Virulence characteristics of Salmonella following deletion of genes encoding the tRNA modification enzymes GidA and MnmE.

Salmonella is an important foodborne pathogen causing major public health problems throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division (gidA) gene significantly altered Salmonella virulence in both in vitro and in vivo models of infection. In Escherichia coli, GidA and MnmE have been shown to modify several bacterial factors by a post-transcriptional mechanism to modify tRNA. Therefore, we hypothesize that GidA and MnmE complex together to modulate virulence genes in Salmonella using a similar mechanism. To test our hypothesis, and to examine the relative contribution of GidA and MnmE in modulation of Salmonella virulence, we constructed gidA and mnmE single mutants as well as a gidA mnmE double mutant strain of Salmonella. Results from the in vitro data displayed a reduction in growth, motility, intracellular replication, and invasion of T84 intestinal epithelial cells in the mutant strains compared to the wild-type Salmonella strain. The in vivo data showed a significant attenuation of the mutant strains as indicated by the induction of inflammatory cytokines and chemokines, as well as in the severity of histopathological lesions in the liver and spleen, compared to mice infected with the wild-type strain. Also, a significant increase in the LD50 was observed in mice infected with the mutant strains, and mice immunized with the mutants were protected against a lethal dose of wild-type Salmonella. A pull-down assay indicated that Salmonella GidA and MnmE bind together, and HPLC analysis revealed that deletion of gidA and/or mnmE altered Salmonella tRNA modification. Overall, the data suggest MnmE and GidA bind together and use a post-transcriptional mechanism to modify tRNA to regulate Salmonella pathogenesis.

Case report: Long-term survival in puppies assessed with echocardiography, electrocardiography and cardiac troponin I after acute death in littermates due to parvoviral myocarditis.

Positive clinical outcomes of a group of surviving puppies from a litter affected by parvoviral myocarditis are detailed in this case report. Past reports focus on the negative outcomes of littermates of puppies who have died of parvoviral myocarditis. In this case, two puppies in a shelter setting, from a litter exposed to parvovirus, died suddenly with parvoviral myocarditis diagnosed at necropsy. The other seven puppies were screened for cardiac health with echocardiogram, electrocardiogram, and cardiac troponin I prior to adoption. All seven puppies had normal echocardiograms, electrocardiograms, and normal initial and recheck cardiac troponin I results. At recheck 2 years after the initial round of testing, two of the puppies were screened and continue to have normal cardiac diagnostics. All seven dogs are alive and thriving at 5 years old in homes with adopters who were given a complete medical history on the dogs prior to adoption. In summary, the outcomes for puppies in litters affected by parvoviral myocarditis are variable but they do not have to be grave. The use of cardiac diagnostics including echocardiogram, electrocardiogram and cardiac troponin I may serve as a prognostic basis for assessing the potential outcomes for the surviving puppies in affected litters.

Chronic wasting disease (CWD) susceptibility of several North American rodents that are sympatric with cervid CWD epidemics.

Chronic wasting disease (CWD) is a highly contagious always fatal neurodegenerative disease that is currently known to naturally infect only species of the deer family, Cervidae. CWD epidemics are occurring in free-ranging cervids at several locations in North America, and other wildlife species are certainly being exposed to infectious material. To assess the potential for transmission, we intracerebrally inoculated four species of epidemic-sympatric rodents with CWD. Transmission was efficient in all species; the onset of disease was faster in the two vole species than the two Peromyscus spp. The results for inocula prepared from CWD-positive deer with or without CWD-resistant genotypes were similar. Survival times were substantially shortened upon second passage, demonstrating adaptation. Unlike all other known prion protein sequences for cricetid rodents that possess asparagine at position 170, our red-backed voles expressed serine and refute previous suggestions that a serine in this position substantially reduces susceptibility to CWD. Given the scavenging habits of these rodent species, the apparent persistence of CWD prions in the environment, and the inevitable exposure of these rodents to CWD prions, our intracerebral challenge results indicate that further investigation of the possibility of natural transmission is warranted.

Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene.

Salmonella enterica serovar Typhimurium is a frequent cause of enteric disease due to the consumption of contaminated food. Identification and characterization of bacterial factors involved in Salmonella pathogenesis would help develop effective strategies for controlling salmonellosis. To investigate the role of glucose-inhibited division gene (gidA) in Salmonella virulence, we constructed a Salmonella mutant strain in which gidA was deleted. Deletion of gidA rendered Salmonella deficient in the invasion of intestinal epithelial cells, bacterial motility, intracellular survival, and induction of cytotoxicity in host cells. Deletion of gidA rendered the organism to display a filamentous morphology compared to the normal rod-shaped nature of Salmonella. Furthermore, a significant attenuation in the induction of inflammatory cytokines and chemokines, histopathological lesions, and systemic infection was observed in mice infected with the gidA mutant. Most importantly, a significant increase in LD(50) was observed in mice infected with the gidA mutant, and mice immunized with the gidA mutant were able to survive a lethal dose of wild-type Salmonella. Additionally, deletion of gidA significantly altered the expression of several bacterial factors associated with pathogenesis as indicated by global transcriptional and proteomic profiling. Taken together, our data indicate GidA as a potential regulator of Salmonella virulence genes.

Biological and virulence characteristics of the YqhC mutant of Salmonella.

Previous work by the present authors indicated a murein lipoprotein mutant of Salmonella shows a marked down-regulation in expression of yqhC. Because YqhC is a putative DNA-binding protein, it is likely involved in modulation of Salmonella genes. Deletion of yqhC renders Salmonella defective in invasion of intestinal epithelial cells, motility, and induction of cytotoxicity. In the present study, further attenuation in induction of inflammatory cytokines/chemokines and histopathological lesions was seen in mice infected with the yqhC mutant. On the other hand, deletion of yqhC did not significantly affect the LD(50) in mice or the ability of Salmonella to survive and replicate in vivo. To better understand how YqhC affects Salmonella virulence and to identify factors potentially modulated by YqhC, comparative transcriptome and proteome analysis of the yqhC mutant and the WT Salmonella was performed. Data from these experiments indicate that deletion of yqhC significantly alters the transcription of several genes associated with the SPI-1 encoded T3SS and flagellar regulons, correlating with the yqhC mutant phenotype. Overall, this study indicates that deletion of the yqhC gene causes a number of virulence-related defects in vitro, but has a modest effect in vivo, despite affecting induction of inflammatory cytokines and histopathology.

Validation of use of rectoanal mucosa-associated lymphoid tissue for immunohistochemical diagnosis of chronic wasting disease in white-tailed deer (Odocoileus virginianus).

The examination of rectoanal mucosa-associated lymphoid tissue (RAMALT) biopsy specimens for the diagnosis of transmissible spongiform encephalopathies has been described in sheep, elk, and small numbers of mule and white-tailed deer. Previous sample numbers have been too small to validate examination of this type of tissue as a viable antemortem diagnostic test. In this study, we examined RAMALT collected postmortem from 76 white-tailed deer removed from a farm in Wisconsin known to be affected by chronic wasting disease (CWD) and from 210 free-ranging white-tailed deer harvested from an area in Wisconsin where the overall prevalence of CWD among the deer was approximately 4 to 6%. The results of immunohistochemical (IHC) staining of the RAMALT sections were compared to the results of IHC staining of sections from the brain stem at the convergence of the dorsal motor nucleus of the vagus nerve, sections of the medial retropharyngeal lymph nodes (RLNs), and sections of tonsil (sections of tonsil only from captive animals were tested). The sensitivities of the IHC staining test with RAMALT sections were 81% for the captive animals and 91% for the free-ranging animals. False-negative results were usually associated with early infection, indicated by a low intensity of immunostaining in the obex and/or a polymorphism at PRNP codon 96. While the RLN remains the tissue of choice for use for the diagnosis of CWD in white-tailed deer, the results of the present study further support the use of RAMALTs collected antemortem as an adjunct to testing of tonsil biopsy specimens and surveillance by necropsy for the screening of farmed deer which have been put at risk through environmental exposure or exposure to deer with CWD.

Comparison of retropharyngeal lymph node and obex region of the brainstem in detection of chronic wasting disease in white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) in Wisconsin was first identified in February 2002. By April 2005, medial retropharyngeal lymph node (RLN) tissues had been examined from over 75,000 white-tailed deer for the presence of CWD by either immunohistochemical (IHC) staining for the prion protein associated with CWD (PrP(res)) or by using enzyme-linked immunosorbent assays with confirmation of positives by IHC staining and had been detected in 469 animals. Obex tissue was also available from 438 of the CWD-positive animals and was CWD positive by IHC staining in 355 (81%). To verify whether false-negative results were possible examining only RLN, both obex and RLN samples were examined for CWD by IHC staining from 4,430 of the white-tailed deer harvested from an area in Wisconsin where the overall deer CWD prevalence was approximately 6.2%. Two hundred and fourteen of the 269 positive deer (79.6%) had deposits of PrP(res) in both obex and lymphoid tissues, 55 (20.4%) had deposits only in lymphoid tissue, and there were no deer that had deposits only in obex.

Chronic wasting disease in a Wisconsin white-tailed deer farm.

In September 2002, chronic wasting disease (CWD), a prion disorder of captive and wild cervids, was diagnosed in a white-tailed deer (Odocoileus virginianus) from a captive farm in Wisconsin. The facility was subsequently quarantined, and in January 2006 the remaining 76 deer were depopulated. Sixty animals (79%) were found to be positive by immunohistochemical staining for the abnormal prion protein (PrP(CWD)) in at least one tissue; the prevalence of positive staining was high even in young deer. Although none of the deer displayed clinical signs suggestive of CWD at depopulation, 49 deer had considerable accumulation of the abnormal prion in the medulla at the level of the obex. Extraneural accumulation of the abnormal protein was observed in 59 deer, with accumulation in the retropharyngeal lymph node in 58 of 59 (98%), in the tonsil in 56 of 59 (95%), and in the rectal mucosal lymphoid tissue in 48 of 58 (83%). The retina was positive in 4 deer, all with marked accumulation of prion in the obex. One deer was considered positive for PrP(CWD) in the brain but not in the extraneural tissue, a novel observation in white-tailed deer. The infection rate in captive deer was 20-fold higher than in wild deer. Although weakly related to infection rates in extraneural tissues, prion genotype was strongly linked to progression of prion accumulation in the obex. Antemortem testing by biopsy of recto-anal mucosal-associated lymphoid tissue (or other peripheral lymphoid tissue) may be a useful adjunct to tonsil biopsy for surveillance in captive herds at risk for CWD infection.

Encephalitis in aborted bovine fetuses associated with Bovine herpesvirus 1 infection.

Brain tissue from 12 aborted bovine fetuses submitted to the Wisconsin Veterinary Diagnostic Laboratory revealed histologic lesions that consisted of glial nodules and variable degrees of mononuclear inflammation, microhemorrhage, neuronal necrosis, and cerebral cortical cavitation. A diagnosis of Bovine herpesvirus 1 (BHV-1) abortion had been made in all of these cases through multiple testing modalities. Brain tissue from 8 of the 12 fetuses was immunohistochemically stained with a monoclonal antibody specific to BHV-1, and, in 5 fetuses, there was positive intralesional staining of neurons, glial cells, and endothelial cells. This preliminary data suggested that herpesviral infection of brain tissue led to the described neurologic lesions. BHV-1 was then amplified from brain tissue in all 12 of the fetuses and was confirmed by partial sequencing of the thymidine kinase and glycoprotein C genes. To the authors' knowledge, neurologic lesions have not previously been described in BHV-1-infected fetuses, nor has BHV-1 previously been identified in bovine fetal brain tissue. The neurologic histopathology attributed to BHV-1 infection in these cases overlaps with the neurologic lesions produced by Neospora caninum, a common etiologic agent of bovine abortion. Therefore, when bovine fetal neurologic lesions are found, both etiologies should be considered and then distinguished by using additional diagnostic tools.

Evaluation of antigen-capture ELISA and immunohistochemical methods for avian surveillance of West Nile virus.

Accurate detection of West Nile virus (WNV) in corvids is essential for monitoring the spread of virus during the mosquito season. Viremia in corvids is very high, with titers approaching 10(8) viral particles/ml. In the presence of such marked viremia, the sensitivity of real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis is unnecessary, and more cost-effective methods should be assessed. To this end, antigen-capture ELISA (ACE) and immunohistochemical (IHC) assays were evaluated. Skin, cloacal swab specimens, and feathers from corvids were tested by use of ACE, and results were compared with results obtained from use of real-time RT-PCR analysis. Of the 3 sample types, skin gave the best sensitivity (98%) and specificity (100%). Skin, brain, kidney, and spleen from corvids were analyzed by IHC, and results were compared with real-time RT-PCR results. Kidney and spleen were more often positive by use of IHC than were brain and skin tissue; however, IHC did not perform as well as ACE in the identification of virus-positive birds. Results of this study support the use of a skin sample in an ACE format as an effective surveillance method for corvids.

Characterization of uptake of 2-deoxy-2-[18F]fluoro-D-glucose by fungal-associated inflammation: the differential uptake ratio for blastomyces-associated lesions is as high as for lymphoma and higher than for turpentine abscesses in experimentally induced lesions in rats.

PURPOSE: The objective of this investigation was to determine the biologic basis and the significance of uptake of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) using experimentally created fungal lesions in rats. PROCEDURES: Uptake of FDG by experimentally induced Blastomyces granulomas was compared with uptake by turpentine abscesses (Group 1) and by lymphomas (Group 2) using the differential uptake ratio (DUR) measured one hour after administration of 2 mCi FDG intravenously. Frozen tissue sections of Blastomyces lesions and turpentine abscesses were placed in contact with radiographic film for macroautoradiography. RESULTS: In rats in Group 1, the median (range) DUR for the Blastomyces granulomas was 1.9 (1.1-2.6) and was significantly higher than the DUR for turpentine abscesses 0.9 (0.6-1.4) and muscle 0.2 (0.1-0.5; P < 0.001). In Group 2, the median (range) DUR for the Blastomyces granulomas, lymphomas, and muscle from the rats in Group 2 were 1.8 (1.2-3.4), 1.9 (1.0-4.0), and 0.2 (0.1-0.3), respectively. There was no significant difference between the DUR of Blastomyces granulomas and lymphomas. Macroautoradiographs of the Blastomyces granulomas revealed intense uptake of FDG in the region occupied by the yeast organisms and the granulomatous inflammation. CONCLUSIONS: Blastomyces granulomas typically have high uptake of FDG associated with the region composed of the granulomatous inflammatory reaction and Blastomyces yeast organisms.

Characterization of uptake of 2-deoxy-2-[18F] fluoro-D-glucose by fungal-associated inflammation: the standardized uptake value is greater for lesions of blastomycosis than for lymphoma in dogs with naturally occurring disease.

PURPOSE: Based on limited reports, fungal lesions can have remarkably high intensity uptake of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) on positron emission tomography (PET) images. The purpose of this investigation was to compare the standardized uptake value (SUV) of naturally occurring lesions of blastomycosis with the SUV of naturally occurring lymphoma in a series of dogs. PROCEDURES: Five dogs with naturally occurring blastomycosis and three dogs with lymphoma underwent whole-body FDG-PET prior to receiving any treatment for their disease. RESULTS: The (mean +/- SD) SUV for 13 blastomycosis lesions was 7.7 +/- 2.0 versus a mean for 17 lymphomas of 4.8 +/- 1.8. These values were significantly different (P = 0.0537). There was overlap between the SUV of Blastomyces-associated lesions versus lymphomas, but a cut-off SUV of 7.0 was 100% specific for Blastomyces lesions. Numerous sites of disease were detected on the FDG-PET images that were not detected clinically. CONCLUSIONS: FDG-PET is useful for determining the extent of disease in dogs with blastomycosis. The SUV for Blastomyces-associated lesions are as high or higher than for malignant lymphoma. Due to the similarities in canine and human blastomycosis and lymphomas, similar results would be predicted in human patients. In regions where blastomycosis is endemic, Blastomyces granulomas should be considered a differential diagnosis for lesions with high intensity uptake of FDG.

Comparison of histologic lesions of endophthalmitis induced by Blastomyces dermatitidis in untreated and treated dogs: 36 cases (1986-2001).

OBJECTIVE: To compare prevalence of organisms and histologic changes in eyes from dogs with blastomycosis that were either untreated or undergoing treatment with itraconazole. DESIGN: Retrospective study. ANIMALS: 36 dogs with endophthalmitis associated with blastomycosis. PROCEDURE: Signalment, results of ophthalmic examination, and duration of treatment with itraconazole were extracted from medical records. Histologic sections from eyes were examined for prevalence and viability (ie, budding) of fungal organisms. A scoring system was devised to assess the degree of inflammation. RESULTS: Clinically, all eyes were blind and had signs of severe endophthalmitis. Histologically, the type and degree of inflammation and prevalence of Blastomyces dermatitidis were not significantly different between dogs treated with itraconazole and untreated dogs or among groups of dogs treated for different time periods (4 to 14, 15 to 28, or 29 to 72 days). Replication of the organisms in vascular tissues as well as avascular spaces in the eyes was similar in treated and untreated dogs. Lens rupture was seen in 12 of 29 (41%) eyes. CONCLUSIONS AND CLINICAL RELEVANCE: Persistence of inflammation in eyes of dogs with naturally occurring blastomycosis is likely attributable to the continued presence of B. dermatitidis, regardless of the duration of treatment with itraconazole. Lens capsule rupture, a common and previously unreported histologic finding, may contribute to cataract formation and continued inflammation.

A technique for creating localized subcutaneous Blastomyces granulomas in rats.

Our purpose was to develop a simple, reliable method for creating subcutaneous Blastomyces dermatitidis nodules in rats and to describe the histologic appearance of these lesions. We used B. dermatitidis isolated from a dog with blastomycosis to prepare a Blastomyces yeast suspension. Four rats were used to test initial dose concentrations of 10(5), 10(6), 10(9), and 10(10) yeast organisms. The dose was administered subcutaneously over the distal tibia in a volume of 0.1 ml. We then inoculated 35 additional rats with 10(9) or 10(10) yeast organisms. Rats were euthanized 7, 10, 14, 21, or 28 days after inoculation, and the histologic appearance of the nodules was described. A full post-mortem examination sought evidence of systemic spread of Blastomyces organisms. We successfully induced subcutaneous Blastomyces abscesses in 34 of 37 rats injected with 10(9) or 10(10) organisms. Nodules first appeared 3 to 7 days after injection and reached 2 to 15 mm in diameter by 7 to 28 days after inoculation. Histologically the lesions were characterized by a necrotic center surrounded by a layer of viable yeast and granulomatous inflammation. Live yeast organisms were recovered from all lesions. No adverse effects or systemic spread of Blastomyces organisms were observed. We conclude that subcutaneous Blastomyces abscesses can be induced safely and reliably in rats after injection of 10(9) and 10(10) organisms. Histologically, the experimentally induced lesions share both similarities to and differences with lesions of naturally occurring blastomycosis.