Search

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.

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

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 (mc2155) 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.

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.

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 .

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.

TheMycobacterium aviumsubsp.paratuberculosis35-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.

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.

The Columbia basin subpopulation of pygmy rabbit Brachylagus idahoensis was listed as endangered by the United States Fish and Wildlife Service in November 2001, and no pygmy rabbits have been seen in the wild since spring 2002. Captive propagation efforts have attempted to increase population size in preparation for reintroduction of animals into central Washington. Disseminated mycobacteriosis due to Mycobacterium avium has been the most common cause of death of adult captive pygmy rabbits. Between June 2002 and September 2004, mycobacteriosis was diagnosed in 28 captive adult pygmy rabbits (representing 29% of the captive population), in contrast to 18 adult pygmy rabbits dying of all other causes in the same time period. Antemortem and postmortem medical records were evaluated retrospectively to describe the clinical course of mycobacteriosis in pygmy rabbits, physical examination findings, and diagnostic test results in the diagnosis of mycobacteriosis in pygmy rabbits. Various treatment protocols, possible risk factors for mortality, and recommendations for prevention of mycobacteriosis were evaluated also. Compromised cell-mediated immunity appears to be the best explanation at this time for the observed high morbidity and mortality from mycobacterial infections in pygmy rabbits.

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°C, 30°C, 37°C, and 39°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°C and 39°C) versus environmental temperatures (20°C and 30°C). Additionally, cultures grown at 30°C then shifted overnight to 37°C were compared with those grown long-term at 30°C and 37°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 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.Key words: mycobacteria, M. avium subsp. paratuberculosis, lateral gene transfer, unique genes, phylogeny.

Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease in cattle and other ruminants. M. avium subsp. paratuberculosis infection of the bovine host is not well understood; however, it is assumed that crossing the bovine intestinal mucosa is important in order for M. avium subsp. paratuberculosis to establish infection. To examine the ability of M. avium subsp. paratuberculosis to infect bovine epithelial cells in vitro, Madin-Darby bovine kidney (MDBK) epithelial cells were exposed to M. avium subsp. paratuberculosis . It was observed that bacteria can establish infection and replicate within MDBK cells. M. avium subsp. paratuberculosis also has been reported to infect mammary tissue and milk, and we showed that M. avium subsp. paratuberculosis infects bovine mammary epithelial cells (MAC-T cell line). Using polarized MAC-T cell monolayers, it was also determined that M. avium subsp. paratuberculosis crosses apical and basolateral surfaces with approximately the same degree of efficiency. Because M. avium subsp. paratuberculosis can be delivered to the naïve host by milk, it was investigated whether incubation of M. avium subsp. paratuberculosis with milk has an effect on invasion of MDBK cells. M. avium subsp. paratuberculosis exposed to milk entered epithelial cells with greater efficiency than M. avium subsp. paratuberculosis exposed to broth medium or water ( P < 0.01). Growth of M. avium subsp. paratuberculosis within MAC-T cells also resulted in augmented ability to subsequently infect bovine MDBK cells ( P < 0.001). Microarray analysis of intracellular M. avium subsp. paratuberculosis RNA indicates the increased transcription of genes which might be associated with an invasive phenotype.

Microarray-based comparisons of three Mycobacterium avium subsp. paratuberculosis isolates, including one sheep strain and two cattle strains, identified three large genomic deletions in the sheep strain, totaling 29,208 bp and involving 24 open reading frames. These deletions may help explain some of the differences in pathogenicity and host specificity observed between the cattle and sheep strains of Mycobacterium avium subsp. paratuberculosis .

Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis . Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (∼1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis . Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis . The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization.

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium , and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis . Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare . A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry ( mce ) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.

The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10 −1 transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis .

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's Disease, an economically important intestinal ailment of ruminants. Due to the considerable genetic and serologic cross-reactivity with closely related and ubiquitous members of the M. avium complex, a species-specific method for the serological diagnosis of Johne's disease is unavailable. Computational and PCR-based analysis of the complete genome sequence of M. avium subsp. paratuberculosis led to the identification of 13 open reading frames with no identifiable homologs. One of these sequences is a putative insertion element present in six copies on the M. avium subsp. paratuberculosis genome. These novel M. avium subsp. paratuberculosis genes were cloned into Escherichia coli expression vectors, and nine were successfully expressed as recombinant fusion proteins. Five of these proteins were purified in sufficient amounts to allow immunoblot analyses of their reactivity with sera from naturally infected cattle as well as mice and rabbits exposed to M. avium subsp. paratuberculosis . Fusion proteins representing MAP0862, MAP3732c, and MAP2963c were recognized by nearly all of the sera tested, including those from cattle in the clinical stages of disease. Notably, further analysis of the protein encoded by MAP0862 showed that it reacted with sera from additional infected cattle but not with sera from uninfected control animals. The fusion product of MAP0860c did not react with any of the sera tested, while the remaining four proteins were variably recognized by sera from M. avium subsp. paratuberculosis -infected cattle. Collectively, the results of this study demonstrate the utility of genomic data to identify potential diagnostic sequences.

The development of immunoassays specific for the diagnosis of Johne's disease in cattle requires antigens specific to Mycobacterium avium subsp. paratuberculosis . However, because of genetic similarity to other mycobacteria comprising the M. avium complex, no such antigens have been found. Through a comparative genomics approach, 21 potential coding sequences of M. avium subsp. paratuberculosis that are not represented in any other mycobacterial species tested ( n = 9) were previously identified (J. P. Bannantine, E. Baechler, Q. Zhang, L. Li, and V. Kapur, J. Clin. Microbiol. 40: 1303-1310, 2002). Here we describe the cloning, heterologous expression, and antigenic analysis of these M. avium subsp. paratuberculosis -specific sequences in Escherichia coli . Nucleotide sequences representing each unique predicted coding region were amplified and cloned into two different E. coli expression vectors encoding polyhistidine or maltose binding protein (MBP) affinity purification tags. All 21 of the MBP fusion proteins were successfully purified under denaturing conditions and were evaluated in immunoblotting studies with sera from rabbits and mice immunized with M. avium subsp. paratuberculosis . These studies showed that 5 of the 21 gene products are produced by M. avium subsp. paratuberculosis and are antigenic. Immunoblot analysis with a panel of sera from 9 healthy cattle and 10 cattle with clinical disease shows that the same five M. avium subsp. paratuberculosis proteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novel M. avium subsp. paratuberculosis antigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johne's disease.

Bacteria often encounter environments where nutrient availability is limited, and they must adapt accordingly. To identify Pasteurella multocida genes that are differentially expressed during nutrient limitation, we utilized whole-genome microarrays to compare levels of gene expression during growth in rich and minimal media. Our analysis showed that the levels of expression of a total of 669 genes, representing approximately one-third of the genome, were detectably altered over the course of the experiment. A large number ( n = 439) of genes, including those involved in energy metabolism, transport, protein synthesis, and binding, were expressed at higher levels in rich medium, suggesting that, upon exposure to a rich environment, P. multocida immediately begins to turn on many energy-intensive biosynthetic pathways or, conversely, turns these genes off when it is exposed to a nutrient-deficient environment. Genes with increased expression in minimal medium ( n = 230) included those encoding amino acid biosynthesis and transport systems, outer membrane proteins, and heat shock proteins. Importantly, our analysis also identified a large number ( n = 164) of genes with unknown functions whose expression was altered during nutrient limitation. Overall, the results of our study show that a wide repertoire of genes, many of which have yet to be functionally classified, undergo transcriptional regulation in P. multocida in response to growth in minimal medium and provide a strong foundation to investigate the transcriptional response of this multispecies pathogen to growth in a nutrient-limited environment.

We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island "modules."

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.

To estimate the effect of telephonic wellness coaching on weight loss in a commercially insured population.Pre-post evaluation design.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.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.Small weight loss was observed for obese/individuals targeted for telephonic wellness coaching.

We present here the complete genome sequence of a common avian clone of Pasteurella multocida , Pm70. The genome of Pm70 is a single circular chromosome 2,257,487 base pairs in length and contains 2,014 predicted coding regions, 6 ribosomal RNA operons, and 57 tRNAs. Genome-scale evolutionary analyses based on pairwise comparisons of 1,197 orthologous sequences between P. multocida , Haemophilus influenzae , and Escherichia coli suggest that P. multocida and H. influenzae diverged ≈270 million years ago and the γ subdivision of the proteobacteria radiated about 680 million years ago. Two previously undescribed open reading frames, accounting for ≈1% of the genome, encode large proteins with homology to the virulence-associated filamentous hemagglutinin of Bordetella pertussis . Consistent with the critical role of iron in the survival of many microbial pathogens, in silico and whole-genome microarray analyses identified more than 50 Pm70 genes with a potential role in iron acquisition and metabolism. Overall, the complete genomic sequence and preliminary functional analyses provide a foundation for future research into the mechanisms of pathogenesis and host specificity of this important multispecies pathogen.

The patient-centered medical home (PCMH) has been recognized as a strategy to redesign and improve the delivery of primary health care. Collaboration between Blue Cross Blue Shield of Michigan (BCBSM) and 39 Physician Organizations in Michigan laid the foundation for a state-wide medical home program.The objective of the study was to describe a unique methodology developed and implemented by BCBSM to designate primary care physician practices as medical homes.Since 2009, practices were designated annually as medical homes on the basis of (1) implementation of PCMH-related capabilities, and (2) performance on quality-of-care and health resource utilization measures. An overall score for each practice was calculated. Practices were ranked relative to each other, with the top portion of the continuum representing an achievable level of performance.The number of practices designated as medical homes more than tripled since the program's inception: 302 (1283 physicians) in 2009, 513 (1876 physicians) in 2010, 772 (2547 physicians) in 2011, and 994 (3028 physicians) in 2012. Designated practices reported implementing more than double the PCMH capabilities of nondesignated practices, yet all practices increased their number of implemented capabilities during the 4 years.This program represents the largest state-based PCMH program in the United States. Over the 4-year period, 1130 unique practices have received designation, representing 3469 unique physicians. An estimated 1.4 million BCBSM members in Michigan received care from these practices. This program will continue to develop, drawing on changes in the health system landscape, collaboration with the physician community, and knowledge gained from PCMH evaluations.

ObjectiveTo 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 SourceWe 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 DesignWe 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 FindingsBased 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. ConclusionsEstimated 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.

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.

PURPOSE Our goal was to describe an approach to patient-centered medical home (PCMH) measurement based on delineating the desired properties of the measurement relative to assumptions about the PCMH and the uses of the measure by Blue Cross Blue Shield of Michigan (BCBSM) and health services researchers. METHODS We developed and validated an approach to assess 13 functional domains of PCMHs and 128 capabilities within those domains. A measure of PCMH implementation was constructed using data from the validated self-assessment and then tested on a large sample of primary care practices in Michigan. RESULTS Our results suggest that the measure adequately addresses the specific requirements and assumptions underlying the BCBSM PCMH program—ability to assess change in level of implementation; ability to compare across practices regardless of size, affiliation, or payer mix; and ability to assess implementation of the PCMH through different sequencing of capabilities and domains. CONCLUSIONS Our experience illustrates that approaches to measuring PCMH should be driven by the measures’ intended use(s) and users, and that a one-size-fits-all approach may not be appropriate. Rather than promoting the BCBSM PCMH measure as the gold standard, our study highlights the challenges, strengths, and limitations of developing a standardized approach to PCMH measurement.

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.

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.

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-κB, 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.

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.

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.

Johne's disease, a chronic inflammatory disease caused by infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), is one of the most prevalent and costly diseases of dairy cattle worldwide. This ruminant pathogen is closely related to the ubiquitous animal and human pathogen Mycobacterium avium subspecies avium (M. avium), confounding the development of specific diagnostic reagents. Exacerbating this problem further is that most existing microbiological, serological, and immunologic assays for the identification of infected animals are inadequate. This is primarily because of the slow-growing nature of the organism, genetic intractability and the previous lack of information on M. paratuberculosis subspecies-specific genes or proteins that may enable the development of specific and sensitive assays. New detection tools are critically needed to definitively answer questions surrounding M. paratuberculosis as a foodborne pathogen as well as aid in determining if it is a contributing factor in Crohn's disease. Thus, the recent characterization of the complete genome sequence of M. paratuberculosis in our laboratories has been a major step forward in meeting this need. We have performed studies that utilize genomic information for the identification of specific DNA sequences and protein antigens in M. paratuberculosis. Based on a preliminary in silico comparison of the M. paratuberculosis genome sequence with that of M. avium, we have now identified at least 35 novel coding sequences that are unique to M. paratuberculosis. These in silico data were then confirmed and expanded by PCR amplification analysis with DNA from several species and isolates of mycobacteria. Finally, these unique sequences have been incorporated into an antigen discovery project that may allow reliable detection of the bacterium in antigen-based diagnostic tests. Application of these new tools in addressing foodborne related issues of M. paratuberculosis is discussed.

Pasteurella multocida was grown in iron-free chemically defined medium supplemented with hemoglobin, transferrin, ferritin, and ferric citrate as iron sources. Whole-genome DNA microarrays were used to monitor global gene expression over seven time points after the addition of the defined iron source to the medium. This resulted in a set of data containing over 338,000 gene expression observations. On average, 12% of P. multocida genes were differentially expressed under any single condition. A majority of these genes encoded P. multocida proteins that were involved in either transport and binding or were annotated as hypothetical proteins. Several trends are evident when the data from different iron sources are compared. In general, only two genes ( ptsN and sapD ) were expressed at elevated levels under all of the conditions tested. The results also show that genes with increased expression in the presence of hemoglobin did not respond to transferrin or ferritin as an iron source. Correspondingly, genes with increased expression in the transferrin and ferritin experiments were expressed at reduced levels when hemoglobin was supplied as the sole iron source. Finally, the data show that genes that were most responsive to the presence of ferric citrate did not follow a trend similar to that of the other iron sources, suggesting that different pathways respond to inorganic or organic sources of iron in P. multocida . Taken together, our results demonstrate that unique subsets of P. multocida genes are expressed in response to different iron sources and that many of these genes have yet to be functionally characterized.

We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island "modules."

Subgenomic DNA microarrays were employed to evaluate the expression of the accessory gene regulator ( agr locus) as well as multiple virulence-associated genes in Staphylococcus aureus . Gene expression was examined during growth of S. aureus in vitro in standard laboratory medium and rabbit serum and in vivo in subcutaneous chambers implanted in either nonimmune rabbits or rabbits immunized with staphylococcal enterotoxin B. Expression of RNAIII, the effector molecule of the agr locus, was dramatically repressed in serum and in vivo, despite the increased expression of secreted virulence factors sufficient to cause toxic shock syndrome (TSS) in the animals. Statistical analysis and clustering of virulence genes based on their expression profiles in the various experimental conditions demonstrated no positive correlation between the expression of agr and any staphylococcal virulence factors examined. Disruption of the agr locus had only a minimal effect on the expression in vivo of the virulence factors examined. An effect of immunization on the expression of agr and virulence factors was also observed. These results suggest that agr activation is not necessary for development of staphylococcal TSS and that regulatory circuits responding to the in vivo environment override agr activity.

Pasteurella multocida is the causative agent of a wide range of diseases in avian and mammalian hosts. Gene expression in response to low iron conditions was analyzed in P. multocida using whole-genome microarrays. The analysis shows that the expression of genes involved in energy metabolism and electron transport generally decreased 2.1- to 6-fold while that of genes used for iron binding and transport increased 2.1- to 7.7-fold in P. multocida during the first 2 h of growth under iron-limiting conditions compared with controls. Notably, 27% of the genes with significantly altered expression had no known function, illustrating the limitations of using publicly available databases to identify genes involved in microbial metabolism and pathogenesis. Taken together, the results of our investigations demonstrate the utility of whole-genome microarray analyses for the identification of genes with altered expression profiles during varying growth conditions and provide a framework for the detailed analysis of the molecular mechanisms of iron acquisition and metabolism in P. multocida and other gram-negative bacteria.

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.

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.