SummaryCopper 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 DctpV mutant survives close to the wild-type levels in both murine and guinea pig models of tuberculosis, animals infected with the DctpV 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.

Johne's disease, caused by Mycobacterium paratuberculosis infection, is a worldwide problem for the dairy industry and has a possible involvement in Crohn's 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 Johne's and Crohn's diseases.Mycobacterium paratuberculosis causes Johne's disease (paratuberculosis), characterized by chronic granulomatous enteritis, in dairy cattle, with an estimated loss of $220 million per year in the United States alone (22). Worldwide, the prevalence of the disease can range from 3 to 4% of herds in regions with low incidence (such as England) (6) to high levels of 50% of herds in some areas within the United States (such as Wisconsin and Alabama) (8, 16). In humans, M. paratuberculosis bacilli have been found in examined tissues from Crohn's disease patients, suggesting a possible role for M. paratuberculosis in developing Crohn's disease (7). Unfortunately, the mechanisms of virulence that control M. paratuberculosis persistence during infection are poorly understood and the key steps for developing paratuberculosis remain elusive. For example, mechanisms responsible for invasion and persistence of M. paratuberculosis inside the intestine remain undefined at the molecular level (42). Both live and dead bacilli are observed in subepithelial macrophages after uptake, most likely in M cells covering the ileum (24). Once inside the macrophages, M. paratuberculosis survives and proliferates inside the phagosomes by using unknown mechanisms (41). In one report examining intestinal invasion, it was suggested that the 35-kDa membrane protein antigen could play a possible role in bacillus invasio...

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.

Abstract. Primary canine gastrointestinal lymphoma has been believed to be of B-cell origin based on the morphology and behavior of the neoplastic cells and the evidence from the human medical field. However, the neoplasms have not to date been characterized as to the origin of the cell population. Forty-four cases diagnosed as canine gastrointestinal lymphoma were retrieved from the records of the Veterinary Teaching Hospitals at the University of Minnesota and the University of Wisconsin-Madison. Four of the cases have been previously identified as epitheliotropic T-cell gastrointestinal lymphoma. Twenty-three of the dogs were female, with 11 intact and 12 neutered, and 21 of the dogs were male, with 12 intact and nine neutered. Sixteen breeds as well as individuals of mixed breeding were represented. The Boxer and the sharpei were the most commonly represented breeds with six individuals each. The age range of the dogs was 1.5-14.66 years, with two dogs identified as adult and two of unknown age. Archived tissue blocks of gastrointestinal samples were sectioned in duplicate and prepared for immunohistochemical staining with CD3 (T-cell marker) and CD20 (B-cell marker). In 75% of the cases examined under light microscopy, 50-95% of the neoplastic cells stained positively with CD3 and exhibited marked epitheliotropic behavior. In three of the cases, from 10% up to 50% of the neoplastic cells stained positively with CD20, with widely scattered CD3(ϩ) cells. In the remainder of the cases, few to none of the neoplastic cells stained with either of the markers. This retrospective study shows that canine primary gastrointestinal lymphoma is more commonly of T-cell origin, rather than B-cell origin.

Infection with Mycobacterium avium subsp. paratuberculosis causes Johne's disease in cattle and is a serious problem for the dairy industry worldwide. Development of models to mimic aspects of Johne's 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 Johne's disease were obtained, and a practical model of mycobacterial invasiveness was developed. A similar approach can be used for other enteric bacteria.Johne's disease (JD) or paratuberculosis in cattle is caused by Mycobacterium avium subsp. paratuberculosis. Virtually all ruminants are believed to be susceptible to infection with this organism, which causes severe economic losses estimated to be around $200 to $250 million a year for the dairy industry in the United States alone (19). Worldwide, the prevalence of the infection can range from 3 to 4% in herds (e.g., in England) (4) to as much as 50% in herds (e.g., in Wisconsin and Alabama) (6,14). A recent report by members of the National Research Council on the status of JD stressed the need to fill several gaps in our knowledge associated with the pathophysiology, immunology, and control of JD (7). JD research is hampered by the low growth rate of M. avium subsp. paratuberculosis and the lack of a reliable animal model to investigate host-pathogen interactions. Despite the introduction of molecular protocols to facilitate JD diagnosis (11, 39), the tools that are available are unreliable for detection of infected cows, especially cows in the early stages of infection (5). Currently, no effective treatment regimen is available, and the control strategies for afflicted herds are bas...