Elsevier

Fish & Shellfish Immunology

Volume 94, November 2019, Pages 769-779
Fish & Shellfish Immunology

Full length article
Functional nutrition modulates the early immune response against viral haemorrhagic septicaemia virus (VHSV) in rainbow trout

https://doi.org/10.1016/j.fsi.2019.09.070Get rights and content

Abstract

Although viruses represent a major threat for cultured fish worldwide, the commercialization of vaccines capable of providing effective and long-lasting protection is still lacking for most of these viral diseases. In this situation, the use of supplemented diets could be a suitable strategy to increase the immune status of the fish and reduce the impact of viral pathogens. Among possible immunostimulants that could be included in these functional feeds, some studies have previously shown that certain β-glucans can significantly increase certain immune parameters of fish and reduce the impact of viral diseases. However, the mechanisms through which β-glucans exert their activity have not been fully elucidated yet. In the current study, we have studied the immune response of different tissues to viral haemorrhagic septicaemia virus (VHSV) in rainbow trout fed with a non-supplemented control diet as well as in fish fed a commercial functional aquafeed (Protec™, Skretting) containing β-glucans, vitamin C, vitamin E and zinc. For this, after 30 days of feeding the fish with one of the two diets, they were subsequently infected with VHSV by bath or mock-infected. After 2 or 6 days post-infection, fish were sacrificed and the levels of transcription of different immune genes such as IgM, IgT, IgD, Mx, interferon γ (IFN γ) and perforin studied in different tissues (kidney, gut and gills). Additionally, the levels of natural IgMs in serum were also determined. Our results demonstrate that fish fed the functional diet were capable of mounting an increased IgM, IgT, IgD and Mx transcriptional response to the virus. Additionally, these fish also showed increased levels of natural IgMs in serum. These results reveal a previously undescribed effect of functional diets on fish Ig production and point to Protec™ as an adequate diet to be incorporated in holistic programs aimed at mitigating the effect of viral diseases.

Introduction

One of the most relevant factors that limits the required expansion of the aquaculture industry is the impact of infectious diseases. As a consequence of intensive rearing conditions in fish farms, with high levels of organic material and low oxygen concentration in the water, cultured fish commonly experience a chronic stress condition that favors the appearance of diseases and increases their susceptibility to pathogens. Thus, infectious diseases severely affect the aquaculture production worldwide provoking major economic losses each year. These losses are not a consequence of the direct fish deaths but are also due to the impact that pathogens have on fish growth, production costs or reproduction cycles. Fish can be infected by different types of pathogens including virus, bacteria, fungi or unicellular or multicellular parasites. Among them, viral infections represent a major threat for cultured fish because the diseases they provoke usually elicit high mortality rates and there are no antivirals authorized for use in aquaculture [1].

Viral haemorrhagic septicaemia virus (VHSV), a member of Rhabdoviridae family included in the genus Novirhabdovirus [2] is responsible for one of the most devastating diseases of aquacultured fish. VHSV was first isolated in rainbow trout (Oncorhynchus mykiss) in 1965 in Denmark [3], however, it now has been now reported to infect more than 80 different fish species from fresh and saltwater [4]. Traditionally, it was thought that VHSV, as other closely related fish rhabdoviruses such as infectious haematopoietic necrosis virus (IHNV) entered the host through the gills, but in 2006, through the use of a luciferase-expressing recombinant rhabdovirus, it was established that these viruses also enter the host through the fin bases, being this the most important early replication site [5]. The most common symptoms of VHS include general haemorrhages, exophthalmia, anaemia and distended abdomen as a consequence of intraperitoneal cavity oedema [6].

Vaccination is, from all points of view, the most adequate way to prevent the appearance of viral diseases. However, few antiviral vaccines are commercially available for use in aquaculture and, in general, vaccines against fish viruses are not capable of generating an effective long-lasting protection in the field. In the case of VHSV and other related rhabdoviruses, DNA vaccination has been shown to generate a robust and long-lasting specific response when administered intramuscularly [7,8]. These DNA vaccines consist of a eukaryotic expression plasmid that encodes the glycoprotein of fish rhabdoviruses under the control of the cytomegalovirus (CMV) promoter. Despite its efficacy, this VHSV DNA vaccine has never been approved by the European authorities due to safety concerns, and thus far, only an IHNV DNA vaccine has been approved for use in Canada (Novartis).

In this situation, an alternative strategy to increase the natural resistance of fish to viral infections could be the introduction of immunostimulants in the diet. Immunostimulants are defined as natural or chemical compounds that have the capacity to activate or modulate non-specific immune responses, thus improving the general immune status of the fish and consequently increasing its natural resistance to pathogens [9]. Numerous studies over the years have confirmed that the use of immunostimulants such us plant or algae extracts, vitamins or oligoelements have the capacity to enhance the innate immune system in different fish species (reviewed in Refs. [9,10]). Among the most frequently used immunostimulants in aquaculture are β-1,3/1,6-glucans [11,12]. These compounds are glucose polymers that make up the cellular wall of plants, fungi and some bacteria [12]. β-glucans have been administered orally, by peritoneal injection or by bath, alone or in combination with vaccines (as adjuvants), and in many cases these compounds generated a positive effect in the immune system [[13], [14], [15],42,43]. In addition, fish fed with diets rich in β-glucans have demonstrated increased resistance against bacterial pathogens such as Vibrio harveyi [16] or Aeromonas hydrophila [17] as well as against some viruses [18,19]. Thus, for example, Pacific herring fed β-glucan-supplemented diets showed a 50–80% increased protection when challenged with VHSV [19]. Despite this, the mechanisms through which supplemented diets exert their activity against viral infections in fish are still not well documented.

Additional supplements such as vitamins or zinc have also been proved to increase the natural defences to pathogens in aquaculture. Thus, for example, vitamin C has been shown to up-regulate several immune functions of rainbow trout leucocytes [20]. Regarding the effect of vitamins on the susceptibility to viral infections, Wahli and colleagues demonstrated that fish fed with a diet supplemented with a combination of both vitamin C and E at high doses had lower susceptibility to VHSV than fish fed with diets supplemented with lower vitamin doses or with either of the vitamins alone [21].

In the current work, we have studied the transcriptional response of different tissues at early time points after VHSV infection comparing the responses of rainbow trout fed with the functional aquafeed Protec™ (Skretting) to that of fish fed with a control non-supplemented feed. Protec™ is a commercial diet supplemented with a combination of glucans, vitamin C, vitamin E and zinc. For this, rainbow trout were fed each of the two diets for 30 days. Thereafter, fish were infected with VHSV through bath exposure and the levels of transcription of different immune genes determined in infected and mock-infected controls at days 2 and 6 post-infection. Additionally, the presence of natural IgMs in serum were also studied in sampled fish. These studies contribute to increase our knowledge on how functional diets exert their antiviral effects and consolidate Protec™ as a suitable diet to diminish the impact of viral diseases.

Section snippets

Experimental fish

Rainbow trout (Oncorhynchus mykiss) of ~25 g were obtained from the Centro de Acuicultura El Molino (Madrid, Spain). Fish were maintained at animal facilities of the Animal Health Research Center of the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (CISA-INIA) in a recirculating water system at 14 °C, with 12:12 h light/dark photoperiod. Prior to any experimental procedure, animals were acclimatized to laboratory conditions for at least 2 weeks. During this period, fish

IgM transcription in kidney, gut and gills of fish fed with Protec™ and challenged with VHSV

We first analysed the levels of transcription of IgM in the tissues obtained from the sampled fish. In the kidney, in mock-infected animals sampled 2 days post-infection, IgM transcription levels were significantly higher in fish fed with the functional diet than in fish fed with the control diet (Fig. 1A). We could consider these basal transcription levels. Upon VHSV infection, only fish fed with Protec™ were able to significantly increase IgM mRNA levels in the kidney in response to the

Discussion

In the current study, we have demonstrated that the administration of the immunostimulants present in the Protec™ diet can have major effects on the early immune response that is mounted against a viral pathogen. In this experiment, the different diets were fed for 30 days, as previous experiments in our laboratory have established that the results obtained after only 15 days of administration were less marked (data not shown). However, after 30 days of administration, this functional diet had

Acknowledgements

The authors want to thank Lucia Gonzalez for technical support. This work was supported by the Spanish Ministry of Science, Innovation and Universities (project AGL2017-85494-C2-1-R).

References (43)

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