Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

Single nucleotide polymorphisms in the Mycobacterium bovis genome resolve phylogenetic relationships.

Mycobacterium bovis isolates carry restricted allelic variation yet exhibit a range of disease phenotypes and host preferences. Conventional genotyping methods target small hypervariable regions of the M. bovis genome and provide anonymous biallelic information that is insufficient to develop phylogeny. To resolve phylogeny and establish trait-allele associations, we interrogated 75 M. bovis and 61 M. tuberculosis genomes for single nucleotide polymorphisms (SNPs), using iPLEX MassArray (Sequenom Inc., CA) technology. We indexed nucleotide variations in 306 genic and 44 intergenic loci among isolates derived from outbreaks in the United States from 1991 to 2010 and isolated from a variety of mammalian hosts. Two hundred six variant SNPs classified the 136 isolates and 4 previously sequenced strains (AF2122/97, BCG Pasteur, H37Rv, and CDC1551) into 5 major "SNP cluster groups." M. bovis isolates clustered into three major lineages based on 118 variant SNPs, while 84 SNPs differentiated the M. bovis BCG lineage from the virulent isolates. Forty-nine of the 51 human M. tuberculosis isolates were identical at all 350 loci studied. Thus, SNP-based analyses resolved the genotypic differences within M. bovis strains and differentiated these strains from M. tuberculosis strains representing diversity in time and space, providing population genetic frameworks that may aid in identifying factors responsible for the wide host range and disease phenotypes of M. bovis.

Bovine tuberculosis in a nebraska herd of farmed elk and fallow deer: a failure of the tuberculin skin test and opportunities for serodiagnosis.

In 2009, Mycobacterium bovis infection was detected in a herd of 60 elk (Cervus elaphus) and 50 fallow deer (Dama dama) in Nebraska, USA. Upon depopulation of the herd, the prevalence of bovine tuberculosis (TB) was estimated at ∼71-75%, based upon histopathology and culture results. Particularly with elk, gross lesions were often severe and extensive. One year ago, the majority of the elk had been tested for TB by single cervical test (SCT), and all were negative. After initial detection of a tuberculous elk in this herd, 42 of the 59 elk were tested by SCT. Of the 42 SCT-tested elk, 28 were TB-infected with only 3/28 reacting upon SCT. After SCT, serum samples were collected from the infected elk and fallow deer from this herd at necropsy and tested by three antibody detection methods including multiantigen print immunoassay, cervidTB STAT-PAK, and dual path platform VetTB (DPP). Serologic test sensitivity ranged from 79 to 97% depending on the test format and host species. Together, these findings demonstrate the opportunities for use of serodiagnosis in the rapid detection of TB in elk and fallow deer.

Disseminated panniculitis in a bottlenose dolphin (Tursiops truncatus) due to Mycobacterium chelonae infection.

A 14-year-old female bottlenose dolphin was diagnosed with mycobacterial panniculitis based on punch biopsy specimens. The necropsy revealed numerous pyogranulomas in the blubber, as well as marked acute multifocal necrosuppurative pneumonia and lymphadenitis. In addition, the animal had marked scoliosis, which had first been noted clinically in the dolphin at about 1 mo of age. The animal had been treated with low-dose dexamethasone for approximately the last 19 mo to reduce perceived discomfort from spondyloarthritis and with the progestational agent altrogenest for approximately 8 yr to prevent pregnancy. Acid-fast positive bacilli were detected in the dermis but not in lung or lymph nodes. Mycobacterium chelonae was isolated from pooled skin, lung, and peripheral lymph-node tissue. Mycobacterial infection may be considered as a differential diagnosis in bottlenose dolphins with generalized cutaneous inflammation, particularly if chronic steroid and progesterone treatments were administered, both of which may have an immunomodulatory effect.

Recovery of Mycobacterium bovis from soft fresh cheese originating in Mexico.

Recent outbreaks of human tuberculosis in the United States caused by Mycobacterium bovis have implicated cheese originating in Mexico as a source of these infections. A total of 203 samples of cheese originating in Mexico were cultured, and M. bovis was recovered from one specimen. Therefore, M. bovis can be recovered from cheese and may be a source of human infections.

Short-sequence-repeat analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium isolates collected from animals throughout the United States reveals both stability of loci and extensive diversity.

We analyzed the multilocus short sequence repeats (SSRs) of 211 and 56 isolates of Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium, respectively. The M. avium subsp. paratuberculosis isolates could be differentiated into 61 genotypes. The M. avium subsp. avium isolates showed limited diversity. These SSRs are stable and suitable for studying the molecular epidemiology of M. avium subsp. paratuberculosis.

Effect of egg yolk on the detection of Mycobacterium avium subsp. paratuberculosis using the ESP II liquid culture system.

Rapid diagnosis of paratuberculosis in infected cattle is important for the successful control of Johne disease within herds. Thus, improving culture methods for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) will aid in the identification of asymptomatic animals. Egg yolk is a component of the media used for growing M. paratuberculosis, but its requirement as a supplement has not been reported. Using the ESP II liquid culture system, 2 different sources and 5 concentrations (3.3%, 1.6%, 0.8%, 0.4%, and 0%) of egg yolk were analyzed. Egg yolk source did not affect either recovery rate or time to detection, but both parameters were significantly improved when the 3.3% egg yolk concentrations (final volume) were used over media containing no egg yolk. This study also assessed the recovery of M. paratuberculosis from fecal samples that were cultured multiple times using Herrold egg yolk agar (HEY). Specimens containing greater than 70 cfu/g feces could routinely be identified as positive for M. paratuberculosis after only 1 culture attempt, whereas specimens with fewer bacteria were only intermittently positive, even after 5 replicate cultures. Therefore, this study indicates that the sensitivity of the Trek Diagnostic ESP II liquid culture system for M. paratuberculosis is affected by egg yolk concentration and that single culture attempts using HEY solid media may not identify specimens containing low numbers of bacteria.

Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants.

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log(10) CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log(10) CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log(10) CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log(10) CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.

Mycobacterium smegmatis D-Alanine Racemase Mutants Are Not Dependent on D-Alanine for Growth.

Mycobacterium smegmatis is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. This study focused on the D-alanine racemase gene (alrA), which is involved in the synthesis of D-alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. M. smegmatis alrA null mutants were generated by homologous recombination using a kanamycin resistance marker for insertional inactivation. Mutants were selected on Middlebrook medium supplemented with 50 mM D-alanine and 20 microg of kanamycin per ml. These mutants were also able to grow in standard and minimal media without D-alanine, giving rise to colonies with a drier appearance and more-raised borders than the wild-type strain. The viability of the mutants and independence of D-alanine for growth indicate that inactivation of alrA does not impose an auxotrophic requirement for D-alanine, suggesting the existence of a new pathway of D-alanine biosynthesis in M. smegmatis. Biochemical analysis demonstrated the absence of any detectable D-alanine racemase activity in the mutant strains. In addition, the alrA mutants displayed hypersusceptibility to the antimycobacterial agent D-cycloserine. The MIC of D-cycloserine for the mutant strain was 2.56 microg/ml, 30-fold less than that for the wild-type strain. Furthermore, this hypersusceptibility was confirmed by the bactericidal action of D-cycloserine on broth cultures. The kinetic of killing for the mutant strain followed the same pattern as that for the wild-type strain, but at a 30-fold-lower drug concentration. This effect does not involve a change in the permeability of the cell wall by this drug and is consistent with the identification of D-alanine racemase as a target of D-cycloserine. This outcome is of importance for the design of novel antituberculosis drugs targeting peptidoglycan biosynthesis in mycobacteria.