Paediatric injuries around the knee: Bony injuries.

The aim of this article is to discuss the diagnosis, management and pitfalls of bony injuries around the skeletally immature knee. Each within their own right is a relatively uncommon injury but associated with potential complications. Distal femoral physeal fractures can result in growth arrest and vascular injury. Tibial spine avulsions can result in an unstable knee. Tibial tubercle fractures can be associated with compartment syndrome and pose a risk to the extensor mechanism of the knee. Fixation can be complicated by growth arrest and subsequent recurvatum deformity. Finally, patella sleeve injuries are often missed and this can also threaten the extensor mechanism. We discuss the approach to clinical and radiological assessment of these injuries, and evidence based recommendations as to how they are best managed to avoid complications.

Identification of aquatic mycobacteria based on sequence analysis of the 16S-23S rRNA internal transcribed spacer region.

PURPOSE: Mycobacteria are common causative agents of bacterial infections in many species of freshwater and marine fish. Identification of mycobacteria to the species level based on phenotypic tests is inappropriate and time consuming. Molecular methods such as partial or entire gene sequence determination in mycobacteria have been employed to resolve these problems. The objective of this study was to assess the use of sequence analysis of the mycobacterial 16S-23S internal transcribed spacer (ITS) region for the identification of different aquatic mycobacteria species. METHODOLOGY: Using published primers, the ITS sequences of 64 field and reference strains were determined.Results/Key findings. The identity of all isolates previously identified as Mycobacterium marinum by RFLP was confirmed as M. marinum by sequence analysis. With the exception of five rapidly growing mycobacteria isolates, all other mycobacteria were easily identified by sequencing of the ITS region. Using this spacer region, it was possible to differentiate between slowly growing and rapidly growing mycobacteria, even before sequence analysis, by the size of the PCR product, although species identification could not be made by size alone. CONCLUSION: Overall, direct sequencing of this genetic element following PCR has been shown to be useful in the identification of aquatic mycobacteria species. With regard to the variability of the ITS region for different mycobacteria isolates, this may be a useful tool in epidemiological studies.

Risk stratification for the recurrence of trigger thumb after surgical release in the paediatric patient.

Trigger thumb, or stenosing tenovaginitis, is a relatively uncommon condition affecting the flexor pollicis longus tendon of children. The condition is characterized by the formation of a nodule within the tendon and thickening of the tendon sheath as it passes through the flexor pulley of the thumb at the level of the metacarpo-phalangeal joint. The optimum age for surgical intervention continues to be discussed. The aim of this study is to establish the temporal relationship and surgical variables to determine factors that may contribute to recurrence of the condition. A retrospective analysis of the entire surgical logbook and patient notes of a stand-alone consultant paediatric orthopaedic practice was scrutinized. 94 patients, 107 thumbs, over a 13-year period were operated on for trigger thumb. The recurrence rate was found to be 5.61 %. The average age of patients at primary release who went on to recurrence was 2.8 years, which is significantly younger than those that did not recur (p = 0.044). Sensitivity analysis revealed that the primary procedure at an age of less than 2.5 years confers a higher risk of recurrence. The data presented here advocate surgical release of trigger thumb after 2½ years of age, a senior surgeon as lead operator and a transverse skin incision at the level of the nodule or a more extensive "zig-zag" one to clearly see the structures to be released. We recommend that the surgeon ensures the stenosing pulley and sheath are released in their entirety.

Puffy skin disease (PSD) in rainbow trout, Oncorhynchus mykiss (Walbaum): a case definition.

Puffy skin disease (PSD) is a disease that causes skin pathology in rainbow trout, Oncorhynchus mykiss (Walbaum). Incidence of PSD in UK fish farms and fisheries has increased sharply in the last decade, with growing concern from both industry sectors. This paper provides the first comprehensive case definition of PSD, combining clinical and pathological observations of diseased rainbow trout from both fish farms and fisheries. The defining features of PSD, as summarized in the case definition, were focal lateral flank skin lesions that appeared as cutaneous swelling with pigment loss and petechiae. These were associated with lethargy, poor body condition, inappetance and low level mortality. Epidermal hyperplasia and spongiosis, oedema of the dermis stratum spongiosum and a mild diffuse inflammatory cellularity were typical in histopathology of skin. A specific pathogen or aetiology was not identified. Prevalence and severity of skin lesions was greatest during late summer and autumn, with the highest prevalence being 95%. Atypical lesions seen in winter and spring were suggestive of clinical resolution. PSD holds important implications for both trout aquaculture and still water trout fisheries. This case definition will aid future diagnosis, help avoid confusion with other skin conditions and promote prompt and consistent reporting.

Gill pathology in Scottish farmed Atlantic salmon, Salmo salar L., associated with the microsporidian Desmozoon lepeophtherii Freeman et Sommerville, 2009.

Gill disorders have emerged in recent years as a significant problem in the production of marine-stage Atlantic salmon Salmo salar L. The multi-aetiological condition 'proliferative gill inflammation' (PGI) has been reported to cause heavy losses in western Norway, yet reports of Scottish cases of the disease have remained anecdotal. In the present study, histopathological material from a marine production site in the Scottish Highlands experiencing mortalities due to a seasonal gill disease with proliferative-type pathology was examined using light microscopy, special staining techniques and transmission electron microscopy (TEM). The microsporidian Desmozoon lepeophtherii Freeman et Sommerville, 2009 (syn. Paranucleospora theridion) was identified by staining using a Gram Twort method and TEM associated with distinctive proliferative and necrotic pathology confined to the interlamellar Malpighian cell areas of the primary filaments. Epitheliocystis was not a feature of the gill pathology observed. It is believed this is the first report of D. lepeophtherii being identified associated with pathology in a Scottish gill disease case, and supports anecdotal reports that a disease at least partly synonymous with PGI as described by Norwegian researchers is present in Scottish aquaculture.

Systemic nocardiosis in a Mediterranean population of cultured meagre, Argyrosomus regius Asso (Perciformes: Sciaenidae).

Marine cultured meagre, Argyrosomus regius Asso, in central and western Greece were affected by an outbreak of systemic granulomatous disease subsequently demonstrated to be nocardiosis. The fish were originally imported as juveniles from hatcheries in France and Italy and on-grown in Greece, the latter also providing broodstock for a small number of local Greek hatcheries for the production of second-generation juveniles. The disease in cage reared fish had been present throughout the year, particularly in the 1+ and 2+ year old fish with a low to variable morbidity and 1-4% total mortality. Multiple lesions were visible externally on the skin of affected fish, with severe ulcerations and necrosis. Internally, multifocal yellowish-white nodules, 0.1-0.5 cm in diameter, were visible on the surface of several internal organs. Histopathology revealed systemic granulomatous inflammation. Fite-Faraco staining clearly demonstrated the presence of Nocardia-like organisms which were Gram-positive, long, rod to beaded filamentous bacteria. Nocardia genus-specific 16s RNA primers NG1 and NG2 were used to generate a 600 bp fragment recovered from affected tissue, confirming the diagnosis of Nocardia spp. To our knowledge, this is the first report of nocardiosis in meagre.

Ultrastructural morphogenesis of salmonid alphavirus 1.

Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE-214). Different stages of viral replication were observed under electron microscopy. Virus-like particles were observed inside membrane-bound vesicles as early as 1 h following contact of the virus with the cells. Membrane-dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome-like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive-sense single-stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by 'fuzzy'-coated membranes was greater in virus-infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post-infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.

Detection and identification of aquatic mycobacteria in formalin-fixed, paraffin-embedded fish tissues.

The isolation of mycobacteria from field samples is problematic, and isolation of the bacterium is sometimes not even attempted. The detection of mycobacteria through traditional histology using formalin-fixed, paraffin-embedded (FFPE) tissues is neither sensitive nor specific. However, detection of mycobacterial DNA from FFPE specimens, suspected of being infected with mammalian mycobacteriosis, is a routine clinical procedure. In the present study, a polymerase chain reaction (PCR)-based method was used to detect and identify mycobacteria in FFPE specimens sampled from fish suspected of being infected with fish mycobacteriosis. A total of 45 fish tissue samples, comprising of 12 tissue samples obtained from experimentally infected fish and the remainder from fish naturally infected with mycobacteria, were analysed using a PCR protocol which amplifies a fragment of the mycobacterial 65 kDa heat-shock protein (hsp65) gene. PCR-restriction enzyme analysis and/or sequencing were employed to further analyse the PCR amplicons. The PCR results were compared with those obtained by histology and culture. Mycobacterial DNA was detected in 34 of the 45 samples examined, of which 16 samples (47%) showed granulomatous reactions on histological examination. Using histology as the gold standard, no false-negative PCR results were obtained. Also, considering the presence or absence of granulomas as a diagnostic criterion, the sensitivity and specificity of PCR in 42 of the FFPE tissues were 16/16 (100%) and 8/26 (approximately 30.8%), respectively. Corresponding microbiological cultures were available for 15 cases, of which 13 were pure Mycobacterium cultures. Of these, 13 were PCR positive (100% sensitivity and 50% specificity). The PCR-based methods used here proved sensitive, specific and rapid for the detection of mycobacteria in routinely processed paraffin wax-embedded and formalin-fixed histological samples, and the results of the study suggest that this method has potential use in retrospective epidemiological studies.

Comparative evaluation of Polymerase Chain Reaction-Restriction Enzyme Analysis (PRA) and sequencing of heat shock protein 65 (hsp65) gene for identification of aquatic mycobacteria.

Traditional identification of mycobacteria based on cultural and biochemical tests can take several weeks and may fail to provide a precise identification. Polymerase Chain Reaction-Restriction Enzyme Analysis (PRA) of the gene encoding heat shock protein 65 kDa (hsp65) gene has been proposed as a rapid and inexpensive alternative approach. Despite being widely used for differentiation of mammalian mycobacteria, this method has only been applied in the identification of a small number of aquatic mycobacteria. The present study aimed to evaluate the potential use of PRA of hsp65 for the identification of aquatic mycobacteria compared with sequence analysis. Seventy one mycobacterial isolates including, 10 type/reference strains and the remainder field isolates, were subjected to PRA of a 441 bp fragment of this gene. For 68 representative isolates, sequence analysis was performed. All rapidly and slowly growing mycobacteria had best matches with 99.3% to 100% similarity with their corresponding species in the databanks. PRA proved to be a simple and rapid method for identifying aquatic mycobacteria. However, the incidence of similar or identical restriction patterns for some species of mycobacteria, and in particular, identification of new species of mycobacteria is a major problem using such a method. In contrast, the nucleic acid sequencing of the hsp65 gene yielded unambiguous results.

Evaluation of the INNO-LiPA mycobacteria v2 assay for identification of aquatic mycobacteria.

Fifty-seven isolates of mycobacteria comprising 10 reference strains, 47 field isolates and one non-Mycobacterium isolate were screened using commercial INNO-LiPA v2 assay kits. All mycobacteria isolates tested hybridized with the Mycobacterium genus probe on the LiPA strip. All M. marinum, M. fortuitum and M. chelonae reference and field strains and three out of the four M. gordonae isolates hybridized to their corresponding species- or complex-specific probes. Two cultures (a type strain and a field isolate) yielded mixed growth of two mycobacterial species, i.e. M. chelonae and M. fortuitum. A Mycobacterium isolate from one of these cultures was subsequently purified and correctly identified with the kit. However, sequence analysis of the 16S-23S rRNA internal transcribed spacer (ITS) region of various mycobacteria isolates revealed a misidentification of M. shottsii and M. pseudoshottsii with the kit because these isolates reacted with the M. marinum/M. ulcerans probe. Moreover, nine of the 13 field isolates presumed to be M. fortuitum from the results of the kit had closer ITS sequence homology with M. conceptionense, a species which, to our knowledge, has never been reported in fish. These findings highlight the need to redesign the M. fortuitum-M. peregrinum probe included in the INNO-LiPA assay and to introduce additional complex-specific probes into the kit. Nevertheless, the kit proved to be a rapid and reliable method for identifying mycobacteria in the aquatic environment and would be particularly useful in laboratories without sequencing facilities.

Identification of B-cell epitopes on the betanodavirus capsid protein.

The pepscan procedure was used to identify betanodavirus B-cell epitopes recognized by neutralizing mouse monoclonal antibodies (MAbs) and serum samples obtained from sea bass, Dicentrarchus labrax, naturally infected with betanodavirus. Pepscan was performed with a panel of thirty-four 12-mer synthetic peptides that mimicked the entire betanodavirus capsid protein. Sea bass serum samples reacted strongly with three regions of the capsid protein comprising amino acid residues 1-32, 91-162 and 181-212. The latter region was also recognized by neutralizing MAbs and coincided with a region of high antigenic propensity identified by an antigen prediction algorithm. These data suggest that a region of the betanodavirus capsid protein spanning amino acid residues 181-212 may represent a neutralization domain that could potentially be used to inform the development of nodavirus vaccines and immunodiagnostic reagents.

Spontaneous resolution of solitary osteochondroma in the young adult.

Spontaneous resolution of a solitary osteochondroma is rare. Such a case is presented in a patient nearing skeletal maturity. Based on a search of the English literature this is the first such report in a patient of this age.

Radiological outcome of innocent infant hip clicks.

Radiographic follow-up is questioned for infants with hip clicks who have normal results from ultrasound scan examination of the hips. Infants whose sole risk factor for developmental dysplasia of the hip was a soft tissue hip click who had a normal ultrasound scan on initial assessment were identified. A follow-up 6-month pelvis radiograph was assessed. The acetabular index, position of femoral ossific nucleus and Shenton's line were measured. Rotated radiographs were excluded. One hundred and seventy-one infants (193 clicking hips) met the criteria for inclusion. All parameters measured were within normal ranges. In this study infants with hip clicks and a normal hip ultrasound scan on initial assessment had a normal radiograph at 6 months.

Characteristics of a new reovirus isolated from epizootic ulcerative syndrome infected snakehead fish.

Epizootic ulcerative syndrome (EUS) has been infecting a wide range of fishes in the South and Southeast Asia for the last 2 decades. One reovirus-like agent (snakehead reovirus, SKRV), isolated from an EUS-infected snakehead fish and investigated in the present study, is the only reovirus so far isolated from an EUS-infected fish. SKRV was characterised by the presence of a double-stranded RNA genome with icosahedral symmetry and double capsid. The virus had an average size of 71 nm, a buoyant density of 1.36 g ml(-1) in CsCl and lacked a lipid-containing envelope. Apart from the above, the presence of a segmented genome and structural proteins falling into 3 specific size classes confirmed that the virus belongs to the family Reoviridae. SKRV differed from aquareoviruses by the lack of a cytopathic effect (CPE) with syncitium formation and in the segmentation pattern of RNA genome. The resistance to pH (3.0 to 9.0) and heat treatment and inability to multiply in mammalian cell lines and haemagglutinate human 'O' red blood cells (RBCs) differentiated SKRV from the rest of the similar genera in the family Reoviridae. Serological comparison indicated the antigenic distinctness of the isolate from selected American and European aquareoviruses. SKRV grew well in SSN-1 and SSN-3 cells at 25 to 30 degrees C but not in the most common Aquareovirus susceptible coldwater fish cell line--CHSE-214.

Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia.

Nodaviruses are widespread causative agents of viral nervous necrosis in fish. Based on the coat protein sequence, fish nodaviruses are categorized into four different genotypes. In this study, we present data on the phylogenetic and antigenic characterization of 12 new isolates, eight European and four of Asian origin, from farmed and wild species of fish. Phylogenetic analysis based on the nucleotide sequence (688 bases) of the coat protein classified the majority of these new isolates to the RGNNV genotype. Geographic or host-species specificities were not revealed by this study. Neutralizing assay experiments, further confirmed the genotypic classification, supporting the possibility that the different nodavirus genotypes can also be serologically distinguishable.

Commercial trials using emamectin benzoate to control sea lice Lepeophtheirus salmonis infestations in Atlantic salmon Salmo salar.

Two trials were conducted at commercial salmon farms to evaluate the efficacy of emamectin benzoate (Slice, 0.2% aquaculture pre-mix, Schering-Plough Animal Health) as a treatment for sea lice Lepeophtheirus salmonis (Krøyer) and Caligus elongatus Nordmann infestations in Atlantic salmon Salmo salar L. Trials were carried out in 15 m2 commercial sea pens, at temperatures of 5.5 to 7.5 degrees C and 10.8 to 13.8 degrees C. Each pen was stocked with 14,000 to 17,500 fish with mean weights of 0.44 to 0.74 and 1.33 to 1.83 kg. Fish were naturally infested with sea lice at the start of each trial. At Day -1, samples of 10 or 15 fish were taken from each pen to determine pre-treatment numbers of lice. Emamectin benzoate was administered in feed, to 4 replicate pens, at a dose of 50 micrograms kg-1 biomass d-1 for 7 consecutive days (Days 0 to 6). Sea lice were counted again, between Days 7 and 77, and comparisons made with untreated control fish. Despite adverse weather conditions, wide variations in fish weights and exposure to new infestations, treatment was effective against chalimus and motile stages of L. salmonis. In the autumn trial, efficacy at Day 27 was 89%, and lice numbers remained lower on treated fish than on control fish 64 d from the start of treatment. In the winter trial, reductions in lice numbers at low temperatures were slower but good efficacy was achieved by Day 35. Although control fish had to be treated with hydrogen peroxide at Day 21, fish treated only with emamectin benzoate on Days 0 to 6 still had 89% fewer lice than control fish at Day 35. There were very few C. elongatus present, but at the end of both trials numbers were lower on treated fish. No adverse effects were associated with treatment of fish with emamectin benzoate.

Observations on the electron-dense bodies of the PKX parasite, agent of proliferative kidney disease in salmonids.

Ultrastructural observations on structures associated with the 'haplosporosome'-like electron-dense bodies (EDBs) of the primary cell of the extrasporogonic stage of PKX are described. Observations made include formation of EDBs by the trans-Golgi network, an additional membrane associated with EDB structure, confronting cisternae, engulfment and presence of EDBs in multivesicular bodies, fusion of EDBs with the plasmalemma, degeneration of EDBs in disintegrating primary cells and endocytosis of PKX cytoplasm by adherent macrophages. Immunogold localisation of a PKX-specific monoclonal antibody (MAb A3) suggests that the EDBs contain periodate-sensitive carbohydrates on their membranes. Tissues prepared for immunogold electron microscopy further suggest that some contain a lipid-rich core. An interpretation is made on their possible function and their relationship with the haplosporosomes and sporoplasmasomes found in members of the Haplosporidea and Myxosporea respectively.

In situ hybridisation identifies the gill as a portal of entry for PKX (Phylum Myxozoa), the causative agent of proliferative kidney disease in salmonids.

PKX (Phylum Myxozoa) is an important pathogen affecting salmonid culture in Western Europe and North America. All of the available oligonucleotide probes developed for the PCR amplification of PKX DNA were examined for their ability to detect PKX in fixed tissue sections using in situ hybridisation. Out of the 12 probes examined, only four stained PKX in tissue sections. The specificity of these probes to PKX was examined by testing them individually against a range of myxosporean infections. One of the probes (1032) cross-reacted with Sphaerospora truttae infecting brown trout kidney and stained this parasite in tissue sections, while probe 6R stained stickleback DNA. The results from these studies allowed for an optimised, relatively rapid, in situ hybridisation protocol to be developed for PKX detection. Using this protocol, a preliminary study was conducted on the life history of the parasite in the rainbow trout Oncorhynchus mykiss. This demonstrated the presence of PKX in the gill arch 3 days after initial exposure in an enzootic river. It is suggested that a portal of entry for PKX is the gill. From here. it migrates to the kidney where the disease progresses as previously described.

Induction of nodavirus disease in seabass, Dicentrarchus labrax, using different infection models.

The aim of this study was to investigate the susceptibility of juvenile and adult seabass, which are generally thought to be refractory to nodavirus. Moreover, preliminary immunological studies were performed to examine the immune response of adult seabass. Successful transmission of the disease was experimentally demonstrated in juvenile and adult seabass as ascertained by the presence of the clinical signs of the disease, re-isolation of the virus in the SSN-1 cell line and subsequent confirmation by histopathology and immunohistochemistry. Bigger seabass not only developed the clinical disease but also suffered mortalities. Serum neutralisation titres were considered low in this study.