Search

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.)

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.

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.

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.

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.

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.

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.

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.

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.