Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.Since the early 1980s, there has been an increase in disease caused by organisms broadly categorized as nontuberculous mycobacteria (NTM), a generic term for mycobacteria not in the Mycobacterium tuberculosis complex and other than M. leprae (32). Of these NTM, Mycobacterium avium complex (MAC) species are the most common cause of human and animal disease globally (6,14,16,24). The clinical relevance of the MAC in humans has been amplified in recent decades with the increasing population of immunocompromised individuals resulting from longer life expectancy, immunosuppressive chemotherapy, and the AIDS pandemic (27 Members of the family Mycobacteriaceae, comprising the MAC, differ in virulence and ecology. Those designated M. avium subsp. hominissuis are genomically diverse, low-virulence, opportunistic pathogens for both animals and humans.
A commercial radiometric medium, BACTEC 12B, was modified by addition of mycobactin, egg yolk suspension, and antibiotics (vancomycin, amphotericin B, and nalidixic acid). Decontaminated bovine fecal specimens were filter concentrated by using 3-,um-pore-size, 13-mm-diameter polycarbonate filters, and the entire filter was placed into the radiometric broth. Comparison of the radiometric technique with conventional methods on 603 cattle from 9 Mycobacterium paratuberculosis-infected herds found that of 75 positive specimens, the radiometric technique detected 92% while conventional methods detected 60% (P < 0.0005). Only 3.9% of radiometric cultures were contaminated. To measure the effect of filter concentration of specimens on the detection rate, 5 cattle with minimal and 5 with moderate ileum histopathology were sampled weekly for 3 weeks. M. paratuberculosis was detected in 33.3% of nonfiltered specimens and 76.7% of filtered specimens (P < 0.005). Detection rates were directly correlated with the severity of disease, and the advantage of specimen concentration was greatest on fecal specimens from cattle with low-grade infections. Detection times were also correlated with infection severity: 13.4 5.9 days with smear-positive specimens, 27.9 8.7 days with feces from cows with typical subclinical infections, and 38.7 ± 3.8 days with fecal specimens from cows with low-grade infections. Use of a cocktail of vancomycin, amphotericin B, and nalidixic acid for selective suppression of nonmycobacterial contaminants was better than the commercial product PANTA (Becton Dickinson Microbiologic Systems, Towson, Md.) only when specimens contained very low numbers of M. paratuberculosis. Radiometric culture of filter-concentrated specimens generally doubled the number of positive fecal specimens detected over conventional methods, making it a useful tool for diagnosis and control of bovine paratuberculosis. Bovine paratuberculosis (Johne's disease) affects at least 2.9% of the 10.3 million dairy cattle in the United States (23) and is equally prevalent in most other countries where laboratory diagnosis of the infection is possible (34). The prevalence of paratuberculosis in some states exceeds 15% (3, 4, 41). The national economic impact has been estimated to be as high as $1.5 billion annually (12). The etiological agent, Mycobacterium paratuberculosis, is among the slowest growing of the cultivable mycobacteria, normally requiring 8 to 16 weeks to produce visible colonies on conventional agar media (40). It infects the terminal ileum of most ruminants and is excreted in the feces. Control of the disease in a dairy cattle herd involves hygienic measures to avoid exposure of calves to the organism or removal of infected animals from the herd (particularly those excreting M. paratuberculosis) or both (5, 10, 20, 29, 36). Damato and Collins reported detection of M. paratuber
Oral and intraperitoneal administration of lactic acid-producing bacteria can significantly augment the immune response in murine models; however, the immunopotentiating effects in these studies differ significantly. Murine macrophagelike cell line J774 was cultured in the presence of cell-free extracts of Lactobacillus acidophilus and Bifidobacterium longum, and the effect on macrophage function was evaluated by measurement of synthesis of selected enzymes and their ability to take up either acrylamide particles or live Salmonella typhimurium. Lysozyme activity of J774 cells was significantly decreased by cell-free extracts of B. longum, but not of L. acidophilus, whereas extracts of both strains induced morphological changes and significantly enhanced phagocytosis of inert particles or viable Salmonella. Whole cell extracts of lactic acid-producing bacteria are therefore capable of altering macrophage function in a strain-dependent manner.
This report describes a simple method for quantifying viable mycobacteria and for determining generation time. We used statistical models and computer analysis of growth curves generated for the slowly growing mycobacterium Mycobacterium paratuberculosis under controlled conditions to derive a mathematical formula relating the dependent variable, growth, to the independent variables, loglo number of organisms in the inoculum (inoculum size) and incubation time. Growth was measured by a radiometric method which detects 910
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