Effects of maternal high-energy diet and spirulina supplementation in pregnant and lactating sows on performance, quality of carcass and meat, and its fatty acid profile in male and female offspring.

The concept of developmental programming suggests that maternal excessive energy intake during intrauterine development could permanently affect the offspring's performance. Spirulina might alleviate adverse programming effects, but currently has only been shown to affect productivity and product quality of livestock when fed directly. Therefore, we investigated effects of supplementing 20 g spirulina/day to 20 gestating and lactating sows fed either a commercial or a high energy diet (HED), on performance and meat quality of their piglets fattened for 4 months. Control and HED offspring did not differ in growth and slaughter performance. Maternal spirulina supplementation impaired growth performance in male but not in female offspring. Physicochemical meat quality was not affected by any treatment. Maternal spirulina intake tended to improve the polyunsaturated fatty acids (FA)/saturated FA ratio without affecting n-6/n-3 FA ratio in offspring meat. The present findings indicate a sex-specific programming effect for offspring growth in response to maternal spirulina but not high energy intake in pigs.

Detection of phospholipase C in nontuberculous mycobacteria and its possible role in hemolytic activity.

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.

Correlation of pncA sequence with pyrazinamide resistance level in BACTEC for 21 mycobacterium tuberculosis clinical isolates.

Mutations in the pncA gene, encoding pyrazinamidase, are considered the major mechanism of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis, but resistant strains containing the wild-type gene have been described. The correlation of pncA sequence with PZA resistance level was examined for 21 M. tuberculosis clinical isolates. Susceptibility patterns were determined for 100, 300, and 900 microg/ml concentrations of the drug in BACTEC. Insertions and deletions and a substitution in the putative promoter region led to high-level resistance, whereas substitutions within the open reading frame seemed to confer variable levels of resistance. Variable resistance levels and PZase activities were also observed among isolates lacking pncA mutations. The high-level resistance (900 microg/ml) in pncA wild-type isolates highlights the clinical significance of these isolates. These data also suggest that there may still be more than one alternative mechanism leading to PZA resistance in M. tuberculosis isolates.

Relationship between pyrazinamide resistance, loss of pyrazinamidase activity, and mutations in the pncA locus in multidrug-resistant clinical isolates of Mycobacterium tuberculosis.

Sixty-two Mycobacterium tuberculosis isolates were tested for pyrazinamidase activity, and their pyrazinamide susceptibility was determined by the radiometric method. Sequencing of pncA genes in the 23 resistant strains revealed mutations in 16 pyrazinamidase-negative strains, 11 of which had not been previously described. Six isolates containing wild-type pncA might possess alternative resistance mechanisms.

Blood and charcoal added to acidified agar media promote the growth of Mycobacterium genavense.

Ten different agar media were tested for the in vitro growth of Mycobacterium genavense in primary cultures and in subcultures from BACTEC vials. These agar media were based on Middlebrook 7H9, 7H10 and 7H11, and supplemented with additives: mycobactin J, yeast extract, charcoal, or defibrinated sheep blood. Some media were acidified with phosphoric acid to a final pH of 6.2 +/- 0.2. Fourteen M. genavense strains from nude mouse organs as well as one decontaminated clinical specimen (from a bird) were tested. The optimal medium for primary cultures of M. genavense was Middlebrook 7H11 acidified to pH 6.2 +/- 0.2 and supplemented with charcoal and sheep blood: on this medium, all strains produced colonies within 6-12 weeks of incubation in numbers approaching the number of bacilli inoculated. It was also the only medium to support the growth of the decontaminated clinical specimen. Added blood and charcoal appeared not as essential for subcultures as for primary cultures. Three media supported the growth of all strains within 1 month incubation: they were acidified, and were supplemented with yeast extract or pancreatic digest of casein, and with either blood or charcoal.

Effect of oxygen on growth of Mycobacterium ulcerans in the BACTEC system.

The effect of low oxygen concentration on the growth of 15 strains of Mycobacterium ulcerans was evaluated in the BACTEC system. Reduced oxygen tension enhanced the growth of M. ulcerans, suggesting that this organism has a preference for microaerobic environments. Application of this observation may improve rates of isolation of M. ulcerans in primary culture from clinical samples and promote isolation of the bacterium from environmental sources.

Microaerophilic conditions promote growth of Mycobacterium genavense.

Our studies show that microaerophilic conditions promote the growth of Mycobacterium genavense in semisolid medium. The growth of M. genavense at 2.5 or 5% oxygen was superior to that obtained at 21% oxygen in BACTEC primary cultures (Middlebrook 7H12, pH 6.0, without additives). By using nondecontaminated specimens, it was possible to detect growth with very small inocula (25 bacilli/ml) of 12 different M. genavense strains (from nude mice) within 6 weeks of incubation under low oxygen tension; conversely, with 21% oxygen, no growth of 8 of 12 (66.7%) M. genavense strains was detected (growth index, <10). The same beneficial effect of 2.5 or 5% oxygen was observed in primary cultures of a decontaminated clinical specimen. Low oxygen tension (2.5 or 5%) is recommended for the primary isolation of M. genavense. Microaerophilic cultivation of other atypical mycobacteria, especially slow-growing (e.g., Mycobacterium avium) and difficult-to-grow (e.g., Mycobacterium ulcerans) species, is discussed.

Inhibitory effects of polyoxyethylene stearate, PANTA, and neutral pH on growth of Mycobacterium genavense in BACTEC primary cultures.

We report on the influences of polyoxyethylene stearate (POES), PANTA, and pH on primary cultures of Mycobacterium genavense in BACTEC vials. As a model for primary cultures from tissue, seven different strains first isolated from AIDS patients (five from Switzerland and two from the United States) were inoculated into nude mice in order to obtain large amounts of bacilli to test different conditions simultaneously. Our results demonstrate that the size of the inoculum (10[6] acid-fast bacilli/vial), an acid pH (pH 6.0), and the absence of additives (POES and PANTA) significantly (P < 0.001) increased the probability of a successful culture in 1 month, considering growth index (GI) of > or =100 or a GI of > or =999 as criterion of success. In logistic regression analysis, all factors maintained a significant (P < 0.001) independent effect, and no interactions were observed between them. The best conditions for the primary cultures of M. genavense were the use of Middlebrook 7H12 medium at pH 6.0 without any additives.

Mycobacteriosis caused by Mycobacterium genavense in birds kept in a zoo: 11-year survey.

We report on a disease in 27 birds (1 bird belonging to the order Coraciiformes, 3 to Piciformes, 4 to Galliformes, 7 to Psittaciformes, and 12 to Passeriformes) caused by fastidious mycobacteria. All birds were caged at the Antwerp Zoo and died suddenly between 1983 and 1994. Seventeen birds had no previous signs of disease, and 10 birds showed emaciation. Gross necropsy findings were generally nonspecific, but all the birds were smear positive for acid-fast bacilli (AFB). Histopathologic evaluation performed on 14 birds revealed predominantly intracellular AFB. Extracellular AFB were more abundant in advanced lesions, especially in necrotic areas. In the intestine the mucosal area was generally heavily infiltrated, suggesting an intestinal origin of the infection. There was extensive invasion of the lungs in most birds. In 11 birds sparse growth was obtained after at least 6 months of incubation on Löwenstein-Jensen medium or on Ogawa medium supplemented with mycobactin. Subculture was unsuccessful in all instances. The 16S rRNA gene sequence of the cultured organisms or tissues from seven birds revealed the characteristic signature sequence for Mycobacterium genavense. Direct bird-to-bird transmission in the zoo was unlikely, and the pathogenicity of M. genavense in birds seems to be limited. The source of M. genavense in nature and the epidemiology of the disease in birds remain obscure. As suspected for human cases of M. genavense infection, an oral route of infection has been suggested, and contaminated local water distribution systems may have been the source of the infection. Our study confirms that infections caused by M. genavense should be suspected in birds (especially in Passeriformes and Psittaciformes orders) that die suddenly without previous symptoms and that have AFB in tissues that are difficult to grow on conventional media.

Cervical lymphadenitis caused by a fastidious mycobacterium closely related to Mycobacterium genavense in an apparently immunocompetent woman: diagnosis by culture-free microbiological methods.

Fastidious mycobacteria usually infect immunocompromised hosts (human immunodeficiency virus-infected or otherwise immunosuppressed patients). We here describe severe lymphadenitis, caused by a fastidious mycobacterium closely related to Mycobacterium genavense, in an apparently immunocompetent woman, whose brother had died from an unidentified mycobacterial infection in 1969. A variety of techniques, including inoculation of nude mice, histopathology, electron microscopy, lipid analysis, ATP measurements, and molecular biology, were used to characterize this mycobacterium. All attempts to culture the etiological agent on many different media failed. The organism multiplied only in congenitally athymic nude mice. Although phenotypically similar to M. genavense, the mycobacterium differs from M. genavense by three nucleotides of the 16S rRNA gene sequence. Various antimycobacterial drugs were administered, including gamma interferon, but multiple relapses occurred. Finally, therapy with a combined regimen of clarithromycin, clofazimine, rifabutin, and ethambutol was curative. To our knowledge, this is the first report of lymphadenitis in an apparently immunocompetent patient, caused by a noncultivable Mycobacterium sp. closely related to M. genavense. This study emphasizes the importance of employing a variety of diagnostic approaches such as the inoculation of laboratory animals, histopathology, electron microscopy, lipid analysis, ATP measurements, and molecular biology to characterize novel microorganisms that cannot be cultured in vitro.

Species-specific Mycobacterium genavense DNA in intestinal tissues of individuals not infected with human immunodeficiency virus.

Mycobacterium genavense species-specific DNA was detected in intestinal tissues from two of nine individuals not infected with human immunodeficiency virus. This newly described microorganism is well documented as a causative agent of disseminated infections in AIDS patients. Our results suggest that it may colonize the guts of individuals not infected with human immunodeficiency virus.