Howard Steinberg

CtpV: A putative copper exporter required for full virulence of Mycobacterium tuberculosis

Copper is a required micronutrient that is also toxic at excess concentrations. Currently, little is known about the role of copper in interactions between bacterial pathogens and their human hosts. In this study, we elucidate a mechanism for copper homeostasis in the human pathogen Mycobacterium tuberculosis via characterization of a putative copper exporter, CtpV. CtpV was shown to be required by M. tuberculosis to maintain resistance to copper toxicity. Furthermore, the deletion of ctpV resulted in a 98‐gene transcriptional response, which elucidates the increased stress experienced by the bacteria in the absence of this detoxification mechanism. Interestingly, although the ΔctpV mutant survives close to the wild‐type levels in both murine and guinea pig models of tuberculosis, animals infected with the ΔctpV mutant displayed decreased lung damage, and mutant‐infected mice had a reduced immune response to the bacteria as well as a significant increase in survival time relative to mice infected with wild‐type M. tuberculosis. Overall, our study provides the first evidence for a connection between bacterial copper response and the virulence of M. tuberculosis, supporting the hypothesis that copper response could be important to intracellular pathogens, in general.

Central Nervous System Neosporosis in a Foal

Cutaneous or subcutaneous cysts of all types are considered rare in cats. A literature search yielded no reports of cutaneous or subcutaneous dermoid cysts in cats. The structures reported here were histologically compatible with the description of the dorsal midline structures in dogs but, because they did not communicate with the spinal canal, these cysts posed no danger or potential danger to the animal from central nervous system infection. These structures were present in the flank instead of along the dorsal midline, possibly as a result of faulty embryologic fusion of adjacent dermatomes. The dermoid cyst in cat no. 2, considering its young age, probably was a congenital disorder; however, cat no. 1 was 10 years old at the time of presentation. It was not known how long the cyst in cat 1 had been present or if it had grown in size immediately prior to presentation. Dermoid cysts have been classified according to depth of penetration of the sinus. Class I cysts extend from the skin to the supraspinous ligament, class II cysts do not extend as deeply but are connected to the supraspinous ligament by a fibrous band, and class III cysts are similar to class II cysts but have no connecting band to the ligament. A fourth class has been proposed, in which the cyst extends to the spinal canal and is attached to the dura mater. This class is analogous to the pilonidal sinus of human beings, which usually occurs in the coccygeal region. The term pilonidal cyst, which by definition means any cyst containing a tuft of hair, is usually used synonymously with the term dermoid cyst in veterinary medicine. Perhaps 1 reason for the confusion over the term dermoid cyst is that many deep anomalous structures can have structural components of epithelium and can be loosely termed dermoid cysts by pathologists. A recent textbook has subclassified these cysts in an effort to more clearly define each type from a histologic standpoint. In this classification system, dermoid cysts are considered a type of follicular tumor. More widespread application of the classification system may be helpful to pathologists and clinicians for separating benign lesions from those with potential for serious complications. The surgical treatment of dermoid cysts is straightforward and involves careful dissection of the cyst. Although dermoid cysts are reported to involve only the skin or subcutaneous tissues, the cyst in cat no. 1 was found isolated between muscle layers of the flank and the sinus in cat no. 2 extended to the peritoneum. When excision of the cyst involves creation of an abdominal wall defect, care should be taken to ensure that anatomic closure of the defect is accomplished to avoid subsequent herniation of abdominal contents.

Identification of Novel Virulence Determinants in Mycobacterium paratuberculosis by Screening a Library of Insertional Mutants

ABSTRACT Johnes disease, caused by Mycobacterium paratuberculosis infection, is a worldwide problem for the dairy industry and has a possible involvement in Crohns disease in humans. To identify virulence determinants of this economically important pathogen, a library of 5,060 transposon mutants was constructed using Tn5367 insertion mutagenesis, followed by large-scale sequencing to identify disrupted genes. In this report, 1,150 mutants were analyzed and 970 unique insertion sites were identified. Sequence analysis of the disrupted genes indicated that the insertion of Tn5367 was more prevalent in genomic regions with G+C content (50.5 to 60.5%) lower than the average G+C content (69.3%) of the rest of the genome. Phenotypic screening of the library identified disruptions of genes involved in iron, tryptophan, or mycolic acid metabolic pathways that displayed unique growth characteristics. Bioinformatic analysis of disrupted genes identified a list of potential virulence determinants for further testing with animals. Mouse infection studies showed a significant decrease in tissue colonization by mutants with a disruption in the gcpE, pstA, kdpC, papA2, impA, umaA1, or fabG2_2 gene. Attenuation phenotypes were tissue specific (e.g., for the umaA1 mutant) as well as time specific (e.g., for the impA mutant), suggesting that those genes may be involved in different virulence mechanisms. The identified potential virulence determinants represent novel functional classes that could be necessary for mycobacterial survival during infection and could provide suitable targets for vaccine and drug development against Johnes and Crohns diseases.

Invasion and Persistence of Mycobacterium avium subsp. paratuberculosis during Early Stages of Johne's Disease in Calves

ABSTRACT Infection with Mycobacterium avium subsp. paratuberculosis causes Johnes disease in cattle and is a serious problem for the dairy industry worldwide. Development of models to mimic aspects of Johnes disease remains an elusive goal because of the chronic nature of the disease. In this report, we describe a surgical approach employed to characterize the very early stages of infection of calves with M. avium subsp. paratuberculosis. To our surprise, strains of M. avium subsp. paratuberculosis were able to traverse the intestinal tissues within 1 h of infection in order to colonize distant organs, such as the liver and lymph nodes. Both the ileum and the mesenteric lymph nodes were persistently infected for months following intestinal deposition of M. avium subsp. paratuberculosis despite a lack of fecal shedding of mycobacteria. During the first 9 months of infection, humoral immune responses were not detected. Nonetheless, using flow cytometric analysis, we detected a significant change in the cells participating in the inflammatory responses of infected calves compared to cells in a control animal. Additionally, the levels of cytokines detected in both the ileum and the lymph nodes indicated that there were TH1-type-associated cellular responses but not TH2-type-associated humoral responses. Finally, surgical inoculation of a wild-type strain and a mutant M. avium subsp. paratuberculosis strain (with an inactivated gcpE gene) demonstrated the ability of the model which we developed to differentiate between the wild-type strain and a mutant strain of M. avium subsp. paratuberculosis deficient in tissue colonization and invasion. Overall, novel insights into the early stages of Johnes disease were obtained, and a practical model of mycobacterial invasiveness was developed. A similar approach can be used for other enteric bacteria.

A/J Mice Are Susceptible and C57BL/6 Mice Are Resistant to Listeria monocytogenes Infection by Intragastric Inoculation

ABSTRACT Previous studies demonstrated that the innate resistance of mice to Listeria monocytogenes infection by intravenous or intraperitoneal inoculation is regulated principally by the Hc locus on mouse chromosome 2. The A/J and C57BL/6 mouse strains were identified as prototype L. monocytogenes-susceptible and -resistant strains, respectively. In the present study, we compared the relative susceptibilities of A/J and C57BL/6 mice to intragastric (i.g.) inoculation with L. monocytogenes. The results of our study indicate that A/J mice are significantly more susceptible than C57BL/6 mice to an i.g. challenge with L. monocytogenes. This was reflected in the estimated 50% lethal doses for the two strains (106 and 108 CFU for A/J and C57BL/6 mice, respectively) and a more rapid and severe dissemination of the infection to the spleen and liver in A/J mice than in C57BL/6 mice. Histopathological examination of tissues from the infected mice confirmed the greater severity of disease in A/J mice. Clearance of a primary infection enhanced the resistance of both A/J and C57BL/6 mice to reinfection with L. monocytogenes via the gastrointestinal tract. However, the relative difference in susceptibility between the two strains was evident even after immunization. The A/J mouse holds promise as a model for investigating the pathogenesis of gastrointestinal listeriosis because of its ability to develop systemic infection following challenge with numbers of organisms similar to those recovered from some L. monocytogenes-contaminated food products.

The Aryl Hydrocarbon Receptor Is Required for Optimal Resistance to Listeria monocytogenes Infection in Mice

The aryl hydrocarbon receptor (AhR) is part of a powerful signaling system that is triggered by xenobiotic agents such as polychlorinated hydrocarbons and polycyclic aromatic hydrocarbons. Although activation of the AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin or certain polycyclic aromatic hydrocarbons can lead to immunosuppression, there is also increasing evidence that the AhR regulates certain normal developmental processes. In this study, we asked whether the AhR plays a role in host resistance using murine listeriosis as an experimental system. Our data clearly demonstrate that AhR null C57BL/6J mice (AhR−/−) are more susceptible to listeriosis than AhR heterozygous (AhR+/−) littermates when inoculated i.v. with log-phase Listeria monocytogenes. AhR−/− mice exhibited greater numbers of CFU of L. monocytogenes in the spleen and liver, and greater histopathological changes in the liver than AhR+/− mice. Serum levels of IL-6, MCP-1, IFN-γ, and TNF-α were comparable between L. monocytogenes-infected AhR−/− and AhR+/− mice. Increased levels of IL-12 and IL-10 were observed in L. monocytogenes-infected AhR−/− mice. No significant difference was found between AhR+/− and AhR−/− macrophages ex vivo with regard to their ability to ingest and inhibit intracellular growth of L. monocytogenes. Intracellular cytokine staining of CD4+ and CD8+ splenocytes for IFN-γ and TNF-α revealed comparable T cell-mediated responses in AhR−/− and AhR+/− mice. Previously infected AhR−/− and AhR+/− mice both exhibited enhanced resistance to reinfection with L. monocytogenes. These data provide the first evidence that AhR is required for optimal resistance but is not essential for adaptive immune response to L. monocytogenes infection.

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

ABSTRACT Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.

ULTRASOUND ATTENUATION AND BACKSCATTER IN THE LIVER DURING PREDNISONE ADMINISTRATION

Ultrasound attenuation and backscatter changes resulting from glucocorticoid administration were investigated in a dog model. Ten beagle dogs were randomized into two groups: five were given 2 mg/kg/day IM injections of prednisone to induce steroid hepatopathy and five served as controls. Histology showed vacuolization in most hepatocytes of treated animals on the third day of treatment, and larger, midzonally distributed vacuoles from day 7 on. An increase in both ultrasonic attenuation and backscatter was observed in treated dogs during in vivo measurements. Pooled data from the two groups suggest that attenuation elevations precede backscatter changes. Attenuation was significantly higher in the treated animals than in the controls by day 7. Both attenuation and backscatter were significantly higher in livers of treated than untreated dogs when measured by direct application of the transducer on the liver following euthanasia. We conclude that attenuation and backscatter coefficients can detect early changes in the liver associated with steroid hepatopathy. This may be a useful model to investigate detection of diffuse liver disease with ultrasound tissue characterization.

Treatment of Feline Gastrointestinal Small-Cell Lymphoma With Chlorambucil and Glucocorticoids

Gastrointestinal (GI) lymphoma is the most frequently diagnosed form of lymphoma in the cat and is categorized into two distinct forms based on the size of neoplastic lymphocytes. Treatments for both large- and small-cell GI lymphoma have been described previously; however, multiple chemotherapy protocols were used, a minimal amount of histopathological characterization was provided, and, in most studies, the majority of diagnoses were obtained via endoscopic pinch biopsies. Twenty-eight cats (24 with full-thickness intestinal biopsies) were diagnosed with small-cell GI lymphoma and treated with a combination of chlorambucil and glucocorticoids. The majority of cases were strongly CD3+, and many displayed epitheliotropism. The overall clinical response rate was 96%, with a median clinical remission duration of 786 days. Follow-up identified seven cats with relapsed disease-all of which were treated with a rescue protocol of cyclophosphamide and glucocorticoids; the response rate was 100%, and four of the 28 cats were diagnosed with a second malignancy.

Dietary supplementation with conjugated linoleic acid does not alter the resistance of mice to Listeria monocytogenes infection

Conjugated linoleic acid (CLA) has been used experimentally as a dietary supplement to increase lean body weight and to modulate inflammation in a variety of animal species. In addition, human use of dietary CLA as a supplement to regulate body fat has received both scientific and public attention. No reports have been published regarding the effects of dietary CLA on antimicrobial resistance. In this study, we provide evidence that feeding CLA for up to 4 wk does not alter host defense against Listeria monocytogenes in mice. These findings suggest that the anti-inflammatory effects of CLA do not impair cellular immunity to this intracellular pathogen.