Functional feed additives can reduce the impact of an Enteromyxum leei infection on performance and disease severity: evidence from an experimental challenge with gilthead sea bream.
Published in International Aquafeed, July 2017
Palenzuela O.1, Del Pozo R.1, Piazzon M.C.1, Isern-Subich M.M.2, Ceulemans S.2, Coutteau P.2, Sitjà-Bobadilla A.1
1Institute of Aquaculture Torre de la Sal (IATS-CSIC). Castellón, Spain (firstname.lastname@example.org)
2Nutriad International, Dendermonde, Belgium (email@example.com)
Concern on the impact of parasite infections in aquaculture has increased in recent years. In addition to visible mortality episodes and increased running costs, some estimates of the world annual grow-out loss in finfish farming due to parasites ranges from 1% to 10% of harvest size, with an annual cost that can reach up to $9.58 billion (Shinn et al., 2015). In Mediterranean fish farming, one of the major parasitic diseases is enteromyxosis, caused by Enteromyxum leei (Fig. 1). This microscopic parasite infects the intestinal tract of fish and sometimes associated organs like gall bladder and liver. Enteromyxum species belong to the Myxozoa, a group of parasites related to Cnidarians that produce economically important fish diseases like whirling disease, proliferative kidney disease (PKD), milky fish liquefaction, proliferative gill disease (PGD), gill sphaerosporosis, swim bladder inflammation (SBI) or ceratomyxosis. In contrast to the complex, 2-host life cycle described for about 50 myxozoan species, spontaneous direct fish-to-fish transmission has been demonstrated for the genus Enteromyxum. This unique mode of horizontal transmission favours the spread of enteromyxoses in cultured fish stocks.
- leei has a wide host and geographical range, including economically important aquacultured species in the Mediterranean and worldwide, like tiger puffer, Japanese flounder, parrot fish, Malabar grouper, various sea breams, or Peruvian fine flounder. The virulence and mortality caused on each host is quite variable, and largely affected by the species susceptibility and the rearing system and environmental conditions. In gilthead sea bream (GSB) (Sparus aurata), enteromyxosis has a chronic course leading to a cachectic syndrome with anorexia, anaemia, weight loss, severe epaxial muscle atrophy and, eventually, death (Sitjà-Bobadilla and Palenzuela, 2012) (Fig. 1). Direct mortality due to enteromyxosis in GSB raised in sea cages is most often moderate, whereas the serious economic impact of enteromyxosis in these facilities is largely due to arrested growth and inability to reach commercial size. This effect is most patent in advanced stages of the growout period.
There are neither vaccines nor effective prescription medicines for enteromyxosis and its control measures are limited to avoidance of risk factors, early diagnosis, and good farm management practices. Therefore, the farming industry needs other solutions to minimize the impact of the infection. Health promoting feed additives are a crucial component of effective disease prevention strategies. A wide range of additives with different mode of actions are currently offered including yeast extracts, phytobiotics, probiotics, prebiotics, organic acids and their derivates. Functional feeds containing gut health promotors deliver with every meal an adequate concentration of natural compounds which can work through multiple mechanisms to reduce the success of parasitic infestations. Natural compounds with anti-parasitic activity can work directly on gut parasites and/or reach the blood and/or mucus to affect ectoparasites, whereas immune modulators can change the composition and thickness of the mucus (Couttteau et al., 2011, 2014, 2016). Many of these strategies target the gut as a primary focus for health and offer maximum benefits in chronic or subchronic infection processes, and thus GSB enteromyxosis constitutes an excellent model to study and to evaluate their potential. The present study evaluated the capacity of a functional feed additive to prevent or mitigate the effect of enteromyxosis in gilthead sea bream experimentally infected with E. leei. All the experiments were run at the indoor experimental facilities of the Institute of Aquaculture Torre de la Sal (IATS) using fiberglass tanks in an open flow-through seawater system.
Feeds and feeding protocol
Naïve GSB fingerlings, free of intestinal parasites were obtained from a local hatchery at 4 g and grown up to 12.9 g before starting the feeding trial. They were allocated into 90-L tanks (25 fish/tank) and acclimated to the basal diet for 11 days until the experimental feeding started. Three diets were tested in 2 replicate tanks/treatment: a basal control feed (Diet A) and the same diet with two different inclusion levels of SANACORE® GM, a natural health promoting feed additive (Nutriad International, Belgium); Diet B “low dosage” and diet C “high dosage” of SANACORE® GM. The basal diet was representative of a commercial feed formulation (45/20% crude protein/fat; 15% LT fishmeal, 12% poultry by-product meal, Soybean meal 25%, soybean protein concentrate 10%, corn gluten 8%, wheat gluten 3.4%, wheat flour 10.5%, soybean oil 9.7%, fish oil 6%, amino acids, vitamin and minerals premix). Fish were fed manually ad libitum twice a day for weekdays and with automatic feeders on weekends, during the whole experiment. Daily food intake was recorded and SGR (specific growth rate) and FCR (feed conversion ratio) calculated. Water temperature ranged from 18 to 26.5 °C over the feeding trial (post challenge period between 22°C and 26.5°C; see Fig. 2A). Salinity of the seawater was 37.5 g/l.
Experimental infection and samplings
After 5 weeks on the experimental diets, fish from groups A, B & C were inoculated with 0.2 ml of a homogenate from intestinal scrapings of infected donor fish via the anal route, as previously described (Estensoro et al., 2010). An additional group of fish fed basal diet A received the same volume of PBS without parasites (non-challenged control fish, CTRL). A non-lethal PCR test of challenged A fish was carried out at 5 weeks post-inoculation (p.i.), to check the progress of the infection after the challenge. The final sampling of all groups was performed 10 weeks p.i., when all fish were sacrificed and intestine samples were taken for E. leei diagnosis by histology (n = 16-20/group) and q-PRC (n = 29-30/group). Since the parasitological study by either method consumed the entire sample, fish analysed by histology were different from those used for molecular diagnosis.
Diagnosis of the infection
Histology. Portions of anterior (AI), middle (MI), and posterior (PI) intestine samples were fixed in 10% buffered formalin and embedded in plastic resin, following routine procedures. Sectioned slides were stained with Giemsa and E. leei infection intensity was semiquantitatively evaluated following a conventional scale from 1+ to 6+. A fish was considered positive for infection when the parasite was found at least in one intestinal segment.
Parasite quantification by qPCR. After necropsy, entire intestines of 30 fish per group (15 per tank) were removed and weighed individually. They were homogenized in a blender with steel beads and DNA was extracted from a 200ul aliquot of the homogenate from each fish. Enteromyxum leei rDNA gene copies were estimated by qPCR. Numbers were interpolated from the cycle thresholds (Cts) of the samples using standard curves with known numbers of a plasmid containing the target gene (covering 6-7 orders of magnitude), run in the same plates on each assay. Usually 2 dilutions of each DNA sample were run. Samples with Cq < 38 were considered positive. The total number of parasite rDNA genes present in each fish was estimated from these values and the amount of tissue in the entire homogenate sample. Quantitative parasitological data analysed included: Prevalence (percentage of infected fish in a sampled group); Mean intensity of Infection (mean number of parasites per infected fish); and Mean parasite abundance (mean number of parasites per fish in a sampled group, i.e., involving the zero values of uninfected animals and thus combining variations in prevalence and intensity data.).
Biometrical and quantitative parasitological data were studied for differences between test groups and controls using appropriate tests for each type of variable, using software packages Sigma-Plot and Prism. Since the data is overdispersed and aggregated, quantitative parasite load estimated by qPCR was normalized by logarithmical transformation (y = Ln (y) for intensity data, and y = Ln (1 + y) for abundance data). Unless specifically stated, statistical significance was considered when P < 0.05.
Results & Discussion
Food intake and Growth performance:
Fish challenged with the parasite suffered a decrease in feed intake, which was noticeable as soon as one week p.i. in Group A, and keep on decreasing along the whole period compared to the non-challenged control (Fig. 2A). In contrast, anorexia started later (3 wks p.i.) in fish fed diet B and it was not noticed in fish receiving diet C with the highest additive dose, which kept a similar feed intake to that of the control fish. Thus, the typical anorexia induced by the parasite (Estensoro et al., 2011) appeared to be mostly overcome with the highest dose of the additive.
The effect of the parasite challenge on biometrical values was quite patent, particularly by the last sampling at 10 wks p.i. (Fig. 2B, 2D). Weight, length and Condition Factor (CF) were significantly reduced in challenged fish fed the basal diet (A) compared to the non-challenged control group on the same diet (CTRL). Interestingly, in fish receiving the supplemented diets the length and weight loss associated to the infection were reduced, and the differences only reached statistical significance in the lowest dose group (B). Group C was not significantly different to CTRL in biometrical parameters, and the CF was not significantly reduced in both treated groups (Fig. 2B and data not shown). As expected, the SGR along the whole trial decreased in all challenged groups, but the decrease vs CTRL fish was mitigated in diet C (10.2%), while it reached 21.1% and 25.9% in diets B and A, respectively (Fig. 2D). Overall, the evolution of biometrical data clearly evidenced the clinical effect of the infection in challenged groups, in consonance with the field studies on E. leei-infected stocks and with the results of previous laboratory challenge studies with this parasite. It is believed that the decreased performance of E. leei-infected fish results from a combined effect of direct structural damage to the intestine resulting in poor nutrient absorption and dehydration, to the diversion of energy invested on mounting an immune response and also to the anorexia triggered by the infectious process (Sitjà-Bobadilla & Palenzuela, 2012). As deduced from the results of this trial, supplemented diets could partially revert these effects and reach similar performance levels to CTRL in the high dose group.
Dropping mortality was detected in challenged groups starting at 16 days p.i. A total of 14 fish died. No significant differences among diets were found in the survival curves, but the maximum cumulative mortality was recorded in A group (20 %). Both supplemented groups B & C reached the same cumulative mortality value (13.3 %).
No fish was 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 5 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 in group 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 (Fig.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 (Fig. 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 overdispersion 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) (Fig. 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 (Fig. 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 difference in the prevalence of infection was 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 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 (Fig. 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 (Fig. 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 5 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 PCR 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 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 program and by the Spanish Ministry of Economy and Competitiveness through ParaFishControl (634429) and AGL-2013-48560-R research projects, respectively.
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