Susceptibility of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae), to viral haemorrhagic septicaemia virus (VHSV) genotype III: Experimental challenge and pathology.

Cleaner fish, such as wrasse, are being increasingly used to combat the sea lice infestation of Atlantic salmon (Salmo salar L.) in many European countries. To determine susceptibility of the goldsinny wrasse (Ctenolabrus rupestris L.) and pathogenesis of the viral haemorrhagic septicaemia virus (VHSV) genotype III isolate 12-654, previously associated with VHSV infection in the Shetland Islands in 2012, fish were experimentally challenged by intraperitoneal injection (IP), bath immersion and cohabitation routes. Cumulative proportion of moribund wrasse reached 17% following the virus immersion challenge while by the IP-route moribunds exceeded 50% within 14days post-challenge. Typical signs of VHS as reported in rainbow trout (Oncorhynchus mykiss), were not observed in moribund goldsinny wrasse. The most pronounced histopathological changes, consistent regardless of the route of infection, were observed within the heart and included atrium myofibril degeneration, focal infiltration and multifocal necrosis, with prominent swelling of the endocardium and occasional detachment. Pathological changes in the atrium were associated with presence of the viral antigen as confirmed by a positive immunohistochemical staining. Virus clearance and heart tissue recovery were noted although further experiments are required to confirm these observations. The results of a cohabitation experiment confirmed that goldsinny wrasse shed viable virus and therefore represent a risk of virus transmission to other VHSV susceptible species. Similarities between the pathology in goldsinny wrasse induced through the controlled experimental challenges and that of wrasse spp. from an infection occurrence in Shetland are discussed.

Host tropism of infectious salmon anaemia virus in marine and freshwater fish species.

The aquatic orthomyxovirus infectious salmon anaemia virus (ISAV) causes a severe disease in farmed Atlantic salmon, Salmo salar L. Although some ISA outbreaks are caused by horizontal transmission of virus between farms, the source and reservoir of the virus is largely unknown and a wild host has been hypothesized. Atlantic salmon are farmed in open net-pens, allowing transmission of pathogens from wild fish and the surrounding environment to the farmed fish. In this study, a large number of fish species were investigated for ISAV host potential. For orthomyxoviruses, a specific receptor binding is the first requirement for infection; thus, the fish species were investigated for the presence of the ISAV receptor. The receptor was found to be widely distributed across the fish species. All salmonids expressed the receptor. However, only some of the cod-like and perch-like fish did, and all flat fish were negative. In the majority of the positive species, the receptor was found on endothelial cells and/or on red blood cells. The study forms a basis for further investigations and opens up the possibility for screening species to determine whether a wild host of ISAV exists.

Immersion challenge with low and highly virulent infectious salmon anaemia virus reveals different pathogenesis in Atlantic salmon, Salmo salar L.

The salmonid orthomyxovirus infectious salmon anaemia virus (ISAV) causes disease of varying severity in farmed Atlantic salmon, Salmo salar L. Field observations suggest that host factors, the environment and differences between ISAV strains attribute to the large variation in disease progression. Variation in host mortality and dissemination of ISAV isolates with high and low virulence (based on a previously published injection challenge) were investigated using immersion challenge. Virus dissemination was determined using real-time PCR and immunohistochemistry in several organs, including blood. Surprisingly, the low virulent virus (LVI) replicated and produced nucleoprotein at earlier time points post-infection compared to the virus of high virulence (HVI). This was particularly noticeable in the gills as indicated by different viral load profiles. However, the HVI reached a higher maximum viral load in all tested organs and full blood. This was associated with a higher mortality of 100% as compared to 20% in the LVI group by day 23 post-infection. Immersion challenge represented a more natural infection method and suggested that specific entry routes into the fish may be of key importance between ISAV strains. The results suggest that a difference in virulence is important for variations in virus dissemination and pathogenesis (disease development).

Interferon response following infection with genetically similar isolates of viral haemorrhagic septicaemia virus (VHSV) exhibiting contrasting virulence in rainbow trout.

Isolates of viral haemorrhagic septicaemia virus (VHSV) were identified which are genetically similar yet, based on their isolation history were considered likely to differ in virulence in juvenile rainbow trout. An experimental infection study was performed in order to verify this hypothesis and provide an experimental infectivity model with which to investigate the basis for susceptibility of rainbow trout to this commercially important virus. Significant differences in mortality were obtained following both intraperitoneal (IP) injection and immersion challenges with an early marine (DK-M.Rhabdo) and early rainbow trout VHSV isolate (DK-F1) respectively. Expression of Type I IFN, Mx1 (an IFN-inducible protein), and viral genes (encoding nucleo-, phospho-, matrix, glyco- and non-viron proteins) was studied in sequential tissue samples using real-time quantitative PCR (QPCR). Resulting data revealed a significant increase in IFN and Mx1 expression detected in fish challenged by IP injection with both isolates. Expression levels of these genes were directly related to the degree of viral replication as measured by the expression of VHSV RNAs. In immersion-challenged fish a significant increase in Mx1 was observed only when using the virulent isolate DK-F1; however no elevated host response was detectable in fish challenged with the marine isolate DK-M.Rhabdo. Quintessentially the inability to detect any virus in trout challenged with the marine isolate via immersion suggests the virus was incapable of establishing infection. The mechanisms for this appear to be more related to initial cellular entry and replication rather than due to the overcoming of initial infection via an elevated host innate immune response.

Expression of Mx mRNA following infection with IPNV is greater in IPN-susceptible Atlantic salmon post-smolts than in IPN-resistant Atlantic salmon parr.

The Mx response was compared in parr and post-smolt Atlantic salmon following intra-peritoneal injection of the same dose of Infectious Pancreatic Necrosis Virus (IPNV) per g of fish. Mx gene expression, measured by quantitative RT-PCR in liver, showed a maximum level 3days after injection in parr with undetectable levels on day 7. In post-smolts, similar levels as in parr were attained on day 3, but levels then continued to rise on day 5 and 7 to about 10 times higher than the peak level in parr. Poly I:C injected parr showed Mx levels similar to IPNV injected post-smolts. Mortality from IPN in post-smolts occurred on days 6 and 7. Levels of IPN VP2 transcripts in parr were very low and did not increase with time, suggesting viral replication was low. Individual variation in levels of Mx and IPN VP2 gene transcripts was very high in post-smolts and although data is limited there was an inverse relationship between the levels of Mx and VP2, suggesting that individuals with high Mx levels on day 5 may be able to prevent viral replication. This contrasts with the response in parr, where IPN-resistance was not associated with a high Mx response.

Expression kinetics of interferon and interferon-induced genes in Atlantic salmon (Salmo salar) following infection with infectious pancreatic necrosis virus and infectious salmon anaemia virus.

Infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV) are economically important pathogens of the salmonid aquaculture industry. Atlantic salmon were challenged by intraperitoneal injection (i.p.) with either virus followed by time-course sampling. Cohabiting fish in the IPNV challenge were also sampled. Kidney tissue was analysed using a TaqMan real-time PCR assay to measure the expression of a range of host immune genes in relation to the endogenous control, elongation factor 1 alpha (ELF). Host genes measured included Mx, type I and type II interferon (IFN), gammaIFN induced protein (gammaIP), interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha). Viral levels were also measured. In i.p. injected fish, both viruses greatly induced expression of Mx, gammaIP, type I and type II IFN by day 6 post-infection, however only ISAV caused substantial mortality. Some differences between the expression kinetics produced by both viruses were noted. Infection with ISAV increased IL-1beta expression following day 6, but no effect was seen in fish infected with IPNV. Neither virus induced TNF-alpha expression. This study confirms the presence of both type I and type II IFN responses and their induced genes in Atlantic salmon upon infection with an orthomyxovirus and a birnavirus.

Development, application and validation of a Taqman real-time RT-PCR assay for the detection of infectious salmon anaemia virus (ISAV) in Atlantic salmon (Salmo salar).

Infectious salmon anaemia (ISA) is a disease of cultured Atlantic salmon (Salmo salar) which was successfully eradicated from Scotland following its emergence in 1998. The rapid deployment of sensitive diagnostic methods for the detection of ISA virus (ISAV) was fundamental to the swift eradication of ISA disease in Scotland and continues to be of crucial importance to surveillance of the aquaculture industry. This study reports the development, validation, application and interpretation of two independent, highly sensitive and specific semi-quantitative Taqman real-time RT-PCR (qRT-PCR) methods for the detection of ISAV. Such technology offers considerable advantages over conventional RT-PCR methods in current routine use for ISAV surveillance. These include an increased sensitivity, enhanced specificity, semi-quantification using endogenous controls, a lack of subjectivity in results interpretation, speed of processing and improved contamination control.

Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei).

Thirty-one gyrodactylid species from five families of freshwater fish were examined and variable region V4 of the 18S small subunit ribosomal RNA gene and ribosomal RNA internal transcribed spacers ITS1 and ITS2 were sequenced. Both the V4 region and spacers ITS1 and ITS2 proved useful for gyrodactylid diagnosis. Sequences of these fragments exhibited interspecific variations and allowed clear determination at the species level. In some cases, the length of the ITS1 PCR fragment provided useful genetic markers. Species that yielded a short ITS1 fragment also showed distinct groupings in ITS2 and V4 sequences that were markedly different to sequences from species that contain a long ITS1. Repetitive sequences located in the ITS1 of Gyrodactylus gobii and Gyrodactylus vimbi accounted for some of the variations in length of PCR products. There was no evidence for intraspecific variation within these regions and short tandem repeats were not found in the other species studied. The number of polymorphic and intraspecific variations in nucleic acid sequences was low, therefore these variations did not affect species determination of gyrodactylids. Minor differences in the sequences between Western and Eastern European populations were detected for Gyrodactylus salaris/Gyrodactylus thymalli, Gyrodactylus teuchis and Gyrodactylus truttae, but these do not affect species diagnosis based on ribosomal DNA sequence. These results confirm the utility of both variable region V4 and the ITS as molecular markers for Gyrodactylus species.

Measurement of bone mineral density by dual-energy x-ray absorptiometry in patients with the Wisconsin hip, an uncemented femoral stem.

Although qualitative evidence of femoral bone remodeling, secondary to total hip arthroplasty (THA), is apparent on radiographs, quantification of change in bone mass from radiographs is limited. Dual-energy x-ray absorptiometry overcomes many of the limitations and yields accurate and precise bone mineral density (BMD) data. In this study, regional changes in femoral BMD were examined in 89 THA patients with a 2-year follow-up period. Thirty-two patients were evaluated initially before surgery and followed through the first 2 postoperative years. A second group was comprised of 57 patients whose surgery had been performed 1 to 6 years prior to entry into the study; they were also followed for 2 years hence. Thus, both immediate and later bone responses were evaluated prospectively. Maximal bone remodeling was seen in the first 6 months after THA and with a near plateau by the end of the first year. A slow yearly decline in BMD appeared to occur as long as 8 years after THA, thus demonstrating the long-term effects of the introduction of a femoral stem. Variance in preoperative BMD was explained by disease only; no other factors (age, weight, sex) showed significant associations, and body weight was the only variable that affected rate of remodeling after THA (not age, weight, sex, prosthesis size, nor disease). All patients were healthy, relatively young individuals who were good candidates for uncemented implantation, and none showed evidence of clinical complications or surgical failure. It is therefore suggested that the patterns and results reported here be viewed as normative data, that is, the typical skeletal adaptation to THA. In future application, observation of disparate BMD results as compared with these "normal" data may be predictive of abnormal response to surgery and potential for later problems.

Imaging barium-free bone cement.

Prior to 1972, polymethylmethacrylate, or PMMA, did not contain barium sulfate and was, therefore, radiolucent. We describe a technique using magnetic resonance imaging that is very useful in imaging barium-free PMMA.

The Wisconsin noncemented femoral stem. A prospective study with 5 to 9 years of follow-up evaluation.

In an attempt to decrease aseptic loosening, total joint components are now being used without cement. Most components are designed to achieve fixation biologically. The radiographic results of 144 primary uncemented total hip arthroplasties and the clinical results of 89 arthroplasties that were performed using a proximally porous-coated titanium alloy femoral stem between November 1983 and June 1989 are reported. On the acetabular side, a threaded component or a hemispherical porous-coated component was used. The patients were followed prospectively for 5 to 9 years postsurgery. During the study period, the threaded acetabular component had a high failure rate. Because it was not possible to determine accurately from which component a particular sign or symptom arose, clinical analysis was restricted to only those hips without a failed or revised cup. Eighty-nine hips in 71 patients with a minimum follow-up period of 5 years were available for clinical review. Radiographic review of the femoral stem was completed on all hips with 5 or more years of radiographic follow-up evaluation regardless of the status of the acetabular component. One hundred twelve hips in 85 patients were analyzed radiographically. At a latest mean follow-up period of 6.7 years, the mean Iowa hip score was 91.4 +/- 8.0 with a 5.4% incidence of thigh pain. Throughout the follow-up period, the patients with thigh pain had a statistically lower mean Iowa pain subscore when compared with those patients without thigh pain (P=.0001). Endosteal erosion was seen in two hips (1.8%) and longitudinal loss of the medial neck greater than 2 mm was noted in two hips. One femoral stem was revised for aseptic loosening. The clinical results of this femoral stem equal or exceed the published accounts of other arthroplasties. The results indicate that the stem is associated with good clinical results, minimal bone loss, and little osteolysis. Continued follow-up evaluation of patients with this femoral stem is necessary to assess the durability of these encouraging results.

The gluteus minimus muscle pedicle graft in the treatment of femoral head avascular necrosis.

The treatment of the nonelderly adult patient with avascular necrosis (AVN) of the femoral head remains a challenge. We treated 16 patients (20 hips) afflicted with Ficat stage II or III femoral head AVN with a pedicle bone-grafting technique using the gluteus minimus. The procedure involves core decompression of the osteonecrotic segment of the femoral head followed by introduction of a corticocancellous bone graft from the anterior femur into the decompressed segment. The graft contains the insertion of the gluteus minimus. Each case was retrospectively evaluated to determine the incidence of clinical and roentgenographic progression of the disease after the procedure. The follow-up period averaged 47 months clinically and 33 months radiographically. Twelve (60%) of the cases were considered a success symptomatically;, 8 (40%) of the hips demonstrated radiographic stabilization. This procedure is bei ng presented as a possible "biologic" option in the treatment of femoral head AVN.

Arthrodesis following failed total knee arthroplasty: comprehensive review and meta-analysis of recent literature.

With the increasing duration of follow up on total knee arthroplasties, more revision arthroplasties are being performed. When revision is not advisable, a salvage procedure such as arthrodesis or resection arthroplasty is indicated. This article provides a comprehensive review of the literature regarding arthrodesis following failed total knee arthroplasty. In addition, a statistical meta-analysis of five studies using modern arthrodesis techniques is presented. A statistically significant greater fusion rate with intramedullary nail arthrodesis compared to external fixation is documented. Gram negative and mixed infections are found to be significant risk factors for failure of arthrodesis.

Hip replacement with a threaded acetabular cup. A follow-up study.

A study was begun in 1983 to determine the efficacy of a threaded acetabular cup. Fifty-five patients who had a total of sixty-eight threaded titanium cups had a complete clinical and radiographic evaluation yearly. Fifty-two of the arthroplasties had been primary and sixteen, revisions. The average duration of follow-up was six years (range, five to nine years). Seventeen cups had to be revised at an average of sixty-two months (range, twenty-seven to 108 months) after the index operation. Nine additional cups were loose and revision was pending at the most recent follow-up examination. Failure was defined as revision or pending revision. Thus, twenty-six (38 per cent) of the sixty-eight cups failed. Sixteen (31 per cent) of the fifty-two primary arthroplasties failed and ten of the sixteen revision arthroplasties failed. Radiographic changes that were evident in patients who had a failed cup consisted of superomedial migration of the cup with osteolysis in Zone 3, as classified by DeLee and Charnley. These radiographic changes preceded symptoms in most patients. Because of the high rate of failure of this acetabular component at six years, we believe that its use is not warranted.

Determination of bone mineral density by dual x-ray absorptiometry in patients with uncemented total hip arthroplasty.

Bone remodeling is an expected sequela with total hip arthroplasty (THA). Although there are several methods of estimating bone response in THA patients from radiographs, there are no accurate and generally accepted methods for quantitative determinations in vivo. In this study, we describe an application of dual x-ray absorptiometry (DXA) for measuring bone mineral content and bone mineral density in the proximal femur following THA. DXA is a noninvasive technique with minimal radiation exposure (< 5 mrem). Various aspects of measurement error (accuracy and reliability) of this application of DXA were determined in a series of studies reported here. Accuracy error (how similar are the measured and actual values) was < 1% determined in bone phantoms of four densities. Precision error (how reproducible are the measurements) was also < 1% at all four densities in the phantoms and was only slightly elevated (0.9-1.5%) in repeated measurements of implanted cadaver femora. Precision error in vivo, determined both from multiple replicates on five patients and from duplicate scans on 30 patients, was further elevated but remained < 5%. Contributions to precision error, rotation of the leg, and interoperator variability were assessed; none was found to elevate precision error appreciably. We suggest that DXA is a feasible method for quantifying bone response following THA, and will allow discrimination of small changes (> 5%) not previously measurable.

Fixation stability of femoral components in a canine hip replacement model.

A canine hip replacement model was used to compare fixation stability in cemented and cementless femoral components. Parameters of comparison were the load-induced positional changes of each prosthesis relative to its proximal femoral cortex, hereafter called relative displacements. Identical femoral components, with the proximal third of their stem porous-coated, were implanted in the right femurs of 10 large, mixed-breed dogs. Five were tightly fit to allow porous ingrowth, and five were cemented into the medullary canal. Four months after implantation, all femurs were harvested. A prosthesis was implanted in the left (normal) femur of each dog ex vivo with fixation identical to the contralateral limb to simulate acute postoperative fixation. Eddy current transducers measured relative displacements under application of static loads, serially applied in the axial, mediolateral, and craniocaudal directions. Thereafter, the femurs were transversely sectioned and morphologically analyzed to correlate bony apposition at the implant surface with relative displacements. We observed no difference in relative displacements between acute and 4-month-cemented groups (e.g., 0.0059 +/- 0.0021 vs. 0.0060 +/- 0.0048 mm, respectively, for 100-N axial loading measured at midstem). With cementless implantation, relative displacements of the acute group were significantly larger (p = 0.007) than those of the 4-month group (e.g., 0.236 +/- 0.257 vs. 0.097 +/- 0.129 mm, respectively, for 100-N axial loading measured at midstem). Cementless components implanted for 4 months were not significantly different than cemented components, but a trend suggested that they were still not as stable as cemented components, particularly for craniocaudal loads. Relative displacements of the 4-month, porous ingrowth group were approximately proportional to the percentage of bony apposition raised to the -1.44 power (r = 0.94).

Canine intersegmental hip joint forces and moments before and after cemented total hip replacement.

Intersegmental forces and moments (i.e. resultant free body forces and moments computed at the joint centers) were studied in canine hindlimbs before and after cemented total hip replacement (THR). Five large, adult, mixed-breed dogs were selected. Their gait was recorded (while leash-walked) before surgery using high-speed cinematography and a force plate. Cemented total hip replacement was unilaterally performed on each dog. Gait was again recorded at one and four months after surgery. Segmental properties (mass, center of mass, and mass moment of inertia) of the hindlimbs were experimentally determined, and an inverse dynamics approach was used to compute intersegmental forces and moments in the sagittal plane. Significant reductions in intersegmental joint forces and moments were observed in the operated hindlimb one month after surgery, although kinematic gait parameters were unaltered. Decreases of 77.0% for vertical forces, 61.9% for craniocaudal forces, and 66.2% for extension moments were determined. Four months after surgery, the joint forces and moments had returned to their preoperative values. This experiment demonstrates that the dynamics of normal walking can be restored in a canine model by four months after THR. It also shows that kinetic (rather than kinematic) parameters are more descriptive of antalgic gait in the canine.

Diagnosis and management of vascular injuries associated with skeletal trauma.

The circulatory status of the extremity distal to skeletal trauma is evaluated routinely, but the implications of subtle physical findings are not always clear. Abnormal clinical findings associated with a fracture or dislocation near a major vessel often warrant intraoperative, "one-shot" angiography. Close cooperation between the orthopaedic and vascular teams is essential to ensure adequate revascularization within six hours of injury. Fracture fixation is usually performed first, using the method dictated by the fracture pattern and the nature of the soft-tissue damage. Fasciotomy should be performed in almost all cases.

K-edge digital subtraction arthrography of the painful hip prosthesis: a feasibility study.

K-edge energy subtraction radiography is a method for detecting the presence of iodinated contrast material by subtracting two digital radiographs produced by X-ray beams with energies above and below the iodine K edge. We performed a feasibility study on the application of K-edge energy digital subtraction arthrography (KEDSA) to painful hip prostheses. During arthrography, loosening of the prosthesis is implied if contrast material is seen dissecting around the prosthesis, an often difficult detection task because of adjacent prosthesis metal or cement. In conventional arthrography a preliminary mask image is thus used from which films obtained after injection of iodinated contrast material are subtracted. Movement by the patient during this process may preclude subsequent subtraction. With KEDSA, since multiple image pairs may be obtained after the injection of contrast material, the problem of patient motion is virtually eliminated. A conventional X-ray tube operating between 55 and 65 kVp was alternately filtered by iodine and cerium filters to produce the KEDSA images. The apparatus was capable of producing a subtracted image within 3 sec. The technique was applied to phantoms and to six patients immediately after hip arthrography that had been positive for prosthesis loosening. Although of lower spatial resolution, the KEDSA images were, in all cases, positive for loosening in a pattern consistent with the conventional arthrographic images. KEDSA was shown to be successful in detecting extraarticular contrast material. During a single study, subtraction in various imaging planes as well as postexercise subtraction imaging can be accomplished-techniques not heretofore possible in routine subtraction arthrography.