Strain structure analysis of Mycobacterium tuberculosis circulating among HIV negative, positive and drug resistant TB patients attending chest clinics in Western Kenya.

BACKGROUND: Despite global tuberculosis (TB) interventions, the disease remains one of the major public health concerns. Kenya is ranked 15th among 22 high burden TB countries globally. METHODS: A cross-sectional study was conducted in Western Kenya, which comprises 10 counties. A multistage sampling method was used where a single sub-county was randomly selected followed by sampling two high volume health facility from each sub-county. Identification of spoligotype profiles and their family distribution and lineage level were achieved by comparison with SITVIT database. RESULTS: Lineage distribution pattern revealed that the most predominant lineage was CAS 220 (39.8%) followed by Beijing 128 (23.1%). The other lineages identified were T, LAM, H, X, S and MANU which were quantified as 87 (15.7%), 67 (12.1%), 16 (2.8%), 10 (1.8%), 8 (1.4%) and 5 (0.9%) respectively. CAS and Beijing strains were the most predominant lineage in both HIV negative and positive TB patients. The Beijing lineage was also the most predominant in resistant M. tuberculosis strains as compared to wild type. A total of 12 (2.0%) were orphaned M. tuberculosis strains which were spread across all the 10 counties of the study site. In multivariate logistic regression adjusting for potential cofounders three potential risk factors were significant. HIV status (OR = 1.52, CI = 0.29-3.68 and P value of 0.001), Alcohol use (OR = 0.59, CI = 0.43-3.12 and P-value =0.001) and cross border travel (OR = 0.61, CI = 0.49-3.87 and P value = 0.026). Most M. tuberculosis clinical isolates showed genetic clustering with multivariate logistic regression indicating three potential risk factors to clustering. HIV status (OR = 1.52, CI = 0.29-3.68 and P value of 0.001), Alcohol use (OR = 0.59, CI = 0.43-3.12 and P-value =0.001) and cross border travel (OR = 0.61, CI = 0.49-3.87 and P value = 0.026). CONCLUSION: There exist diverse strains of M. tuberculosis across the 10 counties of Western Kenya. Predominant distribution of clustered genotype points to the fact that most TB cases in this region are as a result of resent transmission other than activation of latent TB.

Impact of Mycobacterium avium subsp. paratuberculosis infection on bovine IL10RA knockout mammary epithelial (MAC-T) cells.

Mycobacterium avium subsp. Paratuberculosis (MAP) is an intracellular pathogen that causes Johne's disease (JD) in cattle and other ruminants. IL10RA encodes the alpha chain of the IL-10 receptor that binds the cytokine IL-10, and is one of the candidate genes that have been found to be associated with JD infection status. In this study, a previously developed IL10RA knockout (IL10RAKO) bovine mammary epithelial (MAC-T) cell line and wild-type (WT) MAC-T cells were infected with live MAP for 72 h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of IL10RA. Cytokine and chemokine concentrations in culture supernatants were measured by multiplexing immunoassay. Total RNA was extracted from the MAC-T cells, and qPCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of TNF-α, IL-6, CXCL8, CXCL10, CCL2, and CCL3 were significantly induced in WT MAC-T cells and IL-10 was significantly inhibited post-MAP infection. However, IL10RAKO MAC-T cells had greater secretion of TNF-α, IL-6, IFN-γ, CCL3, CCL4, CXCL8, and CXCL10, and lower secretion of VEGF-α. Moreover, the expression of inflammatory genes (TNF-α, IL-1α, IL-6) was also more significantly induced in IL10RAKO cells than in WT MAC-T cells post-MAP-infection, and unlike the WT cells, anti-inflammatory cytokines IL-10 and SOCS3 and chemokines CCL2 were not significantly induced. In addition, the expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP-infection; however, there was no significant induction of these miRNAs in the IL10RAKO cells, which suggests IL10 receptor is somehow involved in regulating the miRNA response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in interleukin signaling, and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of IL10RA in the regulation of innate immune response to MAP.

Role of Toll-Like Receptor 4 in Mycobacterium avium subsp. paratuberculosis Infection of Bovine Mammary Epithelial (MAC-T) Cells In Vitro.

Toll-like receptor 4 (TLR4) encodes an innate immune cell pattern-recognition receptor implicated in the recognition of Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease in ruminants. Polymorphisms in TLR4 have been associated with susceptibility to MAP infection. In this study, a previously developed TLR4 knockout (TLR4KO) bovine mammary epithelial (MAC-T) cell line and wild-type MAC-T cells (WT) were infected with live MAP for 72 h to identify potential immunoregulatory miRNAs, inflammatory genes, and cytokines/chemokines impacted by MAP infection in the presence/absence of TLR4. Cytokines/chemokines production in culture supernatants was measured by multiplexing immunoassay. Total RNA was extracted from the remaining MAC-T cells, and quantitative PCR was performed to determine the expression of inflammatory genes and selected bovine miRNAs. Results showed that the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), CXCL8, CXCL10, CCL4, and CCL3 were significantly induced in WT MAC-T cells during MAP infection. However, TLR4KO MAC-T cells had greater secretion of CCL3, IL-6, vascular endothelial growth factor (VEGF-α), and TNF-α and decreased secretion of CXCL10 and CCL2. Moreover, the expression of inflammatory genes was induced in TLR4KO cells. The expression of miRNAs (miR133b, miR-92a, and miR-184) was increased in WT MAC-T cells post-MAP infection; however, there was no significant induction of these miRNAs in TLR4KO cells, which suggests they are involved in regulating the innate immune response to MAP infection. Target gene function analysis further suggests that miR-92a may be involved in TLR and interleukin signaling and miR-133b and miR-184 may be involved in other signaling pathways. These findings support the involvement of TLR4 in the regulation of innate immune response to MAP. IMPORTANCE Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent for paratuberculosis or Johne's disease (JD) in ruminants, a disease clinically very similar to Crohn's disease in humans. Polymorphisms in the bovine Toll-like receptor genes (TLR1, TLR2, and TLR4) have been shown to affect MAP recognition and host innate immune response and have been associated with increased susceptibility of cattle to paratuberculosis. Our results demonstrated that knocking out the TLR4 gene in bovine MAC-T cells enhanced inflammation in response to MAP. These findings show divergent roles for TLR4 in Escherichia coli lipopolysaccharide and mycobacterial infections, and this may have important consequences for the treatment of these inflammatory diseases and for genetic selection to improve disease resistance. It advances our understanding of the role of TLR4 in the context of MAP infection.

Distribution patterns of drug resistance Mycobacterium tuberculosis among HIV negative and positive tuberculosis patients in Western Kenya.

INTRODUCTION: Globally anti-tuberculosis drug resistance is one of the major challenges affecting control and prevention of tuberculosis. Kenya is ranked among 30 high burden TB countries globally. However, there is scanty information on second line antituberculosis drug resistance among tuberculosis patients. Therefore, this study aimed at determining Mycobacterium tuberculosis drug resistant strain distribution pattern in 10 counties of Western Kenya among HIV positive and negative patients. METHOD: A cross-sectional study was conducted in Western Kenya, which comprises 10 counties. A multistage sampling method was used where a single sub-county was randomly selected followed by sampling one high volume health facility from each sub-county. Consenting study subjects with at least two smear positive sputum at the time of enrolment were randomly selected. The collected sputum was decontaminated with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) and then stained with Ziehl Neelsen Stain before visualizing the presence of bacilli under microscope at ×100 magnification with oil immersion. Further, the identified bacilli were cultured and susceptibility test carried out using known first and second line antimycobacterial tuberculosis. HIV testing was carried out using Determine® HIV-1/2 rapid test (Abbot Diagnostics, Maidenhead, United Kingdom). Those who had smear converted were dropped from the study. Finally, drug susceptibility pattern across the 10 counties of Western Kenya was evaluated. RESULTS: Our study showed that Mycobacterium tuberculosis drug resistance among HIV negative and positive cases in Western Kenya was prevalent in all the 10 counties surveyed. Based on the drug susceptibility tests, 53.2% and 42.7% of the study samples were resistant to at least one antituberculosis drug among HIV negative and HIV positive patients respectively. The data analysis revealed that among the HIV-positive and HIV-negative patients, resistance to INH was predominant (28.5%, and 23.6%, respectively), followed by RIF (16.4% and 14.6% respectively). Second-line drug resistant strains identified among HIV negative patients included Ethionamide (0.3%), Gatifloxacin (0.3%), Amikacin (0.3%) and Capreomycin (0.3%). There was no second line drug monoresistance among HIV positive TB patients. Multi/poly drug resistance were noted among HIV-negative patients in, INH + AMK (0.7%), INH + PZA (1%), INH + GFX (0.7%, INH + ETO (0.7%, STY + ETO (1%), ETH + ETO (1.0%), INH + KAN (0.7%) and INH + CAP (0.7%) strains/cases at 95% confidence interval. Among HIV positive patients INH + GFX (1.1%), INH + ETO (0.4%) and INH + KAN (0.4%) strains of M. tuberculosis were identified with a confidence interval of 95%. Geographical distribution patterns analysis of M. tuberculosis drug polyresistant strains across the 10 counties were recorded. Among HIV TB patients, resistant strains were identified in Nyamira (INH + GFX, INH + KAN), Bungoma ((ETO + STY), Busia (ETH + ETO and STY + ETO) Homabay (RIF + AMK. ETO + ETH and ETO + STY), Kisumu (ETH + ETO and PZA + ETO) and in Kakamega, Kisii and Vihiga (INH + KAN and RIF + AMK). There was no M. tuberculosis polyresistant strain identified in Migori and Siaya counties. Among HIV positive TB patients, M. tuberculosis resistant strains were identified in three counties, Nyamira (INH + KAN) Homabay (INH + GFX and INH + AMK) and Kakamega (INH + GFX). There was no polyresistant M. tuberculosis strain identified in Migori, Bungoma, Kisii, Vihiga, Busia, Siaya and Kisumu Counties. DISCUSSION: The distribution patterns of M. tuberculosis drug resistance among HIV negative and positive TB patients could be as a result of reported high prevalence of HIV in Western Kenya counties especially the area under study. Tuberculosis is one of the opportunistic diseases that have been shown to be the major cause of AIDS among HIV infected patients. Resent reports by National AIDS Control Council shows that Kisumu, Siaya, Homabay, Migori, Busia have the overall leading in HIV prevalence in Kenya. The low prevalence of drug resistant strains among HIV tuberculosis patients could be as a result of drug adherence attitude adopted by HIV patients, availability of continuous counselling and close follow up and notification by healthcare workers and community health volunteers. CONCLUSION: Drug resistant M. tuberculosis strains prevalence is still high among HIV negative and positive patients in Western Kenya with the most affected being HIV negative TB patients. It is therefore probable that the existing control measures are not adequate to control transmission of drug resistant strains. Further, miss diagnosis or delayed diagnosis of TB patients could be contributing to the emergence of M. tuberculosis drug polyresistant strains. RECOMMENDATION: Based on the result of this study, regular TB drug resistance surveillance should be conducted to ensure targeted interventions aimed at controlling increased transmission of the tuberculosis drug resistant strains among HIV/AIDS and HIV negative patients. There is also need for improved drug resistant infection control measures, timely and rapid diagnosis and enhanced and active screening strategies of tuberculosis among suspected TB patients need to be put in place. Further, studies using a larger patient cohort and from counties across the country would shed much needed insights on the true national prevalence of different variants of M. tuberculosis drug resistance.

Detection of Mycobacterium avium Subspecies paratuberculosis (MAP) Microorganisms Using Antigenic MAP Cell Envelope Proteins.

Cell envelope proteins from Mycobacterium avium subspecies paratuberculosis (MAP) that are antigenically distinct from closely related mycobacterial species are potentially useful for Johne's Disease (JD) diagnosis. We evaluated the potential of ELISAs, based on six antigenically distinct recombinant MAP cell envelope proteins (SdhA, FadE25_2, FadE3_2, Mkl, DesA2, and hypothetical protein MAP1233) as well as an extract of MAP total cell envelope proteins, to detect antibodies against MAP in the sera of infected cattle. The sensitivity (Se) and specificity (Sp) of an ELISA based on MAP total cell envelope proteins, when analyzing 153 bovine serum samples, was 75 and 96%, respectively. Analysis of the same samples, using a commercial serum ELISA resulted in a Se of 56% and Sp of 99%. Results of ELISA analysis using plates coated with recombinant cell envelope proteins ranged from a highest Se of 94% and a lowest Sp of 79% for Sdh A to a lowest Se of 67% and a highest Sp of 95% for hypothetical protein MAP1233. Using polyclonal antibodies to MAP total cell envelope proteins, immunohistochemical analysis of intestinal and lymph node tissues from JD-positive cattle detected MAP organisms whereas antibodies to recombinant proteins did not. Finally, polyclonal antibodies to MAP total cell envelope protein and to recombinant SdhA, FadE25_2, and DesA2 proteins immunomagnetically separated MAP microorganisms spiked in PBS. These results suggest that antigenically distinct MAP cell envelope proteins and antibodies to these proteins may have potential to detect MAP infection in dairy cattle.

A Bovine Enteric Mycobacterium Infection Model to Analyze Parenteral Vaccine-Induced Mucosal Immunity and Accelerate Vaccine Discovery.

Mycobacterial diseases of cattle are responsible for considerable production losses worldwide. In addition to their importance in animals, these infections offer a nuanced approach to understanding persistent mycobacterial infection in native host species. Mycobacteriumavium ssp. paratuberculosis (MAP) is an enteric pathogen that establishes a persistent, asymptomatic infection in the small intestine. Difficulty in reproducing infection in surrogate animal models and limited understanding of mucosal immune responses that control enteric infection in the natural host have been major barriers to MAP vaccine development. We previously developed a reproducible challenge model to establish a consistent MAP infection using surgically isolated intestinal segments prepared in neonatal calves. In the current study, we evaluated whether intestinal segments could be used to screen parenteral vaccines that alter mucosal immune responses to MAP infection. Using Silirum® - a commercial MAP bacterin - we demonstrate that intestinal segments provide a platform for assessing vaccine efficacy within a relatively rapid period of 28 days post-infection. Significant differences between vaccinates and non-vaccinates could be detected using quantitative metrics including bacterial burden in intestinal tissue, MAP shedding into the intestinal lumen, and vaccine-induced mucosal immune responses. Comparing vaccine-induced responses in mucosal leukocytes isolated from the site of enteric infection versus blood leukocytes revealed substantial inconsistences between these immune compartments. Moreover, parenteral vaccination with Silirum did not induce equal levels of protection throughout the small intestine. Significant control of MAP infection was observed in the continuous but not the discrete Peyer's patches. Analysis of these regional mucosal immune responses revealed novel correlates of immune protection associated with reduced infection that included an increased frequency of CD335+ innate lymphoid cells, and increased expression of IL21 and IL27. Thus, intestinal segments provide a novel model to accelerate vaccine screening and discovery by testing vaccines directly in the natural host and provides a unique opportunity to interrogate mucosal immune responses to mycobacterial infections.

Regional Dichotomy in Enteric Mucosal Immune Responses to a Persistent Mycobacterium avium ssp. paratuberculosis Infection.

Chronic enteric Mycobacterium avium ssp. paratuberculosis (MAP) infections are endemic in ruminants globally resulting in significant production losses. The mucosal immune responses occurring at the site of infection, specifically in Peyer's patches (PP), are not well-understood. The ruminant small intestine possesses two functionally distinct PPs. Discrete PPs function as mucosal immune induction sites and a single continuous PP, in the terminal small intestine, functions as a primary lymphoid tissue for B cell repertoire diversification. We investigated whether MAP infection of discrete vs. continuous PPs resulted in the induction of significantly different pathogen-specific immune responses and persistence of MAP infection. Surgically isolated intestinal segments in neonatal calves were used to target MAP infection to individual PPs. At 12 months post-infection, MAP persisted in continuous PP (n = 4), but was significantly reduced (p = 0.046) in discrete PP (n = 5). RNA-seq analysis revealed control of MAP infection in discrete PP was associated with extensive transcriptomic changes (1,707 differentially expressed genes) but MAP persistent in continuous PP elicited few host responses (4 differentially expressed genes). Cytokine gene expression in tissue and MAP-specific recall responses by mucosal immune cells isolated from PP, lamina propria and mesenteric lymph node revealed interleukin (IL)22 and IL27 as unique correlates of protection associated with decreased MAP infection in discrete PP. This study provides the first description of mucosal immune responses occurring in bovine discrete jejunal PPs and reveals that a significant reduction in MAP infection is associated with specific cytokine responses. Conversely, MAP infection persists in the continuous ileal PP with minimal perturbation of host immune responses. These data reveal a marked dichotomy in host-MAP interactions within the two functionally distinct PPs of the small intestine and identifies mucosal immune responses associated with the control of a mycobacterial infection in the natural host.

An update on Mycobacterium avium subspecies paratuberculosis antigens and their role in the diagnosis of Johne's disease.

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Johne's disease (JD) or paratuberculosis. Diagnosis of MAP infection by measuring host cell-mediated and humoral immune responses has been a major focus in MAP research. For this purpose, several MAP antigens such as secreted protein, cell envelope protein, cell-mediated immune and lipoprotein antigens have been identified and tested to measure their diagnostic utility with varying degree of success. Identifying the optimal antigen or antigen combinations for diagnosis of infected animals is hindered by the complex nature of the disease, prolonged subclinical infection, the differential expression of antigens and scarcity of well characterized MAP-specific epitopes making selection of a single MAP antigen very difficult. Thus, multiplexing of antigens with larger scale and longitudinal studies may lead to development of cost-effective next generation serodiagnostics. This mini review focuses on the role of different MAP antigens in the diagnosis of JD.

Identification of antigenic proteins from Mycobacterium avium subspecies paratuberculosis cell envelope by comparative proteomic analysis.

Johne's disease (JD) is a contagious, chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The aim of this study was to identify antigenic proteins from the MAP cell envelope (i.e. cell wall and cytoplasmic membranes) by comparing MAP, M. avium subsp. hominissuis (MAH) and M. smegmatis (MS) cell envelope protein profiles using a proteomic approach. Composite two-dimensional (2D) difference gel electrophoresis images revealed 13 spots present only in the image of the MAP cell envelope proteins. Using serum from MAP-infected cattle, immunoblot analysis of 2D gels revealed that proteins in the 13 spots were antigenic. These proteins were identified by liquid chromatography tandem mass spectrometry as products of the following genes: sdhA, fadE25_2, mkl, citA, gapdh, fadE3_2, moxR1, mmp, purC, mdh, atpG, fbpB and desA2 as well as two proteins without gene names identified as transcriptional regulator (MAP0035) protein and hypothetical protein (MAP1233). Protein functions ranged from energy generation, cell wall biosynthesis, protein maturation, bacterial replication and invasion of epithelial cells, functions considered essential to MAP virulence and intracellular survival. Five MAP cell envelope proteins, i.e. SdhA, FadE25_2, FadE3_2, MAP0035 and DesA2 were recombinantly expressed, three of which, i.e. SdhA, FadE25_2 and DesA2, were of sufficient purity and yield to generate polyclonal antibodies. Immunoblot analysis revealed antibodies reacted specifically to the respective MAP cell envelope proteins with minimal cross-reactivity with MAH and MS cell envelope proteins. Identification and characterization of MAP-specific proteins and antibodies to those proteins may be useful in developing new diagnostic tests for JD diagnosis.

Identification of Mycobacterium avium subspecies paratuberculosis strains isolated from dairy goats and dairy sheep in Ontario, Canada.

The main objective of this study was to identify the circulating strains of Mycobacterium avium subspecies paratuberculosis (Map) in fecal isolates obtained from dairy goat (N = 29 farms) and dairy sheep (N = 21 farms) populations in Ontario, Canada. Further subtyping was performed to determine if there was adequate diversity between strains that could be used to establish Map transmission patterns. Type C was the dominant strain of Map isolates (95.2%) identified in dairy goats (n = 21). Sub-typing of the Type C strains, based on variable number tandem repeats (VNTR) and mycobacterial interspersed repetitive units, identified 3 VNTR types: INMV 1 (n = 10), INMV 2 (n = 10), and a type not previously identified (n = 1). Only 2 sheep isolates could be identified; both were Type S, sub-type III. Current typing methods demonstrate little Map diversity in the dairy goat population and are therefore of limited use to investigate infection patterns.

L'objectif principal de la présente étude était d'identifier les souches circulantes de Mycobacterium avium sous-espèces paratuberculosis (MAP) dans des échantillons fécaux obtenus de populations de chèvre laitière (N = 29 fermes) et de brebis laitière (N = 21 fermes) en Ontario, Canada. Du sous-typage supplémentaire a été effectué afin de déterminer s'il y avait suffisamment de diversité entre les souches qui permettrait d'établir des patrons de transmission de MAP. Il a été déterminé que le Type C était la souche dominante d'isolats de MAP (95,2 %) chez les chèvres laitières (n = 21), alors que deux isolats ovins ont été identifiés comme étant du Type S/sous-type III (n = 2). Le sous-typage des souches du Type C, basé sur le nombre variable de séquences répétées en tandem (VNTR) et les unités répétitives entrecoupées des mycobactéries, a permis d'identifier trois types de VNTR : INMV 1 (n = 10), INMV 2 (n = 10), et un type encore non-identifié (n = 1). Les méthodes actuelles de typage ne permettent de démontrer que peu de diversité de MAP dans la population de chèvre laitière et sont ainsi d'utilité limitée pour étudier les patrons d'infection.(Traduit par Docteur Serge Messier).

Prevalence of Toxoplasma gondii and Other Gastrointestinal Parasites in Domestic Cats from Households in Thika Region, Kenya.

Gastrointestinal (GIT) parasites of domestic cats (Felis catus) not only cause morbidity but are also potential zoonotic agents. The current study aimed at establishing the prevalence of GIT parasites in cats kept by households in Thika region, Kenya. Fecal samples were collected randomly from 103 cats and analyzed for presence of parasites using standard parasitological methods. In descending order, the prevalence of the detected protozoa parasites was Isospora spp. 43.7% (95% CI: 40.4-47%), Cryptosporidium spp. 40.8% (95% CI: 37.5-44.1%), Toxoplasma gondii 7.8% (95% CI: 4.5-11.1%), and Entamoeba spp. 2.9% (95% CI: 1.6-6.2%). The prevalence of the observed helminths was Strongyloides stercoralis 43.7% (95% CI: 40.4-47%), Toxocara cati 23.3% (95% CI: 20-26.6%), Ancylostoma spp. 9.7% (95% CI: 6.4-13%), Dipylidium caninum 8.7% (95% CI: 5.4-12.0%), and Acanthocephala spp. 1.9% (95% CI: 1-4.2%). The percentage of cats excreting at least one species of parasite was 73.2% (95% CI = 69.9-76.5%). The study shows that the cats have high spectrum (9) of parasites which are known to affect the cat's health and some are of zoonotic significance.

Marked Differences in Mucosal Immune Responses Induced in Ileal versus Jejunal Peyer's Patches to Mycobacterium avium subsp. paratuberculosis Secreted Proteins following Targeted Enteric Infection in Young Calves.

In cattle, Mycobacterium avium subsp. paratuberculosis infection is primarily mediated through M cells overlying Peyer's patches (PP) in the ileum. The capacity of M. avium subsp. paratuberculosis to invade ileal PP (IPP) versus discrete PP in the jejunum (JPP) and subsequent differences in mucosal immune responses were investigated. Intestinal segments were surgically prepared in both mid-jejunum, containing two JPPs, and in terminal small intestine containing continuous IPP. M. avium subsp. paratuberculosis (109 CFU) was injected into the lumen of half of each intestinal segment when calves were 10-14 days-old and infection confirmed 1-2 months later by PCR and immunohistochemistry. Thirteen recombinant M. avium subsp. paratuberculosis proteins, previously identified as immunogenic, were used to analyze pathogen-specific B- and T-cell responses in PP and mesenteric lymph nodes. IgA plasma cell responses to 9 of 13 recombinant proteins were detected in JPP but not in IPP. Secretory IgA reacting in ELISA with 9 of the 13 recombinant proteins was detected in luminal contents from both jejunal and ileal segments. These observations support the conclusion that pathogen-specific IgA B cells were induced in JPP but not IPP early after a primary infection. The presence of secretory IgA in intestinal contents is consistent with dissemination of IgA plasma cells from the identified mucosa-associated immune induction sites. This is the first direct evidence for M. avium subsp. paratuberculosis uptake by bovine JPP and for local induction of pathogen-specific IgA plasma cell responses after enteric infection. We also provide evidence that bacterial invasion of IPP, a primary B lymphoid tissue, provides a novel strategy to evade induction of mucosal immune responses. Over 60% of PPs in the newborn calf small intestine is primary lymphoid tissue, which has significant implications when designing oral vaccines or diagnostic tests to detect early M. avium subsp. paratuberculosis infections.

Novel secreted antigens of Mycobacterium paratuberculosis as serodiagnostic biomarkers for Johne's disease in cattle.

Johne's disease is a chronic gastroenteritis of cattle caused by Mycobacterium avium subsp. paratuberculosis that afflicts 40% of dairy herds worldwide. M. avium subsp. paratuberculosis-infected cattle can remain asymptomatic for years while transmitting the pathogen via fecal contamination and milk. Current serodiagnosis with enzyme-linked immunosorbent assays (ELISAs) fails to detect asymptomatic M. avium subsp. paratuberculosis-infected cattle due to the use of poorly defined antigens and knowledge gaps in our understanding of M. avium subsp. paratuberculosis components eliciting pathogen-specific immune responses. We set out to (i) define a subset of proteins that contain putative antigenic targets and (ii) screen these antigen pools for immunogens relevant in detecting infection. To accomplish our first objective, we captured and resolved M. avium subsp. paratuberculosis-secreted proteins using a 2-step fractionation method and reverse-phase liquid chromatography to identify 162 unique proteins, of which 66 had not been previously observed in M. avium subsp. paratuberculosis culture filtrates. Subsequent screening of M. avium subsp. paratuberculosis-secreted proteins showed four antigens, of which one or more reacted on immunoblotting with individual serum samples from 35 M. avium subsp. paratuberculosis-infected cows. Moreover, these novel antigens reacted with sera from 6 low M. avium subsp. paratuberculosis shedders and 3 fecal-culture-positive cows labeled as ELISA seronegative. The specificity of these antigens was demonstrated using negative-control sera from uninfected calves (n = 5) and uninfected cows (n = 5), which did not react to any of these antigens in immunoblotting. As three of the four antigens are novel, their characterization and incorporation into an ELISA-based format will aid in detecting asymptomatic cattle in early or subclinical stages of disease.

The sensor kinase MprB is required for Rhodococcus equi virulence.

Rhodococcus equi is a soil bacterium and, like Mycobacterium tuberculosis, a member of the mycolata. Through possession of a virulence plasmid, it has the ability to infect the alveolar macrophages of foals, resulting in pyogranulomatous bronchopneumonia. The virulence plasmid has an orphan two-component system (TCS) regulatory gene, orf8, mutation of which completely attenuates virulence. This study attempted to find the cognate sensor kinase (SK) of orf8. Annotation of the R. equi strain 103 genome identified 23 TCSs encoded on the chromosome, which were used in a DNA microarray to compare TCS gene transcription in murine macrophage-like cells to growth in vitro. This identified six SKs as significantly up-regulated during growth in macrophages. Mutants of these SKs were constructed and their ability to persist in macrophages was determined with one SK, MprB, found to be required for intracellular survival. The attenuation of the mprB- mutant, and its complementation, was confirmed in a mouse virulence assay. In silico analysis of the R. equi genome sequence identified an MprA binding box motif homologous to that of M. tuberculosis, on mprA, pepD, sigB and sigE. The results of this study also show that R. equi responds to the macrophage environment differently from M. tuberculosis. MprB is the first SK identified as required for R. equi virulence and intracellular survival.

Antibody and cell-mediated immune responses to Staphylococcus aureus small colony variants and their parental strains associated with bovine mastitis.

Persistence of bovine Staphylococcus aureus mastitis may be associated with the small colony variant (SCV) form that is adapted to intracellular life and resists elimination by the immune system. This study evaluated antibody-mediated (AMIR) and cell-mediated immune responses (CMIR) to two bovine SCV forms and their parent strains isolated from cows with mastitis. Four groups of healthy cows, five cows/treatment group, were challenged by the intramammary route with naturally occurring bovine SCV Heba3231, its parent strain 3231, a hemB mutant displaying the SCV phenotype or its parent strain, Newbould 305. Blood and milk samples were collected at day 0 before challenge and at days 1, 14, 21 and 36 post-challenge to determine antigen-specific immunoglobulin (Ig) IgG(1) and IgG(2) antibody responses as indicators of type 2 and type 1 responses, respectively. At day 24 post-challenge cows in each group were inoculated with the UV-killed homologous strain intradermally in the neck to induce delayed-type hypersensitivity (DTH) as an indicator of CMIR. The SCV Heba3231 and 3231 strains induced significant IgG(1) and IgG(2) antibody responses in sera and in sera and milk whey, respectively. The hemB SCV mutant and Newbould 305 strains induced significant IgG(1) antibody in milk whey, and in sera and milk whey, respectively. The SCV Heba3231 and 3231 strains induced DTH, the hemB mutant induced intermediate hypersensitivity, and Newbould 305 failed to induce DTH. These results indicate marked differences in immune responses induced by parent and SCV forms of the same strain of S. aureus and by the two wild-type strains. This is the first study to evaluate both AMIR and CMIR in the context of persistent bovine mastitis to different and genetically characterized strains of S. aureus including two SCVs. The findings expand our understanding of immune responses to persistent S. aureus mastitis.

Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle and other farm ruminants, and is also a suspected pathogen of Crohn's disease in humans. Development of diagnostic methods for MAP infection has been a challenge over the last few decades. The objective of this study was to investigate the relationship between different methods for detection of MAP in milk and fecal samples. A total of 134 milk samples and 110 feces samples were collected from 146 individual cows in 14 MAP-infected herds in southwestern Ontario. Culture, IS900 polymerase chain reaction (PCR) and nested PCR methods were used for detecting MAP in milk; results were compared with those of fecal culture. A significant relationship was found between milk culture, direct PCR, and nested PCR (P < 0.05). The fecal culture results were not related to any of the 3 assay methods used for the milk samples (P > 0.10). Although fecal culture showed a higher sensitivity than the milk culture method, the difference was not significant (P = 0.2473). The number of MAP colony-forming units (CFU) isolated by culture from fecal samples was, on average, higher than that isolated from milk samples (P = 0.0083). There was no significant correlation between the number of CFU cultured from milk and from feces (Pearson correlation coefficient = 0.1957, N = 63, P = 0.1243). The animals with high numbers of CFU in milk culture may not be detected by fecal culture at all, and vise versa. A significant proportion (29% to 41%) of the positive animals would be missed if only 1 culture method, instead of both milk and feces, were to be used for diagnosis. This suggests that the shedding of MAP in feces and milk is not synchronized. Most of the infected cows were low-level shedders. The proportion of low-level shedders may even be underestimated because MAP is killed during decontamination, thus reducing the chance of detection. Therefore, to identify suspected Johne's-infected animals using the tests in this study, both milk and feces samples should be collected in duplicate to enhance the diagnostic rate. The high MAP kill rate identified in the culture methods during decontamination may be compensated for by using the nested PCR method, which had a higher sensitivity than the IS900 PCR method used.

Improved template DNA preparation procedure for detection of Mycobacterium avium subsp. paratuberculosis in milk by PCR.

Factors affecting the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by PCR in raw milk and their interactions were investigated. Three day old bulk tank raw milk (50 ml) samples were seeded with MAP at a level of an estimated 30 CFU/ml. Heat-treatment of raw milk before centrifugation significantly affected the partitioning of MAP in the cream, whey and pellet fractions. Based on the IS900 PCR results, MAP preferentially partitioned into the cream fraction in unheated raw milk, and into the pellet fraction in the heat-treated milk. Treatment with 0.75% hexadecylpyridinium chloride (HPC) helped collect MAP in cream fraction. Heat treatment, use of pooled cream and pellet fractions and treatment with HPC improved the detection by PCR significantly, while washing of pellets prior to DNA extraction did not. The limit of detection using our optimized procedure was an estimated 15-50 CFU in 50 ml, or

Development of improved method for isolation of Mycobacterium avium subsp. paratuberculosis from bulk tank milk: effect of age of milk, centrifugation, and decontamination.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease in cattle and it has been suggested that this organism may be associated with Crohn's disease in humans. Cows at the advanced stage of the disease shed this organism into both their milk and feces. The objective of this study was to develop a more efficient procedure for isolating MAP from bulk tank raw milk. Bulk tank raw milk (50 mL) samples 3 to 13 d old after collection without spiking were investigated to evaluate the effects of milk age on the efficacy of decontamination. Milk samples, 2 to 3 d old, were seeded with MAP at levels of 50 to 200 colony forming units/mL in experiments involving factorial design to evaluate 1) the effects of different decontaminating reagents and decontamination procedures on recovery of MAP, and 2) partition MAP in milk fractions after centrifugation in raw milk. Decontamination in 20 mL of 0.75% hexadecylpyridinium chloride (HPC) at room temperature (22 degrees C) for 2 to 5 h, with shaking, at intervals was found to be the most effective procedure for decontaminating milk 2 to 3 d old. Prolonged exposure to decontaminants, additional incubations in antibiotics, or at higher temperature (37 degrees C) significantly reduced recovery of live MAP. Enhanced growth of microbial contaminants was noticed in samples decontaminated overnight at room temperature compared to those decontaminated for 2 to 5 h. Decontamination of 6 d old milk samples required extra incubation in antibiotic brew. Decontamination of milk samples that are 8 d and older was not effective in removing microbial contaminants. The MAP cells preferentially partitioned into the cream fraction after centrifugation, and combining the milk cream and pellet fractions enhanced recovery of MAP. A recovery rate of 16.6% was estimated with the use of our improved protocol.

Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences.

The use of hsp60 gene sequences for phylogenetic study and identification of pathogenic marine vibrios was investigated. A 600-bp partial hsp60 gene was amplified by PCR and sequenced from 29 strains representing 15 Vibrio species within the family Vibrionaceae. Sequence comparison of the amplified partial hsp60 gene revealed 71-82% sequence identity among different Vibrio species and 96-100% sequence identity among epidemiologically distinct strains with the same species designation. This degree of discrimination allows unambiguous differentiation of all Vibrio species included in the current study from each other, as well as from Aeromonas hydrophila and Plesiomonas shigelloides, which are often misidentified as Vibrio species by conventional biochemical methods. Based on the hsp60 gene sequences, two previously unidentified shrimp isolates were found to be more closely related to Vibrio alginolyticus (93-94% sequence identity) than to Vibrio parahaemolyticus (89% sequence identity), whereas 16S rRNA gene analysis was unable to differentiate among these closely related species (95-97% sequence identity). Our results indicate that the hsp60 gene may be a useful alternative target for phylogenetic analysis and species identification of marine Vibrios to complement more conventional identification systems.