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RETRACTED: Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin
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Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin Julia W. Pridgeon ∗ , Phillip H. Klesius Aquatic Animal Health Research Unit, USDA-ARS, 990 Wire Road, Auburn, AL 36832, USA
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Article history: Received 14 January 2013 Received in revised form 25 February 2013 Accepted 28 March 2013 Available online xxx Keywords: Streptococcus agalactiae Sparfloxacin Resistance Attenuation Vaccine
a b s t r a c t To develop attenuated bacteria as potential live vaccines, sparfloxacin was used in this study to modify 40 isolates of Streptococcus agalactiae. Majority of S. agalactiae used in this study were able to develop at least 80-fold resistance to sparfloxacin. When the virulence of the sparfloxacin-resistant S. agalactiae isolates were tested in 10–12 g Nile tilapia by intraperitoneal injection at dose of 2 × 107 CFU/fish, 31 were found to be avirulent to fish. Of the 31 avirulent sparfloxacin-resistant S. agalactiae isolates, 30 provided 75–100% protection to 10–12 g Nile tilapia against challenges with a virulent S. agalactiae isolate Sag 50. When the virulence of the 30 sparfloxacin-resistant S. agalactiae isolates was tested in 3–5 g Nile tilapia by intraperitoneal injection at dose of 2 × 107 CFU/fish, six were found to be avirulent to 3–5 g Nile tilapia. Of the six avirulent sparfloxacin-resistant S. agalactiae isolates, four provided 3–5 g Nile tilapia 100% protection against challenges with homologous isolates, including Sag 97-spar isolate that was non-hemolytic. However, Sag 97-spar failed to provide broad cross-protection against challenges with heterologous isolates. When Nile tilapia was vaccinated with a polyvalent vaccine consisting of 30 sparfloxacin-resistant S. agalactiae isolates at dose of 2 × 106 CFU/fish, the polyvalent vaccine provided significant (P < 0.001) protection to both 3–5 g and 15–20 g Nile tilapia against challenges with 30 parent isolates of S. agalactiae. Taken together, our results suggest that a polyvalent vaccine consisting of various strains of S. agalactiae might be essential to provide broader protection to Nile tilapia against infections caused by S. agalactiae. Published by Elsevier Ltd.
1. Introduction Streptococcus agalactiae affects various fish species, including tilapia [1–3], grouper [4], mullet [5], and pomfret [6]. In 2001, S. agalactiae was responsible for a massive fish kill at the Kuwait Bay, killing over 2500 metric tons of wild mullet (Liza klunzingeri) [5]. Large scale disease outbreaks caused by S. agalacatiae also occurred in 95% farms in China, with 30 −80% cumulative mortality [3]. In addition, S. agalactiae (group B streptococcus) is an important cause of disease in infants, pregnant women, immune-suppressed adults [7], and many cases of acute clinical mastitis in dairy animals [8,9]. To control streptococcosis in aquaculture caused by S. agalactiae, feeding infected fish with antibiotic-medicated feed is a general practice [10]. However, this practice is expensive and usually ineffective as sick fish tend to remain off feed. In addition, there are only three antibiotics currently approved for use in aquaculture: oxytetracycline (Terramycin), sulfadimethoxine (Romet-30), and
∗ Corresponding author. Tel.: +1 334 887 3741; fax: +1 334 887 2983. E-mail addresses: [email protected], [email protected] (J.W. Pridgeon).
florfenicol (Aquaflor). The widespread usage of the limited number of antibiotics for treating bacterial diseases in aquaculture has led to the development of antibiotic resistance in many fish pathogens worldwide [11]. Therefore, alternative control methods are urgently needed for the aquaculture industry. Use of vaccine is an alternative control method to prevent fish bacteria disease. Experimentally, formalin-killed S. agalactiae vaccine has been reported to offer significant protection to 30 g tilapia, but not to 5 g tilapia, with a relative percent of survival (RPS) rate of 80% in vaccinated fish compared to non-vaccinated fish at 30 days post vaccination [12,13]. In addition, an experimental S. agalactiae biotype I (-hemolytic) vaccine was reported to be able to offer protection against lethal challenges with both biotype I and biotype II (non-hemolytic) strains, whereas biotype II failed to offer protection against challenges with biotype I [14]. Recently, Merck Animal Health Inc has obtained regulatory approval in Brazil to begin marketing AQUAVAC® Strep Sa, an inactivated oil-adjuvanted vaccine that provides protection against S. agalactiae infections in tilapia and other susceptible fish species. However, AQUAVAC Strep Sa is only available for fish weighing more than 15 g, but not for fish weighing 3–5 g, the size of a 1–2 weeks old fish that farmers obtain from the hatchery. Therefore, a S. agalactiae vaccine that is safe and efficacious to 3–5 g tilapia is urgently needed.
0264-410X/$ – see front matter. Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.vaccine.2013.03.066
Please cite this article in press as: Pridgeon JW, Klesius PH. Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.03.066
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To develop effective bacterial vaccines, rifampicin-resistant strategy has been successfully used to attenuate E. ictaluri (AquaVac-ESC) and Flavobacterium columnare (AquaVac-COL) [15,16]. In addition, novobiocin-resistant strategy has been successfully used to attenuate E. ictaluri [17], Aeromonas hydrophila [18], and S. iniae [19]. However, it is not clear whether sparfloxacin could also be used to attenuate S. agalactiae for the purpose of novel vaccine development. Sparfloxacin works by inhibiting bacterial DNA gyrase, therefore inhibiting DNA replication and transcription [20]. Due to its controversial safety profiles, sparfloxacin was withdrawn from the US market in 2000. However, it was unclear whether sparfloxacin could be used to attenuate pathogenic S. agalactiae. Therefore, the objectives of this study were: (1) Determine whether sparfloxacin could be used to attenuate different isolates of S. agalactiae; and (2) whether sparfloxain-resistant S. agalactiae could be used as attenuated live vaccines to protect 3–5 g Nile tilapia from infections by virulent S. agalactiae isolates. 2. Materials and methods 2.1. Identification of S. agalactiae by fatty acid methyl ester profiling Forty S. agalactiae isolates (Supplementary Table 1) were obtained from various fish species exhibiting clinical disease signs and from different geographical regions. The archived isolates were recovered from frozen stocks (2 ml aliquots stored at −80 ◦ C) and grown in tryptic soy broth (TSB) (Fisher Scientific, Pittsburgh, PA) for 24 h at 28 ◦ C. Bacteria isolates were identified by gas chromatography analysis of fatty acid methyl ester (FAME) using MIDI microbial identification system (MIDI, Newwark, Delaware). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 2.2. Hemolysis, Lancefield grouping and serotyping Hemolysis of S. agalactiae isolates was determined by streaking and stabbing 5% sheep blood agar (SBA, Remel, Lenexa, KS, USA) with S. agalactiae culture followed by incubation at 28 ◦ C for 24 h. The presence and the absence of a clear zone around the growth of the streaking sites and the stab sites were considered hemolytic and non-hemolytic, respectively. Lancefield grouping test of S. agalactiae was performed using StrepProTM grouping kit (Hardy Diagnostics, Santa Maria, CA). Serotyping of S. agalactiae was performed using Group-B Streptococci Tying Antisera (Denka Seiken, Coventry, UK) by slide agglutination method following manufacturer’s procedures. 2.3. Induction of resistance in S. agalactiae Sparfloxacin was purchased from Sigma-Aldrich (St. Louis, MO). All bacteria strains were cultured in tryptic soy broth (TSB) containing different concentrations of chemicals for 24–48 h at 28 ◦ C. After multiple passages of bacteria in TSB culture media containing same or higher concentration of sparfloxacin, resistant bacteria were obtained. 2.4. Virulence of sparfloxacin-resistant S. agalactiae compared to their parent isolates Similar amount (CFU/fish in a total volume of 0.1 ml) of each parent or sparfloxacin-resistant isolate was exposed to Nile tilapia (Oreochromis niloticus, 10–12 g or 3–5 g) through intraperitoneal (IP) injection. For initial virulence screening, a total of 20 fish were used in each treatment group (10 fish per tank, duplicates). All fish used in this study were raised at the USDA ARS Aquatic Animal
Health Research facility located at Auburn, Alabama. All fish treatment protocols were approved by Institutional Animal Care and Use Committee at the Aquatic Animal Health Research Laboratory following mandated guidelines. Mortalities were recorded daily for 14 days post injection 2.5. Vaccination of fish followed by challenge with virulent isolates of S. agalactiae Fish were vaccinated by IP injection. A total of 20 fish were used in each treatment group (10 fish per tank, duplicates). The vaccination dose for single isolate of S. agalactiae was 2 × 107 CFU/fish in a total volume of 100 l. As sham-vaccination controls, 100 l of TSB were injected into each fish. For initial vaccine efficacy screening, a total of 20 fish were used in each treatment group (10 fish per tank, duplicates). Fish were challenged with a virulent S. agalactiae isolate sag-50P (a non-hemolytic Brazil isolate cultured from diseased Nile tilapia in 2006) through IP injection at 14 days post vaccination (dpv). For efficacy testing of a polyvalent vaccine consisting of 30 sparfloxacin-resistant S. agalactiae isolates that were found to offer 75–100% protection to 10–12 g tilapia, the vaccination dose was 2 × 106 CFU/fish in a total volume of 100 l. Fish were challenged with 30 parent isolates of S. agalactiae at 28 dpv. Mortalities were recorded for 14 days post challenge. Results of bacteria challenge were presented as relative percent of survival (RPS) [21]. RPS was calculated according to the following formula: RPS = {1 − (vaccinated mortality ÷ control mortality)} × 100. 2.6. Backpassage safety studies of polyvalent S. agalactiae vaccine One hundred and eighty tilapia were used in the backpassage safety studies. The 180 fish were divided into two groups (control group and vaccine group) with 90 fish per group. The 90 fish were then divided into 6 fish tanks with 15 fish per tank. One hundred microliters (l) of polyvalent S. agalactiae vaccine were IP injected into each tilapia at dose of 2 × 106 CFU/fish. Control group fish were injected 100 l TSB. Forty-eight hours later, five fish were taken from the first tank and homogenized. A 100 l of the homogenate was then injected into each fish in tank 2. This procedure was repeated five times using the remaining of fish. Mortality or adverse behavior or signs of disease were recorded daily for 21 days post injection. 2.7. Macrophage isolation and reactive oxygen species production Macrophages were isolated from catfish by peritoneal lavage according to published procedures [22]. Briefly, 15–20 g Nile tilapia were injected intraperitoneally with 100 l of TSB or polyvalent vaccine at dose of 2 × 107 CFU/fish. Macrophages were harvested at two days after treatment by peritoneal lavage with 20 ml of sterile ice-cold PBS. Harvested macrophages were centrifuged at 500 × g for 15 min, washed once more by centrifugation in 12 ml of ice-cold PBS, and re-suspended in Hank’s Balanced Salt Solution (HBSS, Invitrogen, Carlsbad, CA, USA). Samples of harvested macrophages were stained with Hema-3 (Biochemical Sciences Inc., Swedesboro, NJ, USA) for total macrophage counts. Viability was assessed with trypan blue exclusion (Sigma) and observed microscopically using a hemacytometer. The chemioluminescence assay was conducted according to published procedures [23] with slight modifications. Briefly, macrophages from vaccinated fish or TSB non-vaccinated fish were seeded at 1 × 105 cells/well in a 96well opaque Luminometer plate (Promega, Madison, WI, USA). Overnight bacterial culture of S. agalactiae at 2 × 104 CFU/well ml were added to each well. Luminol [5-amino-2-3-dihydro-1,4phthalazinedione (Sigma)] in 0.2 M sodium borate pH 9.0 was added at 20 l/well. All tests were conducted in triplicate. Plates
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were read at 2 min and 5 min post addition of luminol with a Glowmax 96 microplate luminometer (Promega). 2.8. Indirect enzyme-linked immunosorbent assay (ELISA) Antibody titer against S. agalactiae-97 was determined using published method [24]. Briefly, 96-well ELISA plates were coated for 1 h at 25 ◦ C with 100 l of S. agalactiae-97 antigen in carbonate buffer (CB), which was prepared by sonication. Plates were washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBS-T) and then blocked with 3% bovine serum albumin in CB for 1 h. Following blocking, plates were washed three times with PBS-T. Serum from control fish or vaccinated fish (n = 10) was then added at a 1:500 dilution in PBS-T to the plate. The plates were incubated for 30 min at 25 ◦ C and then plates were washed three times with PBS-T. Monoclonal anti-tilapia antibody 1H1 was diluted 1:1000 in PBS-T and then added to all wells (100 l/well) for 30 min. Following washing three times with PBS-T, 100 l of peroxidase conjugated anti-mouse IgG (1:5000 in PBS-T) was added and incubated for 15 min. Plates were washed three times with PBST and 100 l substrate tetramethylbenzidine (Pierce, Rockford, IL) was added. After 15 min, the reaction was terminated by adding 50 l of 3 M H2 SO4 to each well and the absorbance was read at 450 nm using a BioRad 680 microplate reader (Biorad, Hercules, CA). Serum from S. agalactiae-97 infected fish at 14- and 28-day post infection was used as positive control and PBS was used as negative controls. 2.9. Statistical analysis All statistical analyses were performed using SigmaStat 3.5 software (Systat Software, Inc, Point Richmond, CA). Differences in cumulative mortality of fish vaccinated with polyvalent vaccine compared to sham-vaccinated fish following challenges with 31 different isolates were analyzed with Mann-Whitney U significance test, one well-known non-parametric statistical hypothesis test for assessing whether one of two samples of independent observations tends to have larger values than the other. Differences in antibody titers and reactive oxygen species production were analyzed with Student t-test and the significance level was defined as P < 0.05. 3. Results
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streptococci. Serotyping results revealed that 40 isolates belonged to either sera type Ia, Ib, or II. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 3.3. Induction of resistance in S. agalactiae Majority of the 40 S. agalactiae isolates used in this study were able to develop resistance to sparfloxacin, with 80–10,000- fold resistance to sparfloxacin, except isolate Sag 50, which was only able to develop 10-fold resistance to sparfloxacin (Supplementary Table 3). Of the 40 S. agalactiae isolates used for selection of resistance to sparfloxacin, Sag 141 and Sag 76 were the most naturally sparfloxacin-resistant and sparfloxacin-sensitive isolate, respectively (Supplementary Table 3). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 3.4. Virulence of sparfloxacin-resistant S. agalactiae to 10-12 g Nile tilapia Virulence of sparfloxacin-resistant S. agalactiae and their respective parents to Nile tilapia by IP injection are shown in Supplementary Table 4. Of the 39 sparfloxacin-resistant isolates, majority (31) were found to be avirulent to 10–12 g Nile tilapia at injection dose of 2 × 107 CFU/fish (100 l of undiluted S. agalactiae culture at optical density of 1.0, absorbance reading at 540 nm). However, six sparfloxacin-resistant isolates were still highly virulent to 10–12 g Nile tilapia, killing 90–100% fish (Supplementary Table 4). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 3.5. Vaccinated 10–12 g Nile tilapia challenged with a virulent S. agalactiae isolate Cumulative mortality and relative percent of survival of vaccinated 10–12 g Nile tilapia challenged with a virulent S. agalactiae isolate Sag-50P are summarized in Table 1. Of the 31 avirulent sparfloxacin-resistant S. agalactiae isolates, 30 provided 75% to 100% protection to 10–12 g Nile tilapia against challenges with a virulent S. agalactiae isolate Sag-50P (Table 1). However, the RPS value provided by isolate Sag 106-spar (sparfloxacin-resistant S. agalactiae Sag 106) was only 25% (Table 1).
3.1. Bacteria identity confirmation FAME analysis confirmed the identities of all the S. agalactiae isolates used in this study. All S. agalactiae isolates shared high similarity indices with known S. agalactiae deposited into the FAME profile database. Representative FAME profiles of hemolytic and non-hemolytic S. agalactiae isolates are shown in Supplementary Fig. 1. All S. agalactiae isolates had the following three major fatty acid peaks: (1) the peak of 16:0 fatty acid at retention time of ∼2.74 min; (2) the peak of 18:0 w9c fatty acid at retention time of ∼3.30 min; (3) the peak of 18:0 fatty acid at retention time of ∼3.37 min (Supplementary Fig. 1). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066.
3.6. Virulence of sparfloxacin-resistant S. agalactiae to 3-5 g Nile tilapia Virulence of sparfloxacin-resistant S. agalactiae and their respective parents to 3–5 g Nile tilapia by IP injection are shown in Table 2. The 30 sparfloxacin-resistant S. agalactiae isolates that provided 75% to 100% protection to 10–12 g Nile tilapia were chosen for this study. Of the 30 isolates, majority (24) were found to be virulent to 3–5 g Nile tilapia at injection dose of 2 × 107 CFU/fish (100 l of undiluted S. agalactiae culture at optical density of 1.0, absorbance reading at 540 nm). However, six isolates were found to be avirulent to 3–5 g Nile tilapia (Table 2).
3.2. Hemolysis, Lancefield grouping, and serotyping
3.7. Vaccinated 3–5 g Nile tilapia challenged with homologous isolates of S. agalactiae
The hemolysis, Lancefield grouping, and serotyping results of the 40 isolates of S. agalactiae are summarized in Supplementary Table 2. Of the 40 isolates used in this study, 7 were hemolytic (-hemolysis) and 33 were non-hemolytic (␥-hemolysis). All S. agalactiae isolates used in this study belonged to Lancefield Group B
Cumulative mortality and relative percent of survival of vaccinated 3–5 g Nile tilapia challenged with virulent parent isolate are summarized in Table 3. Of the 6 avirulent sparfloxacin-resistant S. agalactiae isolates, 4 provided 100% protection against challenges with homologous S. agalactiae parent isolates (Table 3). Of the four
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Table 1 Cumulative mortality and relative percent of survival of vaccinated 10-12 g Nile tilapia challenged with virulent isolate Streptococcus agalactiae Sag 50-P. Isolate used for vaccination
Isolate used for challenge
Challenge dose (CFU/fish)
Mortality of TSB sham (%)
Mortality of vaccinated fish
RPS (%)
Sag 4-spar Sag 5-spar Sag 6-spar Sag 36-spar Sag 41-spar Sag 53-spar Sag 72-spar Sag 76-spar Sag 77-spar Sag 81-spar Sag 89-spar Sag 92-spar Sag 95-spar Sag 96-spar Sag 97-spar Sag 99-spar Sag 100-spar Sag 102-spar Sag 104-spar Sag 106-spar Sag 114-spar Sag 120-spar Sag 121-spar Sag 128-spar Sag 130-spar Sag 131-spar Sag 133-spar Sag 137-spar Sag 138-spar Sag 139-spar Sag 142-spar
Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P
3 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 3 × 107
100 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 100
0 0 0 0 0 0 0 0 10 0 0 0 0 0 10 0 0 0 0 30 10 0 10 0 0 0 0 0 0 10 0
100 100 100 100 100 100 100 100 75 100 100 100 100 100 75 100 100 100 100 25 75 100 75 100 100 100 100 100 100 75 100
isolates that provided 100% protection, Sag 97 parent isolate was the most virulent one in 3–5 g Nile tilapia, killing 60% of TSB-sham vaccinated fish, whereas the other three parent isolates killed ≤40% fish (Table 3). Therefore, sparfloxacin-resistant Sag-97 isolate was Table 2 Virulence of parent and sparfloxacin-resistant S. agalactiae to 3-5 g Nile tilapia by IP injection. Isolate
Injection dose (CFU/fish)
Sag 4 Sag 5 Sag 6 Sag 36 Sag 41 Sag 53 Sag 72 Sag 76 Sag 77 Sag 81 Sag 89 Sag 92 Sag 95 Sag 96 Sag 97 Sag 99 Sag 100 Sag 102 Sag 104 Sag 114 Sag 120 Sag 121 Sag 128 Sag 130 Sag 131 Sag 133 Sag 137 Sag 138 Sag 139 Sag 142
2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107
Cumulative mortality (%) Parent (%)
Mutant (%)
100 100 100 40 100 100 100 100 80 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
100 70 90 0 10 80 60 50 40 20 70 70 50 80 0 0 0 80 30 40 40 60 0 0 40 20 50 80 70 90
subjected to further study in 3–5 g to determine whether it could offer cross-protection against challenges by heterologous isolates of S. agalactiae. 3.8. Sag 97-spar vaccinated 3–5 g Nile tilapia challenged with heterologous isolates Cumulative mortality and relative percent of survival of Sag 97spar vaccinated 3–5 g Nile tilapia challenged with heterologous isolates of S. agalactiae are summarized in Table 3. For challenges with six randomly selected heterologous S. agalactiae isolates, Sag 97-spar failed to provide any protection for five isolates, with RPS of 0 (Table 3). However, Sag 97-spar did provide 100% protection to 3–5 g Nile tilapia against challenges with S. agalactiae Sag 133-P isolate (Table 3). 3.9. Efficacy of polyvalent vaccine in 3–5 g Nile tilapia Cumulative mortality and relative percent of survival of 3–5 g Nile tilapia vaccinated with a polyvalent vaccine challenged with 30 parent isolates of S. agalactiae are summarized in Table 4. The polyvalent vaccine provided 3–5 g Nile tilapia 100% protection against challenges with 15 isolates of S. agalactiae (Table 4). The polyvalent vaccine also provided some protection against 14 isolates of S. agalactiae, with RPS values of 20% to 80% (Table 4). However, the polyvalent vaccine failed to provide 3–5 g Nile tilapia any protection against challenges with isolate Sag 128-P. When cumulative mortalities of vaccinated fish were compared to that of TSB sham-vaccinated fish after challenges with the 30 isolates, TSB sham-vaccinated fish had significantly (P < 0.001) higher mortality than that of polyvalent S. agalactiae-vaccinated fish (Fig. 1). 3.10. Efficacy of polyvalent vaccine in 15–20 g Nile tilapia Cumulative mortality and relative percent of survival of 15–20 g Nile tilapia vaccinated with a polyvalent vaccine challenged with
Please cite this article in press as: Pridgeon JW, Klesius PH. Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.03.066
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Table 3 Cumulative mortality and relative percent of survival of vaccinated 3-5 g Nile tilapia challenged with virulent parent isolates of Streptococcus agalactiae. Isolate used for vaccination
Isolate used for challenge
Challenge dose (CFU/fish)
Mortality of TSB sham (%)
Mortality of vaccinated fish
RPS (%)
Sag 36-spar Sag 97-spar Sag 99-spar Sag 100-spar Sag 128-spar Sag 130-spar Sag 97-spar Sag 97-spar Sag 97-spar Sag 97-spar Sag 97-spar Sag 97-spar
Sag 36-P Sag 97-P Sag 99-P Sag 100-P Sag 128-P Sag 130-P Sag 5-P Sag 6-P Sag 41-P Sag 50-P Sag 100-P Sag 133-P
2 × 107 2 × 107 2 × 106 2 × 106 2 × 107 2 × 107 3 × 107 3 × 107 3 × 107 2 × 106 2 × 107 2 × 107
40 60 20 10 100 90 60 100 90 100 30 30
0 0 0 0 100 90 60 100 100 100 40 0
100 100 100 100 0 0 0 0 0 0 0 100
30 parent isolates of S. agalactiae are summarized in Table 4. The polyvalent vaccine provided 15–20 g Nile tilapia 100% protection against challenges with 10 isolates of S. agalactiae (Table 4). In addition, the polyvalent vaccine provided 50% to 80% protection to 15–20 g Nile tilapia against challenges with 9 isolates of S. agalactiae (Table 4). However, the polyvalent vaccine failed to provide 15–20 g Nile tilapia any protection against challenges with two isolates (Sag 114-P and Sag 76-P). When cumulative mortalities of vaccinated fish were compared to that of TSB sham-vaccinated fish after challenges with the 30 isolates, TSB sham-vaccinated fish had significantly (P < 0.001) higher mortality than that of polyvalent S. agalactiae-vaccinated fish (Fig. 1). 3.11. Backpassage safety results Of all fish exposed to the polyvalent S. agalactiae vaccine through IP injection, no mortality or signs of disease or adverse behavior was observed. No fish died in the backpassage safety studies. 3.12. Reactive oxygen species production The production of reactive oxygen species (ROS) by tilapia macrophages from vaccinated and non-vaccinated are summarized in Fig. 2. Macrophages from polyvalent vaccine-vaccinated fish produced significantly (P < 0.05) more ROS than that from TSB sham-vaccinated fish at both 2 min (Fig. 2A) and 5 min (Fig. 2B) post incubation.
Fig. 1. Cumulative mortality of Nile tilapia vaccinated with or without polyvalent vaccine of Streptococcus agalactiae followed by challenges with 31 different isolates of S. agalactiae. Data are presented as mean ± S.D. from challenges with 31 different isolates of S. agalactiae. Significant differences (P < 0.001) between vaccinated fish and sham-vaccinated fish are marked with asterisk.
3.13. Indirect enzyme-linked immunosorbent assay (ELISA) ELISA results revealed that sham-vaccinated fish had consistent absorbance readings comparable to that of negative PBS control at different time points (Supplementary Fig. 2). At 14 days postvaccination (pre-challenge), vaccinated fish produced significantly (P < 0.001) higher antibody titers than TSB sham vaccinated fish. At 28 dpv, antibody titer of vaccinated fish was lower than that at 14 dpv. However, the antibody titer of vaccinated fish at 28 dpv was still significantly (P < 0.001) higher than that of TSB sham vaccinated fish (Supplementary Fig. 2). Antibody titers of vaccinated fish at 28 dpv were not significantly different (P > 0.05) from that of positive control (i.e., 28 days post infection) (Supplementary Fig. 2). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 4. Discussion In an attempt to attenuate bacteria through selection of resistance to chemicals, sparfloxacin was used in this study. Of the 39 sparfloxacin-resistant S. agalactiae isolates, majority of them had lower virulence compared to their respective parents, indicating that sparfloxacin was an effective chemical in attenuating S. agalactiae. The attenuation of virulence resulting from the resistance to sparfloxacin is not surprising since decreased virulence as a fitness cost has been reported in novobiocin-resistant E. ictaluri [17], A. hydrophila [18], and S. iniae [19]. In addition, decreased virulence of Staphylococcus aureus has been reported to be associated with antibiotic resistance [25]. Differential transcriptome analysis on teicoplanin-resistant S. aureus has revealed that as resistance to antibiotic teicoplanin increased, some virulence-associated genes were down-regulated [25]. These results suggest that the gain of antibiotic resistance could result in the attenuation of virulence in bacteria. Of six sparfloxacin-resistant S. agalactiae isolates that were avirulent to 3–5 g Nile tilapia, only four were able to offer 100% protection against challenges with homologous isolates. When vaccinated fish were challenged with 6 heterologous isolates of S. agalactiae, Sag 97-spar failed to offer protection against challenges with isolate Sag 5, although the serotypes of Sag 97 and Sag 5 were both Ia. It is previously reported that one single S. agalactiae vaccine strain is incapable of protecting tilapia against challenges with other strains, although with the same serotype [26]. In addition, it was reported that tilapia vaccinated with an experimental S. agalactiae biotype 1 (hemolytic) vaccine were protected against lethal challenge with a virulent biotype 1 strain. However, there was no protection against challenge with virulent biotype 2 (non-hemolytic) strain in biotype 1-vaccinated fish, and vice versa [27]. Therefore, the failure of Sag 97-spar (non-hemolytic) to protect fish from challenges with Sag 5, Sag 6, and Sag 41 was expected since Sag 5, Sag 6, and Sag 41 were all hemolytic isolates.
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Table 4 Cumulative mortality and relative percent of survival of polyvalent vaccine-vaccinated Nile tilapia challenged with virulent parent isolates of Streptococcus agalactiae. Isolate used for vaccination
Isolate used for challenge
Challenge dose (CFU/fish)
Tested in 3–5 g tilapia Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent
Sag 4-P Sag 5-P Sag 6-P Sag 36-P Sag 41-P Sag 50-P Sag 53-P Sag 72-P Sag 76-P Sag 77-P Sag 81-P Sag 89-P Sag 92-P Sag 95-P Sag 96-P Sag 97-P Sag 99-P Sag 100-P Sag 102-P Sag 104-P Sag 114-P Sag 120-P Sag 121-P Sag 128-P Sag 130-P Sag 131-P Sag 133-P Sag 137-P Sag 138-P Sag 139-P Sag 142-P
2 × 107 3 × 107 2 × 107 2 × 107 3 × 107 2 × 106 2 × 107 1 × 107 2 × 107 2 × 107 1 × 107 1 × 107 2 × 107 1 × 107 1 × 107 2 × 107 1 × 107 1 × 107 2 × 107 2 × 107 1 × 107 2 × 107 2 × 107 2 × 107 2 × 107 1 × 107 2 × 107 2 × 106 1 × 107 1 × 107 3 × 107
Tested in 15–20 g tilapia Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent
Sag 4-P Sag 5-P Sag 6-P Sag 36-P Sag 41-P Sag 50-P Sag 53-P Sag 72-P Sag 76-P Sag 77-P Sag 81-P Sag 89-P Sag 92-P Sag 95-P Sag 96-P Sag 97-P Sag 99-P Sag 100-P Sag 102-P Sag 104-P Sag 114-P Sag 120-P Sag 121-P Sag 128-P Sag 130-P Sag 131-P Sag 133-P Sag 137-P Sag 138-P Sag 139-P Sag 142-P
2 × 107 2 × 107 2 × 107 1 × 107 3 × 107 2 × 106 1 × 107 2 × 107 2 × 107 2 × 107 2 × 108 4 × 107 4 × 107 1 × 107 2 × 107 2 × 107 2 × 107 2 × 107 3 × 107 2 × 107 1 × 107 3 × 107 2 × 107 2 × 107 1 × 107 2 × 107 1 × 107 2 × 107 1 × 107 2 × 107 2 × 107
However, Sag 97-spar also failed to protect fish from challenges against Sag 50 and Sag 100, although both Sag 50 and Sag 100 were non-hemolytic. This could be due to the fact that the serotypes of Sag 97, Sag 50, and Sag 100 were different (Ia, Ib, and II, respectively), although they were all non-hemolytic. However, Sag 97-spar did offer 100% protection against challenges with isolate Sag 133, although the serotype of Sag 133 was Ib whereas the serotype of Sag 97 was Ia. Taken together, these results suggest that
Mortality of TSB sham (%)
Mortality of vaccinated fish
RPS (%)
30 50 30 50 10 50 50 30 10 60 10 10 10 90 30 20 30 60 70 100 80 10 40 90 10 20 70 60 80 10 40
0 0 20 30 0 40 10 20 0 40 0 0 0 40 10 0 20 40 50 80 30 0 20 90 0 0 0 30 0 0 0
100 100 33 40 100 20 80 33 100 33 100 100 100 78 67 100 33 33 29 20 63 100 50 0 100 100 100 50 100 100 100
40 70 40 60 80 80 60 80 20 100 10 10 10 100 100 100 70 40 30 80 60 90 70 100 90 100 90 80 100 100 60
0 0 0 0 0 60 40 0 30 20 0 0 0 60 70 0 20 20 20 40 70 30 50 30 50 90 40 60 30 60 20
100 100 100 100 100 25 33 100 0 80 100 100 100 40 30 100 71 50 33 50 0 67 29 70 44 10 56 25 70 40 67
a S. agalactiae vaccine consisting of a single strain may only offer protection against a limited number of isolates of S. agalactiae. When a polyvalent vaccine consisting of 30 sparfloxacinresistant S. agalactiae isolates was evaluated in tilapia, the polyvalent vaccine provided 3–5 g Nile tilapia 100% protection against challenges with 15 isolates of S. agalactiae. The polyvalent vaccine also provided 15–20 g Nile tilapia 100% protection against challenges with 10 isolates of S. agalactiae. Overall, the polyvalent
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Fig. 2. Mean chemioluminescence absorbance values measured at 350–650 nm. A: after incubation for 2 min; B: after incubation for 5 min. Data are presented as mean ± S.D. from triplicates.
vaccine provided tilapia statistically significant protection against challenges with 30 parent isolates of S. agalactiae. However, the polyvalent vaccine failed to provide protection to 3–5 g Nile tilapia against challenges with isolate Sag 128-P. Similarly, the polyvalent vaccine failed to provide 15–20 g Nile tilapia any protection against challenges with two isolates (Sag 114-P and Sag 76-P). Taken together, our results suggest that the task to develop a polyvalent S. agalactiae vaccine that will protect all S. agalactiae strains that cause streptococcosis in aquaculture will be a difficult one, if not impossible. ROS results revealed that macrophages from vaccinated fish produced significantly higher ROX levels than that of non-vaccinated fish. ELISA results revealed that antibody titers of vaccinated fish (pre-challenge) at both 14 and 28 dpv were significantly higher than that of sham vaccinated fish. Taken together, our results suggest that both cellular and humoral immunity were involved in the protection elicited by the polyvalent vaccines. However, exactly how the polyvalent vaccine offered protection to Nile tilapia merits further studies. In summary, sparfloxacin was used in this study to modify 40 isolates of S. agalactiae in an attempt to develop attenuated bacterial vaccines. Majority of S. agalactiae used in this study were able to develop resistance to sparfloxacin. Of 31 avirulent sparfloxacinresistant S. agalactiae isolates, 30 provided 75% to 100% protection to 10–12 g Nile tilapia against challenges with a virulent S. agalactiae isolate Sag 50. When the virulence of the 30 sparfloxacinresistant S. agalactiae isolates was tested in 3–5 g Nile tilapia by intraperitoneal injection at dose of 2 × 107 CFU/fish, six were found to be avirulent to 3–5 g Nile tilapia. Of the six avirulent sparfloxacinresistant S. agalactiae isolates, four provided 3–5 g Nile tilapia 100% protection against challenges with homologous isolates, including
Sag 97-spar isolate that was non-hemolytic. However, Sag 97spar failed to provide broad cross-protection against challenges with heterologous isolates. When Nile tilapia was vaccinated with a polyvalent vaccine consisting of 30 sparfloxacin-resistant S. agalactiae isolates at dose of 2 × 106 CFU/fish, the polyvalent vaccine provided significant (P < 0.001) protection to both 3–5 g and 15–20 g Nile tilapia against challenges with 30 parent isolates of S. agalactiae. Taken together, our results suggest that a polyvalent vaccine consisting of various strains of S. agalactiae might be essential to provide broader protection to Nile tilapia against infections caused by S. agalactiae. Acknowledgments We thank Drs. Dunhua Zhang (USDA-ARS) and Mediha Aksoy (Tuskegee University) for critical reviews of the manuscript. We thank Beth Peterman (USDA-ARS) for her excellent technical support. We also thank the management team of the Aquatic Animal Health Research Unit for daily care and management of the fish. This study was supported by the USDA/ARS CRIS project #642032000-024-00D. The use of trade, firm, or corporate names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. References [1] Ye X, Li J, Lu M, Deng G, Jiang X, Tian Y, et al. Identification and molecular typing of Streptococcus agalactiae isolated from pond-cultured tilapia in China. Fish Sci 2011;77:623–32.
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[2] Abuseliana AF, Daud HH, Aziz SA, Bejo SK, Alsaid M. Pathogenicity of Streptococcus agalactiae isolated from a fish farm in Selangor to juvenile red tilapia (Oreochromis sp.). J Anim Veterinary Advances 2011;10:914–9. [3] Chen M, Li LP, Wang R, Liang WW, Huang Y, Li J, et al. PCR detection and PFGE genotype analyses of streptococcal clinical isolates from tilapia in China. Vet Microbiol 2012;159:526–30. [4] Bowater RO, Forbes-Faulkner J, Anderson IG, Condon K, Robinson B, Kong F, et al. Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia. J Fish Dis 2012;35:173–86. [5] Glibert PM, Landsberg JH, Evans JJ, Al-Sarawi MA, Faraj M, Al-Jarallah MA, et al. A fish kill of massive proportion in Kuwait Bay, Arabian Gulf, 2001: the roles of bacterial disease, harmful algae, and eutrophication. Harmful Algae 2002;1:215–31. [6] Al-Marzouk A, Duremdez R, Al-Gharabally H. Efforts to control outbreaks of diseases among cultured silver pomfret Pampus argenteus in Kuwait. J Aqua Trop 2004;19:103–10. [7] Heath PT. An update on vaccination against group B streptococcus. Expert Rev Vaccines 2011;10:685–94. [8] Zdragas A, Tsakos P, Kotzamanidis C, Anatoliotis K, Tsaknakis I. Outbreak of mastitis in ewes caused by Streptococcus agalactiae. J Hellenic Vet Med Soc 2005;56:114–21. [9] Watts JL, Nickerson SC, Pankey JW. A case study of Streptococcus group G infection in a dairy herd. Vet Microbiol 1984;9:571–80. [10] DePaola A, Peeler JT, Rodrick GE. Effect of oxytetracycline-medicated feed on antibiotic resistance of gram-negative bacteria in catfish ponds. Appl Environ Microbiol 1995;61:2335–40. [11] Pridgeon JW, Klesius PH, Mu X, Song L. An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish. J Appl Microbiol 2011;111:114–24. [12] Evans JJ, Klesius PH, Gilbert PM, Shoemaker CA, Al-Sarawi MA, Landsberg J, et al. Characterization of -haemolytic Group B Streptococcus agalactiae in cultured seabream, Sparus auratus L., and wild mullet, Liza klunzingeri (Day), in Kuwait. J Fish Dis 2002;25:505–13. [13] Evans JJ, Klesius PH, Shoemaker CA. Efficacy of Streptococcus agalactiae (group B) vaccine in tilapia (Oreochromis niloticus) by intraperitoneal and bath immersion administration. Vaccine 2004;22:3769–73. [14] Sheehan B, Labrie L, Lee Y, Lim W, Wong F, Chan J, et al. Streptococcal diseases in farmed tilapia. Aquaculture Asia Pacific 2009;5:26–9.
[15] Klesius PH, Shoemaker CA. Development and use of modified live Edwardsiella ictaluri vaccine against enteric septicemia of catfish. Adv Vet Med 1999;41:523–37. [16] Shoemaker CA, Klesius PH, Evans JJ. Immunization of eyed channel catfish, Ictalurus punctatus, eggs with monovalent Flavobacterium columnare vaccine and bivalent F. columnare and Edwardsiella ictaluri vaccine. Vaccine 2007;25:1126–31. [17] Pridgeon JW, Klesius PH. Development of a novobiocin-resistant Edwardsiella ictaluri as a novel vaccine in channel catfish (Ictalurus punctatus). Vaccine 2011;29:5631–7. [18] Pridgeon JW, Yildirim-Aksoy M, Klesius PH, Srivastava KK, Reddy PG. Attenuation of a virulent Aeromonas hydrophila with novobiocin and pathogenic characterization of the novobiocin-resistant strain. J Appl Microbiol 2012;113:1319–28. [19] Pridgeon JW, Klesius PH. Development and efficacy of a novobiocin-resistant Streptococcus iniae as a novel vaccine in Nile tilapia (Oreochromis niloticus). Vaccine 2011;29:5986–93. [20] Elsea SH, Osheroff N, Nitiss JL. Cytotoxicity of quinolones toward eukaryotic cells, Identification of topoisomerase II as the primary cellular target for the quinolone CP-115,953 in yeast. J Biol Chem 1992;267:13150–3. [21] Amend DF. Potency testing of fish vaccines. Dev Biol Stand 1981;49:447–54. [22] Jenkins JA, Klesius PH. Elicitation of macrophages from the peritoneal cavity of channel catfish. J Aquat Anim Health 1998;10:69–74. [23] Leung KY, Low KW, Lam TJ, Sin YM. Interaction of the fish pathogen Aeromonas hydrophila with tilapia, Oreochromis aureus (Steindachner), phagocytes. J Fish Dis 1995;18:435–47. [24] Pasnik DJ, Evans JJ, Panangala VS, Klesius PH, Shelby RA, Shoemaker CA. Antigenicity of Streptococcus agalactiae extracellular products and vaccine efficacy. J Fish Dis 2005;28:205–12. [25] McCallum N, Karauzum H, Getzmann R, Bischoff M, Majcherczyk P, BergerBächi B, et al. In vivo survival of teicoplanin-resistant Staphylococcus aureus and fitness cost of teicoplanin resistance. Antimicrob Agents Chemother 2006;50:2352–60. [26] Chen M, Wang R, Li LP, Liang WW, Li J, Huang Y, et al. Screening vaccine candidate strains against Streptococcus agalactiae of tilapia based on PFGE genotype. Vaccine 2012;30:6088–92. [27] Sheehan B, Labrie L, Lee Y, Lim W, Wong F, Chan J, et al. Streptococcal diseases in farmed tilapia. Aquaculture Asia Pacific 2009;5:26–9.
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Contents lists available at SciVerse ScienceDirect
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Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin Julia W. Pridgeon ∗ , Phillip H. Klesius Aquatic Animal Health Research Unit, USDA-ARS, 990 Wire Road, Auburn, AL 36832, USA
a r t i c l e
i n f o
Article history: Received 14 January 2013 Received in revised form 25 February 2013 Accepted 28 March 2013 Available online xxx Keywords: Streptococcus agalactiae Sparfloxacin Resistance Attenuation Vaccine
a b s t r a c t To develop attenuated bacteria as potential live vaccines, sparfloxacin was used in this study to modify 40 isolates of Streptococcus agalactiae. Majority of S. agalactiae used in this study were able to develop at least 80-fold resistance to sparfloxacin. When the virulence of the sparfloxacin-resistant S. agalactiae isolates were tested in 10–12 g Nile tilapia by intraperitoneal injection at dose of 2 × 107 CFU/fish, 31 were found to be avirulent to fish. Of the 31 avirulent sparfloxacin-resistant S. agalactiae isolates, 30 provided 75–100% protection to 10–12 g Nile tilapia against challenges with a virulent S. agalactiae isolate Sag 50. When the virulence of the 30 sparfloxacin-resistant S. agalactiae isolates was tested in 3–5 g Nile tilapia by intraperitoneal injection at dose of 2 × 107 CFU/fish, six were found to be avirulent to 3–5 g Nile tilapia. Of the six avirulent sparfloxacin-resistant S. agalactiae isolates, four provided 3–5 g Nile tilapia 100% protection against challenges with homologous isolates, including Sag 97-spar isolate that was non-hemolytic. However, Sag 97-spar failed to provide broad cross-protection against challenges with heterologous isolates. When Nile tilapia was vaccinated with a polyvalent vaccine consisting of 30 sparfloxacin-resistant S. agalactiae isolates at dose of 2 × 106 CFU/fish, the polyvalent vaccine provided significant (P < 0.001) protection to both 3–5 g and 15–20 g Nile tilapia against challenges with 30 parent isolates of S. agalactiae. Taken together, our results suggest that a polyvalent vaccine consisting of various strains of S. agalactiae might be essential to provide broader protection to Nile tilapia against infections caused by S. agalactiae. Published by Elsevier Ltd.
1. Introduction Streptococcus agalactiae affects various fish species, including tilapia [1–3], grouper [4], mullet [5], and pomfret [6]. In 2001, S. agalactiae was responsible for a massive fish kill at the Kuwait Bay, killing over 2500 metric tons of wild mullet (Liza klunzingeri) [5]. Large scale disease outbreaks caused by S. agalacatiae also occurred in 95% farms in China, with 30 −80% cumulative mortality [3]. In addition, S. agalactiae (group B streptococcus) is an important cause of disease in infants, pregnant women, immune-suppressed adults [7], and many cases of acute clinical mastitis in dairy animals [8,9]. To control streptococcosis in aquaculture caused by S. agalactiae, feeding infected fish with antibiotic-medicated feed is a general practice [10]. However, this practice is expensive and usually ineffective as sick fish tend to remain off feed. In addition, there are only three antibiotics currently approved for use in aquaculture: oxytetracycline (Terramycin), sulfadimethoxine (Romet-30), and
∗ Corresponding author. Tel.: +1 334 887 3741; fax: +1 334 887 2983. E-mail addresses: [email protected], [email protected] (J.W. Pridgeon).
florfenicol (Aquaflor). The widespread usage of the limited number of antibiotics for treating bacterial diseases in aquaculture has led to the development of antibiotic resistance in many fish pathogens worldwide [11]. Therefore, alternative control methods are urgently needed for the aquaculture industry. Use of vaccine is an alternative control method to prevent fish bacteria disease. Experimentally, formalin-killed S. agalactiae vaccine has been reported to offer significant protection to 30 g tilapia, but not to 5 g tilapia, with a relative percent of survival (RPS) rate of 80% in vaccinated fish compared to non-vaccinated fish at 30 days post vaccination [12,13]. In addition, an experimental S. agalactiae biotype I (-hemolytic) vaccine was reported to be able to offer protection against lethal challenges with both biotype I and biotype II (non-hemolytic) strains, whereas biotype II failed to offer protection against challenges with biotype I [14]. Recently, Merck Animal Health Inc has obtained regulatory approval in Brazil to begin marketing AQUAVAC® Strep Sa, an inactivated oil-adjuvanted vaccine that provides protection against S. agalactiae infections in tilapia and other susceptible fish species. However, AQUAVAC Strep Sa is only available for fish weighing more than 15 g, but not for fish weighing 3–5 g, the size of a 1–2 weeks old fish that farmers obtain from the hatchery. Therefore, a S. agalactiae vaccine that is safe and efficacious to 3–5 g tilapia is urgently needed.
0264-410X/$ – see front matter. Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.vaccine.2013.03.066
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To develop effective bacterial vaccines, rifampicin-resistant strategy has been successfully used to attenuate E. ictaluri (AquaVac-ESC) and Flavobacterium columnare (AquaVac-COL) [15,16]. In addition, novobiocin-resistant strategy has been successfully used to attenuate E. ictaluri [17], Aeromonas hydrophila [18], and S. iniae [19]. However, it is not clear whether sparfloxacin could also be used to attenuate S. agalactiae for the purpose of novel vaccine development. Sparfloxacin works by inhibiting bacterial DNA gyrase, therefore inhibiting DNA replication and transcription [20]. Due to its controversial safety profiles, sparfloxacin was withdrawn from the US market in 2000. However, it was unclear whether sparfloxacin could be used to attenuate pathogenic S. agalactiae. Therefore, the objectives of this study were: (1) Determine whether sparfloxacin could be used to attenuate different isolates of S. agalactiae; and (2) whether sparfloxain-resistant S. agalactiae could be used as attenuated live vaccines to protect 3–5 g Nile tilapia from infections by virulent S. agalactiae isolates. 2. Materials and methods 2.1. Identification of S. agalactiae by fatty acid methyl ester profiling Forty S. agalactiae isolates (Supplementary Table 1) were obtained from various fish species exhibiting clinical disease signs and from different geographical regions. The archived isolates were recovered from frozen stocks (2 ml aliquots stored at −80 ◦ C) and grown in tryptic soy broth (TSB) (Fisher Scientific, Pittsburgh, PA) for 24 h at 28 ◦ C. Bacteria isolates were identified by gas chromatography analysis of fatty acid methyl ester (FAME) using MIDI microbial identification system (MIDI, Newwark, Delaware). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 2.2. Hemolysis, Lancefield grouping and serotyping Hemolysis of S. agalactiae isolates was determined by streaking and stabbing 5% sheep blood agar (SBA, Remel, Lenexa, KS, USA) with S. agalactiae culture followed by incubation at 28 ◦ C for 24 h. The presence and the absence of a clear zone around the growth of the streaking sites and the stab sites were considered hemolytic and non-hemolytic, respectively. Lancefield grouping test of S. agalactiae was performed using StrepProTM grouping kit (Hardy Diagnostics, Santa Maria, CA). Serotyping of S. agalactiae was performed using Group-B Streptococci Tying Antisera (Denka Seiken, Coventry, UK) by slide agglutination method following manufacturer’s procedures. 2.3. Induction of resistance in S. agalactiae Sparfloxacin was purchased from Sigma-Aldrich (St. Louis, MO). All bacteria strains were cultured in tryptic soy broth (TSB) containing different concentrations of chemicals for 24–48 h at 28 ◦ C. After multiple passages of bacteria in TSB culture media containing same or higher concentration of sparfloxacin, resistant bacteria were obtained. 2.4. Virulence of sparfloxacin-resistant S. agalactiae compared to their parent isolates Similar amount (CFU/fish in a total volume of 0.1 ml) of each parent or sparfloxacin-resistant isolate was exposed to Nile tilapia (Oreochromis niloticus, 10–12 g or 3–5 g) through intraperitoneal (IP) injection. For initial virulence screening, a total of 20 fish were used in each treatment group (10 fish per tank, duplicates). All fish used in this study were raised at the USDA ARS Aquatic Animal
Health Research facility located at Auburn, Alabama. All fish treatment protocols were approved by Institutional Animal Care and Use Committee at the Aquatic Animal Health Research Laboratory following mandated guidelines. Mortalities were recorded daily for 14 days post injection 2.5. Vaccination of fish followed by challenge with virulent isolates of S. agalactiae Fish were vaccinated by IP injection. A total of 20 fish were used in each treatment group (10 fish per tank, duplicates). The vaccination dose for single isolate of S. agalactiae was 2 × 107 CFU/fish in a total volume of 100 l. As sham-vaccination controls, 100 l of TSB were injected into each fish. For initial vaccine efficacy screening, a total of 20 fish were used in each treatment group (10 fish per tank, duplicates). Fish were challenged with a virulent S. agalactiae isolate sag-50P (a non-hemolytic Brazil isolate cultured from diseased Nile tilapia in 2006) through IP injection at 14 days post vaccination (dpv). For efficacy testing of a polyvalent vaccine consisting of 30 sparfloxacin-resistant S. agalactiae isolates that were found to offer 75–100% protection to 10–12 g tilapia, the vaccination dose was 2 × 106 CFU/fish in a total volume of 100 l. Fish were challenged with 30 parent isolates of S. agalactiae at 28 dpv. Mortalities were recorded for 14 days post challenge. Results of bacteria challenge were presented as relative percent of survival (RPS) [21]. RPS was calculated according to the following formula: RPS = {1 − (vaccinated mortality ÷ control mortality)} × 100. 2.6. Backpassage safety studies of polyvalent S. agalactiae vaccine One hundred and eighty tilapia were used in the backpassage safety studies. The 180 fish were divided into two groups (control group and vaccine group) with 90 fish per group. The 90 fish were then divided into 6 fish tanks with 15 fish per tank. One hundred microliters (l) of polyvalent S. agalactiae vaccine were IP injected into each tilapia at dose of 2 × 106 CFU/fish. Control group fish were injected 100 l TSB. Forty-eight hours later, five fish were taken from the first tank and homogenized. A 100 l of the homogenate was then injected into each fish in tank 2. This procedure was repeated five times using the remaining of fish. Mortality or adverse behavior or signs of disease were recorded daily for 21 days post injection. 2.7. Macrophage isolation and reactive oxygen species production Macrophages were isolated from catfish by peritoneal lavage according to published procedures [22]. Briefly, 15–20 g Nile tilapia were injected intraperitoneally with 100 l of TSB or polyvalent vaccine at dose of 2 × 107 CFU/fish. Macrophages were harvested at two days after treatment by peritoneal lavage with 20 ml of sterile ice-cold PBS. Harvested macrophages were centrifuged at 500 × g for 15 min, washed once more by centrifugation in 12 ml of ice-cold PBS, and re-suspended in Hank’s Balanced Salt Solution (HBSS, Invitrogen, Carlsbad, CA, USA). Samples of harvested macrophages were stained with Hema-3 (Biochemical Sciences Inc., Swedesboro, NJ, USA) for total macrophage counts. Viability was assessed with trypan blue exclusion (Sigma) and observed microscopically using a hemacytometer. The chemioluminescence assay was conducted according to published procedures [23] with slight modifications. Briefly, macrophages from vaccinated fish or TSB non-vaccinated fish were seeded at 1 × 105 cells/well in a 96well opaque Luminometer plate (Promega, Madison, WI, USA). Overnight bacterial culture of S. agalactiae at 2 × 104 CFU/well ml were added to each well. Luminol [5-amino-2-3-dihydro-1,4phthalazinedione (Sigma)] in 0.2 M sodium borate pH 9.0 was added at 20 l/well. All tests were conducted in triplicate. Plates
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were read at 2 min and 5 min post addition of luminol with a Glowmax 96 microplate luminometer (Promega). 2.8. Indirect enzyme-linked immunosorbent assay (ELISA) Antibody titer against S. agalactiae-97 was determined using published method [24]. Briefly, 96-well ELISA plates were coated for 1 h at 25 ◦ C with 100 l of S. agalactiae-97 antigen in carbonate buffer (CB), which was prepared by sonication. Plates were washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBS-T) and then blocked with 3% bovine serum albumin in CB for 1 h. Following blocking, plates were washed three times with PBS-T. Serum from control fish or vaccinated fish (n = 10) was then added at a 1:500 dilution in PBS-T to the plate. The plates were incubated for 30 min at 25 ◦ C and then plates were washed three times with PBS-T. Monoclonal anti-tilapia antibody 1H1 was diluted 1:1000 in PBS-T and then added to all wells (100 l/well) for 30 min. Following washing three times with PBS-T, 100 l of peroxidase conjugated anti-mouse IgG (1:5000 in PBS-T) was added and incubated for 15 min. Plates were washed three times with PBST and 100 l substrate tetramethylbenzidine (Pierce, Rockford, IL) was added. After 15 min, the reaction was terminated by adding 50 l of 3 M H2 SO4 to each well and the absorbance was read at 450 nm using a BioRad 680 microplate reader (Biorad, Hercules, CA). Serum from S. agalactiae-97 infected fish at 14- and 28-day post infection was used as positive control and PBS was used as negative controls. 2.9. Statistical analysis All statistical analyses were performed using SigmaStat 3.5 software (Systat Software, Inc, Point Richmond, CA). Differences in cumulative mortality of fish vaccinated with polyvalent vaccine compared to sham-vaccinated fish following challenges with 31 different isolates were analyzed with Mann-Whitney U significance test, one well-known non-parametric statistical hypothesis test for assessing whether one of two samples of independent observations tends to have larger values than the other. Differences in antibody titers and reactive oxygen species production were analyzed with Student t-test and the significance level was defined as P < 0.05. 3. Results
3
streptococci. Serotyping results revealed that 40 isolates belonged to either sera type Ia, Ib, or II. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 3.3. Induction of resistance in S. agalactiae Majority of the 40 S. agalactiae isolates used in this study were able to develop resistance to sparfloxacin, with 80–10,000- fold resistance to sparfloxacin, except isolate Sag 50, which was only able to develop 10-fold resistance to sparfloxacin (Supplementary Table 3). Of the 40 S. agalactiae isolates used for selection of resistance to sparfloxacin, Sag 141 and Sag 76 were the most naturally sparfloxacin-resistant and sparfloxacin-sensitive isolate, respectively (Supplementary Table 3). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 3.4. Virulence of sparfloxacin-resistant S. agalactiae to 10-12 g Nile tilapia Virulence of sparfloxacin-resistant S. agalactiae and their respective parents to Nile tilapia by IP injection are shown in Supplementary Table 4. Of the 39 sparfloxacin-resistant isolates, majority (31) were found to be avirulent to 10–12 g Nile tilapia at injection dose of 2 × 107 CFU/fish (100 l of undiluted S. agalactiae culture at optical density of 1.0, absorbance reading at 540 nm). However, six sparfloxacin-resistant isolates were still highly virulent to 10–12 g Nile tilapia, killing 90–100% fish (Supplementary Table 4). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 3.5. Vaccinated 10–12 g Nile tilapia challenged with a virulent S. agalactiae isolate Cumulative mortality and relative percent of survival of vaccinated 10–12 g Nile tilapia challenged with a virulent S. agalactiae isolate Sag-50P are summarized in Table 1. Of the 31 avirulent sparfloxacin-resistant S. agalactiae isolates, 30 provided 75% to 100% protection to 10–12 g Nile tilapia against challenges with a virulent S. agalactiae isolate Sag-50P (Table 1). However, the RPS value provided by isolate Sag 106-spar (sparfloxacin-resistant S. agalactiae Sag 106) was only 25% (Table 1).
3.1. Bacteria identity confirmation FAME analysis confirmed the identities of all the S. agalactiae isolates used in this study. All S. agalactiae isolates shared high similarity indices with known S. agalactiae deposited into the FAME profile database. Representative FAME profiles of hemolytic and non-hemolytic S. agalactiae isolates are shown in Supplementary Fig. 1. All S. agalactiae isolates had the following three major fatty acid peaks: (1) the peak of 16:0 fatty acid at retention time of ∼2.74 min; (2) the peak of 18:0 w9c fatty acid at retention time of ∼3.30 min; (3) the peak of 18:0 fatty acid at retention time of ∼3.37 min (Supplementary Fig. 1). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066.
3.6. Virulence of sparfloxacin-resistant S. agalactiae to 3-5 g Nile tilapia Virulence of sparfloxacin-resistant S. agalactiae and their respective parents to 3–5 g Nile tilapia by IP injection are shown in Table 2. The 30 sparfloxacin-resistant S. agalactiae isolates that provided 75% to 100% protection to 10–12 g Nile tilapia were chosen for this study. Of the 30 isolates, majority (24) were found to be virulent to 3–5 g Nile tilapia at injection dose of 2 × 107 CFU/fish (100 l of undiluted S. agalactiae culture at optical density of 1.0, absorbance reading at 540 nm). However, six isolates were found to be avirulent to 3–5 g Nile tilapia (Table 2).
3.2. Hemolysis, Lancefield grouping, and serotyping
3.7. Vaccinated 3–5 g Nile tilapia challenged with homologous isolates of S. agalactiae
The hemolysis, Lancefield grouping, and serotyping results of the 40 isolates of S. agalactiae are summarized in Supplementary Table 2. Of the 40 isolates used in this study, 7 were hemolytic (-hemolysis) and 33 were non-hemolytic (␥-hemolysis). All S. agalactiae isolates used in this study belonged to Lancefield Group B
Cumulative mortality and relative percent of survival of vaccinated 3–5 g Nile tilapia challenged with virulent parent isolate are summarized in Table 3. Of the 6 avirulent sparfloxacin-resistant S. agalactiae isolates, 4 provided 100% protection against challenges with homologous S. agalactiae parent isolates (Table 3). Of the four
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Table 1 Cumulative mortality and relative percent of survival of vaccinated 10-12 g Nile tilapia challenged with virulent isolate Streptococcus agalactiae Sag 50-P. Isolate used for vaccination
Isolate used for challenge
Challenge dose (CFU/fish)
Mortality of TSB sham (%)
Mortality of vaccinated fish
RPS (%)
Sag 4-spar Sag 5-spar Sag 6-spar Sag 36-spar Sag 41-spar Sag 53-spar Sag 72-spar Sag 76-spar Sag 77-spar Sag 81-spar Sag 89-spar Sag 92-spar Sag 95-spar Sag 96-spar Sag 97-spar Sag 99-spar Sag 100-spar Sag 102-spar Sag 104-spar Sag 106-spar Sag 114-spar Sag 120-spar Sag 121-spar Sag 128-spar Sag 130-spar Sag 131-spar Sag 133-spar Sag 137-spar Sag 138-spar Sag 139-spar Sag 142-spar
Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P Sag 50-P
3 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 3 × 107
100 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 100
0 0 0 0 0 0 0 0 10 0 0 0 0 0 10 0 0 0 0 30 10 0 10 0 0 0 0 0 0 10 0
100 100 100 100 100 100 100 100 75 100 100 100 100 100 75 100 100 100 100 25 75 100 75 100 100 100 100 100 100 75 100
isolates that provided 100% protection, Sag 97 parent isolate was the most virulent one in 3–5 g Nile tilapia, killing 60% of TSB-sham vaccinated fish, whereas the other three parent isolates killed ≤40% fish (Table 3). Therefore, sparfloxacin-resistant Sag-97 isolate was Table 2 Virulence of parent and sparfloxacin-resistant S. agalactiae to 3-5 g Nile tilapia by IP injection. Isolate
Injection dose (CFU/fish)
Sag 4 Sag 5 Sag 6 Sag 36 Sag 41 Sag 53 Sag 72 Sag 76 Sag 77 Sag 81 Sag 89 Sag 92 Sag 95 Sag 96 Sag 97 Sag 99 Sag 100 Sag 102 Sag 104 Sag 114 Sag 120 Sag 121 Sag 128 Sag 130 Sag 131 Sag 133 Sag 137 Sag 138 Sag 139 Sag 142
2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107 2 × 107
Cumulative mortality (%) Parent (%)
Mutant (%)
100 100 100 40 100 100 100 100 80 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
100 70 90 0 10 80 60 50 40 20 70 70 50 80 0 0 0 80 30 40 40 60 0 0 40 20 50 80 70 90
subjected to further study in 3–5 g to determine whether it could offer cross-protection against challenges by heterologous isolates of S. agalactiae. 3.8. Sag 97-spar vaccinated 3–5 g Nile tilapia challenged with heterologous isolates Cumulative mortality and relative percent of survival of Sag 97spar vaccinated 3–5 g Nile tilapia challenged with heterologous isolates of S. agalactiae are summarized in Table 3. For challenges with six randomly selected heterologous S. agalactiae isolates, Sag 97-spar failed to provide any protection for five isolates, with RPS of 0 (Table 3). However, Sag 97-spar did provide 100% protection to 3–5 g Nile tilapia against challenges with S. agalactiae Sag 133-P isolate (Table 3). 3.9. Efficacy of polyvalent vaccine in 3–5 g Nile tilapia Cumulative mortality and relative percent of survival of 3–5 g Nile tilapia vaccinated with a polyvalent vaccine challenged with 30 parent isolates of S. agalactiae are summarized in Table 4. The polyvalent vaccine provided 3–5 g Nile tilapia 100% protection against challenges with 15 isolates of S. agalactiae (Table 4). The polyvalent vaccine also provided some protection against 14 isolates of S. agalactiae, with RPS values of 20% to 80% (Table 4). However, the polyvalent vaccine failed to provide 3–5 g Nile tilapia any protection against challenges with isolate Sag 128-P. When cumulative mortalities of vaccinated fish were compared to that of TSB sham-vaccinated fish after challenges with the 30 isolates, TSB sham-vaccinated fish had significantly (P < 0.001) higher mortality than that of polyvalent S. agalactiae-vaccinated fish (Fig. 1). 3.10. Efficacy of polyvalent vaccine in 15–20 g Nile tilapia Cumulative mortality and relative percent of survival of 15–20 g Nile tilapia vaccinated with a polyvalent vaccine challenged with
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Table 3 Cumulative mortality and relative percent of survival of vaccinated 3-5 g Nile tilapia challenged with virulent parent isolates of Streptococcus agalactiae. Isolate used for vaccination
Isolate used for challenge
Challenge dose (CFU/fish)
Mortality of TSB sham (%)
Mortality of vaccinated fish
RPS (%)
Sag 36-spar Sag 97-spar Sag 99-spar Sag 100-spar Sag 128-spar Sag 130-spar Sag 97-spar Sag 97-spar Sag 97-spar Sag 97-spar Sag 97-spar Sag 97-spar
Sag 36-P Sag 97-P Sag 99-P Sag 100-P Sag 128-P Sag 130-P Sag 5-P Sag 6-P Sag 41-P Sag 50-P Sag 100-P Sag 133-P
2 × 107 2 × 107 2 × 106 2 × 106 2 × 107 2 × 107 3 × 107 3 × 107 3 × 107 2 × 106 2 × 107 2 × 107
40 60 20 10 100 90 60 100 90 100 30 30
0 0 0 0 100 90 60 100 100 100 40 0
100 100 100 100 0 0 0 0 0 0 0 100
30 parent isolates of S. agalactiae are summarized in Table 4. The polyvalent vaccine provided 15–20 g Nile tilapia 100% protection against challenges with 10 isolates of S. agalactiae (Table 4). In addition, the polyvalent vaccine provided 50% to 80% protection to 15–20 g Nile tilapia against challenges with 9 isolates of S. agalactiae (Table 4). However, the polyvalent vaccine failed to provide 15–20 g Nile tilapia any protection against challenges with two isolates (Sag 114-P and Sag 76-P). When cumulative mortalities of vaccinated fish were compared to that of TSB sham-vaccinated fish after challenges with the 30 isolates, TSB sham-vaccinated fish had significantly (P < 0.001) higher mortality than that of polyvalent S. agalactiae-vaccinated fish (Fig. 1). 3.11. Backpassage safety results Of all fish exposed to the polyvalent S. agalactiae vaccine through IP injection, no mortality or signs of disease or adverse behavior was observed. No fish died in the backpassage safety studies. 3.12. Reactive oxygen species production The production of reactive oxygen species (ROS) by tilapia macrophages from vaccinated and non-vaccinated are summarized in Fig. 2. Macrophages from polyvalent vaccine-vaccinated fish produced significantly (P < 0.05) more ROS than that from TSB sham-vaccinated fish at both 2 min (Fig. 2A) and 5 min (Fig. 2B) post incubation.
Fig. 1. Cumulative mortality of Nile tilapia vaccinated with or without polyvalent vaccine of Streptococcus agalactiae followed by challenges with 31 different isolates of S. agalactiae. Data are presented as mean ± S.D. from challenges with 31 different isolates of S. agalactiae. Significant differences (P < 0.001) between vaccinated fish and sham-vaccinated fish are marked with asterisk.
3.13. Indirect enzyme-linked immunosorbent assay (ELISA) ELISA results revealed that sham-vaccinated fish had consistent absorbance readings comparable to that of negative PBS control at different time points (Supplementary Fig. 2). At 14 days postvaccination (pre-challenge), vaccinated fish produced significantly (P < 0.001) higher antibody titers than TSB sham vaccinated fish. At 28 dpv, antibody titer of vaccinated fish was lower than that at 14 dpv. However, the antibody titer of vaccinated fish at 28 dpv was still significantly (P < 0.001) higher than that of TSB sham vaccinated fish (Supplementary Fig. 2). Antibody titers of vaccinated fish at 28 dpv were not significantly different (P > 0.05) from that of positive control (i.e., 28 days post infection) (Supplementary Fig. 2). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.03.066. 4. Discussion In an attempt to attenuate bacteria through selection of resistance to chemicals, sparfloxacin was used in this study. Of the 39 sparfloxacin-resistant S. agalactiae isolates, majority of them had lower virulence compared to their respective parents, indicating that sparfloxacin was an effective chemical in attenuating S. agalactiae. The attenuation of virulence resulting from the resistance to sparfloxacin is not surprising since decreased virulence as a fitness cost has been reported in novobiocin-resistant E. ictaluri [17], A. hydrophila [18], and S. iniae [19]. In addition, decreased virulence of Staphylococcus aureus has been reported to be associated with antibiotic resistance [25]. Differential transcriptome analysis on teicoplanin-resistant S. aureus has revealed that as resistance to antibiotic teicoplanin increased, some virulence-associated genes were down-regulated [25]. These results suggest that the gain of antibiotic resistance could result in the attenuation of virulence in bacteria. Of six sparfloxacin-resistant S. agalactiae isolates that were avirulent to 3–5 g Nile tilapia, only four were able to offer 100% protection against challenges with homologous isolates. When vaccinated fish were challenged with 6 heterologous isolates of S. agalactiae, Sag 97-spar failed to offer protection against challenges with isolate Sag 5, although the serotypes of Sag 97 and Sag 5 were both Ia. It is previously reported that one single S. agalactiae vaccine strain is incapable of protecting tilapia against challenges with other strains, although with the same serotype [26]. In addition, it was reported that tilapia vaccinated with an experimental S. agalactiae biotype 1 (hemolytic) vaccine were protected against lethal challenge with a virulent biotype 1 strain. However, there was no protection against challenge with virulent biotype 2 (non-hemolytic) strain in biotype 1-vaccinated fish, and vice versa [27]. Therefore, the failure of Sag 97-spar (non-hemolytic) to protect fish from challenges with Sag 5, Sag 6, and Sag 41 was expected since Sag 5, Sag 6, and Sag 41 were all hemolytic isolates.
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Table 4 Cumulative mortality and relative percent of survival of polyvalent vaccine-vaccinated Nile tilapia challenged with virulent parent isolates of Streptococcus agalactiae. Isolate used for vaccination
Isolate used for challenge
Challenge dose (CFU/fish)
Tested in 3–5 g tilapia Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent
Sag 4-P Sag 5-P Sag 6-P Sag 36-P Sag 41-P Sag 50-P Sag 53-P Sag 72-P Sag 76-P Sag 77-P Sag 81-P Sag 89-P Sag 92-P Sag 95-P Sag 96-P Sag 97-P Sag 99-P Sag 100-P Sag 102-P Sag 104-P Sag 114-P Sag 120-P Sag 121-P Sag 128-P Sag 130-P Sag 131-P Sag 133-P Sag 137-P Sag 138-P Sag 139-P Sag 142-P
2 × 107 3 × 107 2 × 107 2 × 107 3 × 107 2 × 106 2 × 107 1 × 107 2 × 107 2 × 107 1 × 107 1 × 107 2 × 107 1 × 107 1 × 107 2 × 107 1 × 107 1 × 107 2 × 107 2 × 107 1 × 107 2 × 107 2 × 107 2 × 107 2 × 107 1 × 107 2 × 107 2 × 106 1 × 107 1 × 107 3 × 107
Tested in 15–20 g tilapia Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent Polyvalent
Sag 4-P Sag 5-P Sag 6-P Sag 36-P Sag 41-P Sag 50-P Sag 53-P Sag 72-P Sag 76-P Sag 77-P Sag 81-P Sag 89-P Sag 92-P Sag 95-P Sag 96-P Sag 97-P Sag 99-P Sag 100-P Sag 102-P Sag 104-P Sag 114-P Sag 120-P Sag 121-P Sag 128-P Sag 130-P Sag 131-P Sag 133-P Sag 137-P Sag 138-P Sag 139-P Sag 142-P
2 × 107 2 × 107 2 × 107 1 × 107 3 × 107 2 × 106 1 × 107 2 × 107 2 × 107 2 × 107 2 × 108 4 × 107 4 × 107 1 × 107 2 × 107 2 × 107 2 × 107 2 × 107 3 × 107 2 × 107 1 × 107 3 × 107 2 × 107 2 × 107 1 × 107 2 × 107 1 × 107 2 × 107 1 × 107 2 × 107 2 × 107
However, Sag 97-spar also failed to protect fish from challenges against Sag 50 and Sag 100, although both Sag 50 and Sag 100 were non-hemolytic. This could be due to the fact that the serotypes of Sag 97, Sag 50, and Sag 100 were different (Ia, Ib, and II, respectively), although they were all non-hemolytic. However, Sag 97-spar did offer 100% protection against challenges with isolate Sag 133, although the serotype of Sag 133 was Ib whereas the serotype of Sag 97 was Ia. Taken together, these results suggest that
Mortality of TSB sham (%)
Mortality of vaccinated fish
RPS (%)
30 50 30 50 10 50 50 30 10 60 10 10 10 90 30 20 30 60 70 100 80 10 40 90 10 20 70 60 80 10 40
0 0 20 30 0 40 10 20 0 40 0 0 0 40 10 0 20 40 50 80 30 0 20 90 0 0 0 30 0 0 0
100 100 33 40 100 20 80 33 100 33 100 100 100 78 67 100 33 33 29 20 63 100 50 0 100 100 100 50 100 100 100
40 70 40 60 80 80 60 80 20 100 10 10 10 100 100 100 70 40 30 80 60 90 70 100 90 100 90 80 100 100 60
0 0 0 0 0 60 40 0 30 20 0 0 0 60 70 0 20 20 20 40 70 30 50 30 50 90 40 60 30 60 20
100 100 100 100 100 25 33 100 0 80 100 100 100 40 30 100 71 50 33 50 0 67 29 70 44 10 56 25 70 40 67
a S. agalactiae vaccine consisting of a single strain may only offer protection against a limited number of isolates of S. agalactiae. When a polyvalent vaccine consisting of 30 sparfloxacinresistant S. agalactiae isolates was evaluated in tilapia, the polyvalent vaccine provided 3–5 g Nile tilapia 100% protection against challenges with 15 isolates of S. agalactiae. The polyvalent vaccine also provided 15–20 g Nile tilapia 100% protection against challenges with 10 isolates of S. agalactiae. Overall, the polyvalent
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Fig. 2. Mean chemioluminescence absorbance values measured at 350–650 nm. A: after incubation for 2 min; B: after incubation for 5 min. Data are presented as mean ± S.D. from triplicates.
vaccine provided tilapia statistically significant protection against challenges with 30 parent isolates of S. agalactiae. However, the polyvalent vaccine failed to provide protection to 3–5 g Nile tilapia against challenges with isolate Sag 128-P. Similarly, the polyvalent vaccine failed to provide 15–20 g Nile tilapia any protection against challenges with two isolates (Sag 114-P and Sag 76-P). Taken together, our results suggest that the task to develop a polyvalent S. agalactiae vaccine that will protect all S. agalactiae strains that cause streptococcosis in aquaculture will be a difficult one, if not impossible. ROS results revealed that macrophages from vaccinated fish produced significantly higher ROX levels than that of non-vaccinated fish. ELISA results revealed that antibody titers of vaccinated fish (pre-challenge) at both 14 and 28 dpv were significantly higher than that of sham vaccinated fish. Taken together, our results suggest that both cellular and humoral immunity were involved in the protection elicited by the polyvalent vaccines. However, exactly how the polyvalent vaccine offered protection to Nile tilapia merits further studies. In summary, sparfloxacin was used in this study to modify 40 isolates of S. agalactiae in an attempt to develop attenuated bacterial vaccines. Majority of S. agalactiae used in this study were able to develop resistance to sparfloxacin. Of 31 avirulent sparfloxacinresistant S. agalactiae isolates, 30 provided 75% to 100% protection to 10–12 g Nile tilapia against challenges with a virulent S. agalactiae isolate Sag 50. When the virulence of the 30 sparfloxacinresistant S. agalactiae isolates was tested in 3–5 g Nile tilapia by intraperitoneal injection at dose of 2 × 107 CFU/fish, six were found to be avirulent to 3–5 g Nile tilapia. Of the six avirulent sparfloxacinresistant S. agalactiae isolates, four provided 3–5 g Nile tilapia 100% protection against challenges with homologous isolates, including
Sag 97-spar isolate that was non-hemolytic. However, Sag 97spar failed to provide broad cross-protection against challenges with heterologous isolates. When Nile tilapia was vaccinated with a polyvalent vaccine consisting of 30 sparfloxacin-resistant S. agalactiae isolates at dose of 2 × 106 CFU/fish, the polyvalent vaccine provided significant (P < 0.001) protection to both 3–5 g and 15–20 g Nile tilapia against challenges with 30 parent isolates of S. agalactiae. Taken together, our results suggest that a polyvalent vaccine consisting of various strains of S. agalactiae might be essential to provide broader protection to Nile tilapia against infections caused by S. agalactiae. Acknowledgments We thank Drs. Dunhua Zhang (USDA-ARS) and Mediha Aksoy (Tuskegee University) for critical reviews of the manuscript. We thank Beth Peterman (USDA-ARS) for her excellent technical support. We also thank the management team of the Aquatic Animal Health Research Unit for daily care and management of the fish. This study was supported by the USDA/ARS CRIS project #642032000-024-00D. The use of trade, firm, or corporate names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. References [1] Ye X, Li J, Lu M, Deng G, Jiang X, Tian Y, et al. Identification and molecular typing of Streptococcus agalactiae isolated from pond-cultured tilapia in China. Fish Sci 2011;77:623–32.
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Please cite this article in press as: Pridgeon JW, Klesius PH. Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.03.066