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...

Biofilm formation by pathogenic bacteria plays a key role in their pathogenesis. Previously, the pstA gene was shown to be involved in the virulence of Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease in cattle and a potential risk factor for Crohn's disease. Scanning electron microscopy and colonization levels of the M. ap mutant indicated that the pstA gene significantly contributes to the ability of M. ap to form biofilms. Digital measurements taken during electron microscopy identified a unique morphology for the ΔpstA mutant, which consisted of significantly shorter bacilli than the wild type. Analysis of the lipid profiles of the mycobacterial strains identified a novel lipopeptide that was present in the cell wall extracts of wild-type M. ap, but missing from the ΔpstA mutant. Interestingly, the calf infection model suggested that pstA contributes to intestinal invasion of M. ap. Furthermore, immunoblot analysis of peptides encoded by pstA identified a specific and significant level of immunogenicity. Taken together, our analysis revealed a novel cell wall component that could contribute to biofilm formation and to the virulence and immunogenicity of M. ap. Molecular tools to better control M. ap infections could be developed utilizing the presented findings.

Juvenile bovine patients who present with clinical signs of lameness are commonly evaluated using radiographic techniques both within a hospital setting and in a farm environment. The radiographic development of the juvenile bovine skeleton is currently poorly documented. In this study, the limbs of four heifer calves were sequentially radiographed to assess development of the juvenile bovine appendicular skeleton in the first 12 months of life. Images were acquired at three weeks, three months, six months, nine months and one year of age. The normal radiographic anatomy of the fore limbs and hindlimbs and the changes over the first 12 months are described. The majority of physes remain open throughout this period, with the exception of the proximal physes of the proximal and middle phalanges, the proximal radial physis, and the proximal humeral physis which close radiographically between 9 months and 12 months of age, and fusion of the fourth and central tarsal bones occurs between 9 months and 12 months of age. The results of this study may aid in differentiating normal and abnormal anatomy in the juvenile bovine limb.