No fish were found infected in the CTRL group, and no significant differences in the variables measured were present among replicates within each group. 50% of challenged group A fish (fed the basal diet) were found positive for E. leei at the non-lethal sampling carried out at five weeks p.i., indicating the success and adequate progression of the challenge. According to the molecular diagnosis by qPCR, the prevalence of infection at the end of the experiment was clearly higher ingroup A (72.4%) than in the additive-supplemented groups (B = 44.8%, C = 46.6%). Due to the limited number of replicate tanks, these differences did not reach statistical significance (Figure 3A). However, the reduction in the prevalence of infection in treated vs untreated groups was significant (t-test, P = 0.0236), when grouping the data from all the treated B & C tanks (n = 4) compared to those of A group (n = 2) (data not shown). In the current trial, infection intensity data was clearly lower in both B (x̅ = 12.72; Md = 11.32) and C (x̅ = ¬13.33; Md = 12.65) groups than in A group (x̅ = 14.33; Md = 14.67), although differences were not statistically significant (Figure 3B). The mean intensity of infection only considers the parasite load in infected animals, and as such it can evidence variable degrees of “resistance” to the multiplication of the parasite once it has breached the host barriers. Lowered infection levels can also result from a delayed invasion by the parasite and a more incipient multiplication in the host. Since all challenged fish in this experiment were inoculated experimentally with a single dose, the differences detected could indicate slightly improved ability to constrain parasite development or to clear off parasite numbers more efficiently. However, the over dispersion of intensity data due to individual variability and aggregate parasite distribution masks significant differences at this level, even with normalised data. Mean parasite abundance data takes into consideration the mean parasite quantities in all the fish from the group, thus merging prevalence and intensity data in a single variable. This parameter clearly showed the differences in quantitative parasitological data between treated and non-treated groups. Both supplemented diets resulted in significantly lower mean abundance values (x̅ (B) = 5.70; x̅ (C) = 6.22) compared to group A (x̅ (A) =10.38) (ANOVA P = 0.029). The mean parasite abundance data from both treated groups was also significantly different from that of group A (x̅ (B+C) = 5.97) in a t-test (P = 0.005). Median abundance values for both B & C was 0, resulting significantly lower than the value of the untreated group (Md(A) = 13.85) (Kruskal-Wallis test P = 0.017) (Figure 3C). Since the molecular quantitative tests were carried out on homogenates of the whole intestine, a histopathological analysis of a subsample of fish from the experimental groups was also carried out. By looking separately at the three intestine segments, the differences on the extension of the infections along the intestinal tract were investigated (Figure 4). The overall prevalences obtained by histology are not the same than those by qPCR not only because the fish examined by both techniques are different individuals, but also because of the different methodological sensitivities. Significant differences in the prevalence of infection were found between C (31.3%) and A (64.7%) groups (chi-square test, P = 0.05), whereas no differences were found for group B (68.8 %) (Not shown). Analysing the spatial distribution and extension of parasites, the prevalence at the posterior intestine had the highest values in all
Figure 5: IATS Fish Pathology Research Group.
challenged groups, which corresponds to the known usual patterns of E. leei distribution along the intestinal tract of GSB. Only group C showed lower prevalence values in the three intestinal segments, and the proportion of fish with more than one infected portion was significantly lower in group C vs A (chi-square test, P < 0.05), but not in group B (Figure 4A). As for the mean intensity of infection by intestinal segment, again diet C showed the lowest values, especially at the anterior intestine, though these differences were not statistically significant (Figure 4B).
The experimental challenge with E. leei effectively infected fish and induced the clinical signs of enteromyxosis in fish fed the basal diet A with a dramatic decrease of feed intake (32.7 %), weight (36.2 %) and SGR (25.9 %) after 10 wks of infection. 50% of this group tested positive for the parasite in a non-lethal PCR at the intermediate check sampling on week five post infection, and 72.4% were infected at the final sampling on week 10. These are indicators of exposure to a high infection pressure, even further magnified by the high water temperatures during the trial. The supplementation of a functional feed additive to the feed refrained most of the disease signs when used at the highest dose tested (diet C), i.e. the experimental infection did not impact significantly on feed intake, weight nor growth (SGR). A dose effect was patent and the feed additive mitigated the SGR reduction vs. non-challenged fish, from 25.9% with the basal diet to 10.2% with the high-dose supplemented feed. Quantitative parasitological data on prevalence, intensity, and abundance, as well as histopathological studies on infection extension confirmed the effect of supplemented diets on reducing the infection rate and its severity. According to the molecular quantitative data, both groups receiving the functional feed additive showed lower prevalence and intensity of the infection compared to the challenged group not receiving the feed additive. Although no dose effect of the supplement on the reduction of prevalence and parasite load was observed using the qPCR method, these differences were noticed in the histopathological study, with group C showing the lowest infection levels. The inclusion of a functional feed additive into the diet prior to an experimental infection with E. leei, reduced the impact of the infection on performance as well as the success and severity of the infection.
This study was partially supported by EU H2020 programme and by the Spanish Ministry of Economy and Competitiveness through ParaFishControl (634429) and AGL-2013-48560-R research projects, respectively. References available on request
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