Piscine mycobacteriosis - Involvement of bacterial species and reflection in pathology.

INTRODUCTION: Piscine mycobacteriosis is a lethal disease with zoonotic potential, found worldwide in both fresh and marine fish. More than 20 strains of Mycobacterium spp. are known to persist in fish so far, but the pathogenicity is currently unknown for most of them. However, M. marinum is reported as one of the most pathogenic agents for fish and is involved in zoonotic cases. We examined 47 different cases from two zoological gardens, where fish tuberculosis was identified or previously suspected during the last ten years. We collected PCR and sequencing data, which were then compared to previously collected clinical data and pathology. The clinical signs caused by Mycobacterium spp. were similar in all the cases, except for cases infected by M. marinum, which lacked the presence of skin lesions. Lesions seen in histology caused by M. marinum tended to be more acute and severe compared lesions caused by other Mycobacterium spp. The majority of M. marinum cases have been reported within marine fish. In contrast to previous studies we detected this species to be the predominant bacteria present within freshwater fish. Interestingly, we detected M. holsaticum in one of the seawater systems used in this project, being the first report of this Mycobacterium species shown to be present in a fish.

INTRODUCTION: La mycobactériose du poisson est une maladie létale avec un potentiel zoonotique qui se trouve dans le monde entier chez les poissons d'eau douce et marins. Plus de 20 souches de Mycobacterium spp. sont à ce jour connues chez les poissons, mais la pathogénicité est actuellement inconnue pour la plupart d'entre elles. Cependant, M. marinum est signalé comme l'un des agents les plus pathogènes pour les poissons et il est impliqué dans des cas de zoonoses. Nous avons examiné 47 cas différents provenant de deux jardins zoologiques où la tuberculose du poisson a été identifiée ou suspectée au cours des dix dernières années. Nous avons recueilli des données de PCR et de séquençage qui ont ensuite été comparées aux données cliniques et à la pathologie précédemment collectées. Les signes cliniques causés par Mycobacterium spp. étaient similaires dans tous les cas, à l'exception des cas infectés par M. marinum, chez lesquels manquaient les lésions cutanées. Les lésions histologiques observées dans les infections par M. marinum tendaient à être plus aiguës et graves comparées aux lésions provoquées par d'autres espèces de Mycobacterium spp. La majorité des cas de M. marinum ont été documentés chez des poissons marins. Contrairement aux études précédentes, nous avons constaté que cette espèce était la principale bactérie présente chez les poissons d'eau douce. Fait intéressant, nous avons détecté M. holsaticum dans l'un des systèmes d'eau de mer examinés dans ce projet, ce qui est le premier cas confirmé de la présence de cette espèce de Mycobacterium chez un poisson.

Diagnosis of immediate-type beta-lactam allergy in vitro by flow-cytometric basophil activation test and sulfidoleukotriene production: a multicenter study.

INTRODUCTION: This multicenter study aimed to evaluate the diagnostic value of 2 cellular tests based on basophil reactivity--the basophil activation test (BAT, Flow-CAST) and the sulfidoleukotriene release assay (CAST-ELISA)--in immediate-type beta-lactam allergy, particularly in patients with a clinical history of allergy and a negative skin test result. MATERIAL AND METHODS: In a multicenter study encompassing 10 European centers, 181 patients with a history of immediate-type beta-lactam allergy, and 81 controls, we evaluated the diagnostic efficiency of specific IgE determinations and of 2 cellular tests based on basophil reactivity, the BAT and the sulfidoleukotriene release assay. RESULTS: With Flow-CAST, sensitivity varied for individual beta-lactam allergens from 16% for penicilloyl-polylysine to 33% for amoxicillin, reaching 50% when all 5 allergens were considered. In beta-lactam-allergic patients with negative skin test results (22.8%), Flow-CAST showed positive results for at least 1 of the 5 allergens in 37%. Specificity varied from 89% to 97%, depending on the allergens used. In CAST-ELISA, the overall sensitivity in skin test-positive patients was 41.7%; in patients with negative skin test results it was 27.9%. Both tests were not absolutely correlated, so that when all the results were considered together, sensitivity increased to 64.3% and specificity varied for both tests combined from 73% to 92%. In contrast, specific IgE determinations in the same population yielded a lower sensitivity (28.3%). CONCLUSIONS: A diagnostic algorithm including skin tests and specific IgE, followed by cellular tests in negative patients and controlled challenge enabled us to confirm beta-lactam allergy in 92% of cases. This procedure would also allow us to avoid two-thirds of the required controlled challenges.

Severe scuticociliate (Philasterides dicentrarchi) infection in a population of sea dragons (Phycodurus eques and Phyllopteryx taeniolatus).

Scuticociliatosis is a disease of fish induced by ciliated parasites of the genus Scuticociliatida. It has been described in sea horses (Hippocampus sp.), flounders (Paralichthys olivaceus), and turbots (Scophthalmus maximus). Here we present a case study of a population of sea dragons chronically infected with scuticociliates identified as Philasterides dicentrarchi by histopathology and PCR. Beginning in 2004, over a period of 19 months, 10 sea dragons (Phycodurus eques and Phyllopteryx taeniolatus) were found dead in an aquarium of the Zoological Garden Basle, Switzerland. Clinically, the animals showed only faint symptoms of disease over a short period of time. At necropsy, macroscopic lesions were confined to the skin with multiple, often hemorrhagic, ulcerations. Histologically, epidermal ulcers were associated with necrosis and inflammation of the underlying dermis and musculature. Numerous ciliates, with a morphology consistent with scuticociliates, were present in these lesions. In several animals these ciliates had invaded blood vessels and were detected in gills and internal organs including kidney, thyroid gland, and central nervous system (CNS). In these organs, mild degenerative lesions and inflammatory reactions were evident. The ciliates were identified as Philasterides dicentrarchi based on small-subunit ribosomal RNA (SSUrRNA) gene sequences obtained by polymerase chain reaction (PCR) on DNA extracted from paraffin-embedded tissue sections. Our report shows that scuticociliate infections of sea dragons can develop into a systemic infection and that both species of sea dragons can be affected.

Cellular in vitro assays in the diagnosis of Hymenoptera venom allergy.

BACKGROUND: The current diagnostic procedures of anaphylactic reactions to hymenoptera stings include intradermal tests, venom-specific IgE (sIgE) and possibly sting challenge tests. Sometimes, the culprit insect remains unidentified. The usefulness of the cellular assays CAST-ELISA and Flow-CAST in the management of hymenoptera venom allergy was investigated. METHODS: 134 patients with systemic reactions after a yellow jacket wasp and/or honey bee sting and 44 healthy controls underwent skin tests, as well as determination of sIgE (CAP-FEIA), leukocyte sulfidoleukotriene release (CAST-ELISA) and basophil CD63 expression (Flow-CAST) upon insect venom stimulation. The clinical diagnosis based on the history alone served as reference. Sensitivity, specificity, and positive and negative predictive value of all methods were compared. Concordance and correlations among methods were calculated. RESULTS: Sensitivity and specificity of all in vitro tests were consistently high. The combination of all tests (skin tests, sIgE, combined cellular assays) yielded a positive predictive value of 100% for both venoms, if all 3 were positive, and a negative predictive value of 100%, if at least 1 test was positive. Relative specificities were considerably higher for the cellular assays (honey bee: CAST 91.1%, Flow-CAST 85.7%; yellow jacket wasp: CAST 98.4%, Flow-CAST 92.1%) and allow the detection of the culprit insect in patients with reactivity to both insects. The concordance between methods was good. There is no correlation between severity of clinical reaction and cellular assays. CONCLUSION: CAST-ELISA and Flow-CAST are valuable additional diagnostic tools for establishing the true culprit insect in patients with unclear clinical history or sensitization to both insects.

Evaluation of an in vitro sulphidoleukotriene release test for diagnosis of insect bite hypersensitivity in horses.

REASONS FOR PERFORMING STUDY: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. OBJECTIVES: The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. METHODS: A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluated, and 32 horses were tested repeatedly over one year. The kappa reliability test was used to assess agreement of the test results with different insect extracts. RESULTS: Horses with IBH had significantly higher sLT release than controls with C. nubeculosus and S. vittatum. The highest diagnostic sensitivity and specificity levels were attained when using adult C. nubeculosus extracts with the CAST (78% and 97%, respectively), suggesting that most horses with IBH are sensitised against Culicoides allergens. A proportion of IBH-affected horses was found to be sensitised to allergens of Simulium spp. in addition to those of C. nubeculosus. The CAST with C. nubeculosus had positive and negative predictive values > or = 80% for a true prevalence of IBH of 12-52%. In the follow-up study, the proportion of IBH-affected horses with a positive test result ranged from 90% in November to 68% in March. Severity of clinical signs or age did not influence test results significantly. However, IBH-affected males achieved significantly more positive test results than IBH-affected females. CONCLUSIONS: The CAST with adult C. nubeculosus has high specificity and good sensitivity for diagnosis of IBH. Horses with IBH are mainly sensitised to Culicoides allergens, and some horses are additionally also sensitised to allergens in Simulium spp. POTENTIAL RELEVANCE: The CAST is likely to be a useful test for diagnosis of IBH, even allowing the identification of IBH-affected but asymptomatic horses. This test may also help in further characterisation of allergens involved in this condition.

Chlorhexidine anaphylaxis: case report and review of the literature.

Chlorhexidine is a widely used antiseptic and disinfectant. Compared to its ubiquitous use in medical and non-medical environments, the sensitization rate seems to be low. Multivarious hypersensitivity reactions to the agent have been reported, including delayed hypersensitivity reactions such as contact dermatitis, fixed drug eruptions and photosensitivity reactions. An increasing number of immediate-type allergies such as contact urticaria, occupational asthma and anaphylactic shock have been reported. In the case report, we describe anaphylaxis due to topical skin application of chlorhexidine, confirmed by skin testing and sulfidoleukotriene stimulation test (CAST(R): cellular antigen stimulation test). The potential risk of anaphylactic reactions due to the application of chlorhexidine is well known, especially that application to mucous membranes can cause anaphylactic reactions and was therefore discouraged. The use of chlorhexidine at a 0.05% concentration on wounds and intact skin was so far thought to be safe. Besides our patient, only one other case of severe anaphylactic reaction due to application of chlorhexidine on skin has been reported. Hypersensitivity to chlorhexidine is rare, but its potential to cause anaphylactic shock is probably underestimated. This review should remind all clinicians of an important potential risk of this widely used antiseptic.

Origin of dimeric structure in the ribonuclease superfamily.

To enable application of postgenomic evolutionary approaches to understand the divergence of behavior and function in ribonucleases (RNases), the impact of divergent sequence on the divergence of tertiary and quaternary structure is analyzed in bovine pancreatic and seminal ribonucleases, which differ by 23 amino acids. In a crystal, seminal RNase is a homodimer joined by two "antiparallel" intersubunit disulfide bonds between Cys-31 from one subunit and Cys-32' from the other and having composite active sites arising from the "swap" of residues 1-20 from each subunit. Specialized Edman degradation techniques have completed the structural characterization of the dimer in solution, new cross-linking methods have been developed to assess the swap, and sequence determinants of quaternary structure have been explored by protein engineering using the reconstructed evolutionary history of the protein family as a guide. A single Cys at either position 32 (the first to be introduced during the divergent evolution of the family) or 31 converts monomeric RNase A into a dimer. Even with an additional Phe at position 31, another residue introduced early in the seminal lineage, swap is minimal. A hydrophobic contact formed by Leu-28, however, also introduced early in the seminal lineage, increases the amount of "antiparallel" connectivity of the two subunits and facilitates swapping of residues 1-20. Efficient swapping requires addition of a Pro at position 19, a residue also introduced early in the divergent evolution of the seminal RNase gene. Additional cysteines required for dimer formation are found to slow refolding of the protein through formation of incorrect disulfide bonds, suggesting a paradox in the biosynthesis of the protein. Further studies showed that the dimeric form of seminal RNase known in the crystal is not the only form in vivo, where a substantial amount of heterodimer is known. These data complete the acquisition of the background needed to understand the evolution of new structure, behavior, and function in the seminal RNase family of proteins.

Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA.

Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.

Developing new synthetic catalysts. How nature does it.

Paleomolecular biochemistry is a new field of science that seeks to understand how life emerged and developed in interaction with its geophysical surroundings. It is an experimental science, involving reconstruction of extinct biomolecules in the laboratory, studying their properties in the laboratory, and inferring details of their behavior and function in the context of geological data. An outline is provided of some tools of this field, together with its application to the study of two specific systems, ribonuclease and alcohol dehydrogenase.

Pseudogenes in ribonuclease evolution: a source of new biomacromolecular function?

Bovine seminal ribonuclease (RNase) diverged from pancreatic RNase after a gene duplication ca. 35 million years ago. Members of the seminal RNase gene family evidently remained as unexpressed pseudogene for much of its evolutionary history. Between 5 and 10 million years ago, however, after the divergence of kudu but before the divergence of ox, evidence suggests that the pseudogene was repaired and expressed. Intriguingly, detailed analysis of the sequences suggests that the repair may have involved gene conversion, transfer of information from the pancreatic gene to the RNase pseudogene. Further, the ratio of non-silent to silent substitutions suggests that the pancreatic RNases are divergently evolving under functional constraints, the seminal RNase pseudogenes are diverging under no functional constraints, while the genes expressed in the seminal plasma are evolving extremely rapidly in their amino acid sequences, as if to fulfil a new physiological role.

Reconstructing the evolutionary history of the artiodactyl ribonuclease superfamily.

The sequences of proteins from ancient organisms can be reconstructed from the sequences of their descendants by a procedure that assumes that the descendant proteins arose from the extinct ancestor by the smallest number of independent evolutionary events ('parsimony'). The reconstructed sequences can then be prepared in the laboratory and studied. Thirteen ancient ribonucleases (RNases) have been reconstructed as intermediates in the evolution of the RNase protein family in artiodactyls (the mammal order that includes pig, camel, deer, sheep and ox). The properties of the reconstructed proteins suggest that parsimony yields plausible ancient sequences. Going back in time, a significant change in behaviour, namely a fivefold increase in catalytic activity against double-stranded RNA, appears in the RNase reconstructed for the founding ancestor of the artiodactyl lineage, which lived about 40 million years ago. This corresponds to the period when ruminant digestion arose in the artiodactyls, suggests that contemporary artiodactyl digestive RNases arose from a non-digestive ancestor, and illustrates how evolutionary reconstructions can help in the understanding of physiological function within a protein family.

Amphibious vision in Coryphoblennius galerita L. (Perciformes).

The morphology of the ophthalmic cornea in the blenniid fish Coryphoblennius galerita (Teleostei) shows adaptation to the amphibious life. Amphibious vision is provided by a flattened area within the cornea. Eyes of other, non-amphibious blenniids are compared with those of C. galerita.