Enumeration of Mycobacterium avium subsp. paratuberculosis by quantitative real-time PCR, culture on solid media and optical densitometry

<p>Abstract</p> <p>Background</p> <p>Different approaches are used for determining the number of <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) cells in a suspension. The majority of them are based upon culture (determination of CFU) or visual/instrumental direct counting of MAP cells. In this study, we have compared the culture method with a previously published F57 based quantitative real-time PCR (F57qPCR) method, to determine their relative abilities to count the number of three different MAP isolates in suspensions with the same optical densities (OD). McFarland turbidity standards were also compared with F57qPCR and culture, due to its frequent inclusion and use in MAP studies.</p> <p>Findings</p> <p>The numbers of MAP in two-fold serial dilutions of isolates with respective OD measurements were determined by F57qPCR and culture. It was found that culture provided lower MAP CFU counts by approximately two log₁₀, compared to F57qPCR. The McFarland standards (as defined for <it>E. coli</it>) showed an almost perfect fit with the enumeration of MAP performed by F57qPCR.</p> <p>Conclusions</p> <p>It is recommended to use culture and/or qPCR estimations of MAP numbers in experiments where all subsequent counts are performed using the same method. It is certainly not recommended the use of culture as the standard for qPCR experiments and <it>vice versa</it>.</p

A Novel Multi-Antigen Virally Vectored Vaccine against Mycobacterium avium Subspecies paratuberculosis

BACKGROUND: Mycobacterium avium subspecies paratuberculosis causes systemic infection and chronic intestinal inflammation in many species including primates. Humans are exposed through milk and from sources of environmental contamination. Hitherto, the only vaccines available against Mycobacterium avium subspecies paratuberculosis have been limited to veterinary use and comprised attenuated or killed organisms. METHODS: We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins. HAV was transformed into DNA, human Adenovirus 5 (Ad5) and Modified Vaccinia Ankara (MVA) delivery vectors. Full length expression of the predicted 95 kDa fusion protein was confirmed. PRINCIPAL FINDINGS: Vaccination of naïve and Mycobacterium avium subspecies paratuberculosis infected C57BL/6 mice using DNA-prime/MVA-boost or Ad5-prime/MVA-boost protocols was highly immunogenic resulting in significant IFN-gamma ELISPOT responses by splenocytes against recombinant vaccine antigens and a range of HAV specific peptides. This included strong recognition of a T-cell epitope GFAEINPIA located near the C-terminus of the fusion protein. Antibody responses to recombinant vaccine antigens and HAV specific peptides but not GFAEINPIA, also occurred. No immune recognition of vaccine antigens occurred in any sham vaccinated Mycobacterium avium subspecies paratuberculosis infected mice. Vaccination using either protocol significantly attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection measured by qPCR in spleen and liver and the Ad5-prime/MVA-boost protocol also conferred some protection against subsequent challenge. No adverse effects of vaccination occurred in any of the mice. CONCLUSIONS/SIGNIFICANCE: A range of modern veterinary and clinical vaccines for the treatment and prevention of disease caused by Mycobacterium avium subspecies paratuberculosis are needed. The present vaccine proved to be highly immunogenic without adverse effect in mice and both attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection and conferred protection against subsequent challenge. Further studies of the present vaccine in naturally infected animals and humans are indicated

Specific Immunoassays Confirm Association of Mycobacterium avium Subsp. paratuberculosis with Type-1 but Not Type-2 Diabetes Mellitus

Mycobacterium avium subspecies paratuberculosis (MAP) is a versatile pathogen with a broad host range. Its association with type-1 diabetes mellitus (T1DM) has been recently proposed. Rapid identification of infectious agents such as MAP in diabetic patients at the level of clinics might be helpful in deciphering the role of chronic bacterial infection in the development of autoimmune diseases such as T1DM.We describe use of an ELISA method to identify live circulating MAP through the detection of a cell envelope protein, MptD by a specific M13 phage--fMptD. We also used another ELISA format to detect immune response to MptD peptide. Both the methods were tested with blood plasma obtained from T1DM, type-2 diabetes (T2DM) patients and non-diabetic controls. Our results demonstrate MptD and fMptD ELISA assays to be accurate and sensitive to detect MAP bacilli in a large fraction (47.3%) of T1DM patients as compared to non-diabetic controls (12.6%) and those with confirmed T2DM (7.7%). Comparative analysis of ELISA assays performed here with 3 other MAP antigen preparations, namely HbHA, Gsd and whole cell MAP lysates confirmed comparable sensitivity of the MptD peptide and the fMptD based ELISA assays. Moreover, we were successful in demonstrating positive bacterial culture in two of the clinical specimen derived from T1DM patients.The MptD peptide/fMptD based ELISA or similar tests could be suggested as rapid and specific field level diagnostic tests for the identification of MAP in diabetic patients and for finding the explanations towards the occurrence of type-1 or type-2 diabetes in the light of an active infectious trigger

Emerging technologies for the management of Type 1 diabetes in pregnancy

Purpose of Review: The purpose of the study is to discuss emerging technologies available in the management of type 1 diabetes in pregnancy. Recent Findings: The latest evidence suggests that continuous glucose monitoring (CGM) should be offered to all women on intensive insulin therapy in early pregnancy. Studies have additionally demonstrated the ability of CGM to help gain insight into specific glucose profiles as they relate to glycaemic targets and pregnancy outcomes. Despite new studies comparing insulin pump therapy to multiple daily injections, its effectiveness in improving glucose and pregnancy outcomes remains unclear. Sensor-integrated insulin delivery (also called artificial pancreas or closed-loop insulin delivery) in pregnancy has been demonstrated to improve time in target and performs well despite the changing insulin demands of pregnancy. Summary: Emerging technologies show promise in the management of type 1 diabetes in pregnancy; however, research must continue to keep up as technology advances. Further research is needed to clarify the role technology can play in optimising glucose control before and during pregnancy as well as to understand which women are candidates for sensor-integrated insulin delivery

Where Are All the Mycobacterium avium Subspecies paratuberculosis in Patients with Crohn's Disease?

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic granulomatous inflammation of the intestines, Johne's disease, in dairy cows and every other species of mammal in which it has been identified. MAP has been identified in the mucosal layer and deeper bowel wall in patients with Crohn's disease by methods other than light microscopy, and by direct visualization in small numbers by light microscopy. MAP has not been accepted as the cause of Crohn's disease in part because it has not been seen under the microscope in large numbers in the intestines of patients with Crohn's disease. An analysis of the literature on the pathology of Crohn's disease and on possible MAP infection in Crohn's patients suggests that MAP might directly infect endothelial cells and adipocytes and cause them to proliferate, causing focal obstruction within already existing vessels (including granuloma formation), the development of new vessels (neoangiogenesis and lymphangiogenesis), and the “creeping fat” of the mesentery that is unique in human pathology to Crohn's disease but also occurs in bovine Johne's disease. Large numbers of MAP might therefore be found in the mesentery attached to segments of intestine affected by Crohn's disease rather than in the bowel wall, the blood and lymphatic vessels running through the mesentery, or the mesenteric fat itself. The walls of fistulas might result from the neoangiogenesis or lymphangiogenesis that occurs in the bowel wall in Crohn's disease and therefore are also possible sites of large numbers of MAP. The direct visualization of large numbers of MAP organisms in the tissues of patients with Crohn's disease will help establish that MAP causes Crohn's disease

Exploring the Zoonotic Potential of Mycobacterium avium Subspecies paratuberculosis through Comparative Genomics

A comparative genomics approach was utilised to compare the genomes of Mycobacterium avium subspecies paratuberculosis (MAP) isolated from early onset paediatric Crohn's disease (CD) patients as well as Johne's diseased animals. Draft genome sequences were produced for MAP isolates derived from four CD patients, one ulcerative colitis (UC) patient, and two non-inflammatory bowel disease (IBD) control individuals using Illumina sequencing, complemented by comparative genome hybridisation (CGH). MAP isolates derived from two bovine and one ovine host were also subjected to whole genome sequencing and CGH. All seven human derived MAP isolates were highly genetically similar and clustered together with one bovine type isolate following phylogenetic analysis. Three other sequenced isolates (including the reference bovine derived isolate K10) were genetically distinct. The human isolates contained two large tandem duplications, the organisations of which were confirmed by PCR. Designated vGI-17 and vGI-18 these duplications spanned 63 and 109 open reading frames, respectively. PCR screening of over 30 additional MAP isolates (3 human derived, 27 animal derived and one environmental isolate) confirmed that vGI-17 and vGI-18 are common across many isolates. Quantitative real-time PCR of vGI-17 demonstrated that the proportion of cells containing the vGI-17 duplication varied between 0.01 to 15% amongst isolates with human isolates containing a higher proportion of vGI-17 compared to most animal isolates. These findings suggest these duplications are transient genomic rearrangements. We hypothesise that the over-representation of vGI-17 in human derived MAP strains may enhance their ability to infect or persist within a human host by increasing genome redundancy and conferring crude regulation of protein expression across biologically important regions

Deficit of social cognition in subjects with surgically treated frontal lobe lesions and in subjects affected by schizophrenia

The ability of humans to predict and explain other people’s behaviour by attributing independent mental states such as desires and beliefs to them, is considered to be due to our ability to construct a “Theory of Mind”. Recently, several neuroimaging studies have implicated the medial frontal lobes as playing a critical role in a dedicated “mentalizing” or “Theory of Mind” network in the human brain. In this study we compare the performance of patients with right and left medial prefrontal lobe lesions in theory of mind and in social cognition tasks, with the performance of people with schizophrenia. We report a similar social cognitive profile between patients with prefrontal lobe lesions and schizophrenic subjects in terms of understanding of false beliefs, in understanding social situations and in using tactical strategies. These findings are relevant for the functional anatomy of “Theory of Mind”

The impact of diabetes on the pathogenesis of sepsis

Diabetes is associated with an increased susceptibility to infection and sepsis. Conflicting data exist on whether the mortality of patients with sepsis is influenced by the presence of diabetes, fuelling the ongoing debate on the benefit of tight glucose regulation in patients with sepsis. The main reason for which diabetes predisposes to infection appears to be abnormalities of the host response, particularly in neutrophil chemotaxis, adhesion and intracellular killing, defects that have been attributed to the effect of hyperglycaemia. There is also evidence for defects in humoral immunity, and this may play a larger role than previously recognised. We review the literature on the immune response in diabetes and its potential contribution to the pathogenesis of sepsis. In addition, the effect of diabetes treatment on the immune response is discussed, with specific reference to insulin, metformin, sulphonylureas and thiazolidinediones