Summer pheasant's eye (Adonis aestivalis) poisoning in three horses.

Three horses died as a result of eating grass hay containing summer pheasant's eye (Adonis aestivalis L.), a plant containing cardenolides similar to oleander and foxglove. A 9-year-old thoroughbred gelding, a 20-year-old appaloosa gelding, and a 5-year-old quarter horse gelding initially presented with signs of colic 24-48 hours after first exposure to the hay. Gastrointestinal gaseous distension was the primary finding on clinical examination of all three horses. Two horses became moribund and were euthanatized 1 day after first showing clinical signs, and the third horse was euthanatized after 4 days of medical therapy. Endocardial hemorrhage and gaseous distension of the gastrointestinal tract were the only necropsy findings in the first two horses. On microscopic examination, both horses had scattered foci of mild, acute myocardial necrosis and neutrophilic inflammation associated with endocardial and epicardial hemorrhage. The third horse that survived for 4 days had multifocal to coalescing, irregular foci of acute, subacute, and chronic myocardial degeneration and necrosis. A. aestivalis (pheasant's eye, summer adonis) was identified in the hay. Strophanthidin, the aglycone of several cardenolides present in Adonis spp., was detected by liquid chromatography-mass spectrometry-mass spectrometry in gastrointestinal contents from all three horses. Although Adonis spp. contain cardiac glycosides, cardiac lesions have not previously been described in livestock associated with consumption of adonis, and this is the first report of adonis toxicosis in North America.

Selective perceptual impairments after perirhinal cortex ablation.

It has been suggested that the primate perirhinal cortex contributes exclusively to memory. However, recent studies in macaque monkeys have implied that the perirhinal cortex may also contribute to object perception. To investigate whether the perirhinal cortex does contribute to perception, we devised several perceptual oddity tasks in which monkeys had to choose which stimulus of several presented concurrently on a touch screen was different. Macaques with bilateral perirhinal cortex ablations were selectively impaired relative to controls at perceptually discriminating the odd stimulus when the odd stimulus was a different object and when the discrimination could not be done on the basis of simple differences in features between the stimuli. They remained unimpaired relative to controls on discriminating the odd stimulus when the odd stimulus was a different color, a different shape, or a different size even when these discriminations were extremely difficult. They were also impaired on human and monkey face oddity tasks and oddity tasks with scenes containing objects. Therefore, we reject the notion that the macaque perirhinal cortex has a role exclusive to memory and conclude that the macaque perirhinal cortex does contribute to perception. We argue that the perirhinal cortex is neither specialized for perception nor memory processes alone, but rather, is specialized for processing stimuli that require processing at a more abstract level such as at the level of an object and that the perirhinal cortex contributes to both memory and perception of such stimuli.

Clonal associations among Staphylococcus aureus isolates from various sites of infection.

A molecular epidemiological analysis was undertaken to identify lineages of Staphylococcus aureus that may be disproportionately associated with infection. Pulsed-field gel electrophoresis analysis of 405 S. aureus clinical isolates collected from various infection types and geographic locations was performed. Five distinct S. aureus lineages (SALs 1, 2, 4, 5, and 6) were identified, which accounted for 19.01, 9.14, 22.72, 10.12, and 4.69% of isolates, respectively. In addition, 85 lineages which occurred with frequencies of <2.5% were identified and were termed "sporadic." The most prevalent lineage was methicillin-resistant S. aureus (SAL 4). The second most prevalent lineage, SAL 1, was also isolated at a high frequency from the anterior nares of healthy volunteers, suggesting that its prevalence among clinical isolates may be a consequence of high carriage rates in humans. Gene-specific PCR was carried out to detect genes for a number of staphylococcal virulence traits. tst and cna were found to be significantly associated with prevalent lineages compared to sporadic lineages. When specific infection sites were examined, SAL 4 was significantly associated with respiratory tract infection, while SAL 2 was enriched among blood isolates. SAL 1 and SAL 5 were clonally related to SALs shown by others to be widespread in the clinical isolate population. We conclude from this study that at least five phylogenetic lineages of S. aureus are highly prevalent and widely distributed among clinical isolates. The traits that confer on these lineages a propensity to infect may suggest novel approaches to antistaphylococcal therapy.

In vitro pharmacodynamics of ofloxacin and ciprofloxacin against common ocular pathogens.

PURPOSE: Time-kill curve methodology was used to assess the pharmacodynamics of two fluoroquinolones, ofloxacin and ciprofloxacin, against six strains representing the most common ocular pathogens: Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Serratia marcescens, and Haemophilus influenzae. METHODS: For time-kill studies, ofloxacin and ciprofloxacin solutions were prepared at concentrations of 0.5 x, 1.0 x, 2.0 x, and 3.0 x the MIC (minimal inhibitory concentration) for each respective strain. Inocula were prepared by diluting overnight cultures to final concentrations of 10(2), 10(3), 10(4), and 10(5) cfu (colony-forming units)/mL in each antibiotic solution. Growth controls were included. Viability counts of antibiotic-containing and control bacterial suspensions were performed at 0, 10, 20, 30, 60, 90, 120, and 180 minutes. RESULTS: In general, the kill rates of ofloxacin at 1.0 x, 2.0 x, and 3.0 x the MIC were significantly faster than the kill rates of ciprofloxacin by approximately 30 minutes, regardless of the bacterial concentration tested. At 0.5 x MIC, the kill kinetics of ciprofloxacin and ofloxacin were similar, regardless of the strain tested. At 1.0 x MIC, ofloxacin achieved 99.9% killing of P. aeruginosa and S. aureus within 30 minutes, and S. epidermidis and S. marcescens within 90 minutes. Overall, the kill kinetics of both quinolones for H. influenzae were similar, while neither quinolone achieved 99.9% killing of S. pneumoniae, regardless of the antibiotic concentration tested. CONCLUSION: Time-kill curve analyses in the present study demonstrate that ofloxacin achieved killing of the majority of ocular pathogens tested at rates equivalent to or faster than that of ciprofloxacin. Both fluoroquinolones were more effective against nonencapsulated bacteria than against encapsulated bacteria.

Multiresidue screen for cardiotoxins by two-dimensional thin-layer chromatography.

A two-dimensional thin-layer chromatographic method was developed for the qualitative determination of the cardiotoxins oleandrin, gitoxin, digitoxin, gitoxigenin, and grayanotoxins I, II, and III in gastrointestinal contents (stomach, rumen, colon, and cecum contents), feces, and plant material. The cardiotoxins were extracted with dichloromethane. The extract was cleaned up by charcoal and reverse phase solid-phase extraction columns. Analysis was performed by two-dimensional thin-layer chromatography on silica gel plates and visualized by aluminum chloride followed by chloramine T spray. The method detection limits were 0.05 microg/g for oleandrin, 0.1 microg/g for gitoxin, and 0.2 microg/g for the other toxicants in gastrointestinal contents and feces and were 5 times higher in plant material. Four replicate fortifications of bovine rumen contents, bovine feces, and alfalfa at these levels were all well recovered. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.

A novel means of self-protection, unrelated to toxin activation, confers immunity to the bactericidal effects of the Enterococcus faecalis cytolysin.

Enterococcus faecalis has become a pervasive clinical problem due to the emergence of resistance to most antibiotics. The cytolysin of E. faecalis is a novel bacterial toxin that contributes to the severity of disease. It consists of two structural subunits, which together possess both hemolytic and bactericidal activity. Both toxin subunits are encoded in a complex operon frequently harbored on pheromone-responsive plasmids. E. faecalis strains lacking such plasmids are susceptible to the bactericidal effects of the cytolysin. A novel cytolysin immunity determinant at the 3' end of the pAD1 cytolysin operon is described in the present study. Deletion analysis and specific mutagenesis isolated the immunity function to a single open reading frame. Specific mutagenesis experiments demonstrate that cytolysin immunity is unrelated to cytolysin activator (CylA) expression as previously proposed. Cytolysin immunity is, however, encoded on the same transcript as and 3' to CylA, and previous associations between immunity and CylA can be ascribed to the polar behavior of Tn917 insertion.

Pathogenesis of gram-positive bacterial endophthalmitis.

The severity of endophthalmitis has been associated generally with the virulence of the offending pathogen. However, precisely what constitutes the virulence in intraocular infections remains ill defined. We therefore sought to identify the basis for virulence for three common ocular pathogens (Bacillus cereus, Enterococcus faecalis, and Staphylococcus aureus) in terms of intraocular growth rates, bacterial localization patterns, and the contribution of cell walls and secreted products to the pathogenesis of endophthalmitis. Rabbit eyes were injected intravitreally with (i) viable B. cereus, E. faecalis, or S. aureus, (ii) metabolically inactive B. cereus, E. faecalis, or S. aureus, (iii) sacculus preparations from each strain, or (iv) culture fluid containing products secreted by each strain. Eyes were assessed at various times following injection by slit lamp biomicroscopy, electroretinography (ERG), bacterial and inflammatory cell enumeration, and histology. B. cereus endophthalmitis followed a more rapid and virulent course than E. faecalis or S. aureus endophthalmitis, eliminating retinal responsiveness, as measured by ERG, by 12 h. Analysis of bacterial localization revealed that B. cereus uniquely migrated rapidly from posterior to anterior segment during infection. Although injection of neither metabolically inactive bacteria nor cell wall sacculi greatly affected ERG, significant intraocular inflammation was observed. Injection of B. cereus or S. aureus culture fluids caused both significant reductions in retinal responsiveness and significant intraocular inflammation, paralleling that seen in natural infections. The results demonstrate that toxins, intraocular localization, and, to a lesser extent, the intraocular host response to cell walls all contribute to the pathogenesis of B. cereus, S. aureus, and E. faecalis endophthalmitis in a pathogen-specific manner. The key pathophysiologic differences in these intraocular diseases highlight opportunities for optimizing conventional therapies and deriving new ones.

View-invariant representations of familiar objects by neurons in the inferior temporal visual cortex.

A view-invariant representation of objects in the brain would have many computational advantages. Here we describe a population of single neurons in the temporal visual cortex (IT) that have view-invariant representations of familiar objects. Ten real plastic objects were placed in the monkeys' home cages for a period of time before neurophysiological experiments in which neuronal responses were measured to four views of each object. The macaques performed a visual fixation task, and had never been trained in object discrimination. The majority of the visual neurons recorded were responsive to some views of some objects and/or to the control stimuli, as would be expected from previous studies. However, a small subset of these neurons were responsive to all views of one or more of the objects, providing evidence that these neurons were coding for objects, rather than simply for individual views or visual features within the image. This result was confirmed by information theoretic analyses, which showed that the neurons provided information about which object was being seen, independently of the view. The coding scheme was shown to be sparse distributed, with relatively independent information being provided by the different neurons. Hypotheses about how these view-invariant cells are formed are described.

Molecular epidemiology of Staphylococcus aureus and Enterococcus faecalis in endophthalmitis.

Genomic DNA fingerprint analysis was performed on 39 Staphylococcus aureus and 28 Enterococcus faecalis endophthalmitis isolates collected from multiple clinical centers. Among 21 S. aureus genomic DNA fingerprint patterns identified, five clonotypes were recovered from multiple unrelated patients and accounted for 58.9% (23 of 39) of the isolates analyzed. Compared with strains having unique genomic DNA fingerprint patterns, the S. aureus clonotypes occurring more than once were more likely to result in visual acuities of 20/200 or worse (P = 0.036 [chi2 test]). In contrast to the S. aureus isolates, the E. faecalis endophthalmitis isolates were a clonally diverse population, enriched for the expression of a known toxin, cytolysin, which is plasmid encoded.

Staphylococcal accessory regulator (sar) in conjunction with agr contributes to Staphylococcus aureus virulence in endophthalmitis.

Previous studies showed that an agr mutant strain of Staphylococcus aureus was partially attenuated in virulence compared to a parental strain in experimental endophthalmitis. The purpose of this study was to determine whether the sar locus, either alone or through interactions with agr, contributes to the regulation of virulence in S. aureus endophthalmitis. Experimental endophthalmitis was established by the midvitreous injection of approximately 30 CFU of S. aureus RN6390 or the isogenic attenuated strains RN6911 (agr mutant), ALC136 (sar mutant), and ALC135 (agr sar double mutant). Unexpectedly, the rate of reduction in electroretinographic B-wave amplitude in eyes infected with strain ALC136 (sar mutant) was not significantly different from the parental strain on postinfection day (PID) 5 (10% retention). In contrast, ALC135 (agr sar double mutant)-infected eyes retained 73% of preoperative B-wave amplitude on PID 5. Therefore, unlike agr, a mutation in the sar locus alone does not alter the overall virulence of wild-type S. aureus in experimental endophthalmitis. However, the combined effect of insertional mutations in both the sar and agr global regulators leads to near-complete attenuation of virulence.

Host/parasite interactions in bacterial endophthalmitis.

Bacterial infections within the eye arise as complications of intraocular surgery, penetrating injury, or hematogenous spread from distant anatomical sites. Because: 1) the interior surfaces of the eye are lined with sensitive, nonregenerating tissues, 2) the inner chambers of the eye are relatively sequestered from circulating immunological components, 3) the integrity of blood-ocular barriers provides poor penetration of systemically administered antibiotics, and 4) aqueous and vitreous humor represent rich, relatively acellular culture media; endophthalmitis often progresses rapidly and total loss of vision frequently results. Years of clinical experience have shown that current therapies for endophthalmitis, including antimicrobials, antiinflammatory agents, and vitrectomy, are frequently unsuccessful in ameliorating destruction of intraocular tissues. While bacterial and host factors were thought to play key roles in the course and severity of endophthalmitis, it is only recently that their contributions have been experimentally defined. Molecular-based techniques are gaining increased use in the study of infectious eye diseases. Current findings regarding the host/parasite interactions within the eye are reviewed, and a resulting integrative model of the natural course of endophthalmitis proposed. A molecular-level understanding of the roles of both bacterial and host factors during endophthalmitis will likely reveal potential targets for therapeutic intervention aimed at salvaging vision.

Responses of neurons in primary and inferior temporal visual cortices to natural scenes.

The primary visual cortex (V1) is the first cortical area to receive visual input, and inferior temporal (IT) areas are among the last along the ventral visual pathway. We recorded, in area V1 of anaesthetized cats and area IT of awake macaque monkeys, responses of neurons to videos of natural scenes. Responses were analysed to test various hypotheses concerning the nature of neural coding in these two regions. A variety of spike-train statistics were measured including spike-count distributions, interspike interval distributions, coefficients of variation, power spectra, Fano factors and different sparseness measures. All statistics showed non-Poisson characteristics and several revealed self-similarity of the spike trains. Spike-count distributions were approximately exponential in both visual areas for eight different videos and for counting windows ranging from 50 ms to 5 seconds. The results suggest that the neurons maximize their information carrying capacity while maintaining a fixed long-term-average firing rate, or equivalently, minimize their average firing rate for a fixed information carrying capacity.

Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic.

Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylLL and CylLS, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity.

Accessory gene regulator controls Staphylococcus aureus virulence in endophthalmitis.

PURPOSE: To evaluate the contribution of toxins to the severity of Staphylococcus aureus endophthalmitis. METHODS: Experimental endophthalmitis was established by injecting rabbit eyes with wild type S. aureus ISP479 and the isogenic attenuated strain, ISP546, defective in expression of the global regulator locus agr. agr regulates expression of at least 19 exoproteins that are potentially important in the pathogenesis of endophthalmitis. Infections were evaluated using electroretinography, slit lamp biomicroscopy, and histology. Two concentrations (approximately 10 and 1000 organisms) of bacteria were injected. RESULTS: The agr- strain consistently resulted in slower loss of b-wave response when compared to the wild type strain, irrespective of inoculum size. Clinical signs were less severe among the agr- group at 24 and 48 hours when 10 organisms were injected. However, when the number of bacteria injected was increased to 1000, earlier onset of clinical signs was observed, with both groups showing maximum cell and flare and a white fundal reflex at 48 hours after infection. Histologic examination of eyes enucleated 36 hours after inoculation revealed that the wild type strain induced focal retinal destruction and mild vitritis, whereas eyes infected with the agr- strain remained completely normal. Histologic examination carried out when loss of B-wave response was 100% revealed that retinal changes for both groups could not be distinguished. CONCLUSIONS: These data indicate that toxin production by S. aureus contributes to severity of endophthalmitis by accelerating the rate of onset of retinal damage. Therefore, toxin-targeting therapies instituted early in the course of infection could preserve retinal function.

Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants.

Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylLL, cylLS, cylM, and cylB). The inferred products of two of these open reading frames, CyILL and CyILS, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity.

Molecular characterization of the Enterococcus faecalis cytolysin activator.

The gene encoding component A (cylA), the activator protein of the Enterococcus faecalis cytolysin, has been localized on pAD1, and the nucleotide sequence was determined. cylA consists of a 1,236-bp open reading frame encoding a 412-amino-acid polypeptide. A search of the National Biomedical Research Foundation data base revealed significant homology between the inferred amino acid sequence of component A and subtilisin BPN'. Component A activation of the cytolysin precursor (component L) was observed to be inhibited by the serine protease inhibitor diisopropylfluorophosphate. Mature component A exhibits a molecular weight of approximately 30,000 and an isoelectric point of 4.5. Differences between the size of the primary translation product (45,625 daltons) and the mature enzyme suggest that, as for subtilisin, component A is secreted as a proenzyme. These results provide the basis for a model of component A activation of component L and a role for component A in protecting the cytolysin-producing cell from lysis.

An HlyB-type function is required for expression of the Enterococcus faecalis hemolysin/bacteriocin.

The nucleotide sequence of a 3,422-bp internal restriction fragment from the Enterococcus faecalis pAD1 hemolysin/bacteriocin-encoding region was determined. This fragment was associated with expression of hemolysin/bacteriocin component L and contained a 2,142-bp open reading frame. The inferred amino acid sequence revealed a protein which shared extensive similarity with HlyB of the Escherichia coli alpha-hemolysin operon. The inferred protein, CylB, was observed to be independently expressed in E. coli and capable of complementing an insertion mutation in the cloned hemolysin/bacteriocin operon in trans. Despite the extensive similarity to HlyB, CylB was incapable of complementing an insertion mutation in hlyB. Cytolysin determinants possessing an HlyB-type transport function are widely dispersed throughout gram-negative genera. We believe this to be the first example of an HlyB-type protein encoded within a cytolysin determinant from a gram-positive bacterium.