Introduction
The misuse or overuse of antibiotics has caused the onset of antimicrobial-resistant strains of Aeromonas salmonicida in fish and the aquatic environment. Different strategies have been proposed to boost fish resistance to A. salmonicida, one of the major pathogens of salmonids. Among the preventive approaches explored to increase natural resistance to disease in fish, probiotics, prebiotics, and immunostimulants have emerged (Minářová et al. 2021).
This study conceived an integrated approach to explore the potential immunomodulatory effects of a commercial feed, Protec (Skretting), which contains β-glucans, vitamins C and E, and zinc (Bulfon et al., 2019), and its ability to mitigate the mortality caused by a naturally occurring A . salmonicida outbreak in adult rainbow trout.
Materials and Methods
Adult rainbow trout (~300 g) were farmed in central Italy (Granica, RI). The animals were fed once a day with two commercial pelleted diets: 1) Optiline (Optiline , Skretting), used as a control diet, and 2) Protec (Protec, Skretting). Shortly after starting the commercial feeding trial, the farmer noticed a steady low-level mortality in both groups . After three weeks, 25 Optiline and 33 Protec fish were randomly caught using a fishing wade and immediately sacrificed for further examinations, as follows. External welfare indicators (Noble et al., 2022), biometric and autoptic indices were recorded. Peripheral blood was collected from the caudal vein and immediately transferred to heparinized tubes. The etiologic agent of the outbreak was detected by inoculating Tryptone Soy Agar (TSA) and Blood Agar (BA) plates with target organs (brain, kidney, and spleen). Identification of the isolates was performed phenotypically by MALDI-TOF (MALDI Biotyper , Bruker), while the subspecies was genotypically identified by INgene ® q Aeromonas salmonicida (Gold Standard Diagnostics) through the StepOne Plus (Applied Biosystems). Blood cell count was assessed at 400X magnification under an Axioplan microscope (Zeiss), and innate immunity parameters (leukocyte viability, Reactive Oxygen Species (ROS) production, and phagocytosis activity) were evaluated by flow cytometry with the CytoFLEX instrument (Beckman Coulter) and analysed with Kaluza software v 2.1 (Beckman Coulter).
Cell counts were analysed using the Chi-square test, while the other paramet ers were tested using the non-parametric Mann-Whitney ( Bonferroni-corrected p-value ; p <0.05 was considered significant). Statistical analysis was performed with the open-source software Past v. 4.14.
Result and discussion
After three weeks of the commercial feeding trial, no differences in either external welfare indicators or biometric parameters were observed between the Optiline and Protec groups. However, both groups showed Hepato-Somatic Index (HSI) and Spleen Somatic Index (SSI) values nearly twice the optimal range for rainbow trout (Nabi et al. 2022; Cardinaletti et al. 2022), suggesting that the mortality recorded could be related to the presence of an infectious agent. Mortality was significantly higher in the Optiline group compared to Protec (p<0.001), suggesting an improved immune competence in trout fed with an immunostimulant-rich diet (Redivo et al. 2023).
The bacteriological culture examination of the target organs revealed the presence of systemic A. salmonicida. The subspecies salmonicida was detected in blood samples with no statistical differences in prevalence (28% Optiline vs. 21% Protec). However, the Optiline group displayed a significantly higher A. salmonicida gene copy number compared to the Protec group (p<0.05) , suggesting a less efficient pathogen clearance in individuals fed with the Optiline diet. Consistently, trout fed with Protec showed a higher proportion of blood lymphocytes (p<0.05) and thrombocytes (p<0.05), and a lower number of neutrophils (p<0.05) . Lymphocytosis has been related to greater recruitment and activation of specific immune cells during systemic defensive response against bacteria (Bulfon et al., 2019). Furthermore, a significantly lower number of leukocyte aggregates (leucoagglutination) was detected in the Protec group compared to the Optiline (p<0.01) .
Leukocytes isolated from trout fed with Protec showed significantly higher viability (p <0.01), and lower apoptosis and death (p <0.05), paralleled by lower ROS production (p<0.05) . They also exhibited a greater proportion of phagocytic ROS-producing live myeloid cells (p<0.05). Overall, t hese data suggested that the immunostimulants included may induce a greater pathogen clearance in trout leukocytes (Dalmo and Bøgwald 2008).
Conclusion
Although severe alterations have occurred in fish affected by the pathogen, regardless of the diet administered, the data obtained by the conceived integrated approach suggest that the immunostimulant-enriched diet strengthens rainbow trout’s immune systems, partially mitigating A. salmonicida infection.
References
Bulfon C, Pacorig V, Sarti M, et al (2019) Veterinary Immunology and Immunopathology. https://doi.org/10.1016/j.vetimm.2019.109885
Cardinaletti G, Di Marco P, Daniso E, et al (2022) Animals. https://doi.org/10.3390/ani12131698
Dalmo RA, Bøgwald J (2008) Fish and Shellfish Immunology. https://doi.org/10.1016/J.FSI.2008.04.008
Nabi N, Ahmed I, Wani GB (2022) Saudi Journal of Biological Sciences . https://doi.org/10.1016/j.sjbs.2022.01.019
Minářová H, Bláhová L, Kalina J, et al (2021) Acta Vet Brno . https://doi.org/10.2754/avb202190020233
Redivo B, Derôme N, Kestemont P, Cornet V (2023) Microorganisms . https://doi.org/10.3390/microorganisms11020539