Assessment of the combination of spinosad and milbemycin oxime in preventing the development of canine Angiostrongylus vasorum infections

Angiostrongylus vasorum is an increasingly reported parasite in Europe that develops in dogs after ingestion of infective third stage larvae (L3) that reside in gastropod molluscs which are needed to complete the parasite's life-cycle. Infection can produce a diversity of clinical signs, determined by involvement of the respiratory, neurological, and/or coagulation system, with a likely fatal outcome in the absence of treatment. Few drugs have been shown to reliably prevent infection, and data on treatment of infections is limited. A controlled, randomized, partially blinded laboratory study was therefore executed to evaluate the efficacy and safety of a combination tablet of spinosad/milbemycin oxime in dogs inoculated with approximately 250 A. vasorum L3. Sixteen healthy nematode free adult dogs were randomly allocated to two study groups of 8 dogs each. Thirty days post inoculation (dpi) all dogs in the fed state were treated: dogs in group B were treated with spinosad and milbemycin oxime at the dose rates of 45–60 mg/kg and 0.75–1.0 mg/kg bodyweight, respectively, approximately the lower half portion of the expected full unit dose range; dogs in group A were treated with placebo tablets. All dogs were euthanized and necropsied 56–58 dpi. The heart and lungs were examined to determine the presence of A. vasorum. All placebo group dogs were infected at necropsy with counts ranging from 22 to 98 adult worms and a geometric mean worm count of 55.2. In contrast, the geometric mean worm count in the spinosad/milbemycin oxime group was 0.7 with worm numbers ranging from 0 to 8. The results of this study demonstrate that a single treatment with the tablet combination of spinosad and milbemycin oxime administered 30 dpi provided 98.8% preventive efficacy against development of adult A. vasorum infections. Monthly treatments with spinosad and milbemycin oxime have the potential to prevent the establishment of infections with A. vasorum in dogs.     

The effect of dietary protein on the growth and survival of the shrimp, Penaeus monodon in outdoor tanks

The comparative effect of reducing the protein content of formulated feed on the growth and survival of black tiger shrimp, Penaeus monodon, and on water quality was tested in outdoor tanks. Three diets, 300, 350 and 400 g kg^?1 crude protein (CP), were fed to P. monodon (3.1 g animals, 25 animals per m²) in each of eight replicated outdoor 2500 L tanks in an 8‐week trial. There was no statistical difference (P > 0.05) in shrimp growth rate (1.34–1.50 g week^?1), survival, or final biomass between the treatments. However, when tanks with lower survival were removed from the analysis (<60 and <80% were tested), shrimp growth rate was statistically higher (P < 0.05) in the 350 and 400 g kg^?1 CP diets than in the 300 g kg^?1 CP diet treatment. There were no differences in the nutritional condition of shrimp between treatments, as determined by moisture and protein content, and lipid content of the digestive gland. Using ¹⁵N‐nitrogen isotope tracers, it was determined that shrimp were consuming natural biota, although these were unlikely to have contributed substantially to their nutrition. Total nitrogen (TN) concentrations in the water column increased over the eight week experiment and were statistically different (P < 0.001) between treatments (3.60, 5.17 and 6.45 mg L^?1 in the 300, 350 and 400 g kg^?1 CP treatments, respectively). Concentrations of dissolved organic nitrogen (DON) were also statistically different between treatments and made up 35–40% of the TN in the water column. Concentrations of total ammoniacal nitrogen (TAN) and oxides of nitrogen, and fluorescence were not statistically different between treatments but there was a trend of higher concentrations in treatments with higher protein levels. There was no difference in sediment nutrients between treatments. This study has shown that there is scope to reduce the protein content of P. monodon diets but only by 5–10%. However, further validation of these results in commercial ponds is needed. Reducing the feed protein content may result in cost savings and also has the advantage of improving water quality and reducing nitrogen discharge.     

Identification of Mycobacterium spp. isolated from snakehead, Channa striata (Fowler), and Siamese fighting fish, Betta splendens (Regan), using polymerase chain reaction–reverse cross blot hybridization (PCR–RCBH)

A variety of methods have been used to identify Mycobacterium spp. isolated from snakehead and Siamese fighting fish, including biochemistry, mycolic acid profiles and antibody-based methods. However, these methods are unable to differentiate between different species of Mycobacterium . Polymerase chain reaction (PCR) followed by reverse cross blot hybridization (RCBH) was adapted in this study to speciate aquatic mycobacteria. The method was highly specific for Mycobacterium spp. and identified the bacteria to species level with a detection limit of 100 fg DNA, equivalent to 20 mycobacteria. Twenty-nine isolates previously collected and cultured from Siamese fighting fish (10 isolates) and snakehead (19 isolates) during outbreaks of mycobacteriosis were analysed using PCR–RCBH. Six of the Siamese fighting fish isolates and nine of the snakehead isolates were identified as Mycobacterium fortuitum , while the remainder were classified as M. marinum . Notably, two isolates recovered from snakehead and Siamese fighting fish, previously identified as M. poriferae and M. piscicida , respectively, were confirmed to be M. fortuitum .     

ULTRASONOGRAPHIC CHARACTERISTICS OF LIPIDURIA IN CLINICALLY NORMAL CATS

Echoes are frequently seen in the urinary bladder of cats during abdominal ultrasound. These have been attributed to hematuria, pyuria, crystalluria, and lipid. However, sonographic findings have not been previously correlated with urinalysis. We prospectively evaluated 40 clinically normal cats via ultrasound, serum chemistry, and urinalysis. Thin layer chromatography was performed on the urine to determine the amount (mg) of lipid subfractions including diacylglycerol, triglyceride, phospholipid, free fatty acid, cholesterol, and cholesterol ester. Ninety percent (36/40) of the cats in our population had sonographic echoes suspended in the urinary bladder, with most having a subjective score of mild echoes (n = 20). None of the sonographic echoes were gravity dependent or caused distal acoustic shadowing, reverberation, or twinkle artifact. Of the cats with sonographic echoes in the urine, 66% (24/36) had no significant findings on urinalysis other than the presence of lipid. The total amount of subjective sonographic echoes was not significantly related to the total amount of fat measured on thin layer chromatography or the number of lipid droplets seen on urinalysis. An increased amount of urine diacylglycerol was significantly associated with clumping of echoes (P = 0.02) and the amount of lipid droplets seen on urinalysis (P = 0.04). An association between increased amounts of urine diacylglycerol and the amount of echoes seen on ultrasound approached significance (P = 0.05). Findings from this study support previously published theories that sonographic echoes within the urinary bladder of clinically normal cats may be due to urine lipid.     

A comparison of the antigenicity of the extracellular products and whole-cell sonicates from Mycobacterium spp. in rabbits, mice and fish by immunoblotting and enzyme-linked immunosorbent assay

The antigenicity of extracellular products (ECPs) derived from Mycobacterium spp. isolated from snakehead, Channa striata (Bloch), and Siamese fighting fish, Betta splendens (Regan), were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera collected from immunized rabbits, mice and fish (rainbow trout). All three species responded to a 65-kDa protein present in both the ECPs and whole cell sonicates (WCSs) from a variety of Mycobacterium spp. Cross-reactivity of anti- M. tuberculosis and anti-human heat-shock protein monoclonal antibodies (MAbs), and the presence of fibronectin binding proteins secreted into ECPs of mycobacteria were also examined. The MAbs against human 60-kDa heat-shock protein cross- reacted with the band at 65 kDa in the ECPs of TB1 (isolated from snakehead fish) and the type strain M. marinum, while the anti- M. tuberculosis MAb F29–47 elicited a strong reaction with a band at 21 kDa with most of the ECPs from mycobacterial strains examined. The major fibronectin-binding proteins were located between 21 and 25 kDa. The 65-kDa protein from ECPs of Mycobacterium spp. proved strongly immunogenic to rabbits, mice and fish. Rabbit antiserum against the 65-kDa protein from strain TB267 reacted with many non- Mycobacterium WCSs, and therefore, the 65-kDa protein from Mycobacterium spp. is believed to be a common protein found in many fish bacterial pathogens.     

Ecological interactions between fungi, other biota and forest litter composition in a unique Scottish woodland

The composition of forest litter and understorey layer, andfungal biomass (in terms of ergosterol) were measured in eightsubplots over a winter–spring period (January to April).The sampling site was positioned in a range of woodland habitats(variously dominated by beech, Fagus sylvatica; birch, Betulapendula x pubescens, and oak Quercus petraea) and a clear areacovered with grass (dominated by Holcus lanatus). The resultswere analysed together with data on bacteria and microinvertebratesavailable from parallel research. Levels of ergosterol in individualsubplots ranged between 50 and 160 µg g^–1 DW. Fungalbiomass decreased in March, and then increased significantlyin April. Stepwise regression models for ergosterol indicatedpositive relationships with moisture content (February), bacteria(all but February and March), flagellates (February) and plant-feedingnematodes and flies (January, overall). The relationships withroots, seeds, the collective variable ‘other microinvertebrates’(all March), amoebae (February) and fragments (March, overall)were negative, while the relationship between fungi- and microbial-feedingnematodes changed sign between February (–) and March(+). Results of analysis of covariance for fungal ergosterolwere significant only for January and the combined dataset.In January, fungi were shown to be significantly related toamoebae, bacteria and a collembolan Folsomia candida, whilethe only significant predictor returned by the overall modelwas bacteria. Correlation analysis confirmed some effects alreadynoted, and revealed a number of further interactions. The resultshighlighted the complexity of factors influencing temporal dynamicsand spatial variability of fungal biomass in forest litter.Most of the registered interactions appeared to be transient,and this should be taken into account while interpreting environmentalobservations. Interpretation of the specific relationships isgiven and implications for further research and overall ecosystemfunctioning are discussed.     

Effect of adrenalectomy in the dog on blood gas tensions and oxygen content.

Arterial and venous blood gas tensions and pH were determined in 8 dogs during a control interval, while adrenal deficient, and following replacement therapy with corticosteroids. Additionally, erythrocyte 2,3-diphosphoglycerate (DPG) values were measured, and oxygen tension (mm of Hg) when hemoglobin is half-saturated with oxygen (P50) values were calculated. Mixed venous oxygen tension (PVO2), but not arterial oxygen tension (Pao2), decreased significantly (P less than 0.001) during adrenal insufficiency. This change was reflected in a significantly decreased (P less than 0.001) venous oxygen content and a significant increase (P less than 0.001) of the arterial-venous oxygen content difference. Other changes during adrenal insufficiency were nonsignificant decreases in DPG and P50 values. Treatment with corticosteroids for 2 to 3 days resulted in a significant (P less than 0.001) increase in DPG concentrations, a significant (P less than 0.025) increase in P50, and a reduction in the arterial-venous oxygen content difference compared with those values found in the adrenal-deficient dog.     

Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in nervous and non-nervous organs of European seabass (Dicentrarchus labrax) during the course of an experimental challenge

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain (cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.