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D A Mosier

Publications and source records attributed to D A Mosier.

At least 19 recordsLinked to original sources
PubMed2001

MHCII, Tlr4 and Nramp1 genes control host pulmonary resistance against the opportunistic bacterium Pasteurella pneumotropica.

MHCII, Tlr4, and Nramp1 genes are each independently important in pulmonary immunity. To determine the effect of these genes on host resistance, mice carrying various combinations of functional alleles for these three genes were experimentally challenged with the opportunistic bacterium, Pasteurella pneumotropica. MHCII-/-, Tlr4d/d, and Nramp1s/s mice were significantly more susceptible to experimental infections by P. pneumotropica after intranasal challenge compared to mice carrying functional alleles at only one of those genes. P. pneumotropica were cultured from the lungs of challenged mice, and the severity of the pneumonia strongly correlated with the number of isolated bacteria. Mice with the genotype MHCII-/- Tlr4n/n genotype were less susceptible to pneumonia than MHCII+/+, Tlr4d/d mice. It is interesting that the Nramp1 gene contribution to host resistance was apparent only in the absence of functional MHCII or Tlr4 genes. These data suggest that MHCII, Tlr4, and Nramp1 genes are important to pulmonary bacterial resistance.

Alleles↗
PubMed2000

FnrP interactions with the Pasteurella haemolytica leukotoxin promoter.

Pasteurella haemolytica FnrP is homologous to Fnr, the global transcriptional regulator of anaerobic respiration in Escherichia coli. To investigate the role of O(2) in the expression of P. haemolytica leukotoxin, we tested a lktC::lacZ fusion constructed in E. coli for a FnrP-mediated regulatory effect under aerobic and anaerobic growth conditions. Both E. coli Fnr and FnrP suppressed leukotoxin transcription under aerobic conditions. Under anaerobic conditions, Fnr suppressed transcription, while FnrP increased transcription. These results were confirmed using FnrP*, a mutant form of FnrP that activates anaerobically inducible genes under aerobic conditions. In mobility shift assays, partially purified FnrP bound to a potential regulatory site in a P. haemolytica lktC promoter fragment.

Aerobiosis↗
PubMed2000

Cryptosporidiosis. A global challenge.

During the last 30 years, our concept of cryptosporidiosis has changed from that of a rare, largely asymptomatic disease, to an important cause of diarrhea in animals and humans worldwide. Significant disease first appeared in cattle. Subsequently, the zoonotic danger of the organism was recognized in HIV-infected persons and young children. Cryptosporidium are now ubiquitous and disease has been described in over 79 host species. Cryptosporidiosis has become a major cause of calfhood diarrhea worldwide. In humans it accounts for up to 20% of all cases of childhood diarrhea in developing countries and is a potentially fatal complication of AIDS. Waterborne contamination is a growing concern as a source of widespread outbreaks of disease. Factors that have contributed to the emergence of cryptosporidiosis in animals include biological features of the organism, the lack of an effective treatment or preventative, increased environmental contamination, and trends in livestock production. In humans the zoonotic nature of infection and an increased at-risk population have contributed to disease. Genetic characterization of Cryptosporidium, improved detection methods, and a better understanding of the factors that predispose to disease are important contributions to understanding the epidemiology of cryptosporidiosis.

AIDS-Related Opportunistic Infections↗
PubMed1999

Actin polymerization enhances Pasteurella haemolytica leukotoxicity.

Pasteurella haemolytica leukotoxin is cytotoxic to bovine leukocytes, causing increased cell membrane permeability, osmotic swelling, release of cytosolic proteins and cell lysis. These studies were designed to test if leukotoxin causes release of the cytoskeletal protein, actin, from bovine leukemia cells and if purified actin-influenced bacterial growth or leukotoxin production. Culture supernatants caused a 7-fold decrease in viability of bovine leukemia cells and increased cell permeability that was accompanied by release of beta-actin into the cell culture supernatant. Exposing P. haemolytica to purified actin solutions induced the conversion of monomeric G-actin to polymerized F-actin. This conversion was partially inhibited by bovine P. haemolytica immune, but not pre-immune, serum. Loss of streptomycin resistance following treatment of the organism with acridine orange ablated the polymerizing activity. Incubation of P. haemolytica in the presence of purified F-actin did not affect growth but resulted in culture supernatant that had 3.0-3.9-fold greater leukotoxicity compared to medium alone or medium containing G-actin, heat-denatured actin or albumin. The effect of actin on leukotoxicity was concentration-dependent and directly associated with increases in secreted leukotoxin. The interaction between P. haemolytica and actin is potentially detrimental to the host by inducing polymerization of actin into insoluble filaments and by enhancing leukotoxicity.

Actins↗
PubMed1999

Cloning and characterization of the gene encoding Pasteurella haemolytica FnrP, a regulator of the Escherichia coli silent hemolysin sheA.

A Pasteurella haemolytica A1 gene was identified from a recombinant library clone that expressed hemolysis in host Escherichia coli cells. The gene, designated fnrP, had sequence identity to E. coli fnr, a global transcriptional regulator of genes required for conversion to anaerobic growth. FnrP complemented anaerobic deficiencies of a fnr-null mutant strain of E. coli and increased expression of the Fnr-dependent, anaerobic terminal reductase gene, frdA. FnrP was purified, identified by immunoblotting, and shown to be nonhemolytic. When FnrP was expressed in E. coli DeltasheA, a null mutant of the cryptic hemolysin SheA, the transformants were nonhemolytic, indicating that FnrP activates this silent hemolysin.

Anaerobiosis↗
PubMed1998

Actin enhances the haemolytic activity of Escherichia coli.

Actin is a major cytoskeletal protein of mammalian muscle and non-muscle cells. Exposure of cells to soluble factors that damage cell membranes results in the release of actin into the extracellular spaces. The alpha-haemolysin (HlyA) of Escherichia coli is the prototype RTX (repeat in toxin) toxin and is thought to be important in virulence because of its ability to lyse cells by formation of pores in the cell membrane. These studies were conducted to determine if actin influences growth and haemolytic activity of E. coli. Growth of E. coli in the presence of actin resulted in culture supernatant haemolytic activity that was 2.4-, 2.7- and 3.3-fold greater than that of E. coli grown in medium containing BSA, non-supplemented medium, or medium containing heat-denatured actin, respectively. The enhanced haemolytic activity occurred only when actin was present during the growth phase and there was no effect when actin was added to culture supernatants containing haemolysin. The increased haemolytic activity by actin was concentration-dependent, detectable in early-exponential-phase growth, and associated with increased concentrations of secreted HlyA by Western blotting. Actin induced a 2.9-fold increase in alkaline phosphatase activity in E. coli CC118 with a TnphoA insertion in the hlyB determinant of the recombinant haemolysin plasmid pWAM04. These results indicate that extracellular actin enhances haemolysin production by E. coli and may have implications in the pathogenesis of E. coli infections.

Actins↗
PubMed1998

In vivo effect of Pasteurella haemolytica infection on bovine neutrophil morphology.

OBJECTIVE: To determine whether characteristic changes in neutrophil morphology caused in vitro by Pasteurella haemolytica leukotoxin (LKT) can be observed in vivo by electron microscopic examination of infected tissue chamber fluids and pneumonic lungs. ANIMALS: 7 mixed-breed beef calves. PROCEDURE: Tissue chambers were implanted subcutaneously in 3 calves and were inoculated with P haemolytica or phosphate-buffered saline solution. Chamber fluid samples, obtained at 8 and 32 hours after inoculation, were examined, using electron microscopy. Experimental pneumonia was induced in an additional 4 calves by transthoracic inoculation with P haemolytica. These calves were euthanatized at 6, 12, 24, and 36 hours after inoculation and lung sections were examined, using transmission electron microscopy. RESULTS: On examination, using transmission electron microscopy, neutrophils in lung sections and tissue chamber fluids had cytoplasmic and nuclear changes indicative of irreversible cell injury, including cell swelling, loss of plasma membrane ruffling, mitochondrial swelling, autolytic vacuolation, disruption of plasma membrane, nuclear pyknosis, karyolysis, and karyorrhexis. On examination, using scanning electron microscopy, leukocytes obtained from tissue chambers did not have their typical convoluted surfaces, but appeared rounded and swollen or shrunken with pitted surfaces. CONCLUSIONS: Pasteurella haemolytica-induced changes in neutrophil morphology in vivo were similar to those previously induced by in vitro exposure of neutrophils to LKT. Changes were suggestive of injury initiated by damage to the plasma membrane, which is consistent with the mechanism of action of pore-forming cytolysins. CLINICAL RELEVANCE: Pasteurella haemolytica LKT appears to be an important virulence factor in vivo; a fact that should be addressed in the development of vaccines.

Animals↗
PubMed1998

Comparison of serologic and protective responses induced by two Pasteurella vaccines.

Vaccine development for the prevention of pneumonic pasteurellosis remains a critical issue for the feedlot industry. Most currently available Pasteurella vaccines are formulated to stimulate immunity by either providing an adequate antigenic mass in the administered dose, or by relying on subsequent production of antigens by in vivo growth of live organisms. The ability of these different types of vaccines to stimulate rapid and high titres to key antigens is a key factor that will influence subsequent resistance to disease. The serologic and protective responses to a streptomycin-dependent, modified-live vaccine and a killed (bacterin-toxoid) vaccine against experimental pneumonic pasteurellosis were compared. Calves were vaccinated with a single injection of either a test vaccine or phosphate-buffered saline, challenged 14 d later by transthoracic injection with Pasteurella haemolytica, and euthanized 3 d post-challenge to evaluate the severity of pneumonia. On days 0, 7, and 14, serologic responses to various P. haemolytica antigens, including cell-associated and soluble antigens, were determined by enzyme-linked immunosorbent assays, and anti-leukotoxin antibody levels were determined by leukotoxin neutralization. The bacterin-toxoid elicited significantly greater serologic responses compared to controls for all antigens. The modified-live vaccine elicited a significantly greater response compared to controls for a whole-cell antigen preparation. Lesion scores were significantly smaller (greater protection) in calves that received the bacterin-toxoid, but not the modified-live vaccine, compared to controls.

Animals↗
PubMed1997

Experimental cryptosporidiosis in adult and neonatal rabbits.

Neonatal and adult New Zealand White rabbits were infected experimentally with Cryptosporidium parvum. No histologic evidence of infection was found in adult rabbits. However, increased levels of anti-cryptosporidial serum IgG were present, and multiple antigens were detected by serum on immunoblots. In neonates, variably severe, transient infection was present from Days 3 to 21 postinoculation. Serum IgG was initially elevated, decreased until Day 10 postinoculation, then progressively increased for the remainder of the study. A prominent 15 kilodalton antigen was detected on immunoblots using serum obtained on Days 14 until 28 postinoculation. Neonate anti-cryptosporidial fecal IgA were slightly elevated on Days 14 and 21 postinoculation.

Aging↗
PubMed1997

Bacterial pneumonia.

Bacteria play a critical role in the severe pneumonia and fatalities associated with the bovine respiratory disease complex. Although numerous bacteria have the potential to cause pneumonia, only a small number of these are responsible for the majority of cases of disease. Virulence and immunogenic characteristics of these organisms are important determinants of the host response to infection. These bacterial characteristics are reviewed and applied to a discussion of the epidemiology, pathogenesis, and prevention of bacterial pneumonia is also discussed.

Animals↗
PubMed1996

Differentiation of Cryptosporidium parvum, C. muris, and C. baileyi by PCR-RFLP analysis of the 18S rRNA gene.

We have developed a combined polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay for the detection and differentiation of Cryptosporidium parvum, Cryptosporidium muris, and Cryptosporidium baileyi. The assay utilizes PCR to amplify an approximately 1750 basepair product of the Cryptosporidium 18S rRNA gene. Following double digestion with restriction endonucleases Dral and Vsp1, this PCR product yields DNA fragments that can be resolved electrophoretically into RFLP profiles that are distinct for C. parvum, C. muris, and C. baileyi. Previous PCR-restriction analyses could not simultaneously differentiate all three species. Future application of this technique could include predicting the disease-causing potential of oocyst-contaminated environmental specimens and helping to determine the source of oocyst contamination.

Animals↗
PubMed1996

Simplified method for recovery and PCR detection of Cryptosporidium DNA from bovine feces.

An assay to identify Cryptosporidium DNA in bovine feces has been developed emphasizing standardization of sample preparation and simplification of the DNA recovery process for PCR amplification and DNA hybridization detection. The Cryptosporidium DNA recovery-PCR detection procedure (CR-PCR) can recover DNA suitable for PCR amplification without using or generating hazardous chemicals or wastes. In comparisons with a commercial enzyme-linked immunoassay (Color Vue-Cryptosporidium; Seradyn, Indianapolis, Ind.), the CR-PCR could detect 10(3) to 10(4) times fewer purified oocysts diluted in solution (water or buffered saline) and 10(2) times fewer oocysts from diarrheic feces and showed earlier detectability from solid, nondiarrheic feces in an experimental infection. This assay may prove useful for detecting Cryptosporidium oocysts in feces and in clarifying the role of livestock in waterborne outbreaks of cryptosporidiosis.

Animals↗
PubMed1995

Enhanced karyotype resolution of Cryptosporidium parvum by contour-clamped homogeneous electric fields.

Previous karyotype analysis with contour-clamped homogeneous electric fields (CHEF) indicated that the Cryptosporidium parvum karyotype comprises a minimum of five chromosomes all less than 1.6 Mb in size. In this study we were able to improve the resolution of the karyotype using a modified CHEF gel electrophoretic procedure that allows for the routine identification of seven C. parvum chromosomes ranging in size from about 0.945 to 2.2 Mb. Our data also suggest that an eighth chromosome is present and comigrates with another chromosome at about 1.3 Mb.

Animals↗
PubMed1995

A Cryptosporidium parvum genomic region encoding hemolytic activity.

Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidial sporozoites invade intestinal epithelial cells and subsequently develop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolytic activity was used as a marker to screen a C. parvum genomic expression library. A stable hemolytic clone (H4) containing a 5.5-kb cryptosporidial genomic fragment was identified. The hemolytic activity encoded on H4 was mapped to a 1-kb region that contained a complete 690-bp open reading frame (hemA) ending in a common stop codon. A 21-kDa plasmid-encoded recombinant protein was expressed in maxicells containing H4. Subclones of H4 which contained only a portion of hemA did not induce hemolysis on blood agar or promote expression of the recombinant protein in maxicells. Reverse transcriptase-mediated PCR analysis of total RNA isolated from excysted sporozoites and the intestines of infected adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle.

Amino Acid Sequence↗
PubMed1995

Enteral human serum immunoglobulin treatment of cryptosporidiosis in mice with severe combined immunodeficiency.

The anti-cryptosporidial immunoglobulin G antibodies in two commercially available human serum immunoglobulin (HSIG) products were quantified and characterized. The mean level of Cryptosporidium parvum-specific immunoglobulin G in HSIG was eightfold higher than the antibody level found in the sera of three immunocompetent individuals convalescing from cryptosporidiosis. However, HSIG products displayed no reactivity to cryptosporidial antigens in immunoblot analyses, while convalescent-phase sera demonstrated characteristic banding patterns. When HSIG was given to newborn severe combined immunodeficiency (scid) mice before and shortly after experimental infection, a decreased intensity of infection was observed in the intestines of the mice compared with that of control mice. However, there was no difference in mortality or histopathologic findings in the intestines of HSIG-treated and control mice when treatment was not started until 22 days of age. These results indicate that HSIG may be beneficial when given prophylactically; however, HSIG cannot eradicate cryptosporidia from mucosal surfaces in an established infection.

Adult↗
PubMed1995

Lectin histochemistry of normal and herpesvirus-infected bovine nasal mucosa.

Proliferation of Pasteurella haemolytica serotype 1 in the nasal cavity following stress or viral infection is an important event in the pathogenesis of bovine pneumonic pasteurellosis. Enhanced adhesion of P. haemolytica to nasal mucosa could be one factor that predisposes animals to this proliferation. Nasal mucosa from normal and bovine herpesvirus-1 (BHV1)-infected cattle were examined histochemically for their glycoconjugate composition. Twenty lectins were screened, six of which were chosen for subsequent study. Three of these were specific for N-acetylgalactosamine (NAGal) (Dolichos biflorus, Glycine max, and Vicia villosa), and one each was specific for N-acetylgalactosamine/galactose (Griffonia simplicifolia-I), mannose/glucose (Canavalia ensiformis), and N-acetylglucosamine (Triticum vulgaris). For the surface mucosa and submucosal glands, there was greater reactivity in samples from BHV1-infected than from normal cattle for all six lectins. Reactivity was most prominent for the NAGal-specific lectins. Neuraminidase treatment of samples from normal and BHV1-infected cattle tended to result in greater lectin reactivity. Lectin reactivity was generally more intense in focally inflamed areas, but diffuse reactivity was not substantially affected by inflammation. BHV1-induced alteration of nasal mucosal glycoconjugates could enhance adhesion and colonization of P. haemolytica to nasal surfaces and may be one factor responsible for the increased number of P. haemolytica serotype 1 in the nasal cavity following viral infection.

Animals↗
PubMed1995

Pasteurella haemolytica lipopolysaccharide-induced cytotoxicity in bovine pulmonary artery endothelial monolayers: inhibition by indomethacin.

Exposure of bovine pulmonary artery endothelial cells to Pasteurella haemolytica lipopolysaccharide caused severe morphologic changes. Initially, there was dilatation of the rough endoplasmic reticulum and mitochondrial swelling followed by cell retraction, membrane bleb formation, and cell detachment. The affected endothelial cells had severe membrane damage resulting in the leakage of lactate dehydrogenase. Indomethacin in concentrations of 0.5 mM or greater caused marked decreases in the lipopolysaccharide-induced leakage of lactate dehydrogenase. Indomethacin at 5 mM also caused a marked reduction of the lipopolysaccharide-induced morphologic changes resulting in apparent maintenance of the monolayer integrity for 8 hours versus 1 hour in the lipopolysaccharide-treated control. A marked decrease in the cell and nuclear membrane effects resulted, but the rough endoplasmic reticulum dilatation and mitochondrial changes proceeded. These results indicate that indomethacin does not prevent lipopolysaccharide binding but interferes with later events in lipopolysaccharide-induced cytotoxicity in the bovine pulmonary endothelial cell. The concentration of indomethacin required to produce this inhibition suggests that the primary mechanism is not cyclooxygenase inhibition.

Animals↗