Elsevier

Fish & Shellfish Immunology

Volume 99, April 2020, Pages 73-85
Fish & Shellfish Immunology

Full length article
Effects of recombinant Lactobacillus casei on growth performance, immune response and disease resistance in crucian carp, Carassius auratus

https://doi.org/10.1016/j.fsi.2020.02.008Get rights and content

Highlights

  • Two recombinant L. casei expressing AcrV of A. veronii were constructed .

  • Recombinant L. casei enhanced the growth and activity of ACP, AKP, LZM and SOD and level of IgM and leukocytes phagocytosis in serum.

  • Recombinant L. casei enhanced the disease resistance and expression level of IL-10, IL-β, IFN-γ and TNF-α in liver, spleen, kidney and intestine of crucian carp fish and disease resistance against A. veronii.

Abstract

In the present study, we constructed two recombinant Lactobacillus casei (L. casei) Lc-pPG-1-AcrV (surface-displayed) and Lc-pPG-2-AcrV (secretory) constitutively expressing AcrV protein of Aeromonas veronii (A. veronii). Expression of recombinant AcrV protein was verified by western blot and immunofluorescence technique. Compared with PBS group, the final weight (FW), weight gain (WG) and specific growth rate (SGR) of fish fed Lc-pPG-1-AcrV, Lc-pPG-2-AcrV and Lc-pPG diets after 56 days observed significantly increase (p < 0.05), while the feed conversion ratio (FCR) showed a significantly decrease (p < 0.05). The recombinant L. casei strains were orally administrated to crucian carp, and significant increased (p < 0.05) the immunoglobulin M (IgM), elevated the acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM) and superoxide dismutase (SOD) activity in serum. Moreover, leukocytes phagocytosis percentage and index of the recombinant L. casei were both enhanced. The results demonstrated that the recombinant L. casei could elicit systemic immune responses and increase the serum immunological index. The Interleukin-10 (IL-10), Interleukin-1β (IL-1β), interferon-γ (IFN-γ) and Tumor Necrosis Factor-α (TNF-α) levels in liver, spleen, kidney and intestine have up regulated significantly in tissues (p < 0.05), suggesting that the recombinant L. casei has the ability to induce expression of cytokines and enhance the innate immune response. Higher survival rates were exhibited that crucian carp immunized with Lc-pPG-1-AcrV (67.5%) and Lc-pPG-2-AcrV (52.5%) after challenge with A. veronii. In conclusion, these two recombinant L. casei vaccine were effective in improving crucian carp growth, immunity response and disease resistance. The recombinant L. casei strains may be a promising candidate for the development of an oral vaccine against A. veronii.

Introduction

Aeromonas veronii (A. veronii) is a conditional pathogenic for humans, livestock and aquatic animals, widely found in aquatic environment [1]. There have been increasing reports of crucian carp diseases caused by A. veronii in China, which has led tremendous economic losses to farmers. According to Deng et al. gibel carp (Carassius auratus gibelio) had an outbreak disease characterized by bleeding in Jiangsu province and other major aquaculture areas [2]. The disease with high mortality rates, acute and infectious, which the pathogen has identified as A. veronii [3]. Moreover, some infection symptoms have appeared of crucian carp (Carassius auratus) cultured in a fishery in Jilin Province, such as ulcer, hemorrhage and ascites. A report that the main pathogen aroused this disease was A. veronii through laboratory methods [4]. Conventional antibiotic treatment is no longer advocated for A. veronii diseases because it has led to a great threat to aquatic products quality and human health [5]. Furthermore, the mismanagement of antibiotics by farmers will cause drug resistance of pathogenic bacteria [6]. Vaccination is an effective measure and alternative therapy strategy for controlling bacterial diseases. Until now, lots of efficient protective antigens for vaccines have been developed against Aeromonas hydrophila (A. hydrophila), such as lipopolysaccharides (LPS) and outer membrane proteins (Omps) [[7], [8], [9]]. A number of articles suggested that vaccination has effect on control A. hydrophila diseases [10], while the aquatic vaccine against A. veronii is still relatively little. In recent years, our laboratory has screened some of the promising immunogenic antigens of A. veronii for the development of recombinant lactic acid bacteria (LAB) oral vaccine, including OmpAI [3] and flaB [11]. The currently known virulence factors of A. veronii were reported by Song et al. [12], including LPS, Omps, flagellin, adhesion (aha1), etc. To date, there have been no studies of the relationship between secreted protein (AcrV) and A. veronii virulence. It was reported that the AcrV is present in the type III secretion system (T3SS) of A. hydrophila [13] and Aeromonas salmonicida (A. salmonicida) [14], and both were shown as good immunogenicity. Therefore, we speculate that AcrV may also be present and play a significant role in A. veronii with considerable immunogenicity.

Mucosa is the first barrier that foreign pathogens come into fish, which prevents the entry of pathogenic microorganisms. The main components of mucus in mucosa surface are two mucins, and the main functions of both are related to the prevention of bacterial colonization [15]. Oral vaccines via mucosa immunizations is comparatively convenient way of administration, and have advantages of good compliance, minimal side-effects, more cost-effective and less stress response to the host [16]. Thus, oral immunization is suitable method to treat the aquatic diseases such as A. veronii infection in fish. However, there are difficulty for orally vaccination that the degeneration of antigens in the intestinal environment may prevent stimulating the intestinal mucosa [17]. Hence, the use of live expressing vector is urgently required that can deliver the antigen and survive in the complex environment. Recently, LAB is recognized as effective live vaccine-carrier that can play double role as adjuvant and vector to improve immune response, expressing reactive antigen by recombinant and cytokine-inducing properties [17]. Lots of Lactobacillus-associated model vaccines have been developed in previous studies. The successfully expression of the phytase gene from Aspergillus ficuum was studied by using Lactobacillus casei (L. casei) vector [18]. A recombinant-expression of epsilon toxoid has been constructed successfully by using the L. casei vector and obtained protection against the enterotoxemia disease [19]. Therefore, L. casei is a promising vector vaccine to provide protective immunogenicity against bacterial pathogens, which worldwide induce significant enhancement of heterologous antigens as vaccines in animal models [20]. The recombinant L. casei successfully expressed CK6 fusion T and VP2 protein for oral immunization of rainbow trout (Oncorhynchus mykiss) with good results [21]. A novel recombinant L. casei expressing the most immunogenic E2 antigen of BVDV have been constructed favourably [17].

In the study, we constructed two recombinant L. casei CC16 strain expressing AcrV and investigated the effects of adding recombinant L. casei in the feed on the growth performance, immune responses and disease resistance against A. veronii of crucian carp. We analyzed the final weight (FW), weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR), survival rate (SR) and disease resistance against infection with A. veronii in crucian carp. We detected the activity of immunoglobulin M (IgM), acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM) and superoxide dismutase (SOD), and tested capacity of leukocytes phagocytosis in serum and the expression of immune-related genes in liver, spleen, kidney and intestine.

Section snippets

Bacterial species and culture conditions

The A. veronii TH0426 was isolated from yellow catfish (Pelteobagrus fulvidraco) as described previously [22] and cultured in Rimler-Shotts (RS) medium (Haibo, China) at 28 °C. A plasmid-free strain of L. casei CC16 was screened from the intestine of common carp in our laboratory and cultured in MRS medium (Solarbio, China) at 30 °C anaerobic environment. To cloning the plasmid, Escherichia coli cells MC1061 was cultured in Luria-Bertani (LB) medium at 37 °C with shaking. The Escherichia coli-

Construction of the recombinant L. casei strains

Recombinant L. casei strains were obtained by screening Cm-resistant clones and detected by PCR and sequencing techniques. We found that the sequence was precisely the same as the designed sequence. These results illustrated that the pPG-1-AcrV and pPG-2-AcrV expression plasmid had been successfully constructed and transferred into L. casei by electroporation (Fig. 1A).

Western blot of recombinant AcrV protein

Cell protein lysates from Lc-pPG-1-AcrV and Lc-pPG-2-AcrV and its control (Lc-pPG) were analyzed by SDS-PAGE and Western blot.

Discussion

To our knowledge, this is the first report of the recombinant L. casei expressing AcrV as a T3SS -mediated virulence factor of A. veronii induce immune responses for oral immunized vaccine. AcrV, a homologue in Yersinia V-antigen (LcrV), has been identified as a major structural translocation genes in A. hydrophila [13] and A. salmonicida T3SS [14]. LcrV have reported by many scholars that is an essential virulence factor and a major protective-antigen in Yersinia pestis T3SS. The expression of

Conclusions

In conclusion, this work represents the first use of recombinant L. casei expressing AcrV on fish. These two recombinant L. casei vaccine were effective in improving crucian carp growth, immunity response and disease resistance. The recombinant L. casei strains may be a promising candidate for the development of an oral vaccine against A. veronii.

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (Project No. 2017YFD0501001), the earmarked fund for Modern Agro-industry Technology Research System (CARS-46), the National Natural Science Foundation of China (No. 31372540), the Natural Science Foundation of Science and Technology Department of Jilin Province (project No. 20170101016JC) and the Jilin Provincial Education Department (Project No. JJKH20180694KJ).

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