Labile assembly of a tardigrade protein induces biostasis.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self-assembly of labile CAHS gels.

Surgical implantation of temperature-sensitive transmitters and data-loggers to record body temperature in koalas (Phascolarctos cinereus).

BACKGROUND: Under predicted climate change scenarios, koala distribution in Australia is expected to be adversely affected. Recent studies have attempted to identify suitable habitat, based on models of bioclimatic regions, but to more accurately reflect the thermal tolerance and behavioural adaptations of the various regional populations, the koala's response to periods of heat stress will need to be investigated at the individual animal level. OBJECTIVE: To explore the safety and suitability of temperature-sensitive intra-abdominal implants for monitoring core body temperature in the koala. METHODS: A temperature-sensitive radio transmitter and thermal iButton data-logger, waxed together as a package, were surgically implanted into the abdominal cavity of four captive koalas. In one animal the implant was tethered and in the other three, it was left free-floating. RESULTS: After 3 months, the implants were removed and all four koalas recovered without complications. The tethering of the package in the one koala resulted in minor inflammation and adhesion, so this practice was subsequently abandoned. The free-floating deployments were complication-free and revealed a diurnal body temperature rhythm, with daily ranges of 0.4-2.8°C. The minimum recorded body temperature was 34.2°C and the maximum was 37.7°C. The difference in the readings obtained from the transmitters and iButtons never exceeded 0.3°C. CONCLUSIONS: The suitability of the surgical approach was confirmed, from both the animal welfare and data collection points of view.

Concordance of motor vehicle crash, emergency department, and inpatient hospitalization data sets in the identification of drugs in injured drivers.

OBJECTIVE: Prescription drug overdoses, abuse, and sales have increased dramatically in the United States in the last decade. The purpose of the present study was to link crash data with emergency department (ED) and inpatient hospitalization data to assess the concordance between the data sets in the identification of the presence of drugs among injured motor vehicle drivers (passenger cars, passenger trucks, light trucks, and semi-trucks) in Kentucky. METHODS: Kentucky CRASH data were probabilistically linked to ED data sets for years 2008-2010 and to inpatient hospitalization data sets for years 2000-2010. Statistical analyses were performed. RESULTS: Of the 72,529 linked crash/ED visits, there were 473 drivers with an associated nondependent abuse of drugs diagnosis in the ED, and 930 drivers had drug involvement recorded in the CRASH data (only 163 cases overlapped with drug involvement both recorded in CRASH data and coded as nondependent abuse of drugs in the ED); 64 drivers had multiple drug types present in their system. Of the 20,860 total linked crash/inpatient hospitalization cases, there were 973 drivers diagnosed with nondependent abuse of drugs in the inpatient hospitalization record and 499 drivers had drug involvement recorded in the CRASH data (only 207 overlapped); 250 drivers were diagnosed with multiple drugs in their system. CONCLUSIONS: Surveillance data from multiple public health data sets is necessary to identify the presence of drugs in injured drivers involved in motor vehicle crashes. The use of a single surveillance data set alone may significantly underreport the number of drugged drivers who were injured in a motor vehicle collision.

Partial genome sequencing of Rhodococcus equi ATCC 33701.

Preliminary analysis of a partial (30% coverage) genome sequence of Rhodococcus equi has revealed a number of important features. The most notable was the extent of the homology of genes identified with those of Mycobacterium tuberculosis. The similarities in the proportion of genes devoted to fatty acid degradation and to lipid biosynthesis was a striking but not surprising finding given the relatedness of these organisms and their success as intracellular pathogens. The rapid recent improvement in understanding of virulence in M. tuberculosis and other pathogenic mycobacteria has identified a large number of genes of putative or proven importance in virulence, homologs of many of which were also identified in R. equi. Although R. equi appears to have currently unique genes, and has important differences, its similarity to M. tuberculosis supports the need to understand the basis of virulence in this organism. The partial genome sequence will be a resource for workers interested in R. equi until such time as a full genome sequence has been characterized.

Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins.

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, vapC, -D, and -E, that are tandemly arranged downstream of vapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other. vapC, -D, and -E are found only in R. equi strains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed when R. equi is cultured at 37 degrees C but not at 30 degrees C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins.

DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103.

The virulence plasmids of the equine virulent strains Rhodococcus equi ATCC 33701 and 103 were sequenced, and their genetic structure was analyzed. p33701 was 80,610 bp in length, and p103 was 1 bp shorter; their sequences were virtually identical. The plasmids contained 64 open reading frames (ORFs), 22 of which were homologous with genes of known function and 3 of which were homologous with putative genes of unknown function in other species. Putative functions were assigned to five ORFs based on protein family characteristics. The most striking feature of the virulence plasmids was the presence of a 27,536-bp pathogenicity island containing seven virulence-associated protein (vap) genes, including vapA. These vap genes have extensive homology to vapA, which encodes a thermoregulated and surface-expressed protein. The pathogenicity island contained a LysR family transcriptional regulator and a two-component response regulator upstream of six of the vap genes. The vap genes were present as a cluster of three (vapA, vapC, and vapD), as a pair (vapE and vapF), or individually (vapG; vapH). A region of extensive direct repeats of unknown function, possibly associated with thermoregulation, was present immediately upstream of the clustered and the paired genes but not the individual vap genes. There was extensive homology among the C-terminal halves of all vap genes but not generally among the N-terminal halves. The remainder of the plasmid consisted of a large region which appears to be associated with conjugation functions and a large region which appears to be associated with replication and partitioning functions.

Restriction fragment length polymorphisms of virulence plasmids in Rhodococcus equi.

Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries-Argentina, Australia, Canada, France, and Japan-were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738-740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world.

Protective effect against Rhodococcus equi infection in mice of IgG purified from horses vaccinated with virulence associated protein (VapA)-enriched antigens.

IgG was purified from horses immunized with repeated doses of virulence associated (VapA) enriched antigens extracted with Triton X-114 from the surface of a virulent strain of R. equi. This IgG were administered to mice immunosuppressed by prior treatment with indomethacin. Mice administered the higher dose were completely protected against intraperitoneal infection with R. equi; mice given the lower dose were partially protected. By contrast, mice administered concentrated nonimmune equine IgG were not protected. This study demonstrates that VapA may be an important antigen involved in humoral protective immunity in R. equi infections caused by foal virulent strains.

Assessment of the immunogenic potential of Rhodococcus equi virulence associated protein (VapA) in mice.

The development of immunity to Rhodococcus equi, particularly to a virulence-associated protein (VapA) based antigen preparation, was examined in CD1 and BALB/c mice after intraperitoneal vaccination. Immunization with VapA based antigen without adjuvant markedly enhanced organ clearance in CD1 mice but not in BALB/c mice. Delayed type hypersensitivity response and antibody titres in VapA based antigen immunized BALB/c mice were less than in CD1 mice. By contrast also to CD1 mice, sera from immunized BALB/c mice did not react as strongly with VapA in western blots. Use of adjuvants (aluminium hydroxide, iscoms) interfered markedly with the immunogenic properties of the VapA based antigen, in the case of aluminium hydroxide by apparently driving a Th2 type of response. Unexpectedly, iscom adjuvants also impaired immunity and, despite the highest DTH response, produced a low IgG2a response, suggesting that iscomization of the antigen produced a low interferon gamma and high interleukin 2 response. Passive immunization of BALB/c mice with serum from mice immunized with live virulent strain 103+ resulted in only temporary and slight enhancement of organ clearance, supporting the central importance of cellular immunity to R. equi. Immunization with live virulence plasmid- and VapA-positive R. equi strain 103 resulted in marked liver clearance, in marked DTH response and high antibody titres. By contrast, immunization with live virulence plasmid- and VapA-negative strain 103 resulted in slight but variable enhancement of clearance, but insignificant DTH and antibody. The virulence plasmid, and by implication VapA, was thus shown to be critical in determining a highly effective protection to live organisms.

Use of Rhodococcus equi virulence-associated protein for immunization of foals against R equi pneumonia.

OBJECTIVE: To evaluate use of the virulence-associated protein of Rhodococcus equi in immunizing foals against R equi pneumonia. ANIMALS: Eight (experimental group) and 6 (controls) mares with their foals. PROCEDURE: Virulence-associated protein extracted from R equi was used to prepare an acetone-precipitated. Triton X-extracted (APTX) antigen. After determination of the efficacy of passive immunization, in untreated foals or in foals given plasma from a horse vaccinated with APTX antigen or from a nonvaccinated horse, a field trial was done to evaluate the efficacy of vaccination of 8 mares, twice with APTX before parturition, and of their foals at ages 3 and 5 weeks; 6 mares and their foals served as unvaccinated controls. All 2-day-old foals were given plasma from local donor horses inoculated with a locally produced bacterin. Serum opsonizing activity produced by vaccination with APTX was determined. Passively immunized foals were challenge exposed with an aerosol of virulent R equi. Foals of the field trial were exposed to enzootic R equi infection. RESULTS: Inoculation with APTX resulted in high IgG antibody liters with opsonizing activity. Passive immunization of foals with plasma from an immunized horse enhanced bacterial clearance from the lungs, compared with that in foals not given plasma or given plasma without APTX antibodies. Vaccination of mares and foals exposed to natural infection resulted in development of R equi pneumonia in 4 of 8 vaccinated foals, but in only 1 of 6 unvaccinated foals. CONCLUSIONS: Vaccination with APTX antigen led to high-titer, opsonizing antibody. Plasma from a vaccinated horse appeared to enhance clearance of R equi from the lungs of foals. Paradoxically, vaccination of mares and their foals with APTX antigen did not protect foals and may have enhanced R equi pneumonia in the foals.

Restriction enzyme analysis of the virulence plasmids of VapA-positive Rhodococcus equi strains isolated from humans and horses.

Restriction enzyme digestion patterns of the large virulence plasmids of 8 human and 37 foal isolates of virulence-associated protein (VapA)-positive Rhodococcus equi strains from different sources were compared. Foal isolates came from five continents. Digestion with EcoRI divided these plasmids into three closely related types, and digestion with BamHI divided them into three major types which corresponded to the EcoRI types. The only EcoRI and BamHI type 3 plasmid was from a single foal isolate obtained from Japan. There are thus two major but related virulence plasmids in isolates from foals. Geographic differences were noted, since foal isolates with the EcoRI type 1 plasmid digestion pattern tended to come mostly from the United States, Canada, European countries, India or Zimbabwe and foal isolates with EcoRI type 2 pattern tended to come mostly from Latin American countries. Only 8 of 38 different human isolates, mostly from AIDS patients, were VapA positive, in contrast to 37 of 42 foal isolates. VapA-positive isolates from humans possessed virulence plasmids of either EcoRI type 1 or EcoRI type 2. These results confirm that only a small proportion of human patients with R. equi infections acquire foal virulent R. equi.

Comparison of cytomegalovirus antigenemia and shell vial culture in allogeneic marrow transplantation recipients receiving ganciclovir prophylaxis.

We prospectively monitored 61 allogeneic BMT patients for evidence of CMV infection and disease starting 7 days prior to transplant until day 110 after transplant. Patients receiving pre- and post-transplantation ganciclovir prophylaxis were followed for the incidence of infection by the CMV antigenemia assay and shell vial cultures. The median age of all patients was 32 years (range 5-54 years). Fourteen (25%) of 57 evaluable patients became CMV antigenemia or culture positive. The incidence of culture or antigenemia positivity in CMV seropositive or seronegative patients with a seropositive donor was 29% (14 of 49 patients). The antigenemia assay became positive a median of 29 days (range 12-89 days) after BMT as compared to 46 days (range 26-98 days) by shell vial assay (P < 0.001). There were no cases of CMV disease in the first 110 days after transplant. This study demonstrates that despite the use of prophylactic ganciclovir, BMT patients developed CMV infection but did not progress to disease in this study, the CMV antigenemia assay may be used to monitor for CMV infection during prophylaxis, and the current regimens for CMV prophylaxis with ganciclovir may require further evaluation to determine an optimal regimen to prevent CMV infection.

Cryptosporidium parvum in environmental samples in the Sligo area, Republic of Ireland: a preliminary report.

To detect Cryptosporidium in environmental specimens in the Republic of Ireland, grab samples of river water were prepared by calcium carbonate flocculation, and marine mussel tissue homogenated prior to testing with a fluorescently labelled monoclonal antibody and fluorescence microscopy. The parasite was detected in both river waters and marine mussels (Mytilus edulis). Filter feeders such as Mytilus edulis may be of value as biological monitors for the presence of cryptosporidial oocysts in sea water. The presence of Cryptosporidium in river and marine waters and, in particular, contaminating mussels used for human consumption, has obvious health implications.

Use of a virulence-associated protein based enzyme-linked immunosorbent assay for Rhodococcus equi serology in horses.

An enzyme-linked immunosorbent assay (ELISA) was developed against Rhodococcus equi using Triton X-114 detergent extracted whole cell material, in which the virulence associated protein (VapA) predominated. Enzymelinked immunosorbent assay titres corresponded to antibody reacting with VapA on Western blots. There was considerable variation in antibody titres of nonimmunised mares and in the time when the colostrally derived antibody of their foals had declined to low or undetectable titres. In general, antibodies in foals declined to their lowest levels at age 4-8 weeks. Seroconversion occurred in foals age 8-10 weeks, but the precise time depended on maternal titre and the month in which the foal was born. Foals reaching age 8 weeks in late summer showed more marked seroconversion than foals born earlier. The ELISA was used to follow the response to immunisation with the same Triton X-114 extracted material. Six mares immunised before parturition with the antigen in aluminium hydroxide adjuvant developed high titres, up to > 102,400 and transferred them to their foals through colostrum. Their foals responded to immunisation with 0.5-1.0 mg antigen 3, 5, 7 and 9 weeks after birth. Antibody titres following immunisation with similar dosage reached up to > 102,400 in a separate group of foals of nonimmunised mares. Nonvaccinated control foals seroconverted at age 6-8 weeks. The VapA based ELISA is useful to follow the course of natural infection with R. equi or immunisation with VapA based antigen.

Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity.

Virulent strains of Rhodococcus equi produce plasmid-mediated 15- and 17-kDa proteins, which are thermoregulated and apparently surface-expressed. We demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that R. equi produce three antigenically-related virulence-associated proteins, a diffuse 18-22-kDa, a 17.5-kDa and a 15-kDa protein. Phase partitioning of whole cells of R. equi strain 103 with Triton X-114 (TX-114) and labelling with [3H]-labelled palmitic acid showed that the two higher molecular weight proteins are hydrophobic and lipid modified. The 15-kDa protein did not partition into TX-114 and was not lipid modified. Cloning and expression of a fragment of the R. equi virulence plasmid in Escherichia coli showed that the three proteins were expressed from a single gene. Sequence analysis of this gene (designated vapA) revealed a 570-bp open reading frame encoding a polypeptide of 189 amino acids with a calculated molecular mass of 19,175 Da. The mature, nonlipid modified protein had a calculated mass of 16,246 Da. The 17.5- and 18-22-kDa forms of the protein are therefore due to lipid modification. No significant sequence homology of the vapA gene with other reported nucleotide sequences were found. Opsonization of virulent R. equi with an IgG1 mouse monoclonal antibody (MAb103) to the VapA protein significantly enhanced uptake in the murine macrophage cell line IC-21. Intraperitoneal injection of mice with Mab103 enhanced initial clearance from the liver of mice challenged intravenously with R. equi. Immunization of mice with the lipid-modified VapA purified by SDS-PAGE fractionation or with acetone precipitated VapA protein following TX-114 extraction resulted in significantly enhanced clearance from the liver and spleen following intravenous challenge. The VapA protein of R. equi appears therefore to be a protective immunogen.

Infectious complications in solid organ transplant recipients.

Infections remain a major problem for individuals who undergo solid organ transplantation. The risk of these infections is determined by previous or future environmental exposures as well as the patient's immune status. With the use of prophylactic antibiotics, antifungal agents, and the development of selective immunosuppressive agents, the incidence of infection should decrease. Rapid diagnosis and the institution of appropriate therapy are necessary for cure. Further investigation is required to determine the optimal prophylaxis necessary for disease prevention.

Outer membrane proteins of three pathogenic Leptospira species.

The outer membrane proteins of seven reference strains of pathogenic Leptospira (L. alstoni serovar grippotyphosa, L. borgpetersenii serovar hardjo, and L. interrogans serovars autumnalis, bratislava, canicola, icterohaemorrhagiae, and pomona) were investigated to identify common surface-exposed outer membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sodium-N-lauroylsarcosinate-insoluble outer membrane enriched fractions of the reference serovars and two field isolates of serovars hardjo and pomona revealed six common protein bands with approximate molecular masses of 77, 66, 42, 35.5, 24, and 18 kDa. At times the 35.5 kDa endoflagellar band resolved into two distinct bands, 35.5 kDa and 34 kDa. Immunoblotting of the same fractions using rabbit leptospiral antibodies showed six bands to be common (66, 59.5, 44, 42, 35.5, and 18 kDa). The 44 kDa band stained poorly with Coomassie blue but prominently by immunoblotting. Four reference strains (serovars bratislava, canicola, icterohaemorrhagiae, pomona), and two field isolates of serovar pomona and one of serovar bratislava were grown in low iron media to which the iron chelators 2,2'-dipyridyl or ethylenediaminehydroxyphenylacetic acid were added. No iron-dependent expression of outer membrane proteins was observed. The only difference observed between the outer membrane proteins when reference serovars of canicola or pomona were grown in dialysis bags in the peritoneum of swine or in vitro was the loss of the 77 kDa band from in vivo grown organisms. Treatment of whole leptospires with proteinase K did not remove the 77, 66, 59.5, or 42 kDa protein; these proteins may not be surface expressed or are inaccessible to the proteinase K. The 44 kDa band could not be evaluated by this method and the 18 kDa band was proteinase K resistant.