Immunostimulation of Salmo salar L., and its effect on Lepeophtheirus salmonis (Krøyer) P-glycoprotein mRNA expression following subsequent emamectin benzoate exposure.

Control of sea lice, Lepeophtheirus salmonis, on farmed Atlantic salmon, Salmo salar, relies heavily on chemotherapeutants. However, reduced efficacy of many treatments and need for integrated sea lice management plans require innovative strategies. Resistance to emamectin benzoate (EMB), a major sea lice parasiticide, has been linked with P-glycoprotein (P-gp) expression. We hypothesized that host immunostimulation would complement EMB treatment outcome. Lepeophtheirus salmonis-infected Atlantic salmon were fed immunostimulatory or control feeds. Sea lice were collected for 24-h EMB bioassays 1 and 2 weeks prior to commencement of EMB treatment of the fish. Two weeks after cessation of immunostimulant-treated feed, EMB was administered at 150 µg kg(-1) fish biomass for 7 days. The bioassay revealed stage, gender and immunostimulant-related differences in EMB EC(50) . Sea lice attached to salmon with a history of immunostimulation exhibited significantly greater survival than those on control feeds, despite similar levels of EMB in host tissues. Lepeophtheirus salmonis from salmon with a history of immunostimulation also exhibited higher P-gp mRNA expression as well as greater survivability compared to controls. Administration of immunostimulants prior to EMB treatment caused increased expression of P-gp mRNA which could have consequently caused decreased efficacy of the parasiticide.

Combinatorial effects of administration of immunostimulatory compounds in feed and follow-up administration of triple-dose SLICE® (emamectin benzoate) on Atlantic salmon, Salmo salar L., infection with Lepeophtheirus salmonis.

Several immunostimulatory feed additives have shown the ability to induce protective responses in Atlantic salmon to infection with Lepeophtheirus salmonis. However, even the most encouraging results rarely surpass a 50% protective index in the host. That fact coupled with the well-documented limitations of single-therapy strategies in the effective management of parasitic infections generally make it imperative to identify therapies that can be combined in an integrated pest management approach for sea lice. With this in mind, we hypothesized that immunostimulatory feeds could enhance the protection provided by SLICE® emamectin benzoate (EMB). To test this hypothesis, Atlantic salmon were fed one of two different immunostimulatory feeds (CpG ODN or Aquate®) for c. 7 weeks, challenged with L. salmonis copepodids early within that immunostimulatory feed period and then placed on a triple-dose (150 µg kg(-1) ) feed of SLICE® for 1 week following the completion of the immunostimulatory feeding period. CpG ODN (2 mg kg(-1) ) and the commercial yeast extract (Aquate® 0.2%) inclusion in feeds were able to successfully induce inflammatory gene expression (interleukin-1ß) in the head kidneys of infected fish at 13 and 26 days post-exposure (DPE), and 13 DPE, respectively. Lice burdens were lower on fish fed CpG ODN (18%) or Aquate® (19%) diets; however, due to variability, these were not statistically significant over time. Despite no statistically significant reductions in lice numbers, by 33 DPE fish on immunostimulatory feeds had significantly reduced cortisol levels when compared to infected fish on control diet. Cortisol levels in fish receiving an immunostimulatory diet were no different from initial baseline levels prior to infection, whereas the levels in control diet fish were significantly elevated from all other time points. Despite the positive effects on infection of fish fed immunostimulatory feeds, no synergism was observed with follow-up treatment with SLICE® . In fact, highest survival of lice was observed in fish with prior immunostimulation.

Comparison of metal versus absorbable implants in tension-band wiring: a preliminary study.

The strength of tension-band wiring using bioabsorbable materials versus metal implants was assessed with a rabbit knee fusion model. Ten rabbit knees were osteotomized and rigidly fixed using a tension-band technique: five with metal implants (2 pins and 24-gauge wire) and five with absorbable implants (2-mm pins [Bionx, Blue Bell, Pa] and 1 Maxon [Davis and Geck, Danbury, Conn]). Biomechanical testing of the fixation strength was completed using a servohydraulic mechanical testing machine and a specifically designed four-point bending jig. The parameters assessed were maximal load, relative stiffness, displacement, and bending moment of the constructs. Results of the biomechanical testing showed no statistical difference between the constructs on any of the parameters assessed.

Critically sized osteo-periosteal femoral defects: a dog model.

A 21-mm defect was created in 1 femoral diaphysis each of 15 dogs. Periosteum as well as a cylinder of bone was removed, and the defect was stabilized with a bone plate. Twelve of the defects were filled with an equal volume of autogenous cancellous bone harvested from the ipsilateral ilium. Three defects were left untreated. Cranial to caudal radiographs were taken postoperatively and every 4 weeks for 16 weeks. The radiographs were evaluated for healing using two ordinal scales. At 16 weeks, the dogs were euthanized and the femurs harvested for biomechanical testing and histologic evaluation. Both operated and contralateral not operated femurs were mechanically tested to failure in torsion, and load at failure and stiffness were calculated. All dogs tolerated the procedure well, and were using the operated limb within 1 or 2 days postoperatively. There were no complications noted during the 16 weeks of the study. Unfilled defects did not heal and became atrophic nonunions. The defects filled with autogenous cancellous bone healed in a consistent pattern of consolidation, incorporation, and remodeling, with uniform increases of both ordinal scales used. The femoral cortex opposite the bone plate demonstrated most mature remodeling, evident both radiographically as well as histologically. Unoperated femurs failed at 13.61 +/- 3.88 N-m and grafted femurs failed at 2.96 +/- 1.3 N-m, which was 23% of the measurement of the unoperated femur. Relative stiffness of the unoperated femurs was 5974 +/- 4316 N-m2/radian, and grafted femurs had a relative stiffness of 642 +/- 561 N-m2/radian, which was 10.4% of the measurement of unoperated femur. This model proved to be a critically sized defect, which when left unfilled resulted in an atrophic nonunion, and when filled with cancellous bone resulted in a consistent healing pattern.

Mechanical comparison of two external fixator clamp designs.

OBJECTIVE: To compare two external fixation clamp designs for their ability to resist movement of a fixation pin in relation to the connecting rod. STUDY DESIGN: Two designs of external fixator clamps were attached to connecting rods mounted on a jig for mechanical testing. Fixator pins were placed perpendicular to the connecting rod. A mechanical testing machine was used to deflect each 3.2-mm pin at a distance that was 25 mm from the center of the clamp bolt. Both clamp designs were tightened to 4.4, 6.1, and 7.8 newton-meters (N x m) torque, and loads were applied in a position ramp through 4 mm and resisting loads were measured. Two clamp orientations were used during load application, such that the deflection of the pin tended to tighten the clamp bolt or tended to loosen the clamp bolt. The tests were videotaped to determine mode of failure. Comparisons of the load/displacement curves for the two external fixator clamp designs were made using nonlinear equational curve fitting methods. The resultant plateau and rise coefficients were compared using analysis of variance. RESULTS: Slippage of the pin in relation to the clamp occurred with the Kirschner-Ehmer clamp tightened to 4.4, 6.1, and 7.8 N x m, and slipping of the pin in relation to the clamp occurred with the experimental clamp design tightened to 4.4 and 6.1 N x m but not to 7.8 N m. At 7.8 N x m, the 3.2-mm pin deformed plastically with the experimental clamp design. Increasing the torque of the clamp bolt resulted in superior plateau coefficients for both clamp designs. At each level of tightness and in each clamp orientation to applied pin load, the experimental clamp design provided greater plateau coefficients than did the Kirschner-Ehmer clamp design. At 7.8 N x m of tightness, the Kirschner-Ehmer clamp and bolt bent, whereas only slight plastic deformation of the experimental clamp design occurred. CONCLUSIONS: The experimental external fixator clamp was more secure in resisting fixator pin movement at all levels of tightening compared with the Kirschner-Ehmer-type external fixator clamp. At 7.8 N x m of tightening, the new clamp design did not allow slippage of the pin within the clamp. CLINICAL SIGNIFICANCE: The experimental external fixator clamp should result in greater rigidity of fixator configurations, in addition to providing design features that allow addition of a clamp between two installed clamps, sleeved predrilling of pilot holes for all pins, measurement of pin depth, and placement of positive profile pins at all sites.

Type-II external fixation, using new clamps and positive-profile threaded pins, for treatment of fractures of the radius and tibia in dogs.

A new external fixation system for repair of fractures of the radius and tibia was developed that uses positive-profile threaded pins. This system allows for addition of a fixation clamp between 2 installed clamps, predrilling of pin holes through a drill sleeve, use of positive-profile threaded pins in all locations, and easier application of full pins. Type-II external fixators were applied, using this system, to fractures in 10 client-owned dogs, and outcome was evaluated. All fractures healed without complications. Duration of surgery, mean time until radiographic evidence of a bridging callus, and mean time until removal of the external fixator were shorter, and frequency of pin loosening were less than with other techniques. Pin loosening was uncommon. This system provides an important improvement in external skeletal fixation.

Increased susceptibility and reduced phytoalexin accumulation in drought-stressed peanut kernels challenged with Aspergillus flavus.

Three genotypes of peanut (Arachis hypogaea L.), with ICG numbers 221, 1104, and 1326, were grown in three replicate plots and drought stressed during the last 58 days before harvest by withholding irrigation water. Within each plot there were eight levels of stress ranging from 1.1 to 25.9 cm of water. Kernels harvested from the plots were hydrated to 20% moisture and challenged with Aspergillus flavus. Fungal colonization, aflatoxin content, and phytoalexin accumulation were measured. Fungal colonization of non-drought-stressed kernels virtually ceased by 3 days after inoculation, when the phytoalexin concentration exceeded 50 micrograms/g (fresh weight) of kernels, but the aflatoxin concentration continued to rise exponentially for an additional day. When fungal colonization, aflatoxin production, and phytoalexin accumulation were measured 3 days after drought-stressed material was challenged, the following relationships were apparent. Fungal colonization was inversely related to water supply (r varied from -0.848 to -0.904, according to genotype), as was aflatoxin production (r varied from -0.876 to -0.912, according to genotype); the phytoalexin concentration was correlated with water supply when this exceeded 11 cm (r varied from 0.696 to 0.917, according to genotype). The results are discussed in terms of the critical role played by drought stress in predisposing peanuts to infection by A. flavus and the role of the impaired phytoalexin response in mediating this increased susceptibility.

Circumstantial evidence for phytoalexin involvement in the resistance of peanuts to Aspergillus flavus.

Three stilbene phytoalexins, elicited by slicing and incubating imbibed peanut kernels under aerobic conditions, inhibited spore germination and hyphal extension of Aspergillus flavus with ED50 values in the range 4.9-12.8 micrograms ml-1. Phytoalexin yield was dependent on cultivar, conditions and duration of incubation after slicing, and crop history. The yield of phytoalexin from ten cultivars studied, after slicing and incubating at 25 degrees C for 24 h, ranged from 28 to 935 micrograms per g fresh weight and was negatively correlated with dry kernel colonization by A. flavus [r = -0.868 when plotted as 1n (phytoalexin concn) against 1n (percentage peanut colonization)]. When the incubation period was extended to 96 h there was no such correlation. Reduced phytoalexin yields were obtained when sliced kernels of one cultivar studied were incubated in water or at 37 degrees C, and no phytoalexin was obtained when the slices were incubated under nitrogen gas or frozen before aerobic incubation. Drought stress during pod development in four cultivars studied reduced phytoalexin yields of sliced kernels incubated at 25 degrees C for 24 h by 17-65% compared with non-stressed controls.