The Reduction in ED and Hospital Admissions in Medical Home Practices Is Specific to Primary Care-Sensitive Chronic Conditions.

OBJECTIVE: To determine whether the Patient-Centered Medical Home (PCMH) transformation reduces hospital and ED utilization, and whether the effect is specific to chronic conditions targeted for management by the PCMH in our setting. DATA SOURCES AND STUDY SETTING: All patients aged 18 years and older in 2,218 primary care practices participating in a statewide PCMH incentive program sponsored by Blue Cross Blue Shield of Michigan (BCBSM) in 2009-2012. STUDY DESIGN: Quantitative observational study, jointly modeling PCMH-targeted versus other hospital admissions and ED visits on PCMH score, patient, and practice characteristics in a hierarchical multivariate model using the generalized gamma distribution. DATA COLLECTION: Claims data and PCMH scores held by BCBSM. PRINCIPAL FINDINGS: Both hospital and ED utilization were reduced proportionately to PCMH score. Hospital utilization was reduced by 13.9 percent for PCMH-targeted conditions versus only 3.8 percent for other conditions (p = .003), and ED utilization by 11.2 percent versus 3.7 percent (p = .010). Hospital PMPM cost was reduced by 17.2 percent for PCMH-targeted conditions versus only 3.1 percent for other conditions (p < .001), and ED PMPM cost by 9.4 percent versus 3.6 percent (p < .001). CONCLUSIONS: PCMH transformation reduces hospital and ED use, and the majority of the effect is specific to PCMH-targeted conditions.

Implementation of Patient-Centered Medical Homes in Adult Primary Care Practices.

There has been relatively little empirical evidence about the effects of patient-centered medical home (PCMH) implementation on patient-related outcomes and costs. Using a longitudinal design and a large study group of 2,218 Michigan adult primary care practices, our study examined the following research questions: Is the level of, and change in, implementation of PCMH associated with medical surgical cost, preventive services utilization, and quality of care in the following year? Results indicated that both level and amount of change in practice implementation of PCMH are independently and positively associated with measures of quality of care and use of preventive services, after controlling for a variety of practice, patient cohort, and practice environmental characteristics. Results also indicate that lower overall medical and surgical costs are associated with higher levels of PCMH implementation, although change in PCMH implementation did not achieve statistical significance.

Michigan's fee-for-value physician incentive program reduces spending and improves quality in primary care.

As policy makers and others seek to reduce health care cost growth while improving health care quality, one approach gaining momentum is fee-for-value reimbursement. This payment strategy maintains the traditional fee-for-service arrangement but includes quality and spending incentives. We examined Blue Cross Blue Shield of Michigan's Physician Group Incentive Program, which uses a fee-for-value approach focused on primary care physicians. We analyzed the program's impact on quality and spending from 2008 to 2011 for over three million beneficiaries in over 11,000 physician practices. Participation in the incentive program was associated with approximately 1.1 percent lower total spending for adults (5.1 percent lower for children) and the same or improved performance on eleven of fourteen quality measures over time. Our findings contribute to the growing body of evidence about the potential effectiveness of models that align payment with cost and quality performance, and they demonstrate that it is possible to transform reimbursement within a fee-for-service framework to encourage and incentivize physicians to provide high-quality care, while also reducing costs.

Patient-centered medical home implementation and use of preventive services: the role of practice socioeconomic context.

IMPORTANCE: The patient-centered medical home (PCMH) model of primary care is being implemented in a wide variety of socioeconomic contexts, yet there has been little research on whether its effects differ by context. Clinical preventive service use, including cancer screening, is an important outcome to assess the effectiveness of the PCMH within and across socioeconomic contexts. OBJECTIVE: To determine whether the relationship between the PCMH and cancer screening is conditional on the socioeconomic context in which a primary care physician practice operates. DESIGN, SETTING, AND PARTICIPANTS: A longitudinal study spanning July 1, 2009, through June 30, 2012, using data from the Blue Cross Blue Shield of Michigan Physician Group Incentive Program was conducted. Michigan nonpediatric primary care physician practices that participated in the Physician Group Incentive Program (5452 practice-years) were included. Sample size and outlier exclusion criteria were applied to each outcome. We examined the interaction between practices' PCMH implementation scores and their socioeconomic context. The implementation of a PCMH was self-reported by the practice's affiliated physician organizations and was measured as a continuous score ranging from 0 to 1. Socioeconomic context was calculated using a market-based approach based on zip code characteristics of the practice's patients and by combining multiple measures using principal components analysis. MAIN OUTCOMES AND MEASURES: Breast, cervical, and colorectal cancer screening rates for practices' Blue Cross Blue Shield of Michigan patients. RESULTS: The implementation of a PCMH was associated with higher breast, cervical, and colorectal cancer screening rates across most market socioeconomic contexts. In multivariable models, the PCMH was associated with a higher rate of screening for breast cancer (5.4%; 95% CI, 1.5% to 9.3%), cervical cancer (4.2%; 95% CI, 1.4% to 6.9%), and colorectal cancer (7.0%; 95% CI, 3.6% to 10.5%) in the lowest socioeconomic group but nonsignificant differences in screening for breast cancer (2.6%; 95% CI, -0.1% to 5.3%) and cervical cancer (-0.5%; 95% CI, -2.7% to 1.7%) and a higher rate of colorectal cancer (4.5%; 95% CI, 1.8% to 7.3%) screening in the highest socioeconomic group. Because PCMH implementation was associated with larger increases in screening in lower socioeconomic practice settings, models suggest reduced disparities in screening rates across these contexts. For example, the model-predicted disparity in breast cancer screening rates between the highest and lowest socioeconomic contexts was 6% (77.9% vs 72.2%) among practices with no PCMH implementation and 3% (80.3% vs. 77.0%) among practices with full PCMH implementation. CONCLUSIONS AND RELEVANCE: In our study, the PCMH model was associated with improved cancer screening rates across contexts but may be especially relevant for practices in lower socioeconomic areas.

Dialing in: effect of telephonic wellness coaching on weight loss.

OBJECTIVE: To estimate the effect of telephonic wellness coaching on weight loss in a commercially insured population. STUDY DESIGN: Pre-post evaluation design. METHODS: Self-reported weight was obtained from 2 annual health assessment questionnaires administered during 2008 and 2010. Baseline (T1) information from these questionnaires was used to identify overweight/obese individuals and to determine targets for a 4-call wellness coaching program. Overweight/obese individuals identified at T1 were classified into following groups: (1) targeted for wellness coaching (N = 1448, including 1050 participants and 398 nonparticipants); (2) not targeted for wellness coaching, but targeted for other telephonic wellness care management (WCM) programs (N = 1270); (3) not targeted for any WCM programs (N = 7586). Weight reported on questionnaires a year later (T2) was used to calculate weight change between T1 and T2. Paired t-tests were used to detect significant weight changes over time. Multivariable linear regressions were used to compare weight changes between the groups. Stratified analysis was conducted to determine the effectiveness of telephonic wellness coaching for subgroups based on participants' selected health goals, intensity of the intervention received and initial stage of change. RESULTS: The group targeted for wellness coaching reported an average weight change of -0.44 kg (95% confidence interval [CI], -0.76 to -0.16) at T2, significantly more weight loss than reported by the group not targeted for any WCM programs. Participants who started in preparation stage and completed the program reported weight change of -1.43 kg (95% CI, -2.17 to -0.68), highest among program participants. CONCLUSIONS: Small weight loss was observed for obese/individuals targeted for telephonic wellness coaching.

Association between maternal preventive care utilization and adolescent vaccination: it's not just about Pap testing.

STUDY OBJECTIVE: To examine the association between maternal preventive care utilization and human papillomavirus (HPV) vaccine uptake by their adolescent daughters. DESIGN: A cross-sectional study using immunization records from administrative claims and the state health department's immunization information system from June 2006 through May 2011. PARTICIPANTS: Commercially-insured Michigan females aged 13-17 in May 2011 and their mothers. Mothers were identified using relationship information on the insurance contract. MAIN OUTCOME MEASURES: Using logistic regression, we investigated whether initiating and/or completing the HPV vaccine series were associated with maternal preventive care utilization (Papaniculou testing, mammograms, primary care office visits) independently and using a combined maternal preventive care utilization index. RESULTS: Among 38,604 mother-daughter pairs, 36% of daughters initiated and 22% completed the HPV vaccine series. Maternal utilization of each recommended service was modestly associated with both daughter's initiation and completion of the HPV vaccine. Effect estimates for receipt of Papaniculou test on vaccine initiation (OR = 1.07, 95% CI = 1.06-1.08) were not any higher than for mammograms (OR = 1.10, 95% CI = 1.08-1.11) or primary care office visits (OR = 1.07, 95% CI = 1.06-1.09). Using a maternal preventive care utilization index, vaccine uptake increased with an increasing number of received services. CONCLUSIONS: Maternal receipt of recommended preventive care, which may reflect general attitudes toward prevention, is as or more predictive of daughter's vaccination status than cervical cancer screening alone. Engaging women in broad routine preventive care practices may have additional positive effects on adolescent HPV vaccination beyond those achieved through cervical cancer prevention efforts alone.

Partial and incremental PCMH practice transformation: implications for quality and costs.

OBJECTIVE: To examine the associations between partial and incremental implementation of the Patient Centered Medical Home (PCMH) model and measures of cost and quality of care. DATA SOURCE: We combined validated, self-reported PCMH capabilities data with administrative claims data for a diverse statewide population of 2,432 primary care practices in Michigan. These data were supplemented with contextual data from the Area Resource File. STUDY DESIGN: We measured medical home capabilities in place as of June 2009 and change in medical home capabilities implemented between July 2009 and June 2010. Generalized estimating equations were used to estimate the mean effect of these PCMH measures on total medical costs and quality of care delivered in physician practices between July 2009 and June 2010, while controlling for potential practice, patient cohort, physician organization, and practice environment confounders. PRINCIPAL FINDINGS: Based on the observed relationships for partial implementation, full implementation of the PCMH model is associated with a 3.5 percent higher quality composite score, a 5.1 percent higher preventive composite score, and $26.37 lower per member per month medical costs for adults. Full PCMH implementation is also associated with a 12.2 percent higher preventive composite score, but no reductions in costs for pediatric populations. Incremental improvements in PCMH model implementation yielded similar positive effects on quality of care for both adult and pediatric populations but were not associated with cost savings for either population. CONCLUSIONS: Estimated effects of the PCMH model on quality and cost of care appear to improve with the degree of PCMH implementation achieved and with incremental improvements in implementation.

Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association.

BACKGROUND: The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map. RESULTS: Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the MAP S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human M. avium subsp. hominissuis strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of MAP (JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level. CONCLUSIONS: Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the M. avium complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of MAP. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for M. avium complex strains based on these genome sequences.

Identification and functional characterization of the iron-dependent regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep, has unique iron requirements in that it is mycobactin-dependent for cultivation in vitro. The iron-dependent regulator (IdeR) is a well-characterized global regulator responsible for maintaining iron homeostasis in Mycobacterium tuberculosis (MTB). We identified an orthologous segment in the MAP genome, MAP2827, with >93 % amino acid identity to MTB IdeR. Electrophoretic mobility shift assays and DNase protection assays confirmed that MAP2827 binds the 19 bp consensus motif (iron box) on the MAP genome. Sequencing of MAP2827 from multiple isolates revealed a non-synonymous change (R91G) exclusive to sheep strains. Reporter gene assays and quantitative real-time RT-PCR assays in two diverse MAP strains and in an ideR deletion mutant of M. smegmatis (mc(2)155) suggested that both sheep MAP IdeR (sIdeR) and cattle MAP IdeR (cIdeR) repress mbtB transcription at high iron concentrations and relieve repression at low iron concentrations. On the other hand, bfrA (an iron storage gene) was upregulated by cIdeR when presented with MTB or the cattle MAP bfrA promoter, and was downregulated by sIdeR in the presence of MTB, or sheep or cattle MAP bfrA promoters, at high iron concentrations. The differential iron regulatory mechanisms between IdeR-regulated genes across strains may contribute to the differential growth or pathogenic characteristics of sheep and cattle MAP strains. Taken together, our study provides a possible reason for mycobactin dependency and suggests strong implications in the differential iron acquisition and storage mechanisms in MAP.

Immunogenicity of proteome-determined Mycobacterium avium subsp. paratuberculosis-specific proteins in sheep with paratuberculosis.

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis, a chronic granulomatous enteritis. Detecting animals with paratuberculosis infections is difficult because the currently available tools have low sensitivity and lack specificity; these tools are prone to generating spurious positive test results caused by exposure to environmental M. avium complex organisms. To generate candidate antigens for incorporation into a specific test for paratuberculosis, subspecies-specific proteins were determined by proteomic comparison of M. avium subsp. paratuberculosis and M. avium subsp. avium. Analysis was aimed at revealing proteins only expressed (or predominant) in the protein profile of M. avium subspecies paratuberculosis. Two-dimensional gel electrophoresis resolved approximately 1,000 protein spots from each subspecies. Proteome analysis identified protein spots whose expression profile appeared markedly increased in M. avium subsp. paratuberculosis, and 32 were identified by analysis of their tryptic peptide profile by matrix-assisted laser desorption ionization-time of flight analysis. Thirty of these proteins were cloned, and their recombinant proteins were expressed. Ovine paratuberculosis sera were used to assess their immunoreactivity by enzyme-linked immunosorbent assay (ELISA), Western blotting, and dot blot analysis. Seventeen proteins were detected in at least one of the immunoassays, and eleven proteins were detected by ELISA with an optical density in excess of the cutoff of 0.1 in four of six sera tested. The immunoreactivity of these proteins indicates their potential as unique diagnostic antigens for the development of a specific serological detection of paratuberculosis.

Comparative genomic analysis of Mycobacterium avium subspecies obtained from multiple host species.

BACKGROUND: Mycobacterium avium (M. avium) subspecies vary widely in both pathogenicity and host specificity, but the genetic features contributing to this diversity remain unclear. RESULTS: A comparative genomic approach was used to identify large sequence polymorphisms among M. avium subspecies obtained from a variety of host animals. DNA microarrays were used as a platform for comparing mycobacterial isolates with the sequenced bovine isolate M. avium subsp. paratuberculosis (MAP) K-10. Open reading frames (ORFs) were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to MAP K-10 DNA. Multiple large polymorphic regions were found in the genomes of MAP isolates obtained from sheep. One of these clusters encodes glycopeptidolipid biosynthesis enzymes which have not previously been identified in MAP. M. avium subsp. silvaticum isolates were observed to have a hybridization profile very similar to yet distinguishable from M. avium subsp. avium. Isolates obtained from cattle (n = 5), birds (n = 4), goats (n = 3), bison (n = 3), and humans (n = 9) were indistinguishable from cattle isolate MAP K-10. CONCLUSION: Genome diversity in M. avium subspecies appears to be mediated by large sequence polymorphisms that are commonly associated with mobile genetic elements. Subspecies and host adapted isolates of M. avium were distinguishable by the presence or absence of specific polymorphisms.

Development and use of a partial Mycobacterium avium subspecies paratuberculosis protein array.

As an initial step toward systematically characterizing all antigenic proteins produced by a significant veterinary pathogen, 43 recombinant Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) expression clones were constructed, cataloged, and stored. NC filters were spotted with purified proteins from each clone along with a whole cell lysate of M. paratuberculosis. Spots on the resulting dot array consisted of hypothetical proteins (13), metabolic proteins (3), cell envelope proteins (7), known antigens (4), and unique proteins with no similarity in public sequence databases (16). Dot blot arrays were used to profile antibody responses in a rabbit and mouse exposed to M. paratuberculosis as well as in cattle showing clinical signs of Johne's disease. The M. paratuberculosis heat shock protein DnaK, encoded by ORF MAP3840 and a membrane protein (MAP2121c), were identified as the most strongly immunoreactive in both the mouse and rabbit hosts, respectively. MAP3155c, which encodes a hypothetical protein, was most strongly immunoreactive in sera from Johne's disease cattle. This study has enabled direct comparisons of antibody reactivity for an entire panel of over 40 proteins and has laid the foundation for future high throughput production and arraying of M. paratuberculosis surface proteins for immune profiling experiments in cattle.

Early antibody response against Mycobacterium avium subspecies paratuberculosis antigens in subclinical cattle.

BACKGROUND: Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection. RESULTS: Sera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A M. paratuberculosis specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease. CONCLUSION: Collectively these results demonstrate that M. paratuberculosis proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of M. paratuberculosis infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of M. paratuberculosis antigens.

Profiling bovine antibody responses to Mycobacterium avium subsp. paratuberculosis infection by using protein arrays.

With the genome sequence of Mycobacterium avium subsp. paratuberculosis determined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against M. avium subsp. paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp. paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. avium subsp. paratuberculosis. Also included were previously reported or known M. avium subsp. paratuberculosis antigens to serve as a frame of reference. Sera from healthy control cattle (n = 3) and cattle infected with either M. avium subsp. avium and Mycobacterium bovis were exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. avium subsp. avium proteins that was higher than the degree of cross-reactivity with the more distantly related M. bovis proteins. Finally, sera from naturally infected cattle (n = 3) as well as cattle experimentally infected with M. avium subsp. paratuberculosis (n = 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp. paratuberculosis.

Development and characterization of monoclonal antibodies and aptamers against major antigens of Mycobacterium avium subsp. paratuberculosis.

Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne's disease (JD). To fill this gap in JD research, monoclonal antibodies (MAbs) against M. avium subsp. paratuberculosis were produced from BALB/c mice immunized with a whole-cell extract of M. avium subsp. paratuberculosis. A total of 10 hybridomas producing MAbs to proteins ranging from 25 to 85 kDa were obtained. All MAbs showed some degree of cross-reactivity when they were analyzed against a panel of whole-cell protein lysates comprising seven different mycobacterial species. The MAbs were characterized by several methods, which included isotype analysis, specificity analysis, epitope analysis, reactivity in immunoblot assays, and electron microscopy. The identities of the antigens that bound to two selected MAbs were determined by screening an M. avium subsp. paratuberculosis lambda phage expression library. This approach revealed that MAb 9G10 detects MAP1643 (isocitrate lyase) and that MAb 11G4 detects MAP3840 (a 70-kDa heat shock protein), two proteins present in high relative abundance in M. avium subsp. paratuberculosis. The epitopes for MAb 11G4 were mapped to the N-terminal half of MAP3840, whereas MAb 9G10 bound to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind to specific protein sequences, against the hypothetical protein encoded by MAP0105c were also generated and tested for their binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.

Production and characterization of monoclonal antibodies against a major membrane protein of Mycobacterium avium subsp. paratuberculosis.

The Mycobacterium avium subsp. paratuberculosis 35-kDa major membrane protein (MMP) encoded by MAP2121c is an important membrane antigen recognized in cattle with Johne's disease. In this study, purified recombinant MMP was used to produce two stable monoclonal antibodies, termed 8G2 and 13E1, which were characterized by immunoblotting, epitope mapping, and immunofluorescence microscopy.

Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-kappaB, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M. avium subsp. avium isolate, and they significantly up-regulated proinflammatory genes related to IL-6, T-cell receptor, B-cell receptor, and death receptor signaling within THP-1 cells. Additionally, we demonstrated consistency among infecting genotypes of M. avium subsp. paratuberculosis isolated from diverse hosts [cattle (n=2), human (n=3), sheep (n=2), and bison (n=1)] in quantitative reverse transcription-PCR analysis of seven differentially expressed genes. While the levels of expression induced by the bison isolate were different compared with cattle or human isolates, they followed the common anti-inflammatory, antiapoptotic trend. Our data suggest that the macrophage responses to M. avium subsp. paratuberculosis isolates from cattle and human sources, regardless of genotype, follow a common theme of anti-inflammatory responses, an attribute likely associated with successful infection and persistence. However, these expression patterns differ significantly from those in THP-1 cells infected with sheep isolates of M. avium subsp. paratuberculosis or the M. avium subsp. avium isolate. These data provide a transcriptional basis for a variety of pathophysiological changes observed during early stages of infection by different strains of M. avium subsp. paratuberculosis, a first step in understanding trait-allele association in this economically important disease.

Identification of diagnostic proteins in Mycobacterium avium subspecies paratuberculosis by a whole genome analysis approach.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is an economically significant veterinary pathogen that causes Johne's disease in cattle and sheep. There is a critical need for improved diagnostic tests to detect M. paratuberculosis infection in these animals. As with many other animal diseases, efforts need to be concentrated on the development of simple, rapid, noninvasive tests that can be performed by veterinarians or animal producers without expensive laboratory equipment. With the genome sequence of M. paratuberculosis now complete, we have taken a different strategy to identify novel proteins that are present uniquely in M. paratuberculosis and are antigenic in the context of infected cattle. Through a whole genome comparison of M. paratuberculosis with other sequenced mycobacterial genomes, we identified a collection of more than 90 genes that are present uniquely in M. paratuberculosis. This list has been further trimmed to 39 after amplification using polymerase chain reaction of unique genes using the genomic deoxyribonucleic acid template from several mycobacterial species and isolates. A selection of the remaining genes has been cloned and expressed in Escherichia coli and purified by affinity chromatography. Successfully purified proteins were analyzed using sera from rabbits immunized with M. paratuberculosis. Furthermore, to identify antigens in the context of disease, sera from cattle with Johne's disease as well as healthy control cattle are used in immunoassays. Using this methodology, we identified the first protein antigens specific to M. paratuberculosis.

Effects of temperature on gene expression patterns in Leptospira interrogans serovar Lai as assessed by whole-genome microarrays.

Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic Leptospira spp. from infected animals or contaminated water or soil. The availability of genome sequences for Leptospira interrogans, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression. Leptospira spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20 degrees C, 30 degrees C, 37 degrees C, and 39 degrees C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37 degrees C and 39 degrees C) versus environmental temperatures (20 degrees C and 30 degrees C). Additionally, cultures grown at 30 degrees C then shifted overnight to 37 degrees C were compared with those grown long-term at 30 degrees C and 37 degrees C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.

Lateral gene transfer in Mycobacterium avium subspecies paratuberculosis.

Lateral gene transfer is an integral part of genome evolution in most bacteria. Bacteria can readily change the contents of their genomes to increase adaptability to ever-changing surroundings and to generate evolutionary novelty. Here, we report instances of lateral gene transfer in Mycobacterium avium subsp. paratuberculosis, a pathogenic bacteria that causes Johne's disease in cattle. A set of 275 genes are identified that are likely to have been recently acquired by lateral gene transfer. The analysis indicated that 53 of the 275 genes were acquired after the divergence of M. avium subsp. paratuberculosis from M. avium subsp. avium, whereas the remaining 222 genes were possibly acquired by a common ancestor of M. avium subsp. paratuberculosis and M. avium subsp. avium after its divergence from the ancestor of M. tuberculosis complex. Many of the acquired genes were from proteobacteria or soil dwelling actinobacteria. Prominent among the predicted laterally transferred genes is the gene rsbR, a possible regulator of sigma factor, and the genes designated MAP3614 and MAP3757, which are similar to genes in eukaryotes. The results of this study suggest that like most other bacteria, lateral gene transfers seem to be a common feature in M. avium subsp. paratuberculosis and that the proteobacteria contribute most of these genetic exchanges.