Mycobacteria

Bovine tuberculosis (BTB) is endemic in Ethiopian livestock. Disease prevalence varies greatly by species, geography, husband-ry system and diagnostic methods used. A substantial body of work has been done on BTB prevalence in Ethiopia over the last decade, the true burden of BTB however, is still largely unknown. This review highlighted gaps and weakness in BTB diagnostics and the urgent need to standardize/harmonize testing methods. It discusses the prevalence of M. tuberculosis and Non Tuber-culous Mycobacteria (NTM) in livestock and their possible interference with the BTB diagnostic methods. This paper finally discusses possible control options for the disease, particularly in the intensive milk farm systems in the wake of increased and or emerging dairy farms in the country.

Alois Alzheimer might have mentioned plaques and tangles in a single short paper on pre-senile dementia in 1907, but it was the co-discover of Alzheimer's disease (AD), Oskar Fischer, who in that same year far more extensively reported neuritic plaque in 12 cases of senile dementia, a condition which he and many others refused to differentiate from Alzheimer's " pre-senile " dementia. Fischer, Alzheimer's great rival, speculated that for the most part these plaques, found only in senile demented patients, caused their dementia. Moreover, Fischer felt such cerebral plaque to be the result of an infection and was very specific as to the sort of infection that might be involved. He felt that he had spotted, throughout his brain autopsies, a tubercular-like Actinobacteria then called Streptothrix (Actinomycosis), often and repeatedly confused with the filamentous cell-wall-deficient forms of the tubercular bacilli. At this point for Fischer, this was the possible infectious cause of AD. To be sure, Oskar Fischer was the first on record to suggest that chronic infection might be causative for what we today call AD. Fischer's infectious view never gained immediate popularity, although today, more than a century later, a volume of data supporting such an approach has begun to accumulate. But was Fischer's specific microbe on the right track to discovering the cause of AD to begin with? Documents uncovered since then seem to suggest that he was considerably closer than anyone else—either then or since. In June of 2017, a University of Bristol study in the UK found a 5 to 10-fold increase in Actinobacteria (order Actinomycetales) population in postmortem AD brains compared with controls [1]. Oskar Fischer's Steptothrix was an Actinobacteria as well, as was the filamentous tuberculosis that Streptothrix was so often mistaken for. But specifically, the Bristol group found an Actinobactor called Propionibacterium acnes (P. Acnes) in autopsied AD brains—a common organism, traditional felt to be non-pathogenic and commonly found on our skin which can

IN PRESS

Every seventy-two seconds someone in America develops Alzheimer’s disease (AD). And it has been said that almost everyone living long enough will eventually show evidence of Alzheimer’s disease. Thus far its cause has remained elusive.
Nevertheless, recently, study after study, in which scientists have injected human Alzheimer diseased brain tissue into mice and other laboratory animals that later developed the disease have left little doubt that Alzheimer’s arises from an infectious process, the focus of debate seeming to be which particular disease. But clearly, it must be a disease that is not only statistically widespread in the world today, but that was also prevalent at the time of Dr. Alois Alzheimer.
To be sure, it was German neuropathologist Oskar Fischer of the Prague school of neuropathology, Alzheimer’s great rival, who was the first to suggest that infection might be causative for Alzheimer’s. Fischer’s credentials: he was the co-discoverer of Alzheimer’s disease and tirelessly did autopsies on the brains of patients with Alzheimer’s.
Fischer’s infectious view never gained immediate popularity, although today, more than a century later, a volume of data supporting such an approach has begun to accumulate. But was Fischer’s specific microbe on the right track to discovering the cause of Alzheimer’s to begin with? The evidence uncovered in this book seems to suggest that he was considerably closer than anyone else—either then or since.
In this volume, doctor and medical researcher Lawrence Broxmeyer presents a stunning hypothesis about the bacterial nature of Alzheimer’s disease, forgotten long ago. Immensely readable and enormously well-researched, this work of medical history should change the way we think about, diagnose, and treat this debilitating condition.

KIRKUS REVIEWS: "The author is an internist and a medical researcher, and his double mastery of both the scientific minutiae and historical nuances of his subject matter is breathtaking. This is more than an account of a scientific debate--it's also an examination of the sometimes--unempirical way that such debate proceeds, as it's conducted by human beings with agendas of their own. Although it's a relatively short book--less than 200 pages of text--it is by no means a quick read. Nevertheless, readers with strong science backgrounds will be impressed by the author's undeniable competence, as well as his journalistic approach to chartering the evolution of thought regarding one of our era's most challenging diseases."

The thought that tuberculosis and the mycobacteria could cause diabetes seems farfetched, but is not. The peculiar relationship and frequent association of diabetes mellitus and tuberculosis has been observed for more than 2000 years, yet the reason for this correlation is, to this day, not known. Before the discovery of insulin, a diagnosis of diabetes was a death sentence within 5 years, and the usual cause of that death was tuberculosis. Despite this, in the 5th century, tuberculosis was already being portrayed as a "complication" of diabetes, a view little changed to this day, parroting Root's original 1934 description of "a one-sided relationship": tuberculosis still seen as a common complication of diabetes, while diabetes is thought to be no more common among TB patients than in the population at large. To Nichol's, this was "not logically tenable" and in his study of 178 otherwise healthy, non-diabetic military men with tuberculosis at Fitzsimmons Army Hospital, one-third had abnormal glucose screening tests. But despite his findings and those of Reaud in New York and others, this was not being recognized elsewhere, and Nichols wanted to know why. Nichols concluded that the incidence of diabetes among tuberculosis patients was considerably underestimated and that in tuberculosis patients, diabetes develops quite commonly. Diabetes was easy to detect. Tuberculosis and the mycobacteria were not. The evidence for a mycobacterial cause of diabetes is mounting rapidly. Schwartz and Haas both linked Type-2 diabetes to tuberculosis. And the pancreatic islet amyloid deposits that they found as a by-product of systemic tubercular infection have recently been dissolved by rifampicin, a first line drug against tuberculosis. Engelbach spoke of "transitory" diabetes in TB and Karachunskii noted changes in carbohydrate metabolism in patients with tuberculosis which commonly led to insulin deficiency with persistent hyperglycemia. Furthermore, mycobacterial elements have been shown recently not only to cause "autoimmune" Type-1 diabetes in NOD (non-obese diabetic) mice, but act as a vaccine to stop the inevitable diabetes that would otherwise materialize. The documentation of patient cases where TB has preceded and come before the development of diabetes is extensive yet underplayed and both Lin's and Tsai's studies speak of tuberculosis complicated by diabetes. Diabetes has been around since the first century AD, in a perpetual state of coping and managing. It is time, it is long past time, to cure diabetes. But current models as to its cause are not equipping us to do so.

Mycobacterium avium subsp. paratuberculosis (MAP) is the bacterium that causes Johne’s disease in cattle. Although infected cattle can be identified by examining fecal, blood, or milk samples, the gold standard is identification of MAP in tissue samples postmortem. Although tissue samples are commonly frozen, the ability to detect MAP in frozen–thawed tissue samples has apparently not been reported. We therefore determined the ability to detect MAP in tissue samples following freezing. Tissue samples were collected from calves that were either inoculated (IN) 3 mo prior, or contact-exposed (CE) for 3 mo. Following autopsy, tissues were immediately processed for culture, followed by DNA extraction and detection by qPCR. Samples were categorized as positive or negative based on the cycle threshold (Ct) value. The remaining unprocessed tissue samples were frozen at −80°C. After 18 mo, 50 tissue samples designated MAP-positive were thawed and processed for detection of MAP. Four (8%) samples were qPCR-negative, and Ct values of the remaining 46 samples were higher after freezing. Given the small numerical change in Ct values for MAP-positive samples after 18 mo of frozen storage, freezing and thawing may have had some deleterious effects on MAP detection in tissues. Although the decrease in ability to detect MAP-positive samples was minor for IN calves, there may be a greater effect for CE calves that should be considered when freezing tissue samples.

Background: Tuberculosis (TB) is preventable and completely curable major public health problem in the world. Even though the number of TB death decreased by 22% in the past fifteen years it remains one of the top 10 causes of death and the leading cause from a single infectious agent, ranking above HIV/AIDS. It is the largest killer among communicable diseases in age group of 15 to 49years, when humans are most productive. Ethiopia is among the 22 high TB-burden and among the 27 high MDR-TB burden countries in the world with case detection and treatment outcome vary across regions.

The TSE'S or transmissible spongiform enchephalopathies, include bovine spongiform encephalopathy (also called BSE or " mad cow disease "), Creutzfeldt– Jakob disease (CJD) in humans, and " scrapie " in sheep or goats (caprine spongiform encephalopathy). They remain a mystery, their cause still hotly debated. Current mad cow diagnosis lies solely in the detection of late appearing " prions " , an acronym for hypothesized, geneless, misfolded proteins, somehow claimed to cause the disease. Yet laboratory preparations of prions contain other things, which could include unidentified bacteria or viruses. And the only real evidence that prion originator Stanley Prusiner had in his original paper that the disease agent behind " Scrapie " in sheep and goats was devoid of DNA or RNA– was based upon the fact that he couldn't find any. Furthermore, the rigors of prion purification alone, might, in and of themselves, have killed any causative microorganism and Heino Dringer, who did pioneer work on their nature, candidly predicts " it will turn out that the prion concept is wrong. " Roels and Walravens as well as Hartly traced Mad Cow to Mycobacterium bovis. Moreover, epidemiologic maps of the origins and peak incidence of Mad Cow in the UK, suggestively match those of England's areas of highest bovine tuberculosis, the Southwest. The neurotaxic potential of bovine tuberculosis has for some time been well known. By 1911 Alois Alzheimer called attention to " a characteristic condition of the cortical tissue which Fischer referred to as 'spongy cortical wasting " in Alzheimer's disease (AD). But behind AD, Fischer suspected a microbe called Streptothrix which was constantly being mistaken and confused for tuberculosis. Our present investigation of the TSEs clearly shows cell-wall-deficient (CWD) tubercular mycobacteria present, verified by molecular analysis, ELISA, PCR and microscopy to cause spongiform encephalopathy.

Johne's disease is a worldwide concern, as it causes huge economic losses. The etiological agent, Mycobac-terium avium ssp. paratuberculosis (MAP), has limited genetic diversity, impeding efforts to understand transmission and distribution of strain types. Whole-genome sequencing was previously performed on a representative set of MAP isolates from Canadian dairy herds and 9 divergent clades were identified. Four clades were of particular interest, as they were either MAP types rarely reported in North America, or they represented a substantial proportion of isolates recovered from dairy farms in Canada. One clade included type I/III isolates, whereas the remaining clades included type II isolates. Variant sites in the MAP genome are often separated by thousands of base pairs, limiting use of single nucleotide polymorphism (SNP)-based genotyp-ing on a single genomic region. Therefore, a SNP-PCR assay was developed to facilitate interrogation of 5 SNP in 2 distant regions of the genome, linking them together in a single PCR reaction for subsequent Sanger sequencing. This high-throughput assay enabled discrimination of 602 MAP isolates from 264 herds (from all 10 provinces). More than 1 isolate was cultured from 133 herds, 14 of which included multiple subtypes. A previously identified dominant type included 87% of isolates, whereas the Bison type was more widespread than previously reported. The latter type and isolates from a second clade of interest were overrepresented in Québec and Saskatchewan, respectively. In conclusion , the distribution and relative frequency of MAP subtypes within Canadian dairy herds were assessed using a novel SNP-based typing assay. These findings will contribute to understanding the clinical relevance and transmission dynamics of MAP in this population and elsewhere.

Se denominan micobacterias atípicas todas las especies del gé nero Mycobacterium capaces de producir enfermedades en el hombre, excepto las que forman el complejo M. tuberculosis y M. leprae. Aunque el té rmino «atípicas» ha sido muy criticado y se han propuesto diversas alternativas, como micobacterias ambientales, micobacterias anó nimas y micobacterias no tuberculosas (MNT), el hecho cierto es que sigue siendo ampliamente utilizado en la literatura mé dica y en la prá ctica clínica 1 .

Tuberculosis (TB) caused by Mycobacterium bovis is a re-emerging disease of livestock that is of major economic importance worldwide, as well as being a zoonotic risk. There is significant heritability for host resistance to bovine TB (bTB) in dairy cattle. To identify resistance loci for bTB, we undertook a genome-wide association study in female Holstein–Friesian cattle with 592 cases and 559 age-matched controls from case herds. Cases and controls were categorised into distinct phenotypes: skin test and lesion positive vs skin test negative on multiple occasions, respectively. These animals were genotyped with the Illumina BovineHD 700K BeadChip. Genome-wide rapid association using linear and logistic mixed models and regression (GRAMMAR), regional heritability mapping (RHM) and haplotype-sharing analysis identified two novel resistance loci that attained chromosome-wise significance, protein tyrosine phosphatase receptor T (PTPRT; P=4.8 × 10−7) and myosin IIIB (MYO3B; P=5.4 × 10−6). We estimated that 21% of the phenotypic variance in TB resistance could be explained by all of the informative single-nucleotide polymorphisms, of which the region encompassing the PTPRT gene accounted for 6.2% of the variance and a further 3.6% was associated with a putative copy number variant in MYO3B. The results from this study add to our understanding of variation in host control of infection and suggest that genetic marker-based selection for resistance to bTB has the potential to make a significant contribution to bTB control.

The authors describe a study of the tuberculosis (TB) incidence in cattle exposed to low doses of radiation resulting from the Chernobyl (pronounced ‘Chornobyl’ in Ukrainian) nuclear plant catastrophe in 1986. The purpose of the study was to determine if ionising radiation influences the number of outbreaks of bovine TB and their severity on farms in the Kyiv, Cherkasy and Chernigiv regions of Ukraine. These farms are all located within a 200 km radius of Chernobyl and have had low-dose radiation pollution. Pathological and blood samples were taken from cattle in those regions that had positive TB skin tests. Mycobacterium spp. were isolated, differentiated by PCR, analysed and tested in guinea-pigs and rabbits. Species differentiation showed a significant percentage of atypical mycobacteria, which resulted in the allergic reactions to tuberculin antigen in the skin test. Mixed infection of M. bovis and M. avium subsp. hominissuis was found in three cases. The results concluded that low-dose radiation plays a major role in the occurrence of bovine TB in regions affected by the Chernobyl nuclear disaster.

BACKGROUND AND PURPOSE:
Phagosomal maturation arrest is known to play a central role in the survival of pathogenic mycobacteria within macrophages. The maturation arrest of mycobacterial phagosome results from the retention of tryptophan-aspartate-containing coat protein (TACO) on this organelle, enabling successful replication of the pathogen. We have shown earlier that vitamin D(3) and retinoic acid (RA) down-regulate TACO gene transcription in a dose-dependent manner.
METHODS:
In this study, we analyzed the promoter region of TACO gene using bioinformatics tools and observed that the vitamin D receptor (VDR)/retinoid-X-receptor (RXR) response sequence was highly functional. We also evaluated the effect of treatment with vitamin D(3)/RA on Mycobacterium tuberculosis entry and survival in cultured human macrophages.
RESULTS:
TACO gene down-regulation observed with vitamin D(3)/RA treatment occurred through modulation of this gene via the VDR/RXR response sequence present in the promoter region of TACO gene. Treatment of macrophages with vitamin D(3)/RA allows maturation of mycobacterial phagosome, leading to degradation of the pathogen.
CONCLUSIONS:
Our results elucidate the mechanism of TACO gene down-regulation observed with vitamin D(3)/RA. Furthermore, the results revealed that vitamin D(3)/RA treatment inhibits mycobacterial entry as well as survival within macrophages, possibly through rescue of phagosome maturation arrest. The developing knowledge in this area suggests that vitamin D(3)/RA may be of importance in the treatment of intracellular infection, particularly tuberculosis.

Mycobacterium species are the source of a variety of infectious diseases in a range of hosts. Genome based methods are used to understand the adaptation of each pathogenic species to its unique niche. In this work, we report the comparison of pathogenic and non-pathogenic Mycobacterium genomes. Phylogenetic trees were constructed using sequence of core orthologs, gene content and gene order. It is found that the genome based methods can better resolve the inter-species evolutionary distances compared to the conventional 16S based tree. Phylogeny based on gene order highlights distinct evolutionary characteristics as compared to the methods based on sequence, as illustrated by the shift in the relative position of M. abscessus. This difference in gene order among the Mycobacterium species is further investigated using a detailed synteny analysis. It is found that while rearrangements between some Mycobacterium genomes are local within synteny blocks, few possess global rearrangements across the genomes. The study illustrates how a combination of different genome based methods is essential to build a robust phylogenetic relationship between closely related organisms.

The present study was designed to determine the half-life of gfp m 2+ mRNA, which encodes mycobacterial codon-optimised highly fluorescent GFP m 2+ protein, and to find out whether mycobacterial promoter activity can be quantitated more accurately using the mRNA levels of the reporter gene, gfp m 2+ , than the fluorescence intensity of the GFP m 2+ protein. Quantitative PCR of gfp m 2+ mRNA in the pulse-chased samples of the rifampicin-treated Mycobacterium smegmatis/gfp m 2+ transformant showed the half-life of gfp m 2+ mRNA to be 4.081 min. The levels of the gfp m 2+ mRNA and the fluorescence intensity of the GFP m 2+ protein, which were expressed by the promoters of Mycobacterium tuberculosis cell division gene, ftsZ (MtftsZ), were determined using quantitative PCR and fluorescence spectrophotometry, respectively. The data revealed that quantification of mycobacterial promoter activity by determining the gfp m 2+ mRNA levels is more accurate and statistically significant than the measurement of GFP m 2+ fluorescence intensity, especially for weak promoters.

Mycobacterium avium subsp. paratuberculosis (MAP) is the bacterium that causes Johne’s disease in cattle. Although infected cattle can be identified by examining fecal, blood, or milk samples, the gold standard is identification of MAP in tissue samples postmortem. Although tissue samples are commonly frozen, the ability to detect MAP in frozen–thawed tissue samples has apparently not been reported. We therefore determined the ability to detect MAP in tissue samples following freezing. Tissue samples were collected from calves that were either inoculated (IN) 3 mo prior, or contact-exposed (CE) for 3 mo. Following autopsy, tissues were immediately processed for culture, followed by DNA extraction and detection by qPCR. Samples were categorized as positive or negative based on the cycle threshold (Ct) value. The remaining unprocessed tissue samples were frozen at −80°C. After 18 mo, 50 tissue samples designated MAP-positive were thawed and processed for detection of MAP. Four (8%) sa...

Se comunican dos casos clínicos poco frecuentes de micobacteriosis cutáneas; uno adquirido en la comunidad y uno en medio hospitalario, ambos pacientes del género masculino, con dermatosis crónicas constituidas por úlceras, fístulas y placas de aspecto papilomatoso. El diagnóstico fue tardío y la evolución no fue satisfactoria, a pesar de prescribir tratamiento adecuado contra Mycobacterium chelonae. El propósito de este artículo es mostrar la manifestación clínica de las lesiones para sospecha diagnóstica temprana y oportuna de las diferentes disciplinas médicas que participan en la atención de los pacientes con estas infecciones.

This paper reports the two rare clinical cases of cutaneous mycobacterial infections. One of them acquired the infection within the community while the other was a noso-comial case, both cases were male with chronic ulcers, fistulae and papilloma-like lesions. In both cases diagnosis was late and evolution, despite correct Mycobacterium chelonae treatment, was unsatisfactory. The purpose of this paper is to show the clinical presentation and accurate suspicion of infection of the different medical areas involved in its management.

Rapidly growing mycobacteria (RGM) are common soil saprophytes, but certain strains cause infections in human and animals. The
infections due to RGM have been increasing in past decades and are often difficult to treat. The susceptibility to antibiotics is regularly
evaluated in clinical isolates of RGM, but the data on soil RGM are missing. The objectives of this study was to isolate RGM from four
grassland soils with different impact of manuring, and assess their resistance to antibiotics and the ability to grow at 37°C and 42°C.
Since isolation of RGM from soil is a challenge, a conventional decontamination method (NaOH/malachite green/cycloheximide) and a
recent method based on olive oil/SDS demulsification were compared. The olive oil/SDS method was less efficient, mainly because of
the emulsion instability and plate overgrowing with other bacteria. Altogether, 44 isolates were obtained and 23 representatives of
different RGM genotypes were screened. The number of isolates per soil decreased with increasing soil pH, consistently with previous
findings that mycobacteria were more abundant in low pH soils. Most of the isolates belonged to the Mycobacterium fortuitum group.
The majority of isolates was resistant to 2-4 antibiotics. Multiresistant strains occurred also in a control soil that has a long history
without the exposure to antibiotic-containing manure. Seven isolates grew at 37°C, including the species M. septicum and M. fortuitum
known for infections in humans. This study shows that multiresistant RGM close to known human pathogens occur in grassland soils
regardless the soil history of manuring.

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is the third most common mycobacterial disease after tuberculosis and leprosy. The present treatment options are limited and emergence of treatment resistant isolates represents a serious concern and a need for better therapeutics. Conventional drug discovery methods are time consuming and labor-intensive. Unfortunately, the slow growing nature of M. ulcerans in experimental conditions is also a barrier for drug discovery and development. In contrast, recent advancements in complete genome sequencing, in combination with cheminformatics and computational biology, represent an attractive alternative approach for the identification of therapeutic candidates worthy of experimental research. A computational, comparative genomics workflow was defined for the identification of novel therapeutic candidates against M. ulcerans, with the aim that a selected target should be essential to the pathogen, and have no homology in the human host. Initially, a total of 424 genes were predicted as essential from the M. ulcerans genome, via homology searching of essential genome content from 20 different bacteria. Metabolic pathway analysis showed that the most essential genes are associated with carbohydrate and amino acid metabolism. Among these, 236 proteins were identified as non-host and essential, and could serve as potential drug and vaccine candidates. Several drug target prioritization parameters including druggability were also calculated. Enzymes from several pathways are discussed as potential drug targets, including those from cell wall synthesis, thiamine biosynthesis, protein biosynthesis, and histidine biosynthesis. It is expected that our data will facilitate selection of M. ulcerans proteins for successful entry into drug design pipelines.

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is the third most common mycobacterial disease after tuberculosis and leprosy. The present treatment options are limited and emergence of treatment resistant isolates represents a serious concern and a need for better therapeutics. Conventional drug discovery methods are time consuming and labor-intensive. Unfortunately, the slow growing nature of M. ulcerans in experimental conditions is also a barrier for drug discovery and development. In contrast, recent advancements in complete genome sequencing, in combination with cheminformatics and computational biology, represent an attractive alternative approach for the identification of therapeutic candidates worthy of experimental research. A computational, comparative genomics workflow was defined for the identification of novel therapeutic candidates against M. ulcerans, with the aim that a selected target should be essential to the pathogen, and have no homology in the human host. Initially, a total of 424 genes were predicted as essential from the M. ulcerans genome, via homology searching of essential genome content from 20 different bacteria. Metabolic pathway analysis showed that the most essential genes are associated with carbohydrate and amino acid metabolism. Among these, 236 proteins were identified as non-host and essential, and could serve as potential drug and vaccine candidates. Several drug target prioritization parameters including druggability were also calculated. Enzymes from several pathways are discussed as potential drug targets, including those from cell wall synthesis, thiamine biosynthesis, protein biosynthesis, and histidine biosynthesis. It is expected that our data will facilitate selection of M. ulcerans proteins for successful entry into drug design pipelines.

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be * Corresponding author at: S2-064 Schurman Hall,

The skin of amphibians has unique structural properties and physiologic functions that make amphibians particularly sensitive to environmental perturbations and cutaneous injury. This inherent sensitivity makes the skin a critically important site of evaluation in both clinical and postmortem examination of the amphibian patient. Many of the described amphibian skin diseases can be traced back to factors related to captive husbandry. Cutaneous injuries, alterations in water quality (such as pH, ammonia), and exposure to chemical irritants, are all potential hazards of the captive environment that can lead to skin irritation. Furthermore, many infectious diseases of amphibian skin such as "red leg" syndrome, mycobacteriosis, and saprolegniasis are often secondary to environmental factors and require correction of underlying lapses in husbandry for control. Other amphibian skin diseases, such as chytridiomycosis and systemic iridovirus infections are not yet associated with specific environmental cofactors but have been associated with significant mortalities in captive and/or wild amphibians. Amphibian skin diseases often have a similar gross appearance with cutaneous hyperemia and discoloration, cutaneous papules and nodules, and ulceration being among the most common presentations. This review emphasizes conditions most often encountered in amphibian dermatology as well as recently recognized diseases such as chytridiomycosis.

Mycobacterium kansasii disease present epidemiological, clinical and radiological features similar to Mycobacterium tuberculosis. Mycobacterium kansasii is the second most frequent mycobacteria isolated in human immunodeficiency virus infected patients. Confirmed diagnosis is often difficult according with the American Thoracic Society criteria. Here we describe a patient with AIDS that developed a severe lung compromise due to Mycobacterium kansasii. Epidemiological, clinical, radiological and diagnosis methods are analyzed.

Interleukin-10 is an immunomodulatory cytokine that has been implicated, along with IFN-c, in the disease sequelae of mycobacterial infection. In order to investigate the role of IL-10 in marsupial disease models we sequenced and characterised the IL10 gene in the tammar wallaby (Macropus eugenii) and rufous hare-wallaby (Lagorchestes hirsutus). An isoform IL-10D3, in which an in-frame deletion of exon 3 occurs, was discovered in both macropod species. Analysis of wallaby and other reported marsupial IL-10 homologs suggests that while marsupial IL-10 is comparable to that of human IL-10, the predicted IL-10D3 protein may play a more complicated role in the modulation of IL-10-directed responses. Expression of the canonical gene and splicing variant was confirmed in both wallabies, and the rufous hare-wallaby showed differential expression across lymph node, spleen and liver, with isoform expression detected in the lymph node. This characterisation and expression of IL-10 in de novo tissues provides a basis for further study into the role of IL-10 in disease models in marsupials.

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be * Corresponding author at: S2-064 Schurman Hall,

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be * Corresponding author at: S2-064 Schurman Hall,

Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multidrug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0-7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. , 24390106 (S Matsumoto), 24501015 (Y Ozeki).

Among the costs attributed to Mycobacterium avium ssp. paratuberculosis (MAP) infection in dairy cattle, the effects on reproduction and culling are the least documented. To estimate the cost of MAP infections and Johne's disease in a dairy herd, the rates of calving and culling were calculated for cows in each stage of MAP infection relative to uninfected cows. Data from 6 commercial dairy herds, consisting of 2,818 cows with 2,754 calvings and 1,483 cullings, were used for analysis. Every cow in each study herd was tested regularly for MAP, and herds were followed for between 4 and 7 yr. An ordinal categorical variable for Johne's disease status [test-negative, low-positive (low-shedding or ELISA-positive only), or high-shedding] was defined as a time-dependent variable for all cows with at least 1 positive test result or 2 negative test results. A Cox regression model, stratified on herd and controlling for the time-dependent infection variable, was used to analyze time to culling. Nonshedding animals were significantly less likely to be culled in comparison with animals in the low-shedding or ELISA-positive category, and highshedding animals had nonsignificantly higher culling rates than low-shedding or ELISA-positive animals. Time to calving was analyzed using a proportional rates model, an analog to the Andersen-Gill regression model suitable for recurrent event data, stratifying on herd and weighted to adjust for the dependent censoring caused by the culling effects described above. Highshedding animals had lower calving rates in comparison with low-shedding or ELISA-positive animals, which tended to have higher calving rates than test-negative animals.

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be * Corresponding author at: S2-064 Schurman Hall,

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of nontuberculous mycobacterial (NTM) isolates was evaluated in this study. Overall, 125 NTM isolates were analyzed by MALDI-TOF and GenoType CM/AS. Identification by 16S rRNA/hsp65 sequencing was considered the gold standard. Agreements between MALDI-TOF and GenoType CM/AS with the reference method were, respectively, 94.4% and 84.0%. In 17 cases (13.6%), results provided by GenoType and MALDI-TOF were discordant; however, the reference method agreed with MALDI-TOF in 16/17 cases (94.1%; P = 0.002).

Rapidly growing mycobacteria (RGM) inhabit soil and water but certain strains represent a health risk for human
and animals. Both clinical and soil RGM may be under selection pressure for resistance to tetracycline (TET) antibiotics,
since tetracyclines are administrated to humans and farm animals, and TET residues enter soil through manuring;
however, resistance to TET and the presence of TET-resistance genes have been assessed only in clinical isolates. We
were therefore interested in comparing soil and clinical RGM in terms of TET resistance and the presence of TETresistance
genes. We used 44 RGM from grasslands with different exposure to animal manure, and 38 clinical RGM
from Czech hospitals. There was no difference between the clinical and soil isolates in TET resistance, with >50%
resistant isolates in both groups. otr(A), otr(B), tet(K), tet(L) or tet(M) were not detected in any soil or clinical isolate.
In contrast, most isolates harbored tet(V) and tap, both encoding mycobacterial efflux pumps, including species where
these genes have never been evidenced before. The phylogeny of tet(V) correlated with isolates’ BOX-PCR profiles,
suggesting that this gene evolved along with mycobacterial genomes as a part of the intrinsic resistome. In certain
cases, tet(V) and/or tap were found in TET-sensitive isolates, or inversely, were not found in resistant strains. Concluding,
intrinsic efflux pumps may be more important for TET resistance than horizontally transferred genes in both soil and
clinical RGM. Their simple presence, however, does not attest to resistance, and therefore their diversity, function and
expression merit further research.

We performed a multistage genome-wide association study of melanoma. In a discovery cohort of 1804 melanoma cases and 1026 controls, we identified loci at chromosomes 15q13.1 (HERC2/OCA2 region) and 16q24.3 (MC1R) regions that reached genome-wide significance within this study and also found strong evidence for genetic effects on susceptibility to melanoma from markers on chromosome 9p21.3 in the p16/ARF region and on chromosome 1q21.3 (ARNT/LASS2/ANXA9 region). The most significant single-nucleotide polymorphisms (SNPs) in the 15q13.1 locus (rs1129038 and rs12913832) lie within a genomic region that has profound effects on eye and skin color; notably, 50% of variability in eye color is associated with variation in the SNP rs12913832. Because eye and skin colors vary across European populations, we further evaluated the associations of the significant SNPs after carefully adjusting for European substructure. (C.I.A.); qwei@mda nderson.org (Q.W.) ‡ These authors contributed equally to this work.

Collagen deposition within granulomas formed after Mycobacterium avium infection was analysed on histological sections stained with Masson’s trichrome using acquired computerized image analysis and a program that was specifically designed for that purpose. Comparison was made between immunocompetent C57BL⁄ 6 mice and mice genetically deficient in the inducible nitric oxide (NO) synthase gene (iNOS– ⁄ – mice) infected with either a highly virulent strain or a moderately virulent strain of M. avium. iNOS-deficient mice were more resistant to the highly virulent strain than control C57B1⁄ 6 mice, but both strains were equally susceptible to the less virulent M. avium strain. Collagen distribution in the granuloma was found in the cuff surrounding the granuloma in an area rich in lymphoid cells as well as inside the granuloma itself, conferring a mesh-like structure within that lesion. It was seen that iNOS– ⁄ – mice induced a higher collagen deposition than C57BL ⁄ 6 mice and that such collagen deposition varied with the mycobacterial strain used to
infect the animals.

Among the costs attributed to Mycobacterium avium ssp. paratuberculosis (MAP) infection in dairy cattle, the effects on reproduction and culling are the least documented. To estimate the cost of MAP infections and Johne's disease in a dairy herd, the rates of calving and culling were calculated for cows in each stage of MAP infection relative to uninfected cows. Data from 6 commercial dairy herds, consisting of 2,818 cows with 2,754 calvings and 1,483 cullings, were used for analysis. Every cow in each study herd was tested regularly for MAP, and herds were followed for between 4 and 7 yr. An ordinal categorical variable for Johne's disease status [test-negative, low-positive (low-shedding or ELISA-positive only), or high-shedding] was defined as a time-dependent variable for all cows with at least 1 positive test result or 2 negative test results. A Cox regression model, stratified on herd and controlling for the time-dependent infection variable, was used to analyze time to culling. Nonshedding animals were significantly less likely to be culled in comparison with animals in the low-shedding or ELISA-positive category, and highshedding animals had nonsignificantly higher culling rates than low-shedding or ELISA-positive animals. Time to calving was analyzed using a proportional rates model, an analog to the Andersen-Gill regression model suitable for recurrent event data, stratifying on herd and weighted to adjust for the dependent censoring caused by the culling effects described above. Highshedding animals had lower calving rates in comparison with low-shedding or ELISA-positive animals, which tended to have higher calving rates than test-negative animals.

Restless legs syndrome (RLS) is a sensorimotor disorder with an age-dependent prevalence of up to 10% in the general population above 65 years of age. Affected individuals suffer from uncomfortable sensations and an urge to move in the lower limbs that occurs mainly in resting situations during the evening or at night. Moving the legs or walking leads to an improvement of symptoms. Concomitantly, patients report sleep disturbances with consequences such as reduced daytime functioning. We conducted a genome-wide association study (GWA) for RLS in 922 cases and 1,526 controls (using 301,406 SNPs) followed by a replication of 76 candidate SNPs in 3,935 cases and 5,754 controls, all of European ancestry. Herein, we identified six RLS susceptibility loci of genome-wide significance, two of them novel: an intergenic region on chromosome 2p14 (rs6747972, P = 9.03 6 10 211 , OR = 1.23) and a locus on 16q12.1 (rs3104767, P = 9.4 6 10 219 , OR = 1.35) in a linkage disequilibrium block of 140 kb containing the 59-end of TOX3 and the adjacent non-coding RNA BC034767.

Currently, the Chilean authority has implemented a
National Eradication Program for bovine tuberculosis (bTB),
aimed at controlling and eradicating the disease in Chile. The
area under study has a low within-herd prevalence, has a relatively
low number of infected herds, and is one of the major
milk and beef producing areas in the country. However, so far,
no attempts at eradicating the disease have been successful. It
has been suggested that the diagnostic tests used were either
not sensitive or specific enough. In addition, previous studies
have shown that a great number of herds are infected with
Mycobacterium avium subsp. paratuberculosis (MAP). The
present study estimates the effect of MAP infection under field
conditions, on the diagnostic sensitivity and specificity of routine
tests in live animals for Mycobacterium bovis infection
diagnosis in cattle. In general, the estimations of test accuracy
observed an increase in the sensitivity and specificity on
MAP-infected animals for tuberculin test but observed a
decrease in the sensitivity of gamma interferon tests for
MAP-infected cattle. These results are different from those
of previous studies considering the role of MAP infection as
an interfering infection. More research is needed in order to
understand the complex interactions of the different
mycobacteria that can be found infecting production

Microbial survival in dynamic environments requires the ability to successfully respond to abrupt changes in osmolarity. The mechanosensitive channel of large conductance (MscL) is a ubiquitous channel that facilitates the survival of bacteria and archaea under severe osmotic downshock conditions by relieving excess turgor pressure in response to increased membrane tension. A prominent structural feature of MscL, the cytoplasmic C-terminal domain, has been suggested to influence channel assembly and function. In this report, we describe the X-ray crystal structure and electrophysiological properties of a C-terminal domain truncation of the Mycobacterium tuberculosis MscL (MtMscLΔC). A crystal structure of MtMscLΔC solubilized in the detergent n-dodecyl-β-D-maltopyranoside reveals the pentameric, closed state-like architecture for the membrane spanning region observed in the previously solved full-length MtMscL. Electrophysiological characterization demonstrates that MtMscLΔC retains mechanosensitivity, but with conductance and tension sensitivity more closely resembling full length EcMscL than MtMscL. This study establishes that the C-terminal domain of MtMscL is not required for oligomerization of the full-length channel, but rather influences the tension sensitivity and conductance properties of the channel. The collective picture that emerges from these data is that each MscL channel structure has characteristic features, highlighting the importance of studying multiple homologs. Mechanosensitive (MS) channels transduce mechanical stimuli into a variety of cellular responses. MS channels are widely distributed through all kingdoms of life, including bacteria and archaea, where they play a prominent role in maintaining proper osmoregulation 1-5. Sudden changes in osmolarity, such as osmotic downshock during exposure to hypotonic conditions, increases turgor pressure across the cell membrane. This in turn increases the tension within the lipid bilayer and activates a network of MS channels which respond by opening typically nonselective pores in the membrane to release cell contents and alleviate the pressure 1,5-7. Bacterial MS channels are characterized by their relatively high and non-selective conductances, ranging from ~0.1 nS to ~3 nS under defined experimental conditions 6-8 , which is up to several orders of magnitude greater than typical for ion-selective channels 3,6,9. Of these channels, the mechanosensitive channel of large conductance (MscL) exhibits the largest conductance, with an estimated open state pore diameter of ~25-35 Å-large enough for the passage of molecules up to 9 kDa 4,7,10,11. In view of the high conductance yet small subunit size of MscL (Escherichia coli MscL (EcMscL) has 136 residues; Fig. 1), the oligomeric nature of this channel was appreciated from its initial discovery 11. Considerable efforts have been made to establish the molecular mechanism of MscL gating 3,12 , employing a variety of techniques including electrophysiology, biochemical and genetic studies, molecular dynamics