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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.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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 β'-component (β'-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 β'-c) in catshark.

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.

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.)

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 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.

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 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.

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.

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.

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.

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.

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.

In an effort to identify life-stage specific Boophilus microplus proteins that elicit a humoral response in cattle, soluble proteins were extracted from 10- to 14-day-old larvae and subsequently fractionated by size-exclusion chromatography and reverse-phase high pressure liquid chromatography. Several antigens were identified by Western blotting as potentially shared with other ixodid tick species since antibodies to these proteins were present in sera of calves not previously exposed to B. microplus. Six putative B. microplus-specific antigens were identified by antibodies in the sera of calves repeatedly exposed to B. microplus larvae. One of the antigens, a 19.1 kDa protein, was used in the development of a diagnostic kELISA for previous exposure to B. microplus. The 19.1 kDa protein did not have tryptic protease activity or inhibit bovine trypsin activity, but appeared to be allergenic in that a partially pure fraction elicited immediate-type hypersensitivity responses in calves previously exposed to B. microplus.

Monoclonal antibody against kanamycin was prepared, and competitive direct ELISA and immunochromatographic assay were developed using the antibody to detect kanamycin in animal plasma and milk. The monoclonal antibody produced was identified to be IgG1, which has a kappa light chain. No cross-reactivity of the antibody was detected with other aminoglycosides, indicating that the monoclonal antibody was highly specific for kanamycin. Based on competitive direct ELISA, the detection limits of kanamycin were determined to be 1.1 ng/ml in PBS, 1.4 ng/ml in plasma, and 1.0 ng/ml in milk. The concentration of intramuscularly injected kanamycin was successfully monitored in rabbit plasma with competitive direct ELISA. Based on the colloidal gold-based immunochromatographic assay, the detection limits of kanamycin were estimated to be about 6-8 ng/ml in PBS, plasma, and milk. The immunochromatographic assay would be suitable for rapid and simple screening of kanamycin residues in veterinary medicine. Screened positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could be used to complement each other as well as other laboratory findings. Moreover, instead of slaughtering the animals to obtain test samples, these methods could be applied to determine kanamycin concentration in the plasma of live animals.

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.

Enzyme-linked immunosorbent assays (ELISAs) based on soluble antigens derived from promastigote or amastigote-like stages of Leishmania infantum and on the recombinant rK39 antigen, each in combination with different conjugates [anti-immunoglobulin G1 [IgG1], anti-IgG2, anti-IgG(gamma), and anti-IgG heavy plus light chains], were compared to an immunofluorescent-antibody test (IFAT) and two commercially available rapid test systems (DiaMed-Vet-IT Leish and ID-PaGIA canine leishmaniasis antibody test) for the detection of specific anti-Leishmania antibodies in symptomatic and asymptomatic dogs with proven L. infantum infections. ELISAs based on soluble promastigote and amastigote antigens had very high sensitivities in symptomatic (n = 30; 100%) and asymptomatic dogs (n = 17; 94.1 to 100%), except when combined with the anti-IgG1 conjugate (41.2 to 82.4%). Specificities were high for all combinations (n = 50; 96 to 100%). The rK39 ELISA detected fewer asymptomatic cases (sensitivities, 52.9 to 64.7%) but was highly specific (96 to 100%). The IFAT was 90% sensitive in symptomatic dogs but was significantly less sensitive in asymptomatic cases (29.4%). However, it had an excellent specificity (100%). Test performances of the rapid tests based on the rK39 antigen were comparable to the ELISAs based on the same antigen. ELISAs based on soluble promastigote or amastigote antigens seem to be most suited for the serological diagnosis of canine Leishmania infections in both symptomatic and asymptomatic dogs. IFAT and the rK39 ELISA lack sensitivity in asymptomatic cases but are highly specific. Rapid tests like the rK39 dipstick test or the ID-PaGIA are helpful for confirming clinically suspected cases because of their high specificities in symptomatic animals.

Group A rotaviruses are established agents of disease in buffalo calves. Early epidemiological studies in Italian buffalo herds revealed the predominance of strains with G8 specificity and detected strains with the rare, RRV-like, VP4 P[3] genotype. To acquire additional information on the VP4 and VP7 specificities of buffalo rotaviruses, a total of 125 fecal samples were collected from buffalo calves affected with diarrhoea, in seven dairy farms in Southern Italy. Rotaviruses were detected in 21 samples (16.8%) by an immunochromatographic assay and by reverse transcription-PCR (RT-PCR). Analysis of the VP7 gene revealed that 57% (12 of 21) of the isolates were G6, 23.8% were G8 (5 of 21) and 19% (4 of 21) were G10. Analysis of the VP4 revealed that 71.4% (15 of 21) of the isolates were P[5] and that 28.6% (6 of 21) were P[1]. The most common combination of G and P types was P[5],G6 (57%), followed by P[1],G10 (19%), P[5],G8 (14%) and P[1],G8 (9.5%). While P[5],G6 rotaviruses are very common in Italian bovine herds, the antigenic combination P[1],G10 is unusual and presumably derives from reassortment between P[1] and G10 strains, that appear to be more frequent in buffaloes and bovines, respectively. The presence of bovine-like G and P serotypes suggests that in Italy the epidemiology of buffalo rotaviruses overlaps the epidemiology of bovine rotaviruses, presumably because of the strict species affinity and/or of the intermingled distribution over the same geographical areas of the buffalo and bovine herds.

Objective: To evaluate the specificity of an immunochromatographic test (ICT) for anthrax in cattle., Design: A comparison of an ICT with blood smear and culture in uninfected cattle., Procedure: Two hundred and forty blood samples were collected from dead cattle at two knackeries within Victoria and tested on-site with an ICT for the detection of protective antigen (PA) of Bacillus anthracis. Blood smears were prepared on-site and blood samples transported to the laboratory for aerobic and anaerobic culture. The results of the ICT were compared with blood smear and culture. Animals were regarded as not infected with B anthracis if the organism was not detected in a stained blood smear or on culture. Ten healthy yearling cattle were vaccinated with live spore anthrax vaccine and blood samples collected on days 0 to 7 and day 15 were tested in the ICT for the presence of PA., Results: All blood samples from the 240 knackery cattle were ICT, smear and culture negative. All blood samples from the 10 vaccinated cattle were ICT negative., Conclusion: The ICT is a test with high specificity in cattle (98.5 to 100%; 95% CI) and recent vaccination of cattle does not give rise to positive reactions.

In this paper, the status of the analytical technologies for assaying animal antinutritional compounds, such as raffinose oligosaccharides, pentosans, phytic acid, and glucosinolates, is reviewed in terms of selectivity, sensitivity, and sample throughput. The implementation of simplified sample preparation schemes, use of novel separation approaches, and alternate detector technologies are discussed. The challenges and opportunities posed by these assays are highlighted along with the recommendations for best analytical practices.

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.

Genetically modified corn has been approved as an animal feed in several countries, but information about the fate of genetically modified DNA and protein in vivo is insufficient. Genetically modified corn Bt11 is developed by inserting a recombinant DNA sequence encoding insecticidal Cry1Ab protein from Bacillus thuringiensis subsp. kurstaki. We examined the presence of corn intrinsic and recombinant cry1Ab gene by PCR, and the Cry1Ab protein by immunological tests in the gastrointestinal contents of five genetically modified corn Bt11-fed and five nongenetically modified corn-fed pigs. Fragments of corn zein (242 bp), invertase (226 bp) and of ribulose-1,5-bisphosphate carboxylase/ oxygenase genes (1,028 bp) were detected in the gastrointestinal contents of both Bt11 and nongenetically modified corn-fed pigs. Fragments of recombinant cry1Ab gene (110 bp and 437 bp) were detected in the gastrointestinal contents of the Bt11-fed pigs but not in the control pigs. Neither corn intrinsic nor cry1Ab gene fragments were detected in the peripheral blood by PCR. The gastrointestinal contents were positive for Cry1Ab protein by ELISA, immunochromatography, and immunoblot; however, these methods did not work for blood and precluded conclusions about any potential absorption of the protein. These results suggest that ingested corn DNA and Cry1Ab protein were not totally degraded in the gastrointestinal tract, as shown by their presence in a form detectable by PCR or immunological tests.

The capacity of pig gastrointestinal microflora to metabolise the trichothecenes 3-acetyl-deoxynivalenol (3-acDON) and nivalenol (NIV) was investigated. 3-acDON was deacetylated to DON in anaerobic incubations with pig faeces collected at different pig farms. Furthermore, both 3-acDON and NIV were metabolised to the corresponding deepoxy metabolite in these incubates. Five pigs, in which the gastrointestinal microflora lacked the ability to transform 3-acDON and NIV to their corresponding de-epoxidated metabolites, were given low levels of DON in the feed for seven weeks. The gastrointestinal micro-organisms did not acquire the de-epoxidation ability during the seven week long exposure period. At the end of the exposure period, faeces from pigs with a known de-epoxidation ability was spread out in the pens and left for 24 hours. One week after the faeces had been spread out in the pens, the de-epoxidation ability was found in faecal incubations from four out of five experimental pigs. This change in metabolic ability of the intestinal de-epoxidation ability was not accompanied by any detectable changes in the DNA-profiles of the bacterial community composition. The results show that the intestinal de-epoxidation ability is common at pig farms in the Uppsala area, and that the ability may be transferred between pigs in a stock.

In the present work, two biochemical approaches were used to characterize PAGs isolated from Bos indicus fetal cotyledons removed at different gestational ages. The first procedure included acidic and ammonium sulfate precipitations, anion and cation exchange chromatographies and the second included pepstatin-agarose affinity chromatography. A bovine PAG radioimmunoassay was used to monitor the immunoreactivity throughout the isolation procedures. The most immunoreactive fractions issued from cation exchange and affinity chromatographies were analyzed by SDS-PAGE and Western blotting, before transfer to a polyvinylidene difluoride (PVDF) membrane for NH2-microsequence determination. Use SDS-PAGE and Western blotting, different isoforms of PAG with apparent molecular masses of 51 to 69 kDa and isoelectric points varying from 4.4 to 6.7 were identified in the placentas from different gestational ages. N-terminal microsequencing (10 to 25 aa long) indicates the expression of one single terminal amino acid sequence in the Bos indicus placenta, which is 100% identical to the bovine PAG-1.

The highly glycosylated protein MUC1 was purified from bovine milk-fat globule membranes by a procedure involving detergent extraction, ion-exchange chromatography and reverse-phase chromatography. The identity of the purified mucin protein was confirmed by N-terminal sequencing and partial amino acid sequences obtained by peptide mapping. The complete amino acid sequence of MUC1 was determined by cloning and sequencing the corresponding bovine mammary gland cDNA, which was shown to encode a protein of 580 amino acid residues comprising a cleavable signal peptide of 22 residues. The deduced amino acid sequence demonstrated structural features characteristic for mucins, including an extracellular tandem repeat region with 11 partially conserved repeats (20 amino acids each), a membrane-proximal SEA module, a transmembrane domain, and a cytoplasmic C-terminal region. Monosaccharide composition determinations suggested significant structural differences between O-linked glycans of MUC1 originating from either bovine or human milk. Interspecies differences of the consensus repeat sequence in MUC1 and the physiological functions are discussed.

Foot-and-mouth disease (FMD) is the most contagious animal virus disease of cloven-hoofed livestock and requires reliable and accurate diagnosis for the implementation of measures to control effectively its spread. Routine diagnosis of FMD is carried out at the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright by the combined use of ELISA and virus isolation in cell culture supplemented by reverse transcription polymerase chain reaction (RT-PCR) methods. These techniques require skilled personnel and dedicated laboratory facilities which are expensive. The development of a rapid and simple test for the detection of FMD virus antigen using Clearview chromatographic strip test technology for field application is described. This device detected FMD viral antigen in nasal swabs, epithelial suspensions and probangs from clinical samples submitted from the field, from animals infected experimentally and in supernatant fluids resulting from their passage in cell culture. The test system was more sensitive than ELISA for the diagnosis of all seven serotypes of FMD virus in the epithelial suspensions and nasal swabs and had equivalent sensitivity to the ELISA for the detection of contemporary virus strains in cell culture supernatant fluids. The study demonstrated the potential for this device to confirm a clinical diagnosis at the site of a suspected FMD outbreak, thereby offering the possibility of implementing control procedures more rapidly. Such pen-side diagnosis would have particular benefits in FMD emergencies, relevance to FMD control programmes which operate in endemic regions of the world such as South East Asia and for increasing disease awareness in other areas where efforts to control disease may be difficult. In each circumstance the availability of a pen-side device for diagnosis would reduce the necessity for sending routine diagnostic samples to an FMD laboratory and thereby reduce the delay in diagnosis, which can in some areas be considerable.

The objectives of this study were to analyze glycosaminoglycan (GAG) and mineral composition in the chicken eggshell. Eggshells were decalcified with acetic acid, and GAG was extracted from the decalcified shell by digestion with papain. The eggshell contained an average of 0.024% of its dry weight as uronic acid, a carbohydrate moiety of GAG. The eggshell GAG consisted of approximately 48% hyaluronic acid and and 52% galactosaminoglycan. In the latter, chondroitin sulfate-dermatan sulfate copolymers were the major galactosaminoglycans with dermatan sulfate disaccharide as a relatively minor component. The inorganic material recovered after decalcification accounted for approximately 140% of dry weight of the eggshell and contained 24.11% calcium, 0.04% phosphorous, and 0.23% magnesium, with an undetectable amount of nitrogen.

Bovine lactoferrin was separated into lactoferrin-a and lactoferrin-b from bovine colostrum. Lactoferrin-a was eluted at 0.38 M NaCl and lactoferrin-b was eluted at 0.43 M NaCl by carboxymethyl cation-exchange chromatography at pH 7.7, 0.05 M phosphate buffer. The molecular weights were estimated at 84,000 for lactoferrin-a and 80,000 for lactoferrin-b. Lactoferrin-a contents were 258.0 mg/L and lactoferrin-b contents were 524.3 mg/L of colostrum for cow 19. From colostrum to normal milk, total lactoferrin was from 17.1 to 129.4 mg/L during the normal lactational period; however, lactoferrin did not separate clearly into lactoferrin-a and lactoferrin-b. The lactoferrin-a measured from six cows was 258.0, 114.0, 112.8, 64.0, 59.7, and 22.4 mg/ L and the lactoferrin-b 524.3, 331.8, 184.7, 170.7, 129.3, and 44.0 mg/L, respectively. The average was 105.2 mg (31.3%) for lactoferrin-a and 230.8 mg (68.7%) for lactoferrin-b.

A simple chromatographic strip-test based on Clearview technology, is under development as a pen-side test for the detection of rinderpest antigen in eye swabs taken from cattle in the field. An outbreak of rinderpest occurred in the northern zone of Tanzania from late February to June 1997. The affected cattle exhibited very mild clinical signs, which made clinical diagnosis difficult. One hundred and seven eye swabs were collected from cattle suspected of infection with rinderpest. These were tested in the field using a prototype of the pen-side test and 13 (12.15%) of the samples were found to be positive for the presence of rinderpest antigen. These were confirmed by ICE. The positive cases were predominantly found in the Ngorongoro district. This demonstrates the usefulness of such a simple, rapid pen-side diagnostic assay, particularly when clinically 'mild' strains of rinderpest are present.

Rinderpest is a contagious viral disease of cloven-hoofed domestic and wild animals. Eradication of the virus following outbreaks depends on rapid and accurate diagnosis of infection and the implementation of control measures. Reporting and confirmatory diagnosis precede the implementation of control measures. A number of techniques have been used for diagnosis such as agar gel immunodiffusion, enzyme-linked immunosorbent assay (ELISA), molecular biological techniques such as polymerase chain reaction (PCR) and virus isolation in tissue culture. Many of these methods are both time consuming and require skilled personnel. The development of a rapid pen-side test for the detection of rinderpest virus (RPV) antigen in lachrymal fluid of cattle is described using the Clearview chromatographic strip test technology (Unipath, Bedford). Optimum conditions for binding monoclonal antibody to nitrocellulose and latex microspheres were determined and a prototype device was developed. The device detected viral antigen in lachrymal fluids from experimentally and naturally infected cattle and showed no cross-reactivity with other related viruses. A field trial was carried out at the Landhi Cattle Colony (LCC), Pakistan, to assess the performance of the rinderpest test under field conditions. Ninety-seven animals, some of which were showing various clinical signs, at LCC and neighbouring colonies were sampled and tested at the pen-side by Clearview and later by immunocapture ELISA (IC-ELISA) at IAH, Pirbright. Nineteen animals were positive by Clearview and/or IC-ELISA. Seventeen out of 19 rinderpest positive animals were positive by Clearview and 15 out of 19 were positive by IC-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) confirmed the 19 animals to be rinderpest positive. This simple, rapid, specific test allows for the first time, accurate pen-side diagnosis of rinderpest.

Natural and xenobiotic compounds having sex-related actions have long been used for growth promotion and various changes in carcass quality in meat animals. The first compounds used were synthetic estrogens; however, later on a whole battery of compounds having androgenic, and progestogenic actions have also been involved. In surveying the effects of these compounds in meat-producing animals, it became clear that these drugs increase the growth rate of the treated animals and bring about changes in the carcass that are generally characterized by lower fat content and more lean mass. Extensive studies undertaken in various countries, including the European Economic Community (EEC), have shown that if used according to good husbandry practices, the meat from treated animals does not have excessive amounts of residues compared with the endogenous amount of steroid production in the animals in question and also in human beings. The banning of these compounds in the European community brought a new phenomenon of illegal or black market cocktails. These mixtures of anabolic steroids are injected into the body of the animals rather than implanted in the ears, which is the normal practice in countries where they have not yet been banned. Several screening and confirmatory methods are now available for monitoring programs. However, these programs need excessive resources in terms of manpower, funds, and proper legislation, which in underdeveloped countries is questionable, particularly in the absence of strong scientific evidence for the exercise.

Membrane-associated cross-protection factor(s) (CPF) of in vivo-grown Pasteurella multocida were solubilized by detergent and partially purified by sequential chromatography on ion exchange, gel filtration, and hydroxyapatite columns. The CPF activity was determined by challenge of turkeys vaccinated with different chromatography fractions and challenge of poults passively immunized with serum from the same vaccinated turkeys. Analyses of different chromatography fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots suggested that proteins in the range of 59-65 kDa and a protein of 39-kDa molecular mass were associated with cross-protection. Although CPF activity was found to be associated with protein-containing material, treatment with periodate diminished cross-protection, indicating that carbohydrate determinants may also contribute to immune cross-protection.

Partial purification of hog cholera virus (HCV) using a simple batch-type chromatographic procedure with magnetic ferric oxide (MFO) is described. Infectious HCV was adsorbed from isotonic solutions to MFO and was eluted under conditions of low ionic strength and high pH. Aqueous solutions of 0.01 M sodium cyanide or 0.0003 M ammonium hydroxide effectively dissociated MFO-HCV complexes. The data indicate that 50 to 100% of the original HCV infectivity was recovered concomitant with a 90 to 95% reduction of extraneous organic nitrogen.MFO-purified HCV was concentrated by density gradient type centrifugations in buffered solutions of cesium chloride and sucrose. Prolonged isodensity centrifugations of concentrated MFO-purified HCV indicated a buoyant density of 1.14 to 1.15 gm/ml for the strain of virus used.