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Biomedical subjects

W W Marquardt

Publications and source records attributed to W W Marquardt.

At least 19 recordsLinked to original sources
PubMed1996

Effect of staphylococcal beta toxin on the cytotoxicity, proliferation and adherence of Staphylococcus aureus to bovine mammary epithelial cells.

The effect of staphylococcal beta toxin on the cytotoxicity, proliferation and adherence of S. aureus. to bovine mammary epithelial cells was studied. Bovine erythrocytes and mammary epithelial cells were incubated with purified staphylococcal alpha and beta toxins and with culture supernatants from S. aureus M60 and two mutant strain that are negative for either the production of alpha (DU5789 alpha-) or beta (DU5846 beta-) toxin. Lysis of bovine erythrocytes was due primarily to beta toxin. Alpha toxin increased the lysis of bovine erythrocytes by purified beta toxin, but the presence of alpha toxin in culture supernatants from S. aureus did not increase the lysis of bovine erythrocytes. Purified beta toxin was cytotoxic to mammary secretory epithelial cells, but to a lesser extent than alpha toxin. Together they exhibited an additive effect on mammary epithelial cells. Inactivation of the alpha toxin-gene of S. aureus M60 decreased the cytotoxic effect on mammary epithelial cells to a greater extent than the inactivation of the beta toxin-gene. Also, the relative percentages of DU5789 alpha- and DU5846 beta- adhering to mammary cell monolayers, the number and size of colonies and the number of infected epithelial cells decreased. This in vitro study showed that beta toxin damages bovine mammary secretory epithelial cells, increased the damaging effects of alpha toxin, increases the adherence of S. aureus to mammary epithelial cells and increases the proliferation of S. aureus.

Animals↗
PubMed1996

Role of milk fractions, serum, and divalent cations in protection of mammary epithelial cells of cows against damage by Staphylococcus aureus toxins.

OBJECTIVE: To determine the effect of milk and blood serum constituents on cytotoxicity of Staphylococcus aureus on mammary epithelial cells. DESIGN: In vitro incubation of cells with cytotoxic agents and milk and serum constituents. SAMPLE POPULATION: Mammary cells, milk, and blood obtained from 3 cows. PROCEDURE: Staphylococcal alpha-toxin and culture supernatants from S aureus M60 and an alpha-toxin-negative mutant of M60 were incubated with bovine mammary epithelial cells in the presence of milk fractions, serum, and divalent cations. Propidium iodide fluorescence was used as a measure of cell damage. RESULTS: Skim milk and milk whey inhibited S aureus cytotoxic agents. Skim milk protected against alpha-toxin damage to a greater extent than milk whey. Serum from an adult animal was more protective than was fetal serum. Milk fat and serum albumin had no protective effect. Divalent calcium and Mg2+ were more effective inhibitors of mammary epithelial cell damage caused by alpha-toxin than of damage attributable to M60 culture supernatant. Divalent calcium and Mg2+ at concentrations similar to those of free Ca2+ and Mg2+ in normal bovine milk decreased cytotoxic damage attributable to alpha-toxin. However, concentrations similar to those of total Ca2+ and Mg2+ in normal milk were required to decrease cell damage caused by M60 culture supernatant. The alpha-toxin-negative mutant was less cytotoxic than the M60 parent strain. CONCLUSIONS: Casein, as well as Ca2+ and Mg2+ in bovine milk, inhibit the cytotoxic effect of S aureus on mammary epithelial cells.

Animals↗
PubMed1995

Effect of alpha-toxin and capsular exopolysaccharide on the adherence of Staphylococcus aureus to cultured teat, ductal and secretory mammary epithelial cells.

Cultures of teat, ductal and secretory epithelial cells were used to study the role of alpha-toxin and the capsular exopolysaccharide on the adherence of Staphylococcus aureus to mammary epithelium. The adherence of S aureus to the cells and their susceptibility to damage by alpha-toxin increased from teat to ductal to secretory cells. Alpha-toxin increased the susceptibility of epithelial cell monolayers to adherence by S aureus, and the extent of the adherence increased with the time of exposure to alpha-toxin. The exopolysaccharide capsule deterred the adherence of S aureus to mammary epithelial cells and to collagen. Organisms with a rigid capsule adhered to a smaller extent than those with a flaccid capsule. Both encapsulated and unencapsulated S aureus adhered more readily to collagen than to either healthy monolayers of epithelial cells or monolayers of cells damaged by alpha-toxin.

Animals↗
PubMed1995

Complement component C3b and immunoglobulin Fc receptors on neutrophils from calves with leukocyte adhesion deficiency.

Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence-related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (LAD) on ligand binding and receptor expression. We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from LAD, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (CL) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of CL was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1. A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from LAD-affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and LAD-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between LAD-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from LAD-affected calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1994

Adherence of Staphylococcus aureus to cultured bovine mammary epithelial cells.

Bovine mammary secretory cells, isolated at necropsy, were cultured in vitro and used as a model to study the mode of adherence of Staphylococcus aureus to mammary epithelium. Cultured cells were characterized by their morphology and physiology as secretory epithelial cells. Cells showed characteristic growth patterns when grown on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma. Cells cultured on collagen formed confluent monolayers and were the most suitable for bacterial adherence studies. Cultured cells stained intensely for cytokeratin and for specific milk proteins, i.e., alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin. The effect of frozen storage for 10 mo on cell viability or presence of milk proteins was minimal. Staphylococcus aureus showed large affinity for extracellular matrix components, i.e., fibronectin, laminin, and collagen. Adherence to confluent cell monolayers was minimal. In preconfluent cell monolayers, most S. aureus adhered more readily to the exposed matrix than to the epithelial cells. Overnight exposure to staphylococcal alpha-toxin greatly increased adherence of S. aureus to confluent monolayers. However, whether bacteria adhered to alpha-toxin damaged cells or to exposed matrix is not clear. Unencapsulated S. aureus adhered in larger numbers than did encapsulated S. aureus.

Animals↗
PubMed1994

Modulation of Fc receptors for IgG on bovine polymorphonuclear neutrophils by interferon-gamma through de novo RNA transcription and protein synthesis.

Polymorphonuclear neutrophils (PMN) from 4 cows were preincubated (30 minutes, 37 C) in either actinomycin D (100 micrograms/ml) or puromycin (10 micrograms/ml), inhibitors of mRNA transcription and protein translation, or in medium 199. The PMN were incubated for a further 4.5 hours in medium containing 100 U of recombinant bovine interferon-gamma (rboIfn-gamma). The PMN were then incubated with bovine IgG1, IgG2, IgM, or aggregated IgG (aIgG; 4 C, 12 hours) for flow cytometric analysis, using fluoresceinated isotype-specific antibody. The percentage of PMN binding the ligand and the logarithmic mean fluorescent channel (LMFC), an indicator of the amount of receptor (R) expression, were recorded. Competitive inhibition of ligand binding was measured by incubating PMN with fluoresceinated IgG2 in the presence or absence of 100-fold excess of IgG1, IgG2, and aIgG. Activation with rboIfn-gamma induced a 4.5-fold increase in binding of IgG1 and a fivefold increase in LMFC for IgG2. These increases were inhibited by actinomycin D and puromycin. Percentage of PMN binding aIgG decreased after activation by rboIfn-gamma. Interferon-gamma treatment did not affect binding or LMFC of IgM. However, binding of IgM was reduced by treatment with actinomycin D. Binding of fluoresceinated IgG2 was inhibited by unlabeled IgG1, IgG2, and aIgG. Results indicate that bovine PMN Fc receptors (FcR) for IgG1 and IgG2 were rboIfn-gamma inducible, that induction required de novo transcription and translation, that a heterogeneous population of FcR exist on bovine PMN, and that IgG1 and IgG2 share a common FcR. Further, bovine PMN are capable of gene activation and are responsive to changes in their environment, thus being amenable to modulation for effective pathogen destruction.

Animals↗
PubMed1994

Bovine mammary teat and ductal epithelial cell cultures.

Bovine mammary epithelial cells from teat and ductal tissue were isolated at necropsy and were grown in culture. Cells were characterized by the presence of cytokeratin filaments, cell morphologic features, synthesis of milk proteins, esterase activity, DNA content, and growth patterns on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma. Cultured teat and ductal cells stained intensely for cytokeratin and had similar morphologic features. Both cell types synthesized alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin to variable degrees. Cell type and culture conditions did not affect the DNA content of the cells, as indicated by similar amounts of DNA in G0G1 and G2M phases of the mitotic cycle in cultured cells and in cells from freshly isolated mammary explants. Cells cultured on polystyrene, fibronectin, laminin, and collagen formed pavement-like cell monolayers suitable for cytotoxicity and bacterial adherence studies. Cells cultured on the reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma formed three-dimensional structures closely resembling lactiferous ducts and alveoli, which could be used for studying lactogenesis and galactopoiesis. Freshly isolated cells and cultured cells were stored at -70 C or in liquid nitrogen. The latter storage method affected the cells less than did freezing at -70 C.

Animals↗
PubMed1988

Serologic incidence of Cryptosporidium in Delmarva broiler flocks.

Banked serum samples collected from an industry-wide serologic survey of eighteen Delmarva broiler flocks conducted in 1985 were evaluated for the presence of Cryptosporidium- specific antibodies in an indirect enzyme-linked immunosorbent assay (ELISA). Evaluation of sera collected at 49-days posthatch showed that 38% of the broiler flocks were serologically positive, whereas 50% of samples obtained from flocks at 63-days during an extended growout were positive. From a performance standpoint, the top nine flocks in the study were essentially negative at the 49-day sampling. In order to more firmly establish the serologic prevalence of Cryptosporidium in Delmarva broiler flocks, a second serologic survey was conducted early in 1987. In this study, an estimated 22% of the 454 broiler flocks sampled at approximately 49 days of age were positive for Cryptosporidium. The negative Cryptosporidium serology in the top 25% of these flocks was associated with better performance, but positive Cryptosporidium serology was not clearly correlated with poor performance. Remarkable differences between Cryptosporidium serologic exposure rates of the growout companies were observed, with some companies having exposure rates as high as 40%, and others rates of less than 3%.

Animals↗
PubMed1985

An enzyme-linked immunosorbent assay method for the simultaneous measurement of antibody titer to multiple viral, bacterial or protein antigens.

A model enzyme-linked immunosorbent assay (ELISA) system was developed which permits the user to evaluate replicate single sera dilutions of 46 test and control serum samples for the presence of specific antibody against up to eight different antigens in one assay. The system makes use of a transplanting device for the simultaneous transfer of aliquots of independently diluted replicate test samples along with conjugate and substrate from a serum reservoir plate or from a reagent reservoir onto different antigen coated target plates. Kinetically read, raw absorbance data from tests are transmitted to a microcomputer, where absorbance values from all tests are quickly reduced to predicted titer levels by computer analysis. All titer computations involve the use of a single prescribed standard curve for predicting antibody titer against the different antigens. Between assay repeatability of this procedure is high and its potential for replacing a number of different conventional assays for epidemiological studies has been evaluated.

Animals↗
PubMed1985

Presumptive diagnosis of subclinical infections utilizing computer-assisted analysis of sequential enzyme-linked immunosorbent assays against multiple antigens.

One-hundred-seventy-two serum samples, collected sequentially from four flocks of egg- and meat-type chickens, were evaluated for antibodies to multiple infectious agents by enzyme-linked immunosorbent assay (MELISA). The MELISA system used provided simultaneous measurement of antibody titers against avian infectious bronchitis (IB), infectious bursal disease (BD), Newcastle disease, avian encephalomyelitis and reovirus infections, and Mycoplasma gallisepticum. The use of computer-generated graphic print outs of relative MELISA titers provided immediate visulization of over 740 data points and convenient detection of any temporal changes in median titer class (MTC). The temporally changing MTC, or flock profiles obtained, indicated that negligible or waning IB immunity may be a common occurrence in previously vaccinated commercial chickens. These profiles further suggested that, despite no IB revaccination, these same flocks experienced episodes of reexposure to IB which otherwise may have been difficult to detect by conventional clinical or diagnostic laboratory protocols. MELISA profiles and sequential histologic examinations of bursas of Fabricius also provided evidence of a possible BD vaccination problem in young chickens that also experienced excessive losses from coccidiosis, ulcerative enteritis, and Marek's disease. Short sampling intervals were found to foster the detection and definition of fluctuations in MTC which otherwise may have been missed.

Animals↗
PubMed1978

Immune electron microscopy of avian infectious bronchitis virus serotypes.

An immune electron microscopy agglutination technique in which emphasis is placed upon the importance of antigen-antibody equivalence has been developed as a possible method for the serotyping of avian infectious bronchitis viruses. The Connecticut and Massachusetts 41 serotypes were used as a model system. Stock virus concentrations were standardized by physical particle counts of virions sedimented directly onto electron microscope specimen grids. Suspensions containing approximately 150 virions per grid square were allowed to react with dilutions of homologous and heterologous antisera. Virions in these constant virus-variable serum mixtures were sedimented directly onto electron microscope specimen grids, and the relative degree of aggregation per grid was determined from the mean percent aggregation of five randomly selected grid squares. In homologous assays, regions of relative antibody excess, of equivalence, and of relative antigen excess were clearly evident. At equivalence, the mean percent aggregation was significantly higher than in the regions of relative antibody or antigen excess. In the heterologous systems, the degree of aggregation differed little from that of the virus controls containing no antiserum.

Agglutination Tests↗
PubMed1972

Radial immunodiffusion: a simple and rapid method for detection of Marek's disease antigen(s).

A qualitative radial immunodiffusion technique is described which detects antigen(s) in feathers from live or dead chickens infected with Marek's disease herpesvirus. Antiserum, which is incorporated into a support medium, reacts with antigen(s) in the feather tip producing a radial precipitin ring. Antigen(s) was detected in 93.3% of experimentally inoculated chickens 21 days postinoculation and in 100% of infected birds subsequently tested through 6 weeks. No antigen was detectable in the feathers of uninoculated control chickens. The technique is simple and rapid to perform. Positive tests could be detected after 1 to 2 hours of incubation. Antigen detection by the radial immunodiffusion test correlated well with other criteria of infection. This technique should have application as a laboratory research tool and as an adjunct for a rapid flock diagnosis of Marek's disease.

Animals↗
PubMedDate not supplied

The neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant-virus variable-serum method in chicken tracheal cultures.

The constant-virus variable-serum neutralization test in chicken tracheal organ cultures was employed in cross-neutralization tests with the following infectious bronchitis viruses: Massachusetts 41, Connecticut, Iowa 97, Iowa 609, Holte, JMK, Clark 333, SE 17, Florida, and Arkansas 99. These viruses all proved serologically distinct by this method. Chickens immunized with the Connecticut strain and later challenged with Arkansas 99 were not protected against it.

Animals↗
PubMedDate not supplied

New strains of infectious bronchitis virus isolated in Maine.

Twelve IBV isolants from diseased chickens in Maine were studied by serum-neutralization tests in chicken tracheal culture. Six isolants were neutralized by Mass-type antisera. The serum-neutralizing characteristics of six other isolants from vaccine breaks differed from any of the standard IBV viruses and appear to comprise three new strains, Maine 209, 212,and 246. The data provide additional evidence that strains of IBV responsible for disease probably vary from region to region and that the strains present must be known for a regional vaccination program to be effective.

Animals↗
PubMedDate not supplied

Strains of infectious bronchitis virus on the Delmarva peninsula and in Arkansas.

The constant-virus variable-serum neutralization test in chicken tracheal cultures was used to serotype infectious bronchitis virus (IBV) isolants from the Delmarva peninsula and Arkansas. Of 106 isolants obtained from Delmarva in 1971-1974, 93 were found to be the JMK strain, 5 Massachusetts (Mass), 5 Connecticut (Conn), 2 a new strain designated Maryland (Md) 27, and 1 a new strain designated Md 31. Of 26 isolants obtained from Arkansas between October, 1971, and February, 1973, 17 were Arkansas (Ark) 99, 4 Mass, 1 Conn, and 1 an additional new strain, designated Ark 155. Antisera produced against 2 more isolants each neutralized both Ark 99 and Mass viruses. Antiserum against another isolant neutralized both Ark 155 and Conn viruses. The neutralizing characteristics of the new strains are described.

Animals↗
PubMedDate not supplied

Isolation of a monoclonal antibody with specificity for commonly employed vaccine strains of Newcastle disease virus.

A monoclonal antibody, AVS-I, was produced from a hybridization of murine myeloma cells and splenocytes from mice immunized with the La Sota strain of Newcastle disease virus (NDV). The hybridoma producing AVS-I, selected from 184 NDV-positive supernatants, is one of two supernatants that reacted exclusively with lentogenic strains in an indirect enzyme-linked immunosorbent assay. AVS-I can also be assayed by hemagglutination-inhibition (HI), which was used to test selected reference avian paramyxovirus (PMV) strains of types 1 to 3. NDV vaccines La Sota and B1 and field isolates from chickens, turkeys, pigeons, and cockatoos were also used as antigens. AVS-I had a high binding affinity for all La Sota and B1 strains, including vaccines. The antibody bound with a lower titer to the Australian Queensland V4 and Ulster strains, but it did not bind to the F strain, a lentogenic strain from England. AVS-I was HI-negative against the other PMV reference strains. AVS-I may be valuable for identifying field isolates antigenically similar to La Sota and B1 and rapidly differentiate those vaccine strains from more virulent viruses.

Antibodies, Monoclonal↗
PubMedDate not supplied

An immunoperoxidase monoclonal antibody stain for rapid diagnosis of infectious bursal disease.

Cell smears of chicken-embryo-fibroblast (CEF) cultures and bursa of Fabricius from chickens experimentally infected with six different strains of infectious bursal disease virus (IBDV) were examined for the presence of IBDV by the avidin-biotin-peroxidase complex method of immunoperoxidase (IP) staining using a monoclonal antibody specific for IBDV designated BK70. IBDV of different strains and serotypes were readily detected by the IP method in cell smears prepared from infected CEF cultures and from bursas. Bursal cells were positive for IP stain in most of the infected bursas (87.5%), despite their mild IBD lesions. Positive IP staining of bursal smears was well correlated with the recovery of IBDV from the bursas and with IBD lesions in the bursas. IP stain with a monoclonal antibody (BK70) appeared potentially useful for rapid and definitive diagnosis of IBD.

Animals↗