Development of DNA Vaccines against Mycolicibacterium cyprinidarum Isolated from Diseased Koi Carp

Abstract

We aimed to develop DNA vaccines against Mycolicibacterium cyprinidarum that are commonly isolated from diseased koi carp Cyprinus carpio. Major antigenic genes, Ag85B, Ag85C, Ag85D and ESAT-6 were cloned into a mammalian expression vector and used as DNA vaccines. Common carp were injected intramuscularly with each DNA vaccine individually, multiple mixtures of each DNA vaccine, PBS, or empty vector DNA. Cumulative survival rate after a bacterial challenge was 65.5% in PBS-injected group, while all fish survived from the challenge in the group co-immunized with the plasmids encoding Ag85B, Ag85C, Ag85D, and ESAT-6. The DNA vaccine cocktail may be a promising prevention method against M. cypridarum infection in koi carp aquaculture.

Introduction

Mycobacteriosis of cultured koi carp was first reported in Niigata, Japan (Yamada, 2006), which is the place of origin and the biggest producer of this popular ornamental fish. The disease is usually found in yearlings kept in an indoor pool during winter season, and the diseased koi carp exhibit lethargy, emaciation with pin-head body shape, and atrophy of the posterior chamber of the swim bladder (Machida et al., 2021). Mycobacteriosis in koi carp chronically progresses after infection at earthen ponds where the fish are kept during summer season (Machida et al., 2021; Machida et al., 2024) and the cumulative mortality usually reaches 10%–100%. To date, Mycobacterium nonchromogenicum (Yamada, 2006), M. paragordonae (Machida et al., 2021), Mycolicibacterium cyprinidarum (Machida et al., 2021; Matsumoto et al., 2024) and another Mycolicibacterium sp. that forms white/rough colonies (Machida et al., 2021) were reported to be causative agents. Furthermore, we previously showed that many types of non-tuberculous mycobacteria (NTM) can be the pathogens of the disease, since they were isolated from the trunk kidney, a lymphoid organ of apparently healthy fish (Machida et al., 2024). As a countermeasure, high water temperature treatment at 30°C is known to be effective for the disease (Yamada, 2006). However, economic cost for boiler fuel for the treatment is extremely expensive since Niigata is one of the areas with heaviest snowfall in Japan, where temperatures drop to below the freezing point in winter season. Therefore, it is difficult for the farmers to carry out the high-water temperature treatment appropriately during winter season.

Secretory proteins such as antigen 85 (Ag85) family (Ag85A, B and C), MPT51 as known as Ag85D, early secreted antigenic target of 6 kDa (ESAT-6) are known to be major antigens of M. tuberculosis for mammalian immune system (Pal et al., 2022; Steingart et al., 2009). DNA vaccines encoding Ag85 complex (Huygen et al., 1996; Lozes et al., 1997; Teixeira et al., 2006) showed significant protection against M. tuberculosis, inducing Th1 immune responses. Ag85D was also shown to be a protective antigen against M. tuberculosis infection when it was used as DNA vaccines (Miki et al., 2004; Uchijima et al., 2008) and a recombinant protein vaccine inoculated with CpG DNA (Silva et al., 2009). Co-immunization with DNA plasmids encoding ESAT-6, Ag85B and MPT64 genes, respectively, resulted in a greater degree of protection than that induced by any single plasmid (Kamath et al., 1999). Pasnik and Smith (2005) demonstrated protective efficacies of DNA vaccine encoding Ag85A gene of M. marinum against the corresponding bacteria in hybrid striped bass Morone saxatilis × M. chrysops. Intramuscular injections of DNA vaccines encoding Ag85B, CFP-10 and ESAT-6 conferred significant protection against M. marinum infection in zebrafish Danio rerio (Oksanen et al., 2013). These data suggest that mycobacterial secreted antigens such as Ag85 complex and ESAT-6 were promising antigens for DNA vaccines against fish mycobacteriosis.

In the present study, we aimed to develop DNA vaccines against M. cyprinidarum, a causative agent of mycobacteriosis in koi carp. We first identified four genes that code major mycobacterial antigens from the genome sequence of M. cyprinidarum: Ag85B, Ag85C, Ag85D and ESAT-6. These antigenic genes were cloned into a eukaryotic expression vector downstream of the cytomegalovirus (CMV) promoter and used as DNA vaccines. Protective efficacies of each plasmid and combinations of these DNA vaccines were assessed by intraperitoneal challenge with live M. cyprinidarum.

Materials and Methods

Fish rearing and bacteria propagation

Apparently healthy common carp Cyprinus carpio (average 8.7 g) produced in Yoshida Station of Tokyo University of Marine Science and Technology were reared in a 60-cm tank with a recirculation system. The water temperature was kept at 25°C and fish were fed every day with commercial pellet. All animal experiments were performed in accordance with the “Guideline for the Care and Use of Laboratory Animals” of TUMSAT.

Mycolicibacterium cyprinidarum strain NGTWS-NA01 and NGTWS-1803 isolated from diseased koi carp (Machida et al., 2021; Matsumoto et al., 2024) was cultured on 1% Ogawa medium (Nissui) at 30°C for 4 wk. Bacterial colonies on 1% Ogawa medium were scraped and collected in a 1.5-mL tube, suspended in sterile PBS to a final concentration at 1 mg/mL and used for challenge test. Bacterial suspensions were serially diluted and plated on Middlebrook 7H10 agar (Becton, Dickinson and Company) supplemented with oleic albumin dextrose catalase (OADC) enrichment (Becton, Dickinson and Company) and cultured at 25°C for 4 wk to count colony forming units (CFU).

Identification of the antigenic genes and plasmid construction

Mycobacterial antigenic genes for Ag85B (GenBank Accession Number, GJF09732), Ag85C (GJF09230), Ag85D (GJF09728) and ESAT-6 (GJF14271) were identified from a whole genome shotgun sequence of Mycolicibacterium cyprinidarum NGTWSNA01 (BPRH01000490). Nucleotide sequences of the antigenic genes were amplified using gene specific primer sets as follows: Ag85B (forward, 5′-TGGCTAGTTAAGCTTGTATGAGACTTCTTAACAA-3′; reverse, 5′-CCTC TAGACTCGAGGCCGATGAGGTGCGCCT-3′), Ag85C (forward, 5′- GGCTAGTTAAGCTTCGATGCACGTGGGACTTGC-3′; reverse, 5′- CCCTCTAGACTCGAGGGCGGTGTTCTGAGCCC-3′), Ag85D (forward, 5′-TGGCTAGTTAAGCTTCCATGCGTGCATTGACTCG-3′; reverse, 5′-CCCTCTAGACTCGAGCCTGATGGTTGCGGCTA-3′), and ESAT-6 (forward, 5′-TGGCTAGTTAAGCTTCCATGTCACAGATCATGTA-3′; reverse, 5′-CCCTCTAGACTCGAGGCCCCACTTGGCACCTT-3′). The reaction mixture for PCR containing 2.0 μL of 10× ExTaq buffer (Takara), 1.2 μL of 2.5 mM dNTP, 0.3 μL of each primer (10 μM), 0.15 μL of ExTaq DNA polymerase (Takara), and 1.0 μL of genomic DNA extracted from Mycolicibacterium cyprinidarum NGTWSNA01, was adjusted to 20 μL with distilled water. PCR for the genes was performed for 35 cycles of 95°C for 30 s, 57°C for 1 min and 72°C for 1.5 min with a final extension of 72°C for 5 min. Amplified PCR fragment was cloned into a mammalian expression vector, pcDNA3.1⁺ myc-His (Thermo Scientific) using In-Fusion HD Cloning Kit (Takara), following the manufacturer’s instructions. The inserted sequence was confirmed by Sanger sequencing outsourced to Eurofins Genomics and the coding sequences were analyzed using BLSAT program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The constructed plasmid was then transformed into competent Escherichia coli JM109 (Nippon gene), the bacteria containing the plasmid were large-scale cultured in LB medium, and the plasmid was extracted using GenElute HP Plasmid Maxiprep kit (SIGMA-ALDRICH).

Experiment 1

DNA vaccine for Ag85B, Ag85C, Ag85D and ESAT-6 were adjusted to 200 μg/mL with phosphate buffered saline (PBS). Fifty micro-litter of each DNA vaccine (10 μg/fish) was intramuscularly injected into carp (n = 40) after anesthetizing with 2-phenoxyethanol. Total amount of DNA vaccine injected to fish was determined following Kato et al. (2014). Empty vector DNA (10 μg/fish) and PBS (50 μL) were also intramuscularly injected to fish as negative controls. The vaccinated fish were intraperitoneally challenged with 3.6 × 10⁸ CFU of Mycolicibacterium cyprinidarum NGTWS-1803 at 4 wk post-vaccination, mortalities were recorded for 200 days, and Kaplan-Meier survival curves were generated. Relative percent survival was calculated as follows: RPS (%) = [1- (cumulative mortality of the vaccinated fish/ cumulative mortality of PBS-injected fish)] × 100. Significant difference (p < 0.05) in the cumulative survival rate between DNA-vaccinated group and each control group was detected using Log-rank test.

Experiment 2

Carp was also injected with 10 μg of each DNA vaccine coding Ag85B, Ag85C and Ag85D as above. Three kinds of cocktailed vaccines containing 5 μg of each plasmid were prepared as Ag85B/ESAT-6, Ag85C/ESAT-6 and Ag85D/ESAT-6. In addition, another cocktailed DNA vaccine containing three antigens (3.3 μg of Ag85B, Ag85C and Ag85D, in total 10 μg) and four antigens (2.5 μg of Ag85B, Ag85C, Ag85D and ESAT-6, in total 10 μg) were prepared in this experiment. The cocktailed DNA vaccine was intramuscularly injected into the fish, and the control groups administered with 10 μg of vector DNA or 50 μL of PBS were prepared as well (n = 25–30). After 4 wk of the vaccination, fish were intraperitoneally challenged with 5.4 × 10⁸ CFU of Mycolicibacterium cyprinidarum NGTWS-1803 as above and the mortalities were recorded for 235 days. Survival curves, RPS calculation and statistical analyses were performed as above. Furthermore, challenged fish still alive at 235 days post-challenge was sampled after the anesthesia, and the trunk kidney was weighed and homogenized in PBS using glass potters. The homogenized sample was adjusted to 10 mg/mL with PBS and serially diluted with middlebrook 7H9 broth supplemented with OADC enrichment. Five micro litters of each dilution step were incubated on middlebrook 7H10 agar supplemented with OADC enrichment at 30°C for 2 wk to count colony forming units (CFU) per 10 mg tissue.

Results

Identification of the antigenic genes

Ag85B, Ag85C, Ag85D and ESAT-6 were identified from M. cyprinidarum whole genome sequence. Ag85B, Ag85C and Ag85D of M. cyprinidarum encoding 354, 345 and 303 amino acid residues contained an esterase domain in the sequence. M. cyprinidarum Ag85B, Ag85C and Ag85D showed amino acid similarity at 67.6%, 74.4% and 63.7% with M. tuberculosis Ag85B (WP_003409456), Ag85C (WP_003400908) and Ag85D (CAA05211). M. cyprinidarum ESAT-6 gene encoded 95 amino acid residues, showing 68.3% identity to ESAT-6 of M. tuberculosis (P9WNK7).

Experiment 1

Serial mortalities were observed around 100 days after the challenge and the cumulative survival rate was 62.5%, 52.5%, 72.5%, 85.0%, 92.5% and 87.5%, in PBS, vector DNA, Ag85B, Ag85C, Ag85D and ESAT-6 group, respectively. Kaplan-Meier survival curves for the vaccinated fish were shown in Fig. 1A. Statistically significant differences were detected in the survival curves of Ag85C, Ag85D and ESAT-6-vaccinated group compared to that of PBS-injected group (Table 1). In addition, all experimental groups injected with each DNA vaccine showed significantly longer survival time compared with Vector DNA-injected group (Table 1). The RPS value of Ag85C, Ag85D and ESAT-6 group was 60.0%, 80.0% and 66.7%, respectively (Table 1).

Fig. 1. Kaplan-Meier survival curves of DNA vaccinated fish after Mycolicibacterium cyprinidarum challenge. (A) Kaplan Meier survival curves for carp immunized with a plasmid encoding Ag85B, Ag85C, Ag85D and ESAT-6 in Experiment I. PBS and vector DNA are negative controls. (B) Kaplan Meier survival curves for carp with single DNA vaccination of a plasmid encoding each Ag85 complex gene and with cocktailed DNA vaccinations in combinations of Ag85B+ESAT-6, Ag85C+ESAT-6, Ag85D+ESAT-6 in Experiment II. PBS and vector DNA are negative controls. (C) Kaplan Meier survival curves for carp co-immunized with Ag85B+C+D and Ag85B+C+D+ESAT-6 in Experiment II. PBS and vector DNA are negative controls.

Table 1. Mortality, relative percent survival (RPS), p-value calculated by Log-rank test and bacterial isolation rate at 235 days post-challenge in Ex. 2

Group	RPS	p-value (vs PBS)	p-value (vs Vector DNA)	Bacterial isolation rate at 235 dpib
Ex.1
PBS	NAa	NA	NA	NA
Vector DNA	NA	NA	NA	NA
Ag85B	26.6%	0.393	0.053	NA
Ag85C	60.0%	0.033	0.001	NA
Ag85D	80.0%	0.002	5.46e-05	NA
ESAT-6	66.7%	0.024	0.001	NA
Ex.2
PBS	NA	NA	NA	84.6% (11/13)
Vector DNA	30.0%	0.187	NA	63.6% (7/11)
Ag85B	22.7%	0.522	0.720	NA
Ag85C	53.6%	0.126	0.577	NA
Ag85D	42.0%	0.255	0.862	NA
Ag85B+ESAT-6	51.7%	0.152	0.564	NA
Ag85C+ESAT-6	90.3%	0.002	0.023	NA
Ag85D+ESAT-6	71.0%	0.021	0.161	NA
Ag85B+C+D	50.0%	0.135	0.602	100% (10/10)
Ag85B+C+D+ESAT-6	100%	0.001	0.008	50.0% (7/14)

^a  NA, not applicable

^b  total number of positive fish/total number of test fish

Experiment 2

There is no significant difference in the survival curves between the monovalent DNA vaccine groups (Ag85B, C and D) and PBS-injected or Vector DNA-injected group (Fig. 1B, Table 1). In contrast, Ag85C DNA vaccine injected at a same time with ESAT-6 showed significant protective efficacies (p < 0.01, vs PBS-injected group; p < 0.05, vs Vector DNA-injected group) against the challenge, RPS value for Ag85C+ESAT-6 and Ag85D+ESAT-6 was 90.3% and 71.0%, respectively (Table 1). These two vaccinated groups showed significantly longer survival period than PBS-injected group (Fig. 1B and Table 1). Furthermore, a cocktailed DNA vaccine Ag85B+C+D+ESAT-6 showed the highest protective efficacy against the challenge test (Fig. 1C and Table 1). Mycobacterial colonies were isolated from 84.6%, 63.6%, 100% and 50.0% of the surviving fish at 235 days post-challenge in PBS, Vector DNA, Ag85B+C+D, and Ag85B+C+D+ESAT-6 group, respectively (Table 1). The average value of bacteria counts (CFU/ 10 mg tissue) tended to be higher in PBS, Vector DNA and Ag85B+C+D group than in Ag85B+C+D+ESAT-6 (Fig. 2), although no statistical difference was detected.

Fig. 2. The number of bacteria isolated from the trunk kidney of the challenged fish in Experiment II. Each dot indicates colony forming unit in 10 mg of the trunk kidney tissue from an individual. The horizontal line shows an average value in each group.

Discussion

Infections with non-tuberculous Mycobacterium (NTM) cause severe loss of fish production in aquaculture worldwide. In Japan, outbreaks of M. marinum, M. pseudoshottsii in yellowtail Seriola quinqueradiata (Weerakhun et al., 2007; Nakanaga et al., 2012; Matsumoto et al., 2022), M. stephanolepidis in thread-sail filefish Stephanolepis cirrhifer (Fukano et al., 2015; Fukano et al., 2017), M. chelonae in Japanese puffer fish Takifugu rubripes (Ishii et al., 2019), M. paragordonae and M. cyprinidarum in koi carp (Machida et al., 2022; Matsumoto et al., 2024) have been reported in this decade. However, there is no effective vaccine against fish mycobacteriosis commercially available in Japanese aquaculture. A live attenuated vaccine, M. bovis BCG conferred significant protection against Mycobacterium sp. in amberjack (Kato et al., 2011), while this kind of vaccines have not been approved in Japanese aquaculture since there are concerns in reversion of pathogenicity of the attenuated strain and leakage to the environment (Matsuura et al., 2019). DNA vaccine is an alternative strategy to prevent fish disease caused by intracellular parasitic pathogens and has already been licensed against infectious hematopoietic necrosis virus (Salonius et al., 2007) and salmonid alpha virus subtype 3 (Thorarinsson et al., 2021) in Canada and EU, respectively. In the present study, we showed that combination of DNA vaccines encoding Ag85B, Ag85C, Ag85D, and ESAT-6 conferred significant protection against M. cyprinidarum in carp, so that this is a promising candidate for prevention method against mycobacteriosis in carp aquaculture.

Although there was no significant difference, the bacterial load in the trunk kidney of Ag85B+C+D+ESAT-6 vaccinated group was tended to be lower than the other groups. Isolation rate of M. cyprinidarum from the challenged fish was also the lowest in Ag85B+C+D+ESAT-6 vaccinated group. Bacterial burden in the lymphoid organ such as the spleen is an important indicator to assess the protective efficacy of the vaccine candidates in mammalian M. tuberculosis infection models (Lozes et al., 1997; Kamath et al., 1999; Fan et al., 2007; Liang et al., 2023). In fish, bacterial loads in the spleen of amberjack challenged with Mycobacterium sp. were suppressed in fish vaccinated with a live attenuated vaccine, M. bovis BCG (Kato et al., 2011). DNA vaccine encoding Ag85-like gene of Nocardia seriolae also suppressed the bacterial load in the spleen of amberjack after the challenge experiment (Kato et al., 2014). Present results also suggest that co-immunization of DNA plasmids encoding Ag85B, Ag85C, Ag85D and ESAT-6 of M. cyprinidarum suppressed the bacterial load in the trunk kidney of challenged fish, resulting in the protective efficacy against the disease.

In Experiment 2, enhancement of protective efficacy was observed when DNA plasmid encoding ESAT-6 was co-injected with Ag85C and Ag85B+C+D DNA vaccines, respectively. In mammals, ESAT-6 is a major antigen recognized by CD4⁺ and CD8⁺ T cells (Marei et al., 2005), and the ESAT-6 specific CD4⁺ T cells effectively produces IFN-γ and TNFα to control M. tuberculosis infection (Clemmensen et al., 2020). In fact, DNA vaccine encoding ESAT-6 enhanced the protective efficacy of a live attenuated vaccine, M. bovis BCG against M. tuberculosis infection in mice (Fan et al., 2007). In general, injection with multivalent DNA vaccines or a DNA vaccine encoding multi-epitopes confers higher protection than monovalent or single-epitope DNA vaccine (Mortazavi et al., 2024; Patel, 2024). Although the different challenge doses may have affected to the protections, injection with single DNA vaccine didn’t show enough efficacies against the challenge in the experiment 2. These data suggest that ESAT-6 of M. cyprinidarum is a major antigen recognized by carp immune system and provokes strong protective efficacy against the disease.

In conclusion, co-immunization with Ag85C+ESAT-6 and Ag85B+C+D+ESAT-6 showed significant protection against M. cyprinidarum infection in carp. Especially, co-immunization with all DNA vaccines suppressed bacterial burden in the spleen of carp after M. cyprinidarum challenge. This prevention method using DNA plasmids may be useful for prevention of M. cyprinidarum infection in year-ling koi carp.

Acknowledgment

We thank Mr. Masaki Yokota and Mr. Akihiro Harakawa in Yoshida Station, the Field Science Center, Tokyo University of Marine Science and Technology for producing the experimental fish used in this study.

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