Changes in Mycobacterium spp. population structure and pyrene mineralization in polycyclic aromatic hydrocarbon-amended soils

The effect of pyrene and phenanthrene contamination on soil Mycobacterium spp. community structure was examined using PCR-amplification of 16S rRNA genes with primers specific for the fast-growing group of Mycobacterium spp. and separation of phylotypes by temperature gradient gel electrophoresis (TGGE). The degradative potential of the soil microbial community was measured over time by mineralization of ¹⁴C-pyrene added to the contaminated soils. PCR-TGGE profiles, in combination with band sequencing and phylogenetic analysis of the prominent phylotypes, indicated shifts in the Mycobacterium spp. community during incubation. Reductions in species diversity and enrichments of specific populations were observed in all pyrene- and phenanthrene-treated soils, in contrast to the relatively stable control soil profiles. Mineralization studies indicated the shortest acclimation periods and the highest initial rates of pyrene degradation occurred in soils pre-exposed to phenanthrene, or a mixture of phenanthrene and pyrene, for 14 weeks. Pre-exposure of soil microorganisms to a single dose of pyrene for the same length of time also decreased the acclimation period for the degradation of pyrene. Monthly application of either pyrene or phenanthrene to soils, however, resulted in an increase in pyrene degradative potential 6 weeks after the first pre-exposure, but a decrease in degradative potential 14 weeks after the first pre-exposure. Similar PCR-TGGE profiles were obtained from soils with comparable pyrene mineralization curves or degradative potentials.     

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as the pro-inflammatory cytokine IFN-γ were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-γ stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-γ. Finally, the results suggest an important role of IL-10 and TGF-β on the profile of NO production which may explain the low NO production in MAP clinically infected cows.     

重组牛分支杆菌ESAT-6基因的表达与鉴定

在前期工作的基础上，构建了pGEM—TE—ESAT6重组载体，转化至大肠杆菌BL21（DE3）感受态细胞中，经诱导表达得到了20kD左右大小的重组蛋白，SDS—PAGE／Western Blot显示该重组蛋白是上清表达。为重组ESAT-6抗原的应用奠定了基础。     

Estimates of within-herd incidence rates of Mycobacterium bovis in Canadian cattle and cervids between 1985 and 1994

We analysed the individual-animal data from six of the nine outbreaks of tuberculosis in Canadian cattle and cervids from 1985 to 1994. A “positive/reactor” animal was one which had either a positive culture or a positive or suspicious reaction on a mid-cervical, comparative cervical, or gross or histopathological test for tuberculosis. Individual-animal data were collected only for herds which had one or more positive/reactor animals. Data were collected from the outbreak records in the Regional or District offices of Agriculture and Agri-food Canada’s Animal and Plant Health Directorate. The within-herd spread of Mycobacterium bovis was studied by determining the most-likely date at which the herd was first exposed to M. bovis and the number of reactions which had developed by the time the herd was investigated. The animal-time units at risk in the herd were probably overestimated, resulting in conservative estimates of the within-herd incidence rates. Negative-binomial regression was used to investigate factors which might have influenced the within-herd spread of tuberculosis. Increasing age appeared to be a risk factor for being a positive/reactor animal. When compared to animals 0–12 months old, animals 13–24 months old had an incidence rate ratio (IRR) of 7.6, while animals >24 months old had an IRR of 10.4 (p=0.009). Actual and predicted incidence rates for tuberculosis in mature (>24 months old) animals were calculated. Actual and predicted incidence rates were similar for cervids, within an outbreak. There was more variability between actual and predicted rates in the dairy and beef animals. In the one outbreak (Ontario) where there were positive/reactor cervid, dairy and beef herds, the actual incidence rate for cervids (IR=9.3 cases per 100 animal-years) was almost twice that of dairy cattle (IR=5.0) and three times that of beef cattle (IR=3.1).     

结核分支杆菌MPT83在真核、原核表达载体中的克隆、表达与鉴定

为了克隆、鉴定和表达结核分支杆菌抗原蛋白 MPT83 ,为结核病的诊断以及亚单位疫苗、核酸疫苗的制备和应用奠定基础。以结核分支杆菌标准菌株 H3 7RV基因组 DNA为模板 ,PCR扩增目的基因 mpt83 ,扩增产物酶切后分别克隆到真核、原核表达载体 p JW43 0 3和p ET2 2 b( )中 ,构成重组真、原核表达载体 83eu和 MPT83。经酶切和序列鉴定为正确的重组真核表达载体 83 eu和重组原核表达载体MPT83 ,分别转染 COS7细胞和转化 BL2 1( DE3 ) plys S。结果表明 ,经 SDS-PAGE、Westernblotting检测显示 ,IPTG诱导的原核表达载体MPT83在 2 .6万处有正确而特异的蛋白条带 ;转染 COS7细胞的真核表达载体 83 eu,Westernblotting检测表明也有与结核分支杆菌抗血清特异反应的蛋白条带     

Experimental Infection with Mycobacterium Avium,Serotype 2, in Pigs: 4. Contact Infection from Orally Inoculated Pigs*

In 3 experiments, 13 pigs were inoculated orally with 0.5 mg Mycobacterium avium daily for 5 days (1 mg = 32–68×10⁶ viable units). Five to 8 days after inoculation, 16 non-inoculated pigs were added to the inoculated pigs.Cultures from faeces showed excretion of M. avium from all the inoculated pigs for some time within the period 16 to 65 days after the last inoculation; the greatest numbers of organisms (62000 per 100 g faeces) were found at about the middle of the excretion period (Table 2). Two of the contact pigs were found to excrete M. avium in small numbers, 1 at 15, the other at 37 and 44 days after contact.All the inoculated pigs and 13 of the contact pigs showed positive intradermal tuberculin reactions. Post-mortem examination showed tuberculous lesions in all the inoculated pigs (Table 3). M. avium was transmitted to 15 of 16 pigs by contact. Five of these pigs showed gross lesions, 10 of them microscopic lesions only; in 9 of them the infection was proved by culture.     

Experimental Reproduction of Feline Mycobacterium fortuitum Panniculitis

Resumen— Con el fin de lograr un modelo experimental de la infección por Mycobacterium fortuitum, causante de paniculitis en condiciones naturales en el gato, se inoculóM. fortuitum en el cojinte plantar de ratones o en el tejido adiposo inguinal de conejos y gatos. Los ratones manifestaron una dermatitis crónica y una linfadenitis granulomatosa necrotizante con localizatión intracelular del microorganismo. Los conejos manifestaron inflamaciones granulomatosas supurativas necrotizantes con microrganismos en vacuolas adiposas rodeadas por heterófilos macrófagos epitelioides y/o zonas de necrosis. Los cinco gatos adultos y una de las tres crias mostraron fistulas supurativas, úlceras puntuales o nódulos en el paniculo adiposo de la zona inguinal. La lesión en la región inguinal de estos seis animales consistia en una paniculitis granulomatosa; las otras dos crias presentaban una paniculitis piogranulomatosa necrotizante. Se identificaron bacilos en los cortes histológicos teñidos con Hematoxilina y Eosina en cuatro gatos adultos y en una de las crias. Se aislóMycobacterium fortunitum a partir del tejido adiposo en todos los gatos adultos y en una de las tres crias. La inoculación de 1.4 × 10¹⁰M.fortuitum en el tejido adiposo subcutáneo inguinal en los gatos con grandes masas grasas en esas zonas causó una infección microbacteriana idéntica a la enfermedad felina en condiciones naturales. [Lewis, D. T., Hodgin, E. C, Foil, C. S., Cox, H. U., Roy, A. F., Lewis, D. D. Experimental reproduction of feline Mycobacterium fortuitum panniculitis. (Reproductión experimental de la paniculitis felina por Mycobacterium fortuitum). Abstract— In order to establish an animal model of the naturally occurring feline Mycobacterium fortuitum panniculitis an inoculum of M. fortuitum organisms was injected into the hindlimb footpad of mice or subcutaneous fat of the inguinal area of rabbits and cats. Mice developed chronic dermatitis and necrotizing granulomatous lymphadenitis with intracellular localization of the organism. Rabbits developed necrotizing suppurative granulomatous inflammation with organisms in heterophil-lined fat vacuoles, epithelioid macrophages and/or necrotic areas. All five adult cats and one of three kittens developed draining tracts, punctate ulcers or nodules in the panniculus adiposus of the inguinal area. A pyogranulomatous panniculitis characterized the inguinal region in these six animals; a necrotizing pyogranulomatous panniculitis was present in the remaining two kittens. Rod-shaped bacilli were present on hematoxylin and eosin stained tissue sections in four adult cats and one kitten. Mycobacterium fortuitum was isolated from the inguinal subcutaneous fat in all five adult cats and one of three kittens. Injection of 1.4 × 10¹⁰M. fortuitum organisms into the subcutaneous fat of the inguinal area of cats with extensive inguinal fatpads produced a mycobacterial infection identical to the naturally occurring feline disease.     

Paratuberculosis in European wild rabbits from the Iberian Peninsula

Of the non-ruminant wildlife species known to harbor Mycobacterium avium paratuberculosis (MAP), the rabbit (Oryctolagus cuniculus) is thought to pose the greatest risk of transmission to cattle. We analyzed 80 hunter-harvested wild rabbits from a core study area in southern Spain, and sera from 157 wild rabbits sampled opportunistically on seven additional sites. Gross lesions compatible with paratuberculosis were observed in two of 80 necropsied rabbits. Histopathology revealed focal to diffuse multibacillary MAP-compatible lesions in 8 of 10 rabbits examined. Presence of MAP was confirmed in one rabbit with gross lesions by positive amplification curves for both IS900 and ISMAP02. However, no isolate was obtained from 47 samples by culture. We adapted an indirect ELISA for the detection of MAP antibodies. At the established cut-off of 0.5, 6 of 237 wild rabbit sera (2.5%) yielded a positive ELISA result. Antibodies were detected in rabbits from 3 of 8 sampling sites. Considering the increasing relevance of MAP infection for animal health, these results open a challenging field for future research.     

MIRU-VNTR typing of Mycobacterium avium in animals and humans: heterogeneity of Mycobacterium avium subsp. hominissuis versus homogeneity of Mycobacterium avium subsp. avium strains

Epidemiological studies on Mycobacterium avium are requisite for revealing infection sources and disease transmission. They are based upon genotyping methods like RFLP and MIRU–VNTR. In our study, MIRU–VNTR typing was applied to 121 previously RFLP typed M. avium field isolates to compare the discriminatory power of both methods. The applicability of MIRU–VNTR typing was studied for isolates from a limited geographic area, namely 41 M. avium subsp. avium and 80 M. avium subsp. hominissuis isolates. Among the former, exhibiting 12 IS901 RFLP types, five MIRU–VNTR types were found with discriminatory index (DI) of 0.716. Among the latter, exhibiting 56 IS1245 RFLP types, 18 MIRU–VNTR types were found with DI of 0.866. Concomitant use of both methods increased DI to 0.981 and 0.995, respectively. MIRU–VNTR typing employing the selected markers provided discernible discrimination among M. avium subsp. hominissuis isolates, but more discriminative markers are needed for M. avium subsp. avium isolates.