Bacterial skin flora variation and in vitro inhibitory activity against Saprolegnia parasitica in brown and rainbow trout.

Variations in the number and diversity of bacteria from the skin of brown trout Salmo trutta L. and rainbow trout Oncorhynchus mykiss Walbaum were surveyed from different rivers and fish farms in northern Spain. In addition to determining bacterial populations in skin samples of healthy fish, bacterial populations were determined from skin lesions (of brown trout only) infected with Saprolegnia parasitica, the causal agent of saprolegniosis. Mean bacterial counts from skin lesions of brown trout suffering from saprolegniosis were nearly 1000 times greater than from the skin of uninfected brown and rainbow trout. More than 20 different genera of bacteria were identified, with isolates of Aeromonas and Iodobacter being the predominant genera associated with saprolegniosis lesions. The in vitro inhibitory activity of 72 of these skin isolates was tested against S. parasitica using 3 different assays. These included (1) assessing the inhibition by bacteria of colony growth on agar media, (2) the inhibition of colony growth from colonized hemp seeds in liquid media and (3) the inhibition of cyst germination in liquid media. Finally, the fungicidal effect of the 24 most inhibitory bacterial species, and the inhibitory activity of their culture supernatants, was tested in the same way. Isolates identified as Aeromonas piscicola, A. sobria, Pantoea agglomerans and Pseudomonas fluorescens achieved the highest inhibition against S. parasitica. Many of these inhibitory isolates were obtained primarily from skin lesions of fish with saprolegniosis. It is suggested that some of these isolates might be useful in the biological control of saprolegniosis.

Cross-sectional sero-epidemiological survey of sarcoptic mange in sheep of Western Castile and Leon, Spain.

The aim of the present study was to determine the prevalence of Sarcoptes scabiei-infection of ovine livestock in three provinces (Leon, Zamora and Salamanca) in the Western part of the Castile and Leon region in Spain, and to determine the association between different variables and seropositivity. A total of 3730 sheep sera from 373 flocks (10 sera from each flock) collected from May to September over the course of the years 2006 and 2007 were individually analysed by an indirect antibody ELISA validated for diagnosing sarcoptic mange in sheep. The overall flock-level true prevalence was 22.6% (95% CI: 17.8-27.4), the overall individual-level true prevalence within the total flocks was 7.2% (95% CI: 6.1-8.3) and the overall individual-level true prevalence within the seropositive flocks was 31.3% (95% CI: 27.2-35.4). The apparent prevalences, at flock-level and at individual-level within the total flocks and within the seropositive flocks, were not statistically different (p > 0.05) when the primary production objective of the flock is milk vs. meat, or in smaller (< or = 276 sheep, 50th percentile) vs. larger flocks (> 276 sheep). The apparent prevalences, at flock-level and at individual-level within the seropositive flocks, were, likewise, not statistically different between the three provinces, but the individual-level apparent prevalence within the total flocks showed significant variation from one province to another (p < or = 0.05). Sheep maintained in the Provinces of Zamora and Salamanca had greater odds (OR = 1.7, 95% CI: 1.2-2.6; OR = 1.9, 95% CI: 1.3-2.8, respectively) of being seropositive than those located in Leon Province (OR = 1.0). The findings of the present study clearly show the need to implement in this region effective control measures against sarcoptic mange in sheep.

Development and evaluation of an antibody ELISA for sarcoptic mange in sheep and a comparison with the skin-scraping method.

In this work an indirect ELISA for detecting serum-specific IgG antibodies in sheep was developed using a crude saline extract from Sarcoptes scabiei var. ovis mites and then the repeatability of the ELISA outcomes was estimated. Subsequently, its diagnostic accuracy was evaluated by Receiver Operating Characteristics (ROC) analysis using a sample collected from the entire sheep population of western Castile and Leon region in Spain, and then compared with that of the skin-scraping method. The reference method used was a combination of clinical examination, skin-scraping analysis and epidemiological surveys, but it introduced selection and probably information biases. Furthermore, we attempted to identify biological factors useful to predict the sensitivity or specificity of the ELISA as determined by comparison with the reference method. Additionally, conventional latent-class analysis [Hui, S.L., Walter, S.D., 1980. Estimating the error rates of diagnostic tests. Biometrics 36, 167-171] was also used to estimate accuracy parameters. The between-run coefficient of variation (CV) for a standard serum was 8.8% and the within-run CV 4.3%. No significant deviation between the OD% means and strength positive correlation between the OD% values (r=0.98) were found for the results from two different batches of antigen. When compared to the reference method, the Area Under the ROC curve (AUC) for the reference population was 0.967 (95% CI: 0.949-0.985) for the ELISA and 0.915 (95% CI: 0.863-0.968) for the skin-scraping method. By logistic regression analysis, one explanatory biological factor-result to the skin-scraping method-and four explanatory biological factors-Tyroglyphidae individual status, Trichophyton verrucosum individual status, Oestrus ovis status of the flock and presence of adjacent animals with a clinical disease neighbour to S. scabiei infection-were found for diagnostic sensitivity and specificity of the ELISA, respectively, although this depended on the OD% cut-off value used. Latent-class analysis, carried out for the ELISA at 17.8 OD% cut-off value (mean plus 3 SDs of sheep considered negative to anti-S. scabiei antibodies), showed a marked difference between the estimated diagnostic sensitivity of the ELISA (87.6%) and the skin-scraping method (62.8%), but closer diagnostic specificities (95.9% vs. 100%, respectively). These results demonstrate that the developed ELISA is valid for different applications in clinical as well as in epidemiological contexts.

Clinical evaluation and antibody responses in sheep after primary and secondary experimental challenges with the mange mite Sarcoptes scabiei var. ovis.

In this work the clinical evolution and the specific serum IgG and IgE antibody responses in sheep after primary (n=10) and secondary (n=4) experimental challenges with the mange mite Sarcoptes scabiei var. ovis were studied. The primary infection was characterized by the development of mange lesions in all sheep, a detection of live S. scabiei mites in 70% skin scrapings taken in week 10 post-challenge (PC), strongly raised and sustained specific IgG levels and a more moderate but continuous rise in specific IgE levels. Seroconversion was detected for IgG and IgE by ELISA in 90% and 60% of the sheep in week 8 PC, respectively. By Western-blotting (WB), ten IgG-reactive bands (36-120 kDa) and four IgE-reactive bands (90-180 kDa) were observed in week 8 PC. Following the secondary challenge the ewes developed a smaller area of mange lesion than that seen following primary challenge and live S. scabiei mites were not detected in skin scrapings collected in week 8 PC, suggesting that sheep had developed immunity to re-infection. Compared to primary infection, the specific IgG secondary antibody levels were transient, but in contrast there was an anamnestic IgE response, resulting in an elicitation of specific serum IgE levels in week 2 PC significantly higher than those demonstrated after primary infection. WB analysis revealed one additional IgG-reactive band (180 kDa) and no additional IgE-reactive bands. Determining the immunodiagnostic or vaccination value of the IgG-reactive antigens and IgE-reactive allergens detected requires further studies.

Antibody response of brown trout Salmo trutta injected with pathogenic Saprolegnia parasitica antigenic extracts.

Brown trout Salmo trutta injected with antigenic extracts from a pathogenic isolate of Saprolegnia parasitica developed specific antibodies that were detected by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) and Western blotting (WB), but not by immunodiffusion (ID). Three groups of five 2 yr old brown trout were injected intraperitoneally with 3 different antigenic extracts: small hyphal fragments (HF) and soluble extracts from sonicated mycelia grown in medium with or without beta-sytosterol (SEB and SE, respectively). In the 2 groups injected with SE and SEB, antibodies were found in 66.7 % of the serum samples by ELISA, 54.5% by IF and 48.5% by WB. In the group injected with HF, only 1 trout survived the experiment, and in this fish only 1 sample was positive by ELISA. The results obtained by ELISA and IF were similar and show that there is cross-reaction between the antigens used. By WB, the proteins most frequently recognised were 2 proteins of 25 and 29 kDa. No significant differences were found in the groups injected with SE or SEB.