In situ Raman monitoring of dielectric-heating-enhanced freeze-drying under different electromagnetic wave frequencies.

We studied the effect of dielectric heating on the enhancement of freeze-drying by electromagnetic waves (EMWs) under different frequencies: 2.45 GHz microwaves (MWs), and 27 and 200 MHz radio frequencies (RFs). The irradiation with RFs, particularly at 27 MHz, reduced the duration of freeze-drying by 67%. We further analysed the water structure by in situ Raman spectroscopy during freeze-drying under EMWs. The phase transition from ice to water occurred soon after starting irradiation by MWs at 2.45 GHz, while the ice phase was almost maintained at an RF of 27 MHz.

Genotypic characterization of VapA positive Rhodococcus equi in foals with pulmonary affection and their soil environment on a warmblood horse breeding farm in Germany.

Pulsotypes of VapA positive Rhodococcus equi isolated from foals and soil on a farm in Germany were characterized on the basis of nasal and tracheal samples simultaneously collected in 2003 from 217 foals with sonographic evidence of pneumonia or pulmonary abscesses. Of the 217 double samples, R. equi was isolated in 118 (54%) of the tracheal samples and in 52 of the nasal swab samples (24%) (P<0.001). Furthermore, 37 and 55 isolates were also randomly selected from nasal swabs and the tracheal samples, respectively, and further processed to determine the presence of VapA by colony blot enzyme-linked immunosorbent assay. This method showed that 26 (68%) of the nasal swabs and 40 (73%) of the tracheal samples were VapA-positive. R. equi was isolated from 56 (87%) of the 64 soil samples taken from the paddocks and stables in March and from 17 (68%) of the 25 samples taken in July of 2003. Three (21%) of these randomly selected 14 isolates from March and 13 (81%) of the 16 from July were VapA-positive. The VapA positive isolates from foals and soil were genotyped by plasmid profiling, restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis. Of the 83 isolates, 80 contained an 85-kb type I plasmid and three contained an 87-kb type I plasmid. Pulsed-field gel electrophoresis yielded four distinct VspI profiles dividing 83 isolates into three major (A1, 51; D, 14; and 11 isolates) and three minor (C, 4; A3, 2; and A2, 1 isolates) groups. These results suggest that the majority of foals were exposed to and infected with three pulsotypes of VapA positive R. equi containing an 85-kb type I plasmid.

Plasmid profiles of virulent Rhodococcus equi isolates from soil environment on horse-breeding farms in Hungary.

The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 15 contained an 85-kb type III plasmid which had been uniquely isolated from soil isolates in the United States. This study demonstrates that the virulent form of R. equi is very widespread in the soil environment of these stud farms in Hungary and the plasmid pattern is different from farm to farm.

Sequence of the Rhodococcus equi gene encoding the virulence-associated 15-17-kDa antigens.

The nucleotide sequence of the Rhodococcus equi gene encoding the virulence-associated 15-17-kDa antigens, located on plasmid pREAT701, has been determined. The gene encodes a 19-kDa protein of 189 amino acids, with an Ala-rich leader signal sequence (SS). At least five SS peptidase cleavage sites were found in this region. The molecular diversity of 15-17-kDa antigens might be attributed to the multiple SS peptidase cleavage sites.

Evaluation of the poly(ADP-ribose) polymerase gene in human stroke.

Nitric oxide (NO) and its reactant product, peroxynitrite, have been implied to mediate neuronal damage following cerebral ischemia. However, the cellular targets of these compounds remain unclear. Studies using poly(ADP-ribose) polymerase (PARP) inhibitors and PARP knock-out mice have recently demonstrated that excessive activation of this nuclear enzyme plays an important role in NO-induced neurotoxicity. To evaluate the relevance of this plausible candidate gene to human stroke, we undertook a case-control study in Japanese. Participants comprised 213 cerebral infarction cases and 374 age- and sex-matched controls. As a primary investigation, we screened polymorphic sites of the PARP gene, and newly identified a total of four polymorphisms in 1230-bp 5'-flanking sequence. None of them were, however, located on the known promoter components of the gene. Two bi-allelic polymorphisms selected and a CA-repeat polymorphism were subsequently characterized in the case-control study, but none were significantly associated with cerebral infarction in the present study. Our data thus suggest that the tested PARP polymorphisms do not principally contribute to cerebral infarction, although extensive searches would be required to clarify whether the PARP gene plays an important role in the pathogenesis of human stroke.

Local blood flow and vascular permeability of autonomic ganglion-transplants in the brain.

The purpose of this study was to determine whether the blood vessels of transplanted neural tissue retain their functional characteristics. Quantitative autoradiography was used to measure local blood flow (F) with iodoantipyrine and the blood-to-tissue transfer constant (K) with alpha-aminoisobutyric acid in superior cervical ganglion (SCG) allografted to the surface of ventricle IV and into the cerebellum of the same rat. The F of the intraparenchymal grafts was slightly lower than that of the intraventricular grafts; F decreased between 1 and 4 weeks in SCG grafts at both sites. The permeability-surface area (PS) product of the microvessels and extraction fraction of AIB were calculated from these results and indicated restricted transvascular passage of the amino acid in both the in situ and grafted SCG. Surface area (S) and average length (L) of the microvessels were determined morphometrically and their permeability (P) was calculated from these data. Although K and PS decreased in the grafts compared to in situ SCG, a comparable decrease in S indicated that P was similar for the microvessels of both in situ and 1-week-old SCG transplants: 3.5-4.3 x 10(-6) cm/s. Between 1 and 4 weeks after transplantation, the P of the microvessels decreased to approximately 1.6-2.3 x 10(-6) cm/s without any change in S. Thus, the blood vessels of SCG grafts within or upon the brain initially retain the functional attributes of in situ SCG microvessels, but the average permeability of the graft microvessels decreases to approximately one half of the initial value by 4 weeks after transplantation.

Live virulent Rhodococcus equi, rather than killed or avirulent, elicits protective immunity to R. equi infection in mice.

Mice inoculated intravenously with a sublethal dose of live virulent Rhodococcus equi ATCC 33701 that contained an 85-kb virulence plasmid were immune to a lethal intravenous challenge of ATCC 33701. This immunity depended upon the dose of immunization and developed rapidly: mice primed with 10(5) live ATCC 33701 eliminated the challenged bacteria more rapidly than mice primed with doses ranging from 10(2) to 10(4) bacteria, and mice given 10(5) live ATCC 33701 intravenously withstood the lethal challenge as early as 5 days after the initial inoculation. However, this protective immunity did not develop in mice immunized with doses of heat-killed ATCC 33701 ranging from 10(6) to 10(8), or in mice immunized with doses of live ATCC 33701P-, a plasmid-cured derivative (avirulent), in doses ranging from 10(5) to 10(7). These mice had positive antibody titers against R. equi at the challenge (14 days after priming). Adoptive transfer of resistance to virulent R equi was obtained with spleen cells from mice immunized with live ATCC 33701, but not monoclonal antibody to 15- to 17-kDa virulence-associated antigens. These results revealed that live ATCC 33701P-, a plasmid-cured derivative of virulent R equi, could not elicit protective immunity, and are consistent with previous observations that protective immunity was induced by live virulent, but not killed organisms.

Pathogenesis of Rhodococcus equi infection in mice: roles of virulence plasmids and granulomagenic activity of bacteria.

Virulence of Rhocococcus equi ATCC 33701 and its plasmid-cured derivative ATCC 33701P- was compared in BALB/c and C3H/HeJ mice in terms of bacterial growth kinetics and histological changes in the liver, spleen and lungs, and humoral immune responses. Injection with a sublethal dose of 10(6) ATCC 33701 in mice resulted in microabscess formation after rapid multiplication in the liver and spleen by day 4, and then the bacteria were gradually eliminated with the formation of granuloma and the production of specific antibodies against 15- to 17-kDa antigens of the virulent bacteria. By contrast, ATCC 33701P- was avirulent as shown by early elimination of viable bacteria and no evidence of net multiplication in the organs. Histopathological changes consisted of only slight, transient infiltration of neutrophils and macrophages in the liver. Although live ATCC 33701P- did not evoke any humoral or histological responses in the mice, a large inoculum (10(8)) of killed ATCC 33701 and ATCC 33701P- resulted in the formation of granuloma in the liver and accelerated extramedullary hemopoiesis in the spleen. These results suggest that the pathogenesis of R. equi infection involves at least two important virulence determinants, both of which play critical roles in the disease: one is the virulence plasmid, which is required for R. equi to resist and grow within host cells; and the other is the granulomagenic activity that is related to the lipids and nature of the cell wall of the species, which induces the characteristic pathological changes.

Cutaneous malakoplakia in pigs inoculated with Rhodococcus equi.

Cutaneous malakoplakia was observed in pigs inoculated intramuscularly with Rhodococcus equi strains of intermediate virulence. Macroscopically, the inoculation sites showed the indurated swelling of the skin. Histopathologically, abscess formation with histiocytic granulomatous reaction was observed. Many macrophages contained target or owl-eye shaped hematoxyphil intracytoplasmic inclusions or calcosherites (Michaelis-Gutmann bodies) of various sizes. The Michaelis-Gutmann bodies were also seen outside of the macrophages. Histochemically, most Michaelis-Gutmann bodies stained positively with the von Kossa silver method and periodic acid Schiff. Immunohistochemically, some of Michaelis-Gutmann bodies were stained by two rabbit polyclonal antibodies (rabbit anti-A5 serum and rabbit anti-ATCC 33701 serum) and a mouse monoclonal antibody (anti-20-kDa antigen monoclonal antibody). This is the first report of cutaneous malakoplakia in domestic animals, which also revealed the relationship between R. equi infection and malakoplakia immunohistochemically. This experimental swine model is useful to investigate the morphogenesis of Michaelis-Gutmann bodies in malakoplakia through chronological skin biopsies.

Virulent and avirulent Rhodococcus equi infection in T-cell deficient athymic nude mice: pathologic, bacteriologic and immunologic responses.

We investigated the pathologic, bacteriologic and immunologic responses of BALB/c-nu/nu mice (nude mice) and BALB/c mice (euthymic mice) infected intravenously with virulent and avirulent Rhodococcus equi ATCC 33701, and its plasmid-cured derivative ATCC 33701P-, to evaluate the role of T lymphocytes. Adaptive transfer of immune and normal spleen cells into nude mice was also investigated. Nude and euthymic mice were inoculated with 10(6) ATCC 33701 or 10(6) ATCC 33701P- intravenously (i.v.) and killed at 0, 7, 14, 21, 28 and 35 days post-inoculation, except dead cases. In athymic nude mice infected with ATCC 33701, deteriorating systemic inflammatory responses developed during the experimental period and multiplication of the bacteria continued until the end of the experiment. Nude mice developed splenomegaly and multifocal gross hepatic necrosis with some mortality. Splenomegaly was caused by diffuse proliferation of bacteria-laden macrophages and epithelioid cells, and gross hepatic necrosis was caused by the formation of thromboses and granulomatous lesions. Infection of euthymic mice with a sublethal dose of ATCC 33701 resulted in transient granuloma formation in the liver and spleen, production of specific antibodies against the virulent bacteria and gradual elimination thereof. In contrast, infection with ATCC 33701P- produced few lesions after rapid elimination and no antibody production against bacteria in either normal or athymic nude mice. In nude mice given normal and immune spleen cells, histopathological lesions and granulomas formed only in the liver and spleen, in addition to specific antibodies against 15- to 17-kDa antigens. The pathological lesions observed in the nude mice given immune spleen cells were similar to those seen in the mice given normal spleen cells, but they were less severe than those in mice given normal spleen cells. Mice given immune spleen cells showed a significantly higher elevation of antibody production than mice given normal spleen cells. These results suggested that protection against virulent R. equi in mice depends mainly on cell-mediated immune responses, whereas avirulent R. equi in mice are cleared by innate immune responses.

Parapontine trigeminal cryptic angiomas presenting as trigeminal neuralgia.

Posterior fossa microvascular decompression surgery was attempted in 1257 patients with trigeminal neuralgia (TN), of whom seven had a very unusual cryptic angioma. The lesions were not visualized on preoperative enhanced computerized tomography scans, and serial angiography demonstrated a small vascular stain in only one case. The character of the facial pain was indistinguishable from TN caused by vascular compression and there was no other specific symptomatology. The patients' age and sex distributions were also compatible with classical TN. Cryptic angiomas presenting as typical TN without other symptoms have not been reported before, but they should be kept in mind in the differential diagnosis and surgical management of TN.

Metallothionein induction by cadmium, cytokines, thrombin and endothelin-1 in cultured vascular endothelial cells.

Metallothionein induction was investigated using vascular endothelial cells derived from bovine aorta in a culture system. The induction occurred by cadmium (2 and 5 microM) but not by zinc (10 and 300 microM) after a 24-h incubation of the confluent cultures. It was revealed that cytokines including interleukin-1 beta, interleukin-6, tumor necrosis factor alpha and transforming growth factor beta (1 ng/ml each) have a capacity of metallothionein induction. In these inducers, only cadmium and tumor necrosis factor alpha exhibited significant cytotoxicity, suggesting that metallothionein is not induced simply in response to cytotoxicity. It was found that either thrombin or endothelin-1 which are coagulation factor or anti-fibrinolytic factor, respectively, also induced metallothionein synthesis. It was therefore suggested that metallothionein in endothelial cells may be involved in the regulation of the functions of these cells as well as the protection against cytotoxic agents.

Humoral immune response of foals to experimental infection with Rhodococcus equi.

Humoral immune response to Rhodococcus equi in experimentally infected foals was studied with the enzyme-linked immunosorbent assay (ELISA) method. Class-specific antibodies were measured by ELISA in the sera of foals after intratracheal or oral inoculation with R. equi ATCC 6939 or T 48 and in the lung washings of a foal after intratracheal inoculation or of normal horses. After intratracheal or oral inoculation with R. equi, serum antibodies were first detected in immunoglobulin G (IgG) followed by IgM and IgA classes, but significant levels of IgM and IgA developed only in the foal infected intratracheally with R. equi T 48. Only the foal infected intratracheally with T 48 developed pneumonia. Anti-R. equi IgG and IgA antibodies appeared in lung washings of the intratracheally infected foal. There were differences in the antibody response to R. equi among the intratracheally infected foals, the orally infected foal and the naturally infected foal. These results suggest that the humoral immune response to R. equi may be affected by the type of R. equi strain and the route and extent of R. equi exposure.

Ecology of Rhodococcus equi in horses and their environment on horse-breeding farms.

Quantitative culture of R. equi in the feces of dams and foals, in the air of the stalls and in the soil of the paddocks was carried out on three horse-breeding farms during the foaling season. The isolation rates of R. equi from the feces of dams from the 3 farms suddenly increased to approximately 80% at the end of March, when the snow in the paddocks finished melting, and remained at that level during April and May. The mean number of R. equi and the isolation rate of R. equi from the feces of dams on the farms were investigated for 5 weeks before and 5 weeks after delivery. During the 10 weeks, there were no differences in the isolation rate or in the mean number of R. equi from the feces of dams. R. equi was first isolated from the feces of the foals born in February and the middle of March at 3-4 weeks of age, on the other hand, it was first isolated from the feces of foals born in the end of March and April at 1-2 weeks of age. The number of R. equi in the soil collected from the paddocks used by dams during the winter was approximately 10(2)-10(4) g-1 of soil during the experiment. R. equi was isolated from the air in the stalls at the end of March and the number of R. equi in the air increased particularly on dry and windy days.(ABSTRACT TRUNCATED AT 250 WORDS)

Ecology of Rhodococcus (Corynebacterium) equi in soil on a horse-breeding farm.

The ecology of Rhodococcus (Corynebacterium) equi in soil was studied on a horse-breeding farm. R. equi was cultured from soil at a depth of 0, 10, and 20 cm on the six sites of the farm at monthly intervals for 10 months from March to December of 1983. The highest numbers of R. equi were found in the surface soil. The mean number of bacteria in soil samples at every depth increased remarkably from 0 or 10(2) to 10(4) colony-forming units (CFU) g-1 of soil in the middle of April, and later decreased gradually. R. equi inoculated into six soil exudate broths prepared from surface soils at separate sites yielded suspensions with different optical densities, indicating differences in growth. The distribution of serotypes in the soil was similar to that in the horses on the farm. These findings indicated that R. equi could multiply in the soil and flourish in the cycle existing between horses and their soil environment.

Effect of growth temperature on maintenance of virulent Rhodococcus equi.

Repeated passage of virulent Rhodococcus equi ATCC 33701 and L1 at 38 degrees C resulted in attenuation of the strains as a result of curing the virulence plasmid; at 30 degrees C, repeated passage had no such effect. At a temperature of 38 degrees C the plasmid-bearing cells replicated more slowly than their plasmid-cured derivatives and so were gradually replaced by cells lacking plasmids. In contrast, at a temperature of 30 degrees C the growth rate of either strain was not affected by the presence or absence of the plasmid. No plasmid-cured derivative was recovered from mouse organs at 48 h after inoculation of a mixture of equal numbers of bacteria with and without plasmids. It is concluded that under nonselective conditions growth temperature is an important factor in maintaining the virulence of R. equi.

Pathogenicity and virulence of Rhodococcus equi in foals following intratracheal challenge.

Twelve foals, between 27 and 83 days old, were infected with 2 strains of Rhodococcus equi by intratracheal administration. Ten of the 12 foals were inoculated with 10(4)-10(10) colony forming units (cfu) of ATCC 33701 strain. The other 2 foals were inoculated with 10(9) cfu of a plasmid-cured derivative of the ATCC 33701 strain (ATCC 33701P-). All of the 10 foals challenged with the ATCC 33701 strain showed clinical signs of pulmonary disease within 5-13 days, such as gross lesions associated with acute bronchopneumonia and microscopic lesions associated with granulomatous pneumonia. The two foals challenged with the ATCC 33701P- strain showed neither clinical signs of disease nor gross lesions. Apparently, when lacking plasmid, the virulent Rhodococcus equi lost its pathogenicity.

Comparison of tracheal aspiration with other tests for diagnosis of Rhodococcus equi pneumonia in foals.

The diagnostic value of tracheal aspiration was evaluated through comparison with other diagnostic methods using an experimental model of Rhodococcus equi (R. equi) pneumonia in foals. Pneumonia was induced by spraying of the virulent R. equi strain ATCC 33701 into the trachea of foals. All foals developed fever from 11 to 16 days after bacterial inoculation. One foal was euthanized on day 26 due to its poor prognosis, and other foals euthanized on day 43. During the experiment, some tests for diagnosis of Rhodococcus equi pneumonia such as tracheal aspiration, radiography, serodiagnosis and fecal culture were carried out. R. equi was continually isolated from tracheal aspirates collected via a silicone catheter inserted transnasally on day 8 to day 32 after bacterial inoculation. On the other hand, radiography, serodiagnosis and fecal culture were demonstrated to be valuable diagnostic methods, but to be limited compared with tracheal aspiration. Indirect fluorescent antibody technique (IFA) using a monoclonal antibody against the 15- to 17-kDa virulence-associated antigens (VapA) of R. equi and PCR targeting the structural gene of VapA detected bacteria in tracheal aspirates less sensitively than the isolation technique although they were more rapid. Therefore, we conclude that a combination of tracheal aspiration and bacterial isolation was the most valuable method for routine diagnosis of R. equi pneumonia in foals.

Pathogenicity of Rhodococcus equi expressing a virulence-associated 20 kDa protein (VapB) in foals.

Rhodococcus equi strains of intermediate virulence (IMV) for mice possess a 20kDa protein designated Virulence Associated Protein B (VapB) and a virulence plasmid of 79-100kb, and can be recovered from the submaxillary lymph nodes of pigs. The pathogenicity of such R. equi strains for foals is unknown. In this study, two foals, 42 and 43 days of age, were infected intratracheally with 10(6) and 10(9) cells of R. equi IMV strain A5, respectively. The foal infected with 10(9) cells of strain A5 became clinically ill, with the onset of illness (pyrexia and depression) occurring 21 days after inoculation. R. equi was isolated from the feces and tracheal washings of the foal from 14 to 28 days after inoculation. The foal infected with 10(6) cells of A5 showed no clinical signs, and no R. equi was isolated from any of the samples of feces or tracheal washings during the 28 days of observation. Two foals of 45 and 50 days of age were infected with 10(5) or 10(6) of virulent R. equi ATCC 33701 having 15-17kDa surface proteins designated VapA. Both exhibited severe clinical signs (pyrexia, depression and anorexia) at 12 and 13 days after inoculation. Histopathological examination revealed that strain A5 caused focal granulomatous pneumonia in the foals. R. equi IMV strain A5 was isolated from lung lesions of both foals and from the contents of the intestinal tracts of the foal infected with 10(9) bacteria. These results suggest that IMV R. equi having VapB is less virulent than virulent R. equi having VapA in foals. This finding supports our previous results on the pathogenicities of R. equi strains having these virulence-associated antigens assessed by mouse pathogenicity tests.

Two new variants of the Rhodococcus equi virulence plasmid, 90 kb type III and type IV, recovered from a foal in Japan.

This report describes the discovery of two new virulence plasmid types from a crossbred foal with Rhodococcus equi pneumonia in Kumamoto died with severe R. equi pneumonia and ulcerative enteritis. R. equi was isolated in large numbers and isolates from the foal were investigated for the presence of virulence-associated 15-17 kDa antigens (VapA) by colony blotting, using the monoclonal antibody 10G5, and by gene coding for VapA by PCR. Plasmid DNAs extracted from the isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII. The digestion patterns that resulted divided the plasmids of these isolates into two closely related types. The digestion patterns were then compared with eight representative virulence plasmid types (85 kb types I, II, III and IV, 87 kb types I and II, 90 kb types I and II), which have already been reported. None of the EcoRI and EcoT22I digestion patterns of the eight representative plasmids matched those of the two plasmid types. We tentatively designated these new plasmid types as 90 kb type III and type IV, since HindIII and BamHI digestion patterns of the two plasmid types were identical with those of a 90 kb type I plasmid. This study, demonstrated that there are at least 10 distinct but closely related plasmids present in isolates from horses in the world.