Activation of immune cells in bovine mammary gland secretions by zymosan-treated bovine serum.

Mastitis, caused by bacterial infection of the mammary gland, is a major disease of dairy cattle. The greatest risks of intramammary infection occur at the end of lactation and at the initiation of the next lactation when the cow calves. Treating serum with zymosan (yeast cell wall preparation) causes the complement to cleave, allowing this serum to serve as a source of complement fragment 5a (C5a), a potent chemoattractant and activator of the immune system. Our hypothesis was that intramammary infusion of zymosan-treated serum (ZTS) would recruit polymorphonuclear neutrophils (PMN) and generate prolonged activity in lymphocytes within the mammary gland. Ultimately this could help prevent bacterial infections in cows at dry-off and at the initiation of lactation. Two ipsilateral quarters of the mammary gland of each cow were infused with ZTS (12.5 mL/quarter), and 2 contralateral quarters were infused with saline in 8 cows shortly after lactation ended. Mammary secretions were collected periodically throughout the dry period and the first 2 wk of the next lactation. Activation status of lymphocytes and PMN in those secretions was assessed based on the intracellular presence or absence of IFN-gamma and IL-8 as determined by flow cytometry. The ZTS infusion greatly increased PMN numbers in mammary secretions for the first week only. The percentage of IFN-gamma positive lymphocytes and PMN, and the percentage of IL-8 positive PMN, exhibited a sustained increase in secretions from ZTS-treated quarters through the first 2 wk of lactation. The ZTS can stimulate PMN and lymphocyte-mediated immune defense mechanisms in the mammary gland, which may provide a useful means of preventing new intramammary infections during the dry period as well as at the initiation of lactation.

Diagnosis of preclinical and subclinical scrapie in a naturally infected sheep flock utilizing currently available postmortem diagnostic techniques.

Scrapie is a naturally occurring transmissible encephalopathy of sheep and goats. Currently available methods for diagnosis are the presence of characteristic histopathologic changes and detection of an abnormal form of prion protein (PrPres) in the brains of affected animals. This study documents preclinical and subclinical scrapie in a flock of 16 sheep utilizing histopathology, immunohistochemistry (IHC), western blot, and electron microscopy (for scrapie-associated fibrils) for confirmation of the disease. Prior to necropsy, none of the sheep showed signs of clinical scrapie. Based on the results of histopathology and positive PrPres tests, 3 ewes were found to have subclinical scrapie. An additional ewe, which did not have histopathologic changes in the brain but was positive by IHC and western blot,was considered a preclinical case of scrapie. None of the sheep had amyloid in the brain stem.

Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy.

Transmissible spongiform encephalopathies in humans and in animals are fatal neuro-degenerative diseases with long incubation times. The putative cause of these diseases is a normal host protein, the prion protein, that becomes altered. This abnormal prion protein is found mostly in the brains of infected individuals in later stages of the disease, but also can be found in lymphoid and other tissues in lower amounts. In order to eradicate this disease in animals, it is important to develop a system that can concentrate the abnormal prion protein and an assay that is very sensitive. The sensitivity that can be achieved with capillary electrophoresis makes it possible to detect the abnormal protein in blood. A peptide from the carboxyl terminal region, amino acid positions 218-232, was labeled with fluorescein during the synthesis of the peptide at the amino terminus. Antibodies that have been produced to this peptide were affinity purified and used in a capillary electrophoresis immunoassay. The amount of fluorescein labeled peptide in the capillary was 50 amol. Blood was obtained from normal sheep and elk, from sheep infected with scrapie and elk infected with chronic wasting disease. Buffy coats and plasma were prepared by a conventional method. After treatment with proteinase K, which destroys the normal protein but not the altered one, the blood fractions were extracted and tested in the capillary electrophoresis immunoassay for the abnormal prion protein. The abnormal prion protein was detected in fractions from blood from infected animals but not from normal animals. This assay makes a pre-clinical assay possible for these diseases and could be adapted to test for the abnormal prion protein in process materials that are used for manufacture of pharmaceuticals and products for human consumption.

Capillary isoelectric focusing of the scrapie prion protein.

Prion diseases or transmissible spongiform encephalopathies belong to a group of neurodegenerative diseases that infect both animals and humans. These diseases are associated with an accumulation of fibrils in the brains of infected individuals. These fibrils are composed of an abnormal isoform of a host-encoded glycoprotein that is characterized by its insolubility and partial resistance to proteases. Another characteristic of the scrapie prion protein (PrPsc) is the wide range of isoelectric points (pI values) that have been observed on conventional isoelectrofocusing gels. In this study, we explored the use of capillary isoelectric focusing (cIEF) to characterize the pI values for PrPsc isolated from sheep and hamster brain. We used a Beckman 5500 P/ACE using UV detection at 280 nm. A cIEF 3-10 Kit from Beckman Instruments was used to perform the analysis. The PrPsc was solubilized in 0.01 M Tris-HCl, pH 8.00 containing 2 mM EDTA. 5% SDS and 10% hexafluoroisopropanol at 100 degrees C for 10 min. The solubilized PrPsc was placed over a high-performance hydrophilic interaction column. After elution, the peaks were concentrated and assayed for immunoreactivity with specific antisera. The peaks that contained immunoreactivity were then placed on the cIEF capillary. The samples containing PrPsc were solubilized in 1% n-octylglucoside before isoelectric focusing. The scrapie infected sheep sample had peaks with pI values ranging from 5.2 to 3.00 with a major peak at 3.09. The normal sheep brain had pI values that were higher. The hamster adapted scrapie strain had peaks with pI values ranging from 6.47 to 3.8. These pI values were slightly higher than those obtained for the sheep samples. The use of cIEF to determine the pI values of PrPsc led to the identification of a major species of PrPsc from sheep with a very acidic pI.

A diagnostic test for scrapie-infected sheep using a capillary electrophoresis immunoassay with fluorescent-labeled peptides.

Scrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod-shaped fibrils in the brain that form from an aggregated protein. This protein (PrPSC) is a protease-resistant form of a normal host cell protein. When the aggregated protein is denatured in sodium dodecyl sulfate (SDS) and beta-mercaptoethanol, a monomer form of approximately 27 kDa molecular mass is observed. A competition immunoassay to detect PrPSC from scrapie-infected sheep was developed using free zone capillary electrophoresis with laser-induced fluorescence (LIF) for detection and flourescein-labeled synthetic peptides from PrPSC. Antibodies were made to each respective peptide and used in the competition assay. The fluorescent-labeled peptides bound to the antibody were separated from the unbound peptides using 200 mM Tricine, pH 8.0, containing 0.1% n-octylglucoside and 0.1% bovine serum albumin (BSA). The amount of antibody that would bind approximately 50% of the fluorescent-labeled peptide was determined for each peptide. When unlabeled peptide was added to the assay, approximately 2 fmoles of the peptide could be measured. When PrPSC extracted from infected sheep brain was added to the assay, approximately 135 pg of PrPSC could be detected. When preparations from normal sheep were assayed, there was little or no competition for the bound peptides. Assays using two of the peptides, peptides spanning amino acid positions 142-154 and 155-178, clearly differentiated scrapie-positive sheep from normal animals. This assay is a new method that can be used to diagnose scrapie and, possibly, other transmissible spongiform encephalopathies in animals and in humans.

Use of capillary sodium dodecyl sulfate gel electrophoresis to detect the prion protein extracted from scrapie-infected sheep.

Scrapie in sheep and in goats is the prototype of a group of transmissible spongiform encephalopathies (TSE). A feature of these diseases is the accumulation in the brain of rod shaped fibrils that form from an aggregated protein that is a protease-resistant form of a modified normal host cell protein. In this study, we compared SDS gel capillary electrophoresis to conventional SDS-PAGE and Western blot to detect the monomer of this aggregated protein. This prion protein was extracted from the sheep brain by homogenizing the brain stem (10%, w/v) in 0.32 M sucrose and by using a series of ultracentrifugation steps and treatment with sodium lauroyl sarcosine and proteinase K. After the final centrifugation step, the pellet was resuspended in 0.01 M Tris pH 7.4 in a volume equivalent to 0.1 ml/g of brain used. This resuspended pellet was treated with 1% SDS and 5% 2-mercaptoethanol and boiled for 10 min. The analysis was done in a Beckman P/ACE 5500 using a SDS gel capillary (eCap SDS14-200 Beckman capillary). In infected sheep brain samples, but not normal sheep, a major peak at a molecular mass of 16.1 kDa and a minor peak with a leading shoulder were observed. Since the molecular mass determined for this protein was lower than that estimated on Western blot (22.4 kDa), a Ferguson plot was made to determine if there were abberations in the molecular mass determination. After correction, the major peak was estimated to be 19.2 kDa. This has a better correlation with that determined by SDS-PAGE and Western blot. The equivalent amount of brain sample in the capillary was approximately 50 micrograms. For Western blot, the amount of brain sample was approximately 20 mg. For this assay, this is approximately 100 times less than that needed for Western blot for sheep samples.

Improvements in a competition assay to detect scrapie prion protein by capillary electrophoresis.

Scrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod-shaped fibrils in the brain that form from an aggregated protein. This protein is a protease-resistant form of a normal host cell protein. When the aggregated protein is denatured in SDS and beta-mercaptoethanol, a monomer form (prion protein) with a molecular mass of 27 kDa is observed. Free zone capillary electrophoresis and peptides labeled with fluorescein were used to detect the prion protein through competition for a labeled peptide in immune complex formation. The separation of the immune complexes from the unbound peptide using 200 mM Tricine (pH 8.0) was faster and was better resolved than that obtained with phosphate or borate buffer systems. The amount of immune complex formation was dependent on the amount of antibody in the assay. The amount of bound labeled peptide and unbound labeled peptide could be measured directly by calculating the area of each respective peak. As increasing amounts of unlabeled peptide were added to the assay, a concentration dependent reduction in the immune complex peak was observed. The assay could detect less than 10.0 fmol of unlabeled peptide. There was a quantitative difference in the competition of preparations from scrapie infected sheep brain and normal sheep brain.

Capillary electrophoresis of the scrapie prion protein from sheep brain.

Scrapie in sheep and goats causes a progressive, degenerative disease of the central nervous system and is the prototype of other transmissible spongiform encephalopathies (TSE) found in humans and in animals. In samples of TSE-affected brains, unique rod-shaped structures are found and are infectious. These rods are composed of a protease-resistant, post-translationally modified cellular protein (PrPsc) that has a molecular mass of ca. 27,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laboratory tests used for the diagnosis of scrapie detect PrPsc. The overall concentration of PrPsc in tissues is low. The present methods to diagnose scrapie are lengthy, require relatively large quantities of starting material to detect PrPsc and lack sensitivity. We explored the use of free zone capillary electrophoresis and immunocomplex formation to detect PrPsc in the brain tissue of infected sheep. Brain tissue from both infected (as confirmed by histological and biological tests) and from normal animals was used to prepare the PrPsc. After treatment with proteinase K and non-ionic detergents, PrPsc was solubilized and reacted with a rabbit antiserum specific for a peptide of the prion protein. Immunocomplex formation was observed for the samples from scrapie-infected brain but not for samples from normal brain. When a fluorescein-labeled goat anti-rabbit immunoglobulin was used as a second antibody, the detection of immunocomplex formation was enhanced both by the immunological technique and by using laser-induced fluorescence for detection. This same rabbit antiserum was used on immunoblot analysis. Three bands were observed for material from an infected sheep but none in preparations from brain material from normal sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization by capillary electrophoresis of the surface glycoproteins of ovine lentiviruses before and after treatment with glycosidic enzymes.

Ovine lentiviruses are a group of viruses that infect sheep and goats. These viruses contain a surface glycoprotein (SU) that is very similar among the viral strains. Sera from infected animals react equally well with SU from each strain. Monoclonal antibodies produced to SU can distinguish among some of the viral strains. In order to delineate these differences we treated SU from several viral strains with the glycosidic enzymes. These enzymes included a mixture of exoglycosidases, beta-N-acetyl glucosaminidase, neuraminidase and endoglycosidases D, F and H. After these treatments we observed changes in the reactivities of the monoclonal antibodies that were directed to SU. In order to characterize these changes on the surface epitopes, SU from the different viral strains were subjected to free zone capillary electrophoresis (CZE) using an 0.02 M phosphate buffer at pH 9.0 at a running voltage of 5 kV. Differences were readily seen between SU that had not been treated and SU that had been treated with the glycosidic enzymes. Each viral strain had a characteristic electropherogram. The electropherograms indicated that the heterogeneity of the charge on SU was increased after the enzyme treatments. From these results we have concluded that the carbohydrate moieties play an important role in contributing to the surface charge of SU. This charge affects the nature of its surface epitopes and has an impact on its biological function.

Antigenic relatedness between ovine progressive pneumonia virus (OPPV) and HIV-1.

The antigenic relatedness between ovine progressive pneumonia virus (OPPV), a lentivirus that infects sheep, and human immunodeficiency virus (HIV-1) was examined by immunoblot analysis. Fourteen of 20 sheep sera, that were positive for OPPV antibodies on an immunodiffusion test, reacted with HIV-1 p24 on a commercial blot of HIV-1 and cell lysate proteins (Virostat, Portland, Maine). Sheep OPPV antisera did not bind to cellular antigens on a negative control blot of cellular proteins. There was no correlation between the ability to bind to HIV-1 p24 and the status of disease in the sheep. Twenty human anti-HIV-1 sera and two human HIV-1 negative sera were tested on OPPV and cell lysate (OPPV-CON) and cell lysate (CON) protein blots. None of the human serum samples tested reacted to OPPV specific proteins.

Seroprevalence of ovine progressive pneumonia virus in various domestic and wild animal species, and species susceptibility to the virus.

Ovine progressive pneumonia is caused by a lentivirus of known infectivity only for sheep and goats. Virus susceptibility of 11 other species of animals was examined. Species included cattle, chickens, deer, dogs, goats, hamsters, horses, mice, pigs, rabbits, and rats. Of these species, only goats and rabbits could be experimentally infected with the virus. The infection in rabbits was acute, and virus did not persist or induce antibody production as it does in sheep and goats. Sera obtained from several people working in close contact with the virus and from several wild species, with unknown exposure history, were tested for antibodies to viral antigens. All results were negative. Knowledge of the host range of this virus is important for scientific studies and for virus eradication programs.

Separation of ovine IgG1 and IgG2 on protein A-sepharose.

A method using protein A-Sepharose chromatography was developed to separate and purify ovine IgG1 and IgG2. The IgG1 eluted from protein A-Sepharose at pH 6.8 and IgG2 eluted at pH 4.5. This method was used to show the specific transfer of IgG1 from the colostrum to newborn lambs. After separation on protein A-Sepharose both IgG1 and IgG2 were pure as analyzed by isoelectric focusing, Western Blotting and SDS-PAGE. The isoelectric points for the immunoglobulins were calculated to be 3.5 for IgG2 and a range from 6.2 to 8.1 for IgG1. The subclass, IgG1, was present in the whey and was the subclass that was found in the serum of lambs after being fed colostrum. The ewe sera had a decrease of both IgG1 and IgG2 at the time of lambing compared to 2 weeks prior to parturition.

Ovine progressive pneumonia (maedi-visna) in sheep.

Ovine progressive pneumonia (OPP) is a multi-systemic disease of sheep caused by a nononcogenic exogenous retrovirus belonging to the Lentiviridae subfamily. Characteristics of the disease are chronic lymphocytic pneumonitis, encephalitis, arthritis, mastitis and vasculitis associated with progressive wasting, dyspnea, lameness, indurated udder and, rarely, paralysis. Any one or all of the characteristics may be manifest. Transmission of the virus is predominantly through the colostrum to newborn lambs, however, transmission can occur by contact and in utero. Treatment of the disease is only symptomatic and prevention of infection is only by avoiding the virus.

Failure of experimental vaccines to protect against infection with ovine progressive pneumonia (maedi-visna) virus.

Cell culture medium was harvested from cells infected with ovine progressive pneumonia (OPP) virus and used to prepare killed virus vaccines. Virus was inactivated by either heat, formalin, or ethyleneimine and used either without adjuvant, with Freund incomplete adjuvant, or with aluminum hydroxide adjuvant to vaccinate sheep. The sheep produced precipitating antibody against the virus but were not protected against infection when challenged with live OPP virus.

Detection of bovine virus diarrhea virus in cell culture using an immunoperoxidase technique.

An indirect immunoperoxidase staining technique was developed for identifying cell cultures infected with bovine virus diarrhea virus. Infected cell monolayers stained intensely while uninfected monolayers remained colorless. Immunoperoxidase staining was as sensitive as direct immunofluorescence in detecting endpoint dilutions of virus suspensions. Using the immunoperoxidase technique, infected monolayers were detectable by macroscopic, as well as microscopic, observation.

Preparation of sheep and cattle immunoglobulins with antibody activity by high-performance liquid chromatography.

High-performance anion-exchange chromatography (HPIEC) and high-performance size-exclusion chromatography (HPSEC) were used to purify serum IgM and IgG from sheep and cattle. Pooled serum from normal cattle and sheep and serum from sheep, infected with two different viruses, were prepared for HPIEC by chromatography on CM-Affi-Gel Blue. After HPIEC, fractions containing IgG and IgM were pooled and concentrated and further purified by HPSEC. The purity of fractions from HPIEC and HPSEC were evaluated by immunoelectrophoresis, protein-A-Sepharose affinity chromatography, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Monoclonal antibody specific for bovine IgG2 was used to assay for IgG2 and IgG2 contamination of other fractions. The antibody activity to ovine adenovirus 5 and to ovine progressive pneumonia virus was assayed by neutralization and immunodiffusion. Antibody activity was retained against both viruses in the fractions containing IgG1 and IgM. This high-performance liquid chromatography procedure was a rapid preparative method to produce specific immunoglobulins and could be used to evaluate the purity of immuno-reagents.

Conditions for binding bovine IgG1 to protein A-Sepharose.

Conditions were established so that both subclasses of bovine IgG were bound to Protein A-Sepharose. Increasing the pH of the starting buffer to pH 8.0 from pH 7.0 and increasing the starting phosphate concentration of the buffer to 0.5 M from 0.2 M enhanced the separation. Using these modifications in the buffer system, IgG1 was eluted from pH 7.0 to 7.8 and IgG2 at pH 5.0. Two major peaks were associated with IgG1 activity indicating heterogeneity of binding to protein A-Sepharose. One peak was found for IgG2. The molecular weights of the fractions were determined to be that of IgG by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Blood from bovine leukemia virus-infected cattle: antigen production correlated with infectivity.

Leukocytes from 1 ml of blood from cattle seropositive to the bovine leukemia virus were cultured for 3 days and then tested by radioimmunoassay for antigen production. Infectivity of blood from each animal was also tested by calf inoculation and subsequent serologic detection of bovine leukemia virus transmission by agar-gel immunodiffusion. In a preliminary experiment, blood from each of 3 antigen-positive cattle was inoculated intracutaneously into 2 calves in volumes of 20 or 100 microliter. Blood from each of 4 antigen-negative cattle was similarly inoculated into 3 calves in volumes of 20, 100, or 500 microliter. At the termination of the experiment (8 weeks after inoculation), all 6 calves given blood from antigen-positive cattle had seroconverted, and 11 of 12 calves given blood from antigen-negative cattle had seroconverted. In a 2nd experiment, blood from each of 2 antigen-positive and 2 antigen-negative cattle was inoculated into pairs of calves in volumes of 1, 10, or 20 microliter. At the end of the experiment (12 weeks after inoculation), all calves inoculated with blood from antigen-positive cattle had seroconverted, but only 6 of 12 calves that had been given blood from antigen-negative cattle had seroconverted. The relative infectivity of blood was best illustrated by comparing results from the 1-microliter inoculations. At that volume, the 4 calves given blood from antigen-positive cattle were infected, whereas none of 4 calves given blood from antigen-negative cattle was infected.

Expression of type 1 pili by Escherichia coli strains of high and low virulence in the intestinal tract of gnotobiotic turkeys.

Highly virulent (strain 1) and weakly virulent (strain 3) Escherichia coli were examined using immunofluorescent and electron microscopic techniques to determine their ability to express type 1 pili in the intestinal tract of 3-week-old gnotobiotic turkeys. Turkeys were necropsied on postinoculation day (PID) 1, 2, 5, 8, and 12. Nonpiliated forms of strains 1 and 3 were more numerous than piliated forms in cecal and colonic contents examined by negative staining electron microscopy. A piliated form of strain 1 was seen in intestinal contents on each PID and was more numerous in cecal contents than in colonic contents. The mucus blanket of the cecum and colon contained large numbers of bacteria, although organisms were rarely intimately associated with the intestinal epithelium. Immunofluorescent staining indicated large numbers of piliated forms of strains 1 and 3 within the mucus blanket of the cecum and colon on PID 2, 5, 8, and 12. Piliated bacteria were infrequently seen in the ileal mucus blanket. Serum antibody titers to type 1 pili increased markedly by PID 5 and persisted in turkeys inoculated with strain 1. In contrast, antibody titers in turkeys exposed to strain 3 increased gradually and varied markedly among birds at each PID. Type 1 pili may not be important for adherence of pathogenic E coli to intestinal epithelium of turkeys.