Stat-Pak) was evaluated for the detection of Trypanosoma cruzi antibodies in 4 species of wildlife reservoirs. Antibodies to T. cruzi were detected in raccoons (Procyon lotor) (naturally and experimentally infected) and degus (Octodon degu) (experimentally-infected) using the Chagas Stat-Pak. In naturally exposed wild raccoons, the Chagas Stat-Pak had a sensitivity and specificity of 66.7-80.0% and 96.3%, respectively. Compared with indirect immunofluorescent antibody assay results, seroconversion as determined by Chagas Stat-Pak was delayed for experimentally infected raccoons, but occurred sooner in experimentally infected degus. The Chagas Stat-Pak did not detect antibodies in naturally or experimentally infected Virginia opossums (Didelphis virginiana) or in experimentally infected short-tailed opossums (Monodelphis domestica). These data suggest that the Chagas Stat-Pak might be useful in field studies of raccoons and degus when samples would not be available for more-conventional serologic assays. Because this assay did not work on either species of marsupial, the applicability of the assay should be examined before it is used in other wild species.

Development and laboratory validation of a lateral flow device for the detection of foot-and-mouth disease virus in clinical samples.

A lateral flow device (LFD) for the detection of all seven serotypes of foot-and-mouth disease virus (FMDV) was developed using a monoclonal antibody (Mab 1F10) shown to be pan-reactive to FMDV strains of each serotype by ELISA. The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia (304 positive and 1003 negative samples) from suspected cases of vesicular disease collected from 86 countries between 1965 and 2008 and negative samples collected from healthy animals. The diagnostic sensitivity of the LFD for FMDV was similar at 84% compared to 85% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 99.9% for the ELISA. The device recognized FMDV strains of wide diversity of all seven serotypes but weaker reactions were often evident with those of type SAT 2, several viruses of which were not detected. Reactions with the viruses of swine vesicular disease and vesicular stomatitis that produce clinically indistinguishable syndromes in pigs and cattle, did not occur. The test procedure was simple and rapid, and typically provided a result within 1-10min of sample addition. Simple homogenizers that could be used in field conditions for preparing epithelial suspensions were demonstrated to be effective for LFD application. These data illustrate the potential for the LFD to be used next to the animal in the pen-side diagnosis of FMD and for providing rapid and objective support to veterinarians in their clinical judgment of the disease.

Applicability of a rapid chromatographic immunoassay for analysis of the distribution of PrPBSE in confirmed BSE cases.

The Prionics-Check PrioSTRIP is a rapid chromatographic immunoassay for bovine spongiform encephalopathy (BSE) approved by the European Union in 2004. In this study, the PrioSTRIP was used to analyse PrP(BSE) in 16 different brain areas of nine confirmed BSE cases. The levels of PrP(BSE) in the different brain areas were plotted to give the brain PrP(BSE) distribution curve (BPDC) and compared with the BPDC obtained previously by Western blotting and enzyme-linked immunosorbent assay (ELISA) methods on the same samples. The distribution of PrP(BSE) in different areas of the brain was similar, irrespective of the test applied, indicating that each test could be used for the characterisation of BSE cases.

Development of an immunochromatographic test with recombinant BgSA1 for the diagnosis of Babesia gibsoni infection in dogs.

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 convenient immunochromatographic test strip for rapid diagnosis of yellow head virus infection in shrimp.

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.