Derek A. Mosier

The CsgA and Lpp Proteins of an Escherichia coli O157:H7 Strain Affect HEp-2 Cell Invasion, Motility, and Biofilm Formation

ABSTRACT In Escherichia coli O157:H7 strain ATCC 43895, a guanine-to-thymine transversion in the csgD promoter created strain 43895OR. Strain 43895OR produces an abundant extracellular matrix rich in curli fibers, forms biofilms on solid surfaces, invades cultured epithelial cells, and is more virulent in mice than strain 43895. In this study we compared the formic acid-soluble proteins expressed by strains 43895OR and 43895 using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and identified two differentially expressed proteins. A 17-kDa protein unique to strain 43895OR was identified from matrix-assisted laser desorption ionization-time of flight analysis combined with mass spectrometry (MS) and tandem MS (MS/MS) as the curli subunit encoded by csgA. A <10-kDa protein, more highly expressed in strain 43895, was identified as the Lpp lipoprotein. Mutants of strain 43895OR with disruption of lpp, csgA, or both lpp and csgA were created and tested for changes in phenotype and function. The results of this study show that both Lpp and CsgA contribute to the observed colony morphology, Congo red binding, motility, and biofilm formation. We also show that both CsgA and Lpp are required by strain 43895OR for the invasion of cultured HEp-2 cells. These studies suggest that in strain 43895OR, the murein lipoprotein Lpp indirectly regulates CsgA expression through the CpxAR system by a posttranscriptional mechanism.

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model.

The suitability of a rabbit seizure model for studying the neuropathogenesis of bovine herpesvirus type 5 (BHV-5) encephalitis was evaluated. Intranasal administration of BHV-5 (strain TX89) together with intramuscular administration of dexamethasone produced seizures in 70% of rabbits tested and meningo-encephalitis in 100%. Infectious BHV-5 was consistently isolated from the following sites: olfactory bulb; anterior cortex, containing the frontal cortex, olfactory tract and anterior portion of the olfactory cortex; posterior cortex, containing the temporal, parietal, piriform, entorhinal and occipital cortices; amygdala; hippocampus. Less frequently, BHV-5 was isolated from the midbrain and diencephalon, the pons and medulla, the cerebellum, and the trigeminal ganglia. Rabbits similarly infected with the Cooper strain of bovine herpesvirus type 1 showed no neurological signs or meningo-encephalitis, and virus was not recovered from the brain. The brains of BHV-5-infected rabbits showed neuronal degeneration, leptomeningitis, gliosis and perivascular cuffing, predominantly in the olfactory cortex (piriform and entorhinal cortices), amygdala and hippocampus. Mild lymphocytic meningitis was seen in the olfactory bulb and focal lymphocytic infiltration was sometimes present in the medulla and cerebellum. BHV-5, specific antigens and nucleic acids were detected in the olfactory cortex, amygdala and hippocampus by immunohistochemical methods and in-situ hybridization. The results suggested that, after intranasal BHV-5 inoculation, the virus spread to the central nervous system via the olfactory and trigeminal pathways. The olfactory pathway was more susceptible than the trigeminal pathway to neuropathogenic effects.

CRYPTOSPORIDIOSIS A GLOBAL CHALLENGE

Abstract: 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.

Serial evaluation of physiologic, pathological, and behavioral changes related to disease progression of experimentally induced Mannheimia haemolytica pneumonia in postweaned calves

OBJECTIVE To determine the usefulness of physiologic, behavioral, and pathological changes as objective indicators of early respiratory disease in calves with Mannheimia haemolytica pneumonia. ANIMALS 14 crossbred beef steers. PROCEDURES Disease was experimentally induced in healthy calves through endoscopic pulmonary inoculation of M haemolytica. Calves were necropsied on days 1, 2, 3, 5, 7, and 9 after inoculation. Physical examination variables (rectal temperature, heart rate, and respiration characteristics), clinical illness score, and degree of activity were assessed 3 times daily beginning 4 days prior to inoculation and continuing throughout the study. Twice before inoculation and on days 1, 2, 3, 5, 7, and 9, arterial blood gas measurements, serum biochemical analyses, and CBCs were performed. Pedometers and accelerometers were used to monitor cattle behavior and activity throughout the trial. RESULTS All calves became clinically ill after inoculation and had gross and histopathologic signs of bronchopneumonia. No variable was a reliable indicator of disease progression as judged by percentage of pulmonary involvement. However, activity as measured by total steps taken in a 24-hour period was lower after versus before disease induction. CONCLUSIONS AND CLINICAL RELEVANCE This single-pathogen challenge model successfully yielded clinical signs and pathological effects consistent with naturally acquired respiratory disease. Routine laboratory variables and subjective measures were not reliable indicators of lung involvement or the progression of pneumonia. However, activity, objectively measured with pedometers and accelerometers, appeared to be a promising indicator for early recognition of bovine respiratory disease.

Spread of bovine herpesvirus type 5 (BHV-5) in the rabbit brain after intranasal inoculation

Following intranasal inoculation of wild-type BHV-5 in rabbits, we studied the sequential transneuronal passage of the virus in the CNS by immunocytochemistry, histopathology, and virus isolation. At 4 and 6 days postinfection (d.p.i.), rabbits had no or mild neurological signs, and virus was isolated only from the olfactory bulbs. At 8 and 9 d.p.i., infected rabbits had severe neurological signs, and virus could be isolated from multiple regions of the brain segments. In these rabbits, high titers of virus were consistently present in the anterior and posterior cortices, including frontal, piriform/entorhinal, temporal, parietal, and occipital cortices, the hippocampus and the amygdala. Virus was isolated occasionally from the midbrain/diencephalon and pons/medulla. Virus was not isolated from the cerebellum and trigeminal ganglion of rabbits examined from 2-12 d.p.i. Immunocytochemistry revealed virus-specific antigens at 4 d.p.i. within the glomerular layer, external plexiform layer, and mitral cell layer of the main olfactory bulb. At 6 d.p.i., virus-specific antigens were also present within the inner granular layer of the main olfactory bulb. At 8 and 9 d.p.i., widespread BHV-5-specific staining occurred in the areas of the brain connected to the main olfactory bulb, including the frontal/cingulate cortex, anterior olfactory nucleus, lateral olfactory tubercle, piriform/entorhinal cortex, hippocampus, amygdala, dorsal raphe, and locus coeruleus. In the trigeminal ganglion, specific staining was detected within a few neurons at 2,4, 6, 8 d.p.i. However, further spread of the virus along the trigeminal pathway was not evident. These data indicate that BHV-5 replicates and spreads preferentially in the olfactory pathway following intranasal instillation and that this viral spread correlated with the severity of neurological symptoms and histopathological lesions.

Toll-Like Receptor 4-Positive Macrophages Protect Mice from Pasteurella pneumotropica-Induced Pneumonia

ABSTRACT This study investigates Toll-like receptor 4 (TLR4)-positive macrophages in early recognition and clearance of pulmonary bacteria. TLR4 is a trans-membrane receptor that is the primary recognition molecule for lipopolysaccharide of gram-negative bacteria. The TLR4Lps-del mouse strains C57BL10/ScN (B10) and STOCK Abbtm1TLR4Lps-delSlc11a1s(B10 × C2D) are susceptible to pulmonary infections and develop pneumonia when naturally or experimentally infected by the opportunistic bacterium Pasteurella pneumotropica. Since these mice have the TLR4Lps-del genotype, we hypothesized that reconstitution of mice with TLR4-positive macrophages would provide resistance to this bacterium. A cultured macrophage cell line (C2D macrophages) and bone marrow cells from C2D mice were adoptively transferred to B10 and B10 × C2D mice by intraperitoneal injection. C2D macrophages increased B10 and B10 × C2D mouse resistance to P. pneumotropica. In C2D-recipient mice there was earlier transcription of tumor necrosis factor alpha and chemokines JE and macrophage inflammatory protein 2 (MIP-2) in the lungs of B10 and B10 × C2D mice, and there was earlier transcription of KC and MIP-1α in B10 × C2D mice. In addition, the course of inflammation following experimental Pasteurella challenge was altered in C2D recipients. C2D macrophages also protected B10 × C2D mice, which lack CD4+ T cells. These data indicate that macrophages are critical for pulmonary immunity and can provide host resistance to P. pneumotropica. This study indicates that TLR4-positive macrophages are important for early recognition and clearance of pulmonary bacterial infections.

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 byP. 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.

2009 Pandemic H1N1 Influenza Virus Causes Disease and Upregulation of Genes Related to Inflammatory and Immune Responses, Cell Death, and Lipid Metabolism in Pigs

ABSTRACT There exists limited information about whether adaptation is needed for cross-species transmission of the 2009 pandemic H1N1 influenza virus (pH1N1). Here, we compare the pathogenesis of two pH1N1 viruses, one derived from a human patient (A/CA/04/09 [CA09]) and the other from swine (A/swine/Alberta/25/2009 [Alb09]), with that of the 1918-like classical swine influenza virus (A/swine/Iowa/1930 [IA30]) in the pig model. Both pH1N1 isolates induced clinical symptoms such as coughing, sneezing, decreased activity, fever, and labored breathing in challenged pigs, but IA30 virus did not cause any clinical symptoms except fever. Although both the pH1N1 viruses and the IA30 virus caused lung lesions, the pH1N1 viruses were shed from the nasal cavities of challenged pigs whereas the IA30 virus was not. Global gene expression analysis indicated that transcriptional responses of the viruses were distinct. pH1N1-infected pigs had an upregulation of genes related to inflammatory and immune responses at day 3 postinfection that was not seen in the IA30 infection, and expression levels of genes related to cell death and lipid metabolism at day 5 postinfection were markedly different from those of IA30 infection. These results indicate that both pH1N1 isolates are more virulent due in part to differences in the host transcriptional response during acute infection. Our study also indicates that pH1N1 does not need prior adaptation to infect pigs, has a high potential to be maintained in naïve swine populations, and might reassort with currently circulating swine influenza viruses.

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.

Effect of Mannheimia haemolytica pneumonia on behavior and physiologic responses of calves during high ambient environmental temperatures1

The objective of this study was to determine the effect of pneumonia during conditions of high (maximum ≥ 32°C) ambient temperatures on physiological and behavioral responses of calves. Eighteen black beef heifers averaging 240 kg were blocked by BW and randomly assigned to 1 of 2 treatment groups: 1) pneumonia induced by bronchoselective endoscopic inoculation with Mannheimia haemolytica (MH; n = 10) and 2) noninoculated controls (CN; n = 8). Nasal passage and rectal temperatures were measured every 2 h for 24 h after challenge and then twice daily for 9 d. Accelerometers, pedometers, and positioning devices monitored cattle behavior within the pen for 9 d after challenge. Blood samples were collected on trial d 0, 0.5, 1, 2, 3, 7, and 9 and were analyzed to determine the concentration of substance P, cortisol, haptoglobin, and metalloproteinase. All calves in the MH group were euthanized and necropsied on trial d 9. All MH calves became clinically ill postchallenge. A treatment × time interaction (P < 0.05) was evident for nasal and rectal temperatures, behavior, weight, and blood analysis. Rectal temperatures in MH were higher (P < 0.01) than CN during the period from 6 to 24 h after challenge. Conversely, nasal passage temperatures were less in MH calves compared with CN at 12 to 22 h after challenge. Calves in MH spent less time at the grain bunk, less time at the hay feeder, and more time lying down during the early pneumonia period compared with CN calves. Also, MH calves had significantly greater concentrations of blood biomarkers of pain (substance P) on d 0.5 (P < 0.01); stress (cortisol) on d 0.5 and 1 (P < 0.01); haptoglobin on d 0.5, 1, 2, 3, 7 (P < 0.01); and metalloproteinase on d 1, 2, and 3 (P < 0.01) compared with CN calves. At necropsy, all MH calves had right cranioventral bronchopneumonia (median lung lesions = 6.8%). Mannheimia haemolytica pneumonia caused significantly more changes in behavior and increased biomarkers during high (maximum ≥32°C) ambient temperatures compared with control calves. The results of this study may guide research in the development of objective assessment tools for management of cattle affected with bovine respiratory disease during extreme summer conditions.