A PCR-based method for tuberculosis detection in wildlife.

Bovine tuberculosis (TB) is a major worldwide zoonotic disease. Foremost of the drawbacks in the control campaigns is the slow growth of its causative agent, M. bovis, as bacteriology remains the "gold standard" for the diagnosis of this disease. Rapid alternative molecular biology methods for TB diagnosis have long been hampered due to mycobacterial-linked difficulties when conventional DNA extraction techniques are applied. Moreover, the correct specificity is difficult to achieve because of the similarities in genetic background between M. bovis and other ubiquitous mycobacterial species. Nevertheless, much technological progress has been achieved in recent years, allowing the development of accurate molecular diagnosis. One of the main problems for bovine TB control is the existence of M. bovis wildlife reservoirs, a source of re-contamination in bovine TB-free herds. PCR seems an interesting alternative method for rapidly screening these species in epidemiological enquiries and immediate decision-making to avoid transmission to livestock. We describe here the validation process for a PCR diagnostic method compared to bacteriology in a wildlife TB survey.

Hematopoietic stem cell- and marrow stromal cell-specific requirements for gamma irradiation leukemogenesis in vitro.

The hematopoietic and stromal cell-specific properties of the cells involved in gamma irradiation leukemogenesis in vitro were defined. Cocultivation of clonal factor-dependent (FD), interleukin 3 (IL-3)-dependent cell lines 32D cl 3 or B6SUtA, or dual IL-3-/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell lines FDC-P1 or bg/bg d64 was carried out with clonal stromal cell lines D2XRII, GB1/6, +/+ 2.4, or Sld3. FD cell lines were added to control or 5000-cGy-irradiated plateau phase monolayer cultures of each stromal cell line, and parameters of stem cell engraftment and malignant transformation in vitro were quantitated. Cobblestone island formation by FD cells, cumulative production of nonadherent hematopoietic cells, and evolution of tumorigenic factor-independent (FI) subclonal lines were quantitated over 5-8 weeks. There was no detectable evolution of FI sublines with 32D cl 3, B6SUtA, or bg/bg d64 cells cocultivated with control or irradiated Sld3 stromal cells. IL-3-dependent cell lines 32D cl 3 or B6SUtA formed small 10- to 49-cell cobblestone "clusters" at low frequency on control or irradiated D2XRII, showed limited proliferation for less than 1 week, and showed no detectable evolution of FI cell lines. Subclones of 32D cl 3 derived by transfection and expression of recombinant oncogenes v-sis, or c-myc, or the epidermal growth factor receptor remained factor dependent and did not transform to factor independence after cocultivation with irradiated stromal cell lines. In contrast, cell line bg/bg d64, and each of seven subclonal lines of FDC-P1, including subclones selected for growth in GM-CSF, formed abundant cobblestone island colonies of greater than or equal to 50 cells on irradiated D2XRII stromal cells, produced non-adherent cells over 5-8 weeks, and showed evolution of tumorigenic FI subclonal lines. The data provide evidence for stable biological differences in both the hematopoietic and stromal cell components of the in vitro model of gamma irradiation leukemogenesis.

Recombinant murine GM-CSF increases resistance of some factor dependent hematopoietic progenitor cells to low-dose-rate gamma irradiation.

The effects of murine recombinant IL-3 (multi-CSF) and murine recombinant GM-CSF (granulocyte-macrophage colony stimulating factor) on the radiation biology of clonal hematopoietic progenitor cell lines were evaluated. Four clonal cell lines with growth response to either IL-3 or GM-CSF (FDCP-1JL26, and bg/bg d64) or exclusively dependent on IL-3 (32D cl 3 and B6SUtA), were pre-incubated in IL-3, or GM-CSF, for 7 days prior to gamma irradiation, then washed and irradiated at 5 cGy/min, or 116 cGy/min, and transferred to semisolid medium supplemented with either IL-3, or GM-CSF, for assay of 7 day greater than or equal to 50 cell colonies. The cell lines demonstrated similar radiosensitivity and lack of a detectable dose-rate effect when grown in IL-3 (FDCP-1JL26: D0 154, n 1.05 at 5 cGy/min, and D0 138, n 1.16 at 116 cGy/min; bg/bg d64: D0 95.7, n 1.16 at 5 cGy/min, and D0 97.7 n .993 at 116 cGy/min; B6SUtA: D0 101, n 1.29 at 5 cGy/min, D0 100, n 1.27 at 116 cGy/min; and cell line 32D cl 3: D0 123, n 1.65 at 5 cGy/min, and D0 126, n 1.17 at 116 cGy/min). In contrast, FDCP-1JL26 cells demonstrated a significant relative radioresistance at low-dose-rate when grown in recombinant GM-CSF, (D0 217, n 1.27 at 5 cGy/min, D0 138, n 1.34 at 116 cGy/min, p less than .005). The increase in radioresistance of FDCP-1 cells at low-dose-rate was induced either by preincubation in GM-CSF with transfer to IL-3, or by preincubation in IL-3 and transfer to recombinant GM-CSF. Growth factor independent malignant subclones of lines B6SUtA and FDCP-1JL26 demonstrated a significant increase in radioresistance at low-dose-rate (B6SUtA EL4JL: D0 187, n 1.39 at 5 cGy/min, and D0 133, n 1.73 at 116 cGy/min (p. less than .05); and FDCP-1JL26 F7 cl 2: D0 191, n 1.17 at 5 cGy/min, and D0 150, n 1.31 at 116 cGy/min [p less than .05]). Thus, some hematopoietic progenitor cell lines are induced by GM-CSF to grow after irradiation at low-dose-rate similar to the growth of clonal malignant cell lines. The data may have implications for the radiation biology of normal hematopoietic progenitor cells in two circumstances: (a) selective survival of GM-CSF responsive cells after total body irradiation, and (b) selective survival of some hematopoietic progenitors in vivo during clinical recombinant GM-CSF infusion.

Expression of transfected recombinant oncogenes increases radiation resistance of clonal hematopoietic and fibroblast cell lines selectively at clinical low dose rate.

To determine the effect of oncogene expression on gamma radiation sensitivity of hematopoietic compared to fibroblastic cells, we selected clonal sublines of an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell line 32D cl 3 and NIH/3T3 embryo fibroblastic cells following transfection with each oncogene linked to the mycophenolic acid resistance gene. Each mycophenolic acid-resistant subclone demonstrated high levels of specific poly(A)+ mRNA for each oncogene. The parent line 32D cl 3 demonstrated similar radiosensitivity at 116 cGy/min (D0 126, n 1.17) compared to 5 cGy/min (D0 123, n 1.65). This pattern was not altered in subclones of 32D cl 3 cells transfected with the epidermal growth factor (EGF) receptor gene and grown in EGF (at 116 cGy/min D0 104, n 0.998, at 5 cGy/min D0 115, n 1.09), or in 32D cl 3 cells expressing the v-sis oncogene (at 116 cGy/min D0 122.4, n 1.79, at 5 cGy/min D0 135, n 1.43). In contrast, expression of the transfected oncogenes v-erb-B, v-abl, or v-src conferred significant radioresistance at 5 cGy/min dose rate (D0 194, n 1.77; D0 165.5, n 1.56; D0 171, n 1.28, respectively). With the exception of v-sis, oncogene expression resulted in nonautocrine factor independence of 32D cl 3 subclones, and production of donor origin tumors in syngeneic new-born or adult mice. Two rare spontaneous factor-independent subclones of 32D cl 3 were also tested. Nonautocrine clone 32D cl 2 demonstrated significantly increased radioresistance at low dose rate (D0 186, n 1.63), while autocrine (IL-3 producing) subclone 32D cl 4 revealed no significant increase in radioresistance at 5 cGy/min. The parent fibroblast cell line NIH/3T3 showed an intrinsic relative radioresistance at low dose rate (at 5 cGy/min D0 157.3, n 1.81, compared to 116 cGy/min D0 134.3, n 1.57). Expression in NIH/3T3 of transfected oncogenes v-abl, v-fms, v-fos, or H-ras increased radioresistance at low dose rate (D0 208.6, n 1.61; D0 206.6, n 1.51; D0 167.5, n 1.85; and D0 206.8, n 1.08, respectively). Thus expression of each of several oncogenes induces resistance to gamma irradiation at 5 cGy/min in hematopoietic and fibroblast cell lines. These data may help explain the clinical recurrence of oncogene-expressing leukemia and lymphoma cells after marrow stem cell ablative doses of low-dose-rate total-body irradiation.

[Use of the in vitro enzymatic amplification method for the detection of Mycobacterium paratuberculosis in feces]. / Utilisation de la méthode d'amplification enzymatique in vitro pour la détection de Mycobacterium paratuberculosis dans les fèces.

A polymerase chain reaction was developed, using as target sequence an insertion element of 1,451 base pairs (IS 900), specific for Mycobacterium paratuberculosis (15-20 copies per genome). The test was performed in three stages: (1) extraction of bacterial deoxyribonucleic acid (DNA), from faeces stored at +4 degrees C, -20 degrees C, in 70% ethanol or in a buffer solution; (2) amplification of the target DNA by means of thermostable DNA polymerase; (3) detection of the amplified DNA by electrophoresis, confirmed by dot blot assay after hybridisation with an internal labelled oligonucleotide of digoxigenin. Reproducible results were obtained with DNA extracted from faeces stored at -20 degrees C or in 70% ethanol. The sensitivity and specificity of the method used, particularly double amplification and hybridisation, are discussed by comparing the results obtained by bacterial culture from faeces.

[Eales's disease with neurological involvement (author's transl)]. / Maladie de eales avec manifestations neurologiques.

A moroccan male aged 26, with Eales's disease since 6 years, develops a low thoracic level paraplegia over 2 months. Examination then also points out an horizontal nystagmus. CSF changes are important: 292 cells/mm3 (96 p. 100 lymphocytes), 3,80 g/l proteins. Slight improvement is obtained by corticosteroid therapy. This case is compared with those of the literature, mostly myelopathies. The pathogenetic problems of immuno-allergic type are discussed.

[Detection of viral hemorrhagic septicemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss) by reverse transcription followed by polymerase chain reaction. Diagnostic validation]. / Détection du virus de la septicémie hémorragique virale (SHV) de la truite arc-en-ciel (Oncorhynchus mykiss) par une transcription inverse suivie d'une amplification en chaîne par polymérase. Validations diagnostiques.

Viral haemorrhagic septicaemia virus (VHSV) is a fish pathogen that attacks rainbow trout (Oncorhynchus mykiss) farming. The only diagnostic method recognised by the European community for VHS is based on the detection of the viral agent in cell culture followed by an immunological identification of the pathogen. Reverse transcription followed by a double amplification of the polymerase chain reaction (RT-PCR) for the gene encoding the viral glycoprotein G is proposed here as an alternative method to the virus assay in cell culture. The RT-PCR was found to have three advantages over the viral assay method. First, the RT-PCR was found to be more rapid than the virus assay method (24 h compared to 72-120 h). Second, this method was found to be more sensitive than the virus assay (2.10(-2) pfu of virus were detected per millilitre of viral suspension compared to 2.10(-1) pfu.mL-1 by inoculation of the cell lines EPC and RTG2). Third, the RT-PCR was shown to be specific towards the VHS virus assay (represented by its four serotypes VHS I, VHS II, VHS 23/75 and VHS IV). Moreover no amplification was obtained with the other rhabdoviruses used: infectious haematopoietic necrosis virus (American strain Amend 72, WRAC, RB, SRVC) and French reference strain 69/87, eel viruses, spring viraemia of carp virus and pike fry virus. The validation of this method was performed on organs removed from experimentally infected rainbow trout and ovarian fluid samples from farmed broodfish from D0 to D150. By using RT-PCR between D30 and D60, 13 samples from nine experimentally infected trout (ten kidney-spleen pools and three brains) tested positive, whereas only nine samples (four kidney-spleen pools and five brains) from six fish were positive at D30. The last positive response was obtained by RT-PCR at D60 for kidney-spleen pools from three fish. At D150, all the results were negative. From the 60 ovarian fluid samples tested, 28 were VHS positive by the RT-PCR versus 15 by the virus assay method. Eleven out of the 60 broodfish had neutralised anti-VHS, six were negative by RT-PCR and by the virus assay, four were positive by RT-PCR and negative by virus assay and one positive by both methods. The specificity, sensitivity and rapidity of the RT-PCR method makes it an attractive alternative to classical virological methods currently recommended by European Fish Health Surveillance Programmes.

[Acute toxoplasmosis with necrotizing vasculitis (author's transl)]. / Toxoplasmose aiguë et vascularite nécrosante.

Acute toxoplasmosis was diagnosed in a Japanese woman aged 31 years after the discovery of a raised lysis titre and agglutinins resistant to 2-ME, as well as early increases in specific IgM followed later by increases in specific IgG. A high fever was present and signs of mainly distal polymyositis. The muscle lesion was confirmed by EMG examination. No increase in muscle enzyme levels was noted at any stage of the disease. Muscle biopsy demonstrated inflammatory lesions in interstitial tissue and perimysium, and, more particularly, segmental necrotizing arteritis in several arterioles. All the arteriolar lesions were at the same stage of development. After prednisolone (60 mg/day) had failed to produce improvement, spiramycin was given and caused apyrexia in 48 hours and definite disappearance of all muscle signs within several days. Recovery was complete and there was no return of symptoms 18 months later. The authors discuss the association of acute toxoplasmosis, polymyositis, and necrotizing vasculitis, and suggest a possible pathogenic role for the immune complexes deposited on the arterial walls.