Antonie van Leeuwenhoek (2014) 105:423–430 DOI 10.1007/s10482-013-0079-y
SHORT COMMUNICATION
Identification of a Proteus penneri isolate as the causal agent of red body disease of the cultured white shrimp Penaeus vannamei and its control with Bdellovibrio bacteriovorus Haipeng Cao • Shan He • Liqun Lu Xianle Yang • Baiyao Chen
•
Received: 25 September 2013 / Accepted: 15 November 2013 / Published online: 22 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Bacteriosis has become a major economic problem in the farming of the Pacific white shrimp Penaeus vannamei. However, no definitive data are available about Proteus penneri infection in cultured P. vannamei and its control. In this study, a virulent strain NC was isolated from diseased P. vannamei suffering from red body disease and identified as a P. penneri isolate through phylogenetic analysis and ATB 32GN system. A phylogenetic constructed tree using the neighbour-joining method identified the NC isolate as a P. penneri strain. In addition, Bdellovibrio
bacteriovorus conferred significant protection against P. penneri: it exhibited significant bacteriolytic effects on the pathogenic P. penneri, had a wide prey range towards Proteus pathogens, and displayed a good protective efficacy on experimental P. penneri infection in P. vannamei. To the best of our knowledge, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus. Keywords Penaeus vannamei Proteus penneri Bdellovibrio bacteriovorus Red body disease Control
Haipeng Cao and Shan He are contributed equally to this work.
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0079-y) contains supplementary material, which is available to authorized users. H. Cao (&) S. He L. Lu X. Yang Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture of P. R. China, Shanghai Engineering Research Center of Aquaculture, Shanghai University Knowledge Service Platform, Shanghai Ocean University Aquatic Animal Breeding Center (ZF1206), National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, People’s Republic of China e-mail: [email protected] B. Chen (&) Marine and Fisheries Research Institute of Lianyungang, Lianyungang 222044, Jiangsu, People’s Republic of China e-mail: [email protected]
The Pacific white shrimp Penaeus vannamei is widely distributed and cultivated in Hawaii, South Carolina, Texas, China, Thailand, Vietnam, Brazil, Mexico, Venezuela, Peru and Puerto Rico (Briggs et al. 2004; Wakida-Kusunoki et al. 2011). Especially in China, P. vannamei has become the most important commercial shrimp species and has brought a great profit in recent years with annual production of nearly 23.8 million tons (Ministry of Agriculture and Fisheries Bureau of China 2011). However, bacterial infection has become a major economic problem in P. vannamei farming (Wang et al. 2013). For example, Vibrio harveyi has been recognized as an important and destructive pathogen for P. vannamei, causing widespread outbreaks of the bacterial white tail disease (Robertson
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et al. 1998; Zhou et al. 2012). Thus, bacteriosis should be given more attention for the sustainable development of P. vannamei farming industry. Proteus species have been well documented to be associated with the mortality of P. orientalis (Xu et al. 1992), Rana catasbeiana (Shu et al. 1997), Trionyx sinensis (Wang and Wang 1998), Channa punctatus (Mandal et al. 2002), Pseudosciaena crocea (Zhang et al. 2005) and Silurus meridionalis Chen (Cao et al. 2007). The diseases caused by Proteus species are usually associated with their production of virulence factors, such as adherence, invasiveness, swarming phenomenon, hemolytic activity, urea hydrolysis, proteolysis and endotoxicity (Ro´zalski et al. 2007). P. penneri is an invasive but often ignored pathogen and is capable of causing major infectious diseases such as human bacteremia and subcutaneous abscess (Engler et al. 1990; Kaistha et al. 2011; Kishore 2012). However, there are no definitive data on P. penneri infection in cultured P. vannamei. At present, Proteus infections can be partially controlled by farmers with the crude application of antibiotics such as norfloxacin and gentamycin (Zhang et al. 2005; Cao et al. 2007). However, antibiotic treatment is cost-prohibitive to farmers in many undeveloped and developing countries, and may be detrimental to the environment and human health (Harikrishnan et al. 2010). Bdellovibrio species have been well regarded as predatory bacteria which attach to Gram-negative bacteria, penetrate through the cell wall to form a bdelloplast and multiply (Snyder et al. 2002). These bacteria play important roles in reducing microbial density and altering microbial communities through predation (Cao et al. 2012). Because of their intrinsic ability to lyse prey cells, these bacteria have been considered as potential agents in controlling aquatic pathogens (Qi et al. 2009). However, little information is available about Bdellovibrio strains as biocontrol agents for Proteus pathogens. In this paper, a virulent P. penneri strain was isolated from diseased P. vannamei suffering from red body disease in Xiaoshan, Zhejiang China. Phenotypic characterization, taxonomic position and control of the strain with the predatory bacterium Bdellovibrio bacteriovorus were examined. As far as we know, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus.
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Materials and methods White shrimp samples As recommended by Jayasinghe et al. (2008), 26 diseased P. vannamei (averaging 15.1 ± 1.3 g in weight) suffering from red body disease, characterized by the reddening of the shrimp body as described by Chen et al. (2012), were taken into sterile bags, kept in ice during transport to the laboratory from one single site of a P. vannamei farm in Xiaoshan, Zhejiang China in July 2012. In this farm, with 280 acres of ponds, P. vannamei was stocked at an initial rearing density of 60,000 juvenile shrimps per acre. The water quality of the outbreak ponds was pH 8.16, 6.5 mg L-1 of dissolved oxygen, 0.45 mg L-1 of total ammonia and 0.3 mg L-1 of nitrite. This was the farm’s first disease outbreak and it couldn’t be effectively controlled although penicillin, enrofloxacin, norfloxacin had been used. Isolation of bacteria Each sampled moribund P. vannamei was disinfected externally with 75 % alcohol and dissected in the laboratory. After a careful microscopic examination of organs such as gills and hepatopancreas for parasites, and viruses by electron microscropy, a 0.2 g hepatopancreas sample of each shrimp was cut and directly streaked onto nutrient agar (NA) plates (Sinopharm Chemical Reagent Co., Ltd.) as recommended by Geng et al. (2010). After incubation for 24–48 h at 28 °C, the dominant uniform isolates were purified by streaking and re-streaking onto NA plates. Only the dominant isolates with dense virtually pure culture growth on NA plates were obtained, as recommended by Orozova et al. (2012). Pure isolates were stored at -80 °C supplemented with 15 % glycerol. A representative stock, strain NC, was used as the test organism in the present study. Molecular identification of bacteria The extraction of genomic DNA of isolate NC, as well as PCR amplification of its 16S rRNA gene and sequencing were performed according to Cao et al. (2010). The near complete 16S rRNA gene sequence was assembled using MegAlign, Editseq and Seqman
Antonie van Leeuwenhoek (2014) 105:423–430
software with a Power Macintosh computer. A search was done against the National Centre for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST) program. The phylogenetic tree from the near complete 16S rRNA gene sequence of the isolate and its homologous sequences was further constructed using the neighbour-joining method. Phenotypic identification of the isolate using the ATB 32GN system The isolate NC was identified phenotypically using the ATB 32GN system as recommended by Altwegg and Zollinger-Iten (1987). Briefly, isolate NC was grown on NA plates (Sinopharm Chemical Reagent Co., Ltd.) at 28 °C for 24 h, and a bacterial suspension was then used to inoculate the API ID32GN strip (Bio-Merieux, SA) following the manufacturer’s instruction. The strip was incubated at 28 °C and observed after 48 h for checking against the API identification index and database. The type strain P. penneri ATCC35198 was used as the control. Bacterial virulence assay After a careful examination of external appearance, gut condition, growth situation, physical behaviour and feeding trends as recommended by the Marine Products Export Development Authority & Network of Aquaculture Centres in Asia–Pacific (2003), 240 healthy white shrimps (averaging 10.1 ± 0.6 g in weight) were obtained from Pinghu Aquaculture Co., Ltd. in Zhejiang China, and were respectively maintained in 6 aquaria (40 shrimps per aquarium) supplied with 100 L aerated filtered farm water at 28 °C for 14 days. Prior to the bacterial virulence assay, P. penneri strain NC was inoculated into 100 mL nutrient broth (NB) according to Cao et al. (2010), incubated at 28 °C with shaking at 200 rpm for 24 h, enumerated by tenfold serial dilutions in sterile distilled water and spread on NA plates. 40 healthy shrimps were challenged by being immersed in 100 L of water containing P. penneri isolate NC at a final cell density of 5.0 9 106 CFU mL-1 as described by Zhang et al. (2009). Another 40 healthy shrimps were unchallenged as the control. Each treatment was conducted in triplicate. The experimental shrimps were kept at 28 °C and observed
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daily for 7 days. Any dead shrimps were removed immediately and sent to the laboratory for pathogen examination according to Bucke (1989) to find if the challenging strain was re-isolated, and the signs and mortalities were recorded. In vitro bacteriolytic effect of B. bacteriovorus on the isolate Bdellovibrio bacteriovorus strain H16, previously isolated from the gut of Acipenser baerii (identified morphologically and molecularly by Cao et al. (2012), and stored at -80 °C supplemented with 15 % glycerol), was used in our study. Its cell density was determined using the double-layer agar plating method as described by Stolp and Starr (1963). Briefly, B. bacteriovorus H16 was diluted serially tenfold in sterile distilled water. 100 lL aliquots of serial tenfold dilutions and 200 lL of a suspension of isolate NC (1.0 9 1010 CFU mL-1) were added to 10 mL of 0.6 % agar melted and kept at 55 °C, mixed and poured onto a pre-prepared 1.5 % agar plate until solidification. The double-layer agar was then incubated at 30 °C for 4 days, and the plaques were recorded. The in vitro bacteriolytic assay was conducted in triplicate and was carried out in six 250 mL glass flask with 98 mL of autoclaved diluted nutrient broth (DNB). In each flask of the treatment group, 1 mL of a suspension of isolate NC (with a final cell density of 5 9 106 CFU mL-1) and 1 mL of B. bacteriovorus H16 (with a final cell density of 5 9 103 PFU mL-1), were independently inoculated into 98 mL of DNB as recommended by Cao et al. (2012), then the mixtures were incubated at 30 °C with shaking at 200 rpm. Each flask in the control group only consisted of the isolate at a final cell density of 5 9 106 CFU mL-1, incubated as described above. Cell growth of the isolate and B. bacteriovorus H16 were respectively measured using the serial dilution and spread plate method described above and the double-layer agar plating method at 24 h intervals. Lytic activity of B. bacteriovorus against Proteus strains The lysis assay of B. bacteriovorus against Proteus strains was conducted in triplicate. Eight pathogenic
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strains of Proteus species (P. mirabilis strain ZL003, P. mirabilis strain ZXS02, P. mirabilis strain BYK64285, P. mirabilis strain BYK64291, P. vulgaris strain TWN3, Proteus sp. strain ZL0057, Proteus sp. strain BYK000419 and Proteus sp. strain BYK00098) were obtained from the National Pathogen Collection Centre for Aquatic Animals, P.R. China. The bacteriolytic activity of B. bacteriovorus H16 at a density of 5 9 103 PFU mL-1 was checked against the eight pathogenic Proteus strains by the double-layer agar plating method (Stolp and Starr 1963). The bacteriolytic activity of B. bacteriovorus H16 against the NC isolate served as the control. Plaques were observed and recorded on double-layer agar plates after 4 days of incubation at 30 °C. Protective efficacy assay The protective efficacy assay was conducted in triplicate. Healthy shrimps (averaging 10.1 ± 0.6 g in weight) were obtained from Pinghu Aquaculture Co., Ltd. in Zhejiang China, and maintained in 9 glass aquaria supplied with aerated filtered farm water at 28 °C throughout the experiment. Each aquarium, containing 100 L farm water without water recirculation, was stocked with 40 healthy shrimps selected at random. Immediately after all the shrimps were bath challenged through exposure to isolate NC at a final cell density of 5 9 106 CFU mL-1, 0, 5 and 10 mL of B. bacteriovorus H16 (at a final concentration of 3 9 108 PFU mL-1) were added to the 100 L aerated filtered farm water in the control and two treatment groups (ca. 0, 5 9 103 and 1 9 104 PFU mL-1 in water). Each group contained 3 aquaria. The test shrimps were observed daily for 7 days. Dead shrimps were removed immediately and sent to the laboratory for pathogen examination according to Bucke (1989) to find if the challenging strain was re-isolated, and mortalities were recorded each day for 7 days. Relative percentage survivals were calculated according to Baulny et al. (1996).
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Results Identification of the pathogenic isolate A single dominant strain, NC, was isolated from the diseased shrimps and identified by molecular and phenotypic methods as P. penneri. Its near complete 16S rRNA gene sequence (1,459 nucleotides) has been submitted to GenBank database with the accession no. KC113042. Similarities between the 16S rRNA gene sequence of the NC isolate and those of P. penneri strains in the GenBank database were 99 % and the phylogenetic tree constructed using the neighbourjoining method demonstrated isolate NC to be a P. penneri strain (Fig. 1). In addition, the ATB 32GN system identified isolate NC as a P. penneri strain (Table 1) as it showed an identity of 96.9 % with the type strain ATCC35198 in phenotypic characterization, which confirmed the molecular identification. No parasites or viruses were detected in the diseased shrimps from which isolate NC was obtained. The NC isolate was confirmed as the pathogen of this disease according to Koch’s postulates: (A) the NC isolate was virulent to healthy shrimps in an experimental challenge (see Methods). The death of the challenged shrimps began to occur after 1 day and the rate increased gradually over time. In total 93 % of the shrimps challenged with the NC isolate died acutely with the signs of reddening of the shrimp body, similar to the originally infected shrimps with red body disease, and no acute mortality or visible changes were 95 Proteus hauseri strain FFL10 [JN092597] Proteus hauseri strain FFL13 [JN092599] 96
98
Proteus mirabilis strain FUA1267 [JN102561] Proteus mirabilis strain FFL2 [JN092590]
80 30
Proteus mirabilis strain PPB3 [HM771658]
Proteus vulgaris type strain DSM 13387T [HE978268] 99
Proteus vulgaris strain ATCC 29905 [DQ885257] the NC isolate
99
Proteus penneri strain M2 [HQ259936] Proteus penneri strain NCTC 12737 [DQ885258] Proteus penneri strain ALK514 [KC456567]
60
100 Proteus penneri strain ALK515 [KC456568]
Statistical analysis Data are presented as the mean ± standard deviation (S.D.) for the indicated number of each assay. P \ 0.05 is considered statistically significant using one-way analysis of variance.
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0.002
Fig. 1 A 16S rRNA gene tree of 11 known bacteria and the NC isolate constructed using the neighbour-joining method. The bootstrap values (%) are shown besides the clades, accession numbers are indicated beside the names of strains, and scale bars represent distance values
Antonie van Leeuwenhoek (2014) 105:423–430 Table 1 Phenotypic characterization of isolate NC in comparison with the type strain P. penneri ATCC 35198
427
Test item
Reaction
Rhamnose Mannite
-
D-Ribose
R?
R?
Valerate
R-
R-
-
-
R
?
R?
R
-
R-
R
?
R?
-
R-
?
R
-
R
?
R
-
R
?
DL-Lactate Citrate L-Alanine
R
Histidine
R
L-Fucose
R-
R-
5-keto-gluconate
R-
R-
?
?
2-keto-gluconate
R
-
R-
R
-
R-
R
-
R-
R
-
R-
?
R? R?
R
-
R
-
Itaconic acid
R
-
L-Arabinose
R
-
R
-
R
-
R
-
R
-
Glycogen 3-hydroxy-butyrate 4-hydroxy-benzoate
Suberate
R
R
L-Serine
R
Propionate
R-
R-
L-Proline
R?
than that in the control (P \ 0.05) when strain H16 was inoculated at a final cell density of 5 9 103 PFU mL-1; the cell density of the NC isolate was reduced by 99.9 % after incubation for 7 days, compared with the control group. At the same time, the growth of the predator B. bacteriovorus H16 strain was improved at the expense of the NC isolate and a maximum cell density was reached after 3 days incubation. Thus, B. bacteriovorus has potential to be used for exclusion of P. penneri.
C
6 5 4 3 2 1 3
-
R
B
2
R
Sucrose
7
1
R-
-
R
RR?
8
0
Caprate
R-
-
R R?
R
?
R-
3-hydroxy-benzoate Acetate
9
0
R
Malonate
R R?
D-Sorbitol
Logarithm of cell density -1 (lg cells mL )
R-
-
ATCC35198a
R R?
Maltose
A
R-
NC
N-Acetylglucosamin D-Glucose
D-Melibiose
10
ATCC35198
Reaction
?
Inositol
The data for the type strain are in accordance with those previously reported (Dong and Cai 2001); ?positive reaction; negative reaction
NC
R
Salicin
a
Test item a
4
5
6
7
Days
Fig. 2 Effect of B. bacteriovorus strain H16 on the growth of P. penneri strain NC. A: strain NC only in the control group, B: strain NC in the treatment group, C: strain H16 in the treatment group
observed in the control shrimps (Online Supplementary Fig. 1); (B) the same bacterial strain NC, identified phenotypically and by molecular analysis, was re-isolated from the experimentally diseased shrimps. In vitro bacteriolytic effect The in vitro effect of B. bacteriovorus strain H16 on the growth of the NC isolate is shown in Fig. 2. The cell density of the NC isolate was significantly lower
Prey range The lytic activity of B. bacteriovorus strain H16 against Proteus strains is shown in Fig. 3. The data indicated that strain H16 had good abilities to lyse Proteus strains besides strain NC. The maximum plaque no. was recorded with strain NC, followed by Proteus sp. ZL0057 (279 PFU), P. vulgaris TWN3 (270 PFU), Proteus sp. BYK000419 (238 PFU), P. mirabilis ZL003 (233 PFU), P. mirabilis ZXS02 (202 PFU), P. mirabilis BYK64291 (187 PFU), Proteus sp. BYK00098 (186 PFU) and P. Mirabilis BYK64285 (107 PFU). Therefore, B. bacteriovorus has a bacteriolytic activity against a range of pathogenic Proteus strains.
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can confer significant protection against P. penneri infection in farmed P. vannamei.
450
Plaques (PFU)
400 350 300 250
Discussion
200 150 100 50
C N
42
91 TW N 3 ZL 00 BY 57 K0 00 41 BY 9 K0 00 98
BY
K6
85
2 BY
K6
42
S0 ZX
ZL
00
3
0
Strain
Fig. 3 Bacteriolytic activity of B. bacteriovorus strain H16 against Proteus pathogens
100
Control Treatment 1 Treatment 2
Cumulative mortality (%)
90 80 70 60 50 40 30 20 10 0 0
1
2
3
4
5
6
7
Days
Fig. 4 Protective efficacy of B. bacteriovorus strain H16 against P. penneri infection in Pacific white shrimps. Control: 0 PFU mL-1 B. bacteriovorus in water; Treatment 1: 5 9 103 PFU mL-1 B. bacteriovorus in water; Treatment 2: 1 9 104 PFU mL-1 B. bacteriovorus in water
Protective efficacy The protective efficacy of B. bacteriovorus strain H16 against P. penneri infection in shrimps is shown in Fig. 4. The data suggested a positive protective effect of B. bacteriovorus strain H16 in controlling P. penneri infection. Relative percentage survivals of 58.0 and 78.6 % were obtained against the challenge with the NC isolate in shrimps at concentrations of 5 9 103 and 1 9 104 PFU mL-1 for 7 days, respectively. The death of all the test shrimps was caused by isolate NC as determined by bacterial isolation and identification (data not shown). Thus, B. bacteriovorus
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Red body disease is known as a significant economic damage in the cultured shrimp industry in China, India, the Philippines and other countries in Southeast and East Asia (Alapide-Tendencia and Dureza 1997; Jayasree et al. 2006; Zheng et al. 2011; Felix et al. 2011). Thus, more attention should be paid to this pathogen and its control. So far, several virulent bacteria such as V. alginolyticus and V. parahaemolyticus have been reported to cause red body disease of farmed P. vannamei (Zhou et al. 2003; Fan et al. 2006; Chen et al. 2012). However, no information is available about P. penneri infection in farmed shrimps. In this study, we isolated a virulent strain, P. penneri NC, that caused healthy shrimps to die acutely with the typical red body disease symptom of body reddening (Chen et al. 2012) (Online Supplementary Fig. 1). In addition, we further assayed the bacteriolytic effect and predatory spectrum of B. bacteriovorus towards the Proteus strains, as well as its protective efficacy towards P. penneri infection in shrimps. As far as we know, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus. Proteus strains are well documented as shrimp and fish pathogens. These bacteria have numerous virulence factors including fimbriae, flagella, outer membrane proteins, lipopolysaccharide, capsule antigen, urease, immunoglobulin A proteases, hemolysins and amino acid deaminases, which enable them to cause pathological events (Ro´zalski et al. 1997). In the present study, the NC isolate was found to be virulent to healthy P. vannamei with a mortality of 93 % in a challenge study. This further demonstrates the potential threat of Proteus species to aquaculture. Apart from the virulence of the NC isolate, there might be other causes underlying the incidence of red body disease, such as over utilization of shrimp production areas, intensity of stocking rate and over use of antibiotics and chemicals (Rajinikanth et al. 2010); these should also be raised as concerns. There is limited information on the treatment of diseases caused by P. penneri as a result of its multidrug resistance (Stock 2003). The predatory behaviour
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of Bdellovibrio spp. makes them attractive candidates for a number of applications in the biological control of bacterial contaminations (Yair et al. 2003), i.e. reducing Vibrio populations in aquatic waters (Wen et al. 2009), as well as in the improvement of shrimp performances (Xu et al. 2007). Thus, the use of Bdellovibrio is widely expected to prevent and control bacterial diseases in shrimps (Zhou et al. 2009). For application of such bacteria as probiotics during shrimp farming, it is essential to obtain data about their bacteriolytic activity and protection against P. penneri. In our study, B. bacteriovorus was found to significantly reduce the cell density of P. penneri by 99.9 % after inoculation at a final cell density of 5 9 103 PFU mL-1 (Fig. 2) and to be capable of lysing all the tested pathogenic Proteus strains (Fig. 3). This is presumably due to its ability to penetrate and lyse the prey cell wall (Crossman et al. 2013). In addition, B. bacteriovorus also led to a relative percent survival of 58.0 and 78.6 % against experimental P. penneri infection in P. vannamei (Fig. 4). This can be presumptively attributed to reduction of the pathogen numbers due to bacteriolysis by B. bacteriovorus H16. In conclusion, the present study for the first time reports a P. penneri infection in P. vannamei and supports the proposal of B. bacteriovorus as a potential probiotic agent for the biocontrol of P. penneri infection in shrimps. Acknowledgments This work has been financially supported by the Shanghai University Laboratory Technical Team Construction Program, Shangxi Science and Technology Attack Program (No. 20120311025-4), and Jiangsu Agricultural Science and Technology Support Program (No. BE2013366).
References Alapide-Tendencia EV, Dureza LA (1997) Isolation of Vibrio spp. from Penaeus monodon (Fabricius) with red disease syndrome. Aquaculture 154(2):107–114 Altwegg M, Zollinger-Iten J (1987) Identification of Enterobacteriaceae, Aeromonas spp. and Plesiomonas shigelloides with ATB 32GN system. J Microbiol Meth 7:103–109 Baulny MOD, Quentel C, Fournier V, Lamour F, Gouvello RL (1996) Effect of long-term oral administration of b-glucan as an immunostimulant or an adjuvant on some non-specific parameters of the immune response of turbot Scophthalmus maximus. Dis Aquat Organ 26:139–147
429 Briggs M, Funge-Smith S, Subasinghe R, Phillips M (2004) Introductions and movement of Penaeus vannamei and Penaeus stlirostris in Asia and the Pacific. FAO Fisheries Technical Paper, 1-12 pp Bucke D (1989) Histology. In: Austin B, Austin DA (eds) Methods for the microbiological examination of fish and shellfish. Wiley, New York, pp 69–97 Cao H, Li Y, Lei Y, Wu J, Xu H (2007) Isolation, identification, phylogenetic analysis and related properties of a pathogen in Silurus meridionalis Chen. Acta Microbiol Sin 47(1):1–6 Cao H, He S, Lu L, Hou L (2010) Characterization and phylogenetic analysis of the bitrichous pathogenic Aeromonas hydrophila isolated from diseased Siberian sturgen (Acipenser baerii). Isr J Aquacult-Bamid 62:182–189 Cao H, He S, Wang H, Hou S, Lu L, Yang X (2012) Bdellovibrios, potential biocontrol bacteria against pathogenic Aeromonas hydrophila. Vet Microbiol 154:413–418 Chen J, Zhu N, Kong L, Ding X, Zheng T, Du J (2012) Molecular characteristics and antimicrobial sensitivity of bacterial pathogen from the outbreak of Litopenaeus vannamei red-body disease. J Fish China 36(12):1891–1900 Crossman LC, Chen H, Cerden´o-Ta´rraga A, Brooks K, Quail MA, Pineiro SA, Hobley L, Sockett RE, Bentley SD, Parkhill J, Williams HN, Stine OC (2013) A small predatory core genome in the divergent marine Bacteriovorax marinus SJ and the terrestrial Bdellovibrio bacteriovorus. ISME J 7:148–160 Dong XZ, Cai MY (2001) General manual of systematic and determinative bacteriology. Science Press, Beijing 97 pp Engler HD, Troy K, Bottone EJ (1990) Bacteremia and subcutaneous abscess caused by Proteus penneri in a neutropenic host. J Clin Microbiol 28:1645–1646 Fan JF, Li WZ, Zang HM, Wang B, Song L (2006) The pathogen of red body disease in Litopenaeus vannamei. Acta Hydrobiol Sin 30(6):742–746 Felix F, Nugroho T, Silalahi S, Octavia Y (2011) Molecular characteristics of Vibrio sp. causing Black Tiger Prawn (Penaeus monodon) disease in Sumatra and Java shrimp ponds by 16S rDNA sequencing. J Agr Tech 7(3):679–694 Geng Y, Wang K, Chen D, Huang X, He M, Yin Z (2010) Stemptrophomonas maltophilia, an emerging opportunist pathogen for cultured channel catfish, Ictalurus punctatus, in China. Aquaculture 308:132–135 Harikrishnan R, Balasundaram C, Heo M (2010) Effect of probiotics enriched diet on Paralichthys olivaceus infected with lymphocystis disease virus (LCDV). Fish Shellfish Immunol 29:868–874 Jayasinghe CVL, Ahmed SBN, Kariyawasam MGIU (2008) The isolation and identification of Vibrio species in marine shrimps of Sri Lanka. J Food Agric 1(1):36–44 Jayasree L, Janakiram P, Madhavi R (2006) Characterization of Vibrio spp. associated with diseased shrimp from culture ponds of Andhra Pradesh (India). J World Aquacult Soc 37(4):523–532 Kaistha N, Bansal N, Chander J (2011) Proteus penneri lurking in the intensive care unit: an important often ignored nosocomial pathogen. Indian J Anaesth 55:411–413 Kishore J (2012) Isolation, identification & characterization of Proteus penneri-a missed rare pathogen. Indian J Med Res 135(3):341–345
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430 Mandal S, Mandal M, Pal NK, Halder PK, Basu PS (2002) R-factor in Proteus vulgaris from ulcerative disease of fish Channa punctatus. Indian J Exp Biol 40(5):614–616 Marine Products Export Development Authority, Network of Aquaculture Centres in Asia-Pacific (2003) Shrimp Health Management Extension Manual. MPEDA house, Cochin, India. pp 23 Ministry of Agriculture and Fisheries Bureau of China (2011) China Fisheries Statistical Yearbook. China Agriculture Press, Beijing, China. pp28 Orozova P, Sirakov I, Petkov I, Crumlish M, Austin B (2012) Recovery of Aeromonas hydrophila associated with bacteraemia in captive snakes. FEMS Microbiol Lett 334(1):22–26 Qi Z, Zhang X, Boon N, Bossier P (2009) Probiotics in aquaculture of China—Current state, problems and prospect. Aquaculture 290:15–21 Rajinikanth T, Ramasamy P, Ravi V (2010) Efficacy of probiotics, growth promotors and disinfectants in shrimp grow out farms. World J Fish Mar Sci 2(3):208–215 Robertson PAW, Calderon J, Carrera L, Stark JR, Zherdmant M, Austin B (1998) Experimental Vibrio harveyi infections in Penaeus vannamei larvae. Dis Aquat Organ 32:151–155 Ro´zalski A, Sidorczyk Z, Kotełko K (1997) Potential virulence factors of Proteus bacilli. Microbiol Mol Biol Rev 61(1):65–89 Ro´zalski A, Kwil I, Torzewska A, Baranowska M, Staczek P (2007) Proteus bacilli: features and virulence factors. Postepy Hig Med Dosw 61:204–219 Shu XH, Xiao KY, Jin XL, Chen KY, Huang ZJ (1997) Studies on the pathogenic bacteria of the rotten-skin disease of bullfrog. J Fish China 21(suppl):71–76 Snyder AR, Williams HN, Baer ML, Walker KE, Stine OS (2002) 16S rDNA sequence analysis of environmental Bdellovibrio-and-like organisms (BALO) reveals extensive diversity. Int J Syst Evol Microbiol 52:2089–2094 Stock I (2003) Natural antibiotic susceptibility of Proteus spp., with special reference to P. mirabilis and P. penneri strains. J Chemother 15(1):12–26 Stolp H, Starr MP (1963) Bdellovibrio bacteriovorus gen. et sp. n., a predatory, ectoparasitic and bacteriolytic microorganism. Antonie Van Leeuwenhoek 29:217–248 Wakida-Kusunoki AT, Amador-del Angel LE, Alejandro PC, Brahms CQ (2011) Presence of Pacific white shrimp
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Antonie van Leeuwenhoek (2014) 105:423–430 Litopenaeus vannamei (Boone, 1931) in the Southern Gulf of Mexico. Aquat Invasions 6(Suppl):S139–S142 Wang G, Wang Y (1998) Pathology of the caverned disease in soft-shelled turtle (Trionyx sinensis). Acta Univ. Agric 26(4):82–86 Boreali-occidentalis Wang L, Chen Y, Huang H, Huang Z, Chen H, Shao Z (2013) Isolation and identification of Vibrio campbellii as a bacterial pathogen for luminous vibriosis of Litopenaeus vannamei. Aquac Res. doi:10.1111/are.12191 Wen C, Lai X, Xue M, Huang Y, Li H, Zhou S (2009) Molecular typing and identification of Bdellovibrio-and-like organisms isolated from seawater shrimp ponds and adjacent coastal waters. J Appl Microbiol 4:1154–1162 Xu B, Ji WH, Xu H (1992) Proteus vulgaris: a new pathogen in Penaeus orientalis. J Fish China 16(2):130–136 Xu Q, Li J, Liu Q, Wang Q (2007) Effects of Bdellovibrio bacteriovorus and Rhodotorula glutinis on growth and non-specific immune competence of Fenneropenaeus chinensis. Mar Fish Res 28(5):42–47 Yair S, Yaacov D, Susan K, Jurkevitch E (2003) Small eats big: ecology and diversity of Bdellovibrio and like organisms, and their dynamics in predator-prey interactions. Agronomie 23:433–439 Zhang Q, Xiong Q, Xiao L, Yang H, Yang X (2005) A pathogen isolated from skin-ulcer Pseudosciaena crocea-Proteus mirabilis ZXS02 strain. J Fish China 29(6):824–830 Zhang X, Chen C, Yan B, Fang H, Qin G, Xu J (2009) Phenotypic and molecular characterization of pathogenic Vibrio parahaemolyticus isolated from Penaeus vannamei. Oceanologia et Limnologia Sini 5:654–662 Zheng T, Kong L, Zhu N (2011) Epidemiology and control strategy of red body disease of Penaeus vannamei in Zhejiang, China. Sci Fish Farm 12:48–49 Zhou Y, Zhang B, Chen X, Qian J (2003) Preliminary studies on the red body disease in Penaeus vannamei. Mar Sci 27(5):61–65 Zhou Q, Li K, Xie J, Liu B (2009) Role and functions of beneficial microorganisms in sustainable aquaculture. Bioresour Technol 100:3780–3786 Zhou J, Fang W, Yang X, Zhou S, Hu L, Li X, Qi X, Su H, Xie L (2012) A nonluminescent and highly virulent Vibrio harveyi strain is associated with ‘‘bacterial white tail disease’’ of Litopenaeus vannamei Shrimp. PLoS ONE 7(2):e29961
SHORT COMMUNICATION
Identification of a Proteus penneri isolate as the causal agent of red body disease of the cultured white shrimp Penaeus vannamei and its control with Bdellovibrio bacteriovorus Haipeng Cao • Shan He • Liqun Lu Xianle Yang • Baiyao Chen
•
Received: 25 September 2013 / Accepted: 15 November 2013 / Published online: 22 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Bacteriosis has become a major economic problem in the farming of the Pacific white shrimp Penaeus vannamei. However, no definitive data are available about Proteus penneri infection in cultured P. vannamei and its control. In this study, a virulent strain NC was isolated from diseased P. vannamei suffering from red body disease and identified as a P. penneri isolate through phylogenetic analysis and ATB 32GN system. A phylogenetic constructed tree using the neighbour-joining method identified the NC isolate as a P. penneri strain. In addition, Bdellovibrio
bacteriovorus conferred significant protection against P. penneri: it exhibited significant bacteriolytic effects on the pathogenic P. penneri, had a wide prey range towards Proteus pathogens, and displayed a good protective efficacy on experimental P. penneri infection in P. vannamei. To the best of our knowledge, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus. Keywords Penaeus vannamei Proteus penneri Bdellovibrio bacteriovorus Red body disease Control
Haipeng Cao and Shan He are contributed equally to this work.
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0079-y) contains supplementary material, which is available to authorized users. H. Cao (&) S. He L. Lu X. Yang Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture of P. R. China, Shanghai Engineering Research Center of Aquaculture, Shanghai University Knowledge Service Platform, Shanghai Ocean University Aquatic Animal Breeding Center (ZF1206), National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, People’s Republic of China e-mail: [email protected] B. Chen (&) Marine and Fisheries Research Institute of Lianyungang, Lianyungang 222044, Jiangsu, People’s Republic of China e-mail: [email protected]
The Pacific white shrimp Penaeus vannamei is widely distributed and cultivated in Hawaii, South Carolina, Texas, China, Thailand, Vietnam, Brazil, Mexico, Venezuela, Peru and Puerto Rico (Briggs et al. 2004; Wakida-Kusunoki et al. 2011). Especially in China, P. vannamei has become the most important commercial shrimp species and has brought a great profit in recent years with annual production of nearly 23.8 million tons (Ministry of Agriculture and Fisheries Bureau of China 2011). However, bacterial infection has become a major economic problem in P. vannamei farming (Wang et al. 2013). For example, Vibrio harveyi has been recognized as an important and destructive pathogen for P. vannamei, causing widespread outbreaks of the bacterial white tail disease (Robertson
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et al. 1998; Zhou et al. 2012). Thus, bacteriosis should be given more attention for the sustainable development of P. vannamei farming industry. Proteus species have been well documented to be associated with the mortality of P. orientalis (Xu et al. 1992), Rana catasbeiana (Shu et al. 1997), Trionyx sinensis (Wang and Wang 1998), Channa punctatus (Mandal et al. 2002), Pseudosciaena crocea (Zhang et al. 2005) and Silurus meridionalis Chen (Cao et al. 2007). The diseases caused by Proteus species are usually associated with their production of virulence factors, such as adherence, invasiveness, swarming phenomenon, hemolytic activity, urea hydrolysis, proteolysis and endotoxicity (Ro´zalski et al. 2007). P. penneri is an invasive but often ignored pathogen and is capable of causing major infectious diseases such as human bacteremia and subcutaneous abscess (Engler et al. 1990; Kaistha et al. 2011; Kishore 2012). However, there are no definitive data on P. penneri infection in cultured P. vannamei. At present, Proteus infections can be partially controlled by farmers with the crude application of antibiotics such as norfloxacin and gentamycin (Zhang et al. 2005; Cao et al. 2007). However, antibiotic treatment is cost-prohibitive to farmers in many undeveloped and developing countries, and may be detrimental to the environment and human health (Harikrishnan et al. 2010). Bdellovibrio species have been well regarded as predatory bacteria which attach to Gram-negative bacteria, penetrate through the cell wall to form a bdelloplast and multiply (Snyder et al. 2002). These bacteria play important roles in reducing microbial density and altering microbial communities through predation (Cao et al. 2012). Because of their intrinsic ability to lyse prey cells, these bacteria have been considered as potential agents in controlling aquatic pathogens (Qi et al. 2009). However, little information is available about Bdellovibrio strains as biocontrol agents for Proteus pathogens. In this paper, a virulent P. penneri strain was isolated from diseased P. vannamei suffering from red body disease in Xiaoshan, Zhejiang China. Phenotypic characterization, taxonomic position and control of the strain with the predatory bacterium Bdellovibrio bacteriovorus were examined. As far as we know, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus.
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Materials and methods White shrimp samples As recommended by Jayasinghe et al. (2008), 26 diseased P. vannamei (averaging 15.1 ± 1.3 g in weight) suffering from red body disease, characterized by the reddening of the shrimp body as described by Chen et al. (2012), were taken into sterile bags, kept in ice during transport to the laboratory from one single site of a P. vannamei farm in Xiaoshan, Zhejiang China in July 2012. In this farm, with 280 acres of ponds, P. vannamei was stocked at an initial rearing density of 60,000 juvenile shrimps per acre. The water quality of the outbreak ponds was pH 8.16, 6.5 mg L-1 of dissolved oxygen, 0.45 mg L-1 of total ammonia and 0.3 mg L-1 of nitrite. This was the farm’s first disease outbreak and it couldn’t be effectively controlled although penicillin, enrofloxacin, norfloxacin had been used. Isolation of bacteria Each sampled moribund P. vannamei was disinfected externally with 75 % alcohol and dissected in the laboratory. After a careful microscopic examination of organs such as gills and hepatopancreas for parasites, and viruses by electron microscropy, a 0.2 g hepatopancreas sample of each shrimp was cut and directly streaked onto nutrient agar (NA) plates (Sinopharm Chemical Reagent Co., Ltd.) as recommended by Geng et al. (2010). After incubation for 24–48 h at 28 °C, the dominant uniform isolates were purified by streaking and re-streaking onto NA plates. Only the dominant isolates with dense virtually pure culture growth on NA plates were obtained, as recommended by Orozova et al. (2012). Pure isolates were stored at -80 °C supplemented with 15 % glycerol. A representative stock, strain NC, was used as the test organism in the present study. Molecular identification of bacteria The extraction of genomic DNA of isolate NC, as well as PCR amplification of its 16S rRNA gene and sequencing were performed according to Cao et al. (2010). The near complete 16S rRNA gene sequence was assembled using MegAlign, Editseq and Seqman
Antonie van Leeuwenhoek (2014) 105:423–430
software with a Power Macintosh computer. A search was done against the National Centre for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST) program. The phylogenetic tree from the near complete 16S rRNA gene sequence of the isolate and its homologous sequences was further constructed using the neighbour-joining method. Phenotypic identification of the isolate using the ATB 32GN system The isolate NC was identified phenotypically using the ATB 32GN system as recommended by Altwegg and Zollinger-Iten (1987). Briefly, isolate NC was grown on NA plates (Sinopharm Chemical Reagent Co., Ltd.) at 28 °C for 24 h, and a bacterial suspension was then used to inoculate the API ID32GN strip (Bio-Merieux, SA) following the manufacturer’s instruction. The strip was incubated at 28 °C and observed after 48 h for checking against the API identification index and database. The type strain P. penneri ATCC35198 was used as the control. Bacterial virulence assay After a careful examination of external appearance, gut condition, growth situation, physical behaviour and feeding trends as recommended by the Marine Products Export Development Authority & Network of Aquaculture Centres in Asia–Pacific (2003), 240 healthy white shrimps (averaging 10.1 ± 0.6 g in weight) were obtained from Pinghu Aquaculture Co., Ltd. in Zhejiang China, and were respectively maintained in 6 aquaria (40 shrimps per aquarium) supplied with 100 L aerated filtered farm water at 28 °C for 14 days. Prior to the bacterial virulence assay, P. penneri strain NC was inoculated into 100 mL nutrient broth (NB) according to Cao et al. (2010), incubated at 28 °C with shaking at 200 rpm for 24 h, enumerated by tenfold serial dilutions in sterile distilled water and spread on NA plates. 40 healthy shrimps were challenged by being immersed in 100 L of water containing P. penneri isolate NC at a final cell density of 5.0 9 106 CFU mL-1 as described by Zhang et al. (2009). Another 40 healthy shrimps were unchallenged as the control. Each treatment was conducted in triplicate. The experimental shrimps were kept at 28 °C and observed
425
daily for 7 days. Any dead shrimps were removed immediately and sent to the laboratory for pathogen examination according to Bucke (1989) to find if the challenging strain was re-isolated, and the signs and mortalities were recorded. In vitro bacteriolytic effect of B. bacteriovorus on the isolate Bdellovibrio bacteriovorus strain H16, previously isolated from the gut of Acipenser baerii (identified morphologically and molecularly by Cao et al. (2012), and stored at -80 °C supplemented with 15 % glycerol), was used in our study. Its cell density was determined using the double-layer agar plating method as described by Stolp and Starr (1963). Briefly, B. bacteriovorus H16 was diluted serially tenfold in sterile distilled water. 100 lL aliquots of serial tenfold dilutions and 200 lL of a suspension of isolate NC (1.0 9 1010 CFU mL-1) were added to 10 mL of 0.6 % agar melted and kept at 55 °C, mixed and poured onto a pre-prepared 1.5 % agar plate until solidification. The double-layer agar was then incubated at 30 °C for 4 days, and the plaques were recorded. The in vitro bacteriolytic assay was conducted in triplicate and was carried out in six 250 mL glass flask with 98 mL of autoclaved diluted nutrient broth (DNB). In each flask of the treatment group, 1 mL of a suspension of isolate NC (with a final cell density of 5 9 106 CFU mL-1) and 1 mL of B. bacteriovorus H16 (with a final cell density of 5 9 103 PFU mL-1), were independently inoculated into 98 mL of DNB as recommended by Cao et al. (2012), then the mixtures were incubated at 30 °C with shaking at 200 rpm. Each flask in the control group only consisted of the isolate at a final cell density of 5 9 106 CFU mL-1, incubated as described above. Cell growth of the isolate and B. bacteriovorus H16 were respectively measured using the serial dilution and spread plate method described above and the double-layer agar plating method at 24 h intervals. Lytic activity of B. bacteriovorus against Proteus strains The lysis assay of B. bacteriovorus against Proteus strains was conducted in triplicate. Eight pathogenic
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strains of Proteus species (P. mirabilis strain ZL003, P. mirabilis strain ZXS02, P. mirabilis strain BYK64285, P. mirabilis strain BYK64291, P. vulgaris strain TWN3, Proteus sp. strain ZL0057, Proteus sp. strain BYK000419 and Proteus sp. strain BYK00098) were obtained from the National Pathogen Collection Centre for Aquatic Animals, P.R. China. The bacteriolytic activity of B. bacteriovorus H16 at a density of 5 9 103 PFU mL-1 was checked against the eight pathogenic Proteus strains by the double-layer agar plating method (Stolp and Starr 1963). The bacteriolytic activity of B. bacteriovorus H16 against the NC isolate served as the control. Plaques were observed and recorded on double-layer agar plates after 4 days of incubation at 30 °C. Protective efficacy assay The protective efficacy assay was conducted in triplicate. Healthy shrimps (averaging 10.1 ± 0.6 g in weight) were obtained from Pinghu Aquaculture Co., Ltd. in Zhejiang China, and maintained in 9 glass aquaria supplied with aerated filtered farm water at 28 °C throughout the experiment. Each aquarium, containing 100 L farm water without water recirculation, was stocked with 40 healthy shrimps selected at random. Immediately after all the shrimps were bath challenged through exposure to isolate NC at a final cell density of 5 9 106 CFU mL-1, 0, 5 and 10 mL of B. bacteriovorus H16 (at a final concentration of 3 9 108 PFU mL-1) were added to the 100 L aerated filtered farm water in the control and two treatment groups (ca. 0, 5 9 103 and 1 9 104 PFU mL-1 in water). Each group contained 3 aquaria. The test shrimps were observed daily for 7 days. Dead shrimps were removed immediately and sent to the laboratory for pathogen examination according to Bucke (1989) to find if the challenging strain was re-isolated, and mortalities were recorded each day for 7 days. Relative percentage survivals were calculated according to Baulny et al. (1996).
Antonie van Leeuwenhoek (2014) 105:423–430
Results Identification of the pathogenic isolate A single dominant strain, NC, was isolated from the diseased shrimps and identified by molecular and phenotypic methods as P. penneri. Its near complete 16S rRNA gene sequence (1,459 nucleotides) has been submitted to GenBank database with the accession no. KC113042. Similarities between the 16S rRNA gene sequence of the NC isolate and those of P. penneri strains in the GenBank database were 99 % and the phylogenetic tree constructed using the neighbourjoining method demonstrated isolate NC to be a P. penneri strain (Fig. 1). In addition, the ATB 32GN system identified isolate NC as a P. penneri strain (Table 1) as it showed an identity of 96.9 % with the type strain ATCC35198 in phenotypic characterization, which confirmed the molecular identification. No parasites or viruses were detected in the diseased shrimps from which isolate NC was obtained. The NC isolate was confirmed as the pathogen of this disease according to Koch’s postulates: (A) the NC isolate was virulent to healthy shrimps in an experimental challenge (see Methods). The death of the challenged shrimps began to occur after 1 day and the rate increased gradually over time. In total 93 % of the shrimps challenged with the NC isolate died acutely with the signs of reddening of the shrimp body, similar to the originally infected shrimps with red body disease, and no acute mortality or visible changes were 95 Proteus hauseri strain FFL10 [JN092597] Proteus hauseri strain FFL13 [JN092599] 96
98
Proteus mirabilis strain FUA1267 [JN102561] Proteus mirabilis strain FFL2 [JN092590]
80 30
Proteus mirabilis strain PPB3 [HM771658]
Proteus vulgaris type strain DSM 13387T [HE978268] 99
Proteus vulgaris strain ATCC 29905 [DQ885257] the NC isolate
99
Proteus penneri strain M2 [HQ259936] Proteus penneri strain NCTC 12737 [DQ885258] Proteus penneri strain ALK514 [KC456567]
60
100 Proteus penneri strain ALK515 [KC456568]
Statistical analysis Data are presented as the mean ± standard deviation (S.D.) for the indicated number of each assay. P \ 0.05 is considered statistically significant using one-way analysis of variance.
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0.002
Fig. 1 A 16S rRNA gene tree of 11 known bacteria and the NC isolate constructed using the neighbour-joining method. The bootstrap values (%) are shown besides the clades, accession numbers are indicated beside the names of strains, and scale bars represent distance values
Antonie van Leeuwenhoek (2014) 105:423–430 Table 1 Phenotypic characterization of isolate NC in comparison with the type strain P. penneri ATCC 35198
427
Test item
Reaction
Rhamnose Mannite
-
D-Ribose
R?
R?
Valerate
R-
R-
-
-
R
?
R?
R
-
R-
R
?
R?
-
R-
?
R
-
R
?
R
-
R
?
DL-Lactate Citrate L-Alanine
R
Histidine
R
L-Fucose
R-
R-
5-keto-gluconate
R-
R-
?
?
2-keto-gluconate
R
-
R-
R
-
R-
R
-
R-
R
-
R-
?
R? R?
R
-
R
-
Itaconic acid
R
-
L-Arabinose
R
-
R
-
R
-
R
-
R
-
Glycogen 3-hydroxy-butyrate 4-hydroxy-benzoate
Suberate
R
R
L-Serine
R
Propionate
R-
R-
L-Proline
R?
than that in the control (P \ 0.05) when strain H16 was inoculated at a final cell density of 5 9 103 PFU mL-1; the cell density of the NC isolate was reduced by 99.9 % after incubation for 7 days, compared with the control group. At the same time, the growth of the predator B. bacteriovorus H16 strain was improved at the expense of the NC isolate and a maximum cell density was reached after 3 days incubation. Thus, B. bacteriovorus has potential to be used for exclusion of P. penneri.
C
6 5 4 3 2 1 3
-
R
B
2
R
Sucrose
7
1
R-
-
R
RR?
8
0
Caprate
R-
-
R R?
R
?
R-
3-hydroxy-benzoate Acetate
9
0
R
Malonate
R R?
D-Sorbitol
Logarithm of cell density -1 (lg cells mL )
R-
-
ATCC35198a
R R?
Maltose
A
R-
NC
N-Acetylglucosamin D-Glucose
D-Melibiose
10
ATCC35198
Reaction
?
Inositol
The data for the type strain are in accordance with those previously reported (Dong and Cai 2001); ?positive reaction; negative reaction
NC
R
Salicin
a
Test item a
4
5
6
7
Days
Fig. 2 Effect of B. bacteriovorus strain H16 on the growth of P. penneri strain NC. A: strain NC only in the control group, B: strain NC in the treatment group, C: strain H16 in the treatment group
observed in the control shrimps (Online Supplementary Fig. 1); (B) the same bacterial strain NC, identified phenotypically and by molecular analysis, was re-isolated from the experimentally diseased shrimps. In vitro bacteriolytic effect The in vitro effect of B. bacteriovorus strain H16 on the growth of the NC isolate is shown in Fig. 2. The cell density of the NC isolate was significantly lower
Prey range The lytic activity of B. bacteriovorus strain H16 against Proteus strains is shown in Fig. 3. The data indicated that strain H16 had good abilities to lyse Proteus strains besides strain NC. The maximum plaque no. was recorded with strain NC, followed by Proteus sp. ZL0057 (279 PFU), P. vulgaris TWN3 (270 PFU), Proteus sp. BYK000419 (238 PFU), P. mirabilis ZL003 (233 PFU), P. mirabilis ZXS02 (202 PFU), P. mirabilis BYK64291 (187 PFU), Proteus sp. BYK00098 (186 PFU) and P. Mirabilis BYK64285 (107 PFU). Therefore, B. bacteriovorus has a bacteriolytic activity against a range of pathogenic Proteus strains.
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Antonie van Leeuwenhoek (2014) 105:423–430
can confer significant protection against P. penneri infection in farmed P. vannamei.
450
Plaques (PFU)
400 350 300 250
Discussion
200 150 100 50
C N
42
91 TW N 3 ZL 00 BY 57 K0 00 41 BY 9 K0 00 98
BY
K6
85
2 BY
K6
42
S0 ZX
ZL
00
3
0
Strain
Fig. 3 Bacteriolytic activity of B. bacteriovorus strain H16 against Proteus pathogens
100
Control Treatment 1 Treatment 2
Cumulative mortality (%)
90 80 70 60 50 40 30 20 10 0 0
1
2
3
4
5
6
7
Days
Fig. 4 Protective efficacy of B. bacteriovorus strain H16 against P. penneri infection in Pacific white shrimps. Control: 0 PFU mL-1 B. bacteriovorus in water; Treatment 1: 5 9 103 PFU mL-1 B. bacteriovorus in water; Treatment 2: 1 9 104 PFU mL-1 B. bacteriovorus in water
Protective efficacy The protective efficacy of B. bacteriovorus strain H16 against P. penneri infection in shrimps is shown in Fig. 4. The data suggested a positive protective effect of B. bacteriovorus strain H16 in controlling P. penneri infection. Relative percentage survivals of 58.0 and 78.6 % were obtained against the challenge with the NC isolate in shrimps at concentrations of 5 9 103 and 1 9 104 PFU mL-1 for 7 days, respectively. The death of all the test shrimps was caused by isolate NC as determined by bacterial isolation and identification (data not shown). Thus, B. bacteriovorus
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Red body disease is known as a significant economic damage in the cultured shrimp industry in China, India, the Philippines and other countries in Southeast and East Asia (Alapide-Tendencia and Dureza 1997; Jayasree et al. 2006; Zheng et al. 2011; Felix et al. 2011). Thus, more attention should be paid to this pathogen and its control. So far, several virulent bacteria such as V. alginolyticus and V. parahaemolyticus have been reported to cause red body disease of farmed P. vannamei (Zhou et al. 2003; Fan et al. 2006; Chen et al. 2012). However, no information is available about P. penneri infection in farmed shrimps. In this study, we isolated a virulent strain, P. penneri NC, that caused healthy shrimps to die acutely with the typical red body disease symptom of body reddening (Chen et al. 2012) (Online Supplementary Fig. 1). In addition, we further assayed the bacteriolytic effect and predatory spectrum of B. bacteriovorus towards the Proteus strains, as well as its protective efficacy towards P. penneri infection in shrimps. As far as we know, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus. Proteus strains are well documented as shrimp and fish pathogens. These bacteria have numerous virulence factors including fimbriae, flagella, outer membrane proteins, lipopolysaccharide, capsule antigen, urease, immunoglobulin A proteases, hemolysins and amino acid deaminases, which enable them to cause pathological events (Ro´zalski et al. 1997). In the present study, the NC isolate was found to be virulent to healthy P. vannamei with a mortality of 93 % in a challenge study. This further demonstrates the potential threat of Proteus species to aquaculture. Apart from the virulence of the NC isolate, there might be other causes underlying the incidence of red body disease, such as over utilization of shrimp production areas, intensity of stocking rate and over use of antibiotics and chemicals (Rajinikanth et al. 2010); these should also be raised as concerns. There is limited information on the treatment of diseases caused by P. penneri as a result of its multidrug resistance (Stock 2003). The predatory behaviour
Antonie van Leeuwenhoek (2014) 105:423–430
of Bdellovibrio spp. makes them attractive candidates for a number of applications in the biological control of bacterial contaminations (Yair et al. 2003), i.e. reducing Vibrio populations in aquatic waters (Wen et al. 2009), as well as in the improvement of shrimp performances (Xu et al. 2007). Thus, the use of Bdellovibrio is widely expected to prevent and control bacterial diseases in shrimps (Zhou et al. 2009). For application of such bacteria as probiotics during shrimp farming, it is essential to obtain data about their bacteriolytic activity and protection against P. penneri. In our study, B. bacteriovorus was found to significantly reduce the cell density of P. penneri by 99.9 % after inoculation at a final cell density of 5 9 103 PFU mL-1 (Fig. 2) and to be capable of lysing all the tested pathogenic Proteus strains (Fig. 3). This is presumably due to its ability to penetrate and lyse the prey cell wall (Crossman et al. 2013). In addition, B. bacteriovorus also led to a relative percent survival of 58.0 and 78.6 % against experimental P. penneri infection in P. vannamei (Fig. 4). This can be presumptively attributed to reduction of the pathogen numbers due to bacteriolysis by B. bacteriovorus H16. In conclusion, the present study for the first time reports a P. penneri infection in P. vannamei and supports the proposal of B. bacteriovorus as a potential probiotic agent for the biocontrol of P. penneri infection in shrimps. Acknowledgments This work has been financially supported by the Shanghai University Laboratory Technical Team Construction Program, Shangxi Science and Technology Attack Program (No. 20120311025-4), and Jiangsu Agricultural Science and Technology Support Program (No. BE2013366).
References Alapide-Tendencia EV, Dureza LA (1997) Isolation of Vibrio spp. from Penaeus monodon (Fabricius) with red disease syndrome. Aquaculture 154(2):107–114 Altwegg M, Zollinger-Iten J (1987) Identification of Enterobacteriaceae, Aeromonas spp. and Plesiomonas shigelloides with ATB 32GN system. J Microbiol Meth 7:103–109 Baulny MOD, Quentel C, Fournier V, Lamour F, Gouvello RL (1996) Effect of long-term oral administration of b-glucan as an immunostimulant or an adjuvant on some non-specific parameters of the immune response of turbot Scophthalmus maximus. Dis Aquat Organ 26:139–147
429 Briggs M, Funge-Smith S, Subasinghe R, Phillips M (2004) Introductions and movement of Penaeus vannamei and Penaeus stlirostris in Asia and the Pacific. FAO Fisheries Technical Paper, 1-12 pp Bucke D (1989) Histology. In: Austin B, Austin DA (eds) Methods for the microbiological examination of fish and shellfish. Wiley, New York, pp 69–97 Cao H, Li Y, Lei Y, Wu J, Xu H (2007) Isolation, identification, phylogenetic analysis and related properties of a pathogen in Silurus meridionalis Chen. Acta Microbiol Sin 47(1):1–6 Cao H, He S, Lu L, Hou L (2010) Characterization and phylogenetic analysis of the bitrichous pathogenic Aeromonas hydrophila isolated from diseased Siberian sturgen (Acipenser baerii). Isr J Aquacult-Bamid 62:182–189 Cao H, He S, Wang H, Hou S, Lu L, Yang X (2012) Bdellovibrios, potential biocontrol bacteria against pathogenic Aeromonas hydrophila. Vet Microbiol 154:413–418 Chen J, Zhu N, Kong L, Ding X, Zheng T, Du J (2012) Molecular characteristics and antimicrobial sensitivity of bacterial pathogen from the outbreak of Litopenaeus vannamei red-body disease. J Fish China 36(12):1891–1900 Crossman LC, Chen H, Cerden´o-Ta´rraga A, Brooks K, Quail MA, Pineiro SA, Hobley L, Sockett RE, Bentley SD, Parkhill J, Williams HN, Stine OC (2013) A small predatory core genome in the divergent marine Bacteriovorax marinus SJ and the terrestrial Bdellovibrio bacteriovorus. ISME J 7:148–160 Dong XZ, Cai MY (2001) General manual of systematic and determinative bacteriology. Science Press, Beijing 97 pp Engler HD, Troy K, Bottone EJ (1990) Bacteremia and subcutaneous abscess caused by Proteus penneri in a neutropenic host. J Clin Microbiol 28:1645–1646 Fan JF, Li WZ, Zang HM, Wang B, Song L (2006) The pathogen of red body disease in Litopenaeus vannamei. Acta Hydrobiol Sin 30(6):742–746 Felix F, Nugroho T, Silalahi S, Octavia Y (2011) Molecular characteristics of Vibrio sp. causing Black Tiger Prawn (Penaeus monodon) disease in Sumatra and Java shrimp ponds by 16S rDNA sequencing. J Agr Tech 7(3):679–694 Geng Y, Wang K, Chen D, Huang X, He M, Yin Z (2010) Stemptrophomonas maltophilia, an emerging opportunist pathogen for cultured channel catfish, Ictalurus punctatus, in China. Aquaculture 308:132–135 Harikrishnan R, Balasundaram C, Heo M (2010) Effect of probiotics enriched diet on Paralichthys olivaceus infected with lymphocystis disease virus (LCDV). Fish Shellfish Immunol 29:868–874 Jayasinghe CVL, Ahmed SBN, Kariyawasam MGIU (2008) The isolation and identification of Vibrio species in marine shrimps of Sri Lanka. J Food Agric 1(1):36–44 Jayasree L, Janakiram P, Madhavi R (2006) Characterization of Vibrio spp. associated with diseased shrimp from culture ponds of Andhra Pradesh (India). J World Aquacult Soc 37(4):523–532 Kaistha N, Bansal N, Chander J (2011) Proteus penneri lurking in the intensive care unit: an important often ignored nosocomial pathogen. Indian J Anaesth 55:411–413 Kishore J (2012) Isolation, identification & characterization of Proteus penneri-a missed rare pathogen. Indian J Med Res 135(3):341–345
123
430 Mandal S, Mandal M, Pal NK, Halder PK, Basu PS (2002) R-factor in Proteus vulgaris from ulcerative disease of fish Channa punctatus. Indian J Exp Biol 40(5):614–616 Marine Products Export Development Authority, Network of Aquaculture Centres in Asia-Pacific (2003) Shrimp Health Management Extension Manual. MPEDA house, Cochin, India. pp 23 Ministry of Agriculture and Fisheries Bureau of China (2011) China Fisheries Statistical Yearbook. China Agriculture Press, Beijing, China. pp28 Orozova P, Sirakov I, Petkov I, Crumlish M, Austin B (2012) Recovery of Aeromonas hydrophila associated with bacteraemia in captive snakes. FEMS Microbiol Lett 334(1):22–26 Qi Z, Zhang X, Boon N, Bossier P (2009) Probiotics in aquaculture of China—Current state, problems and prospect. Aquaculture 290:15–21 Rajinikanth T, Ramasamy P, Ravi V (2010) Efficacy of probiotics, growth promotors and disinfectants in shrimp grow out farms. World J Fish Mar Sci 2(3):208–215 Robertson PAW, Calderon J, Carrera L, Stark JR, Zherdmant M, Austin B (1998) Experimental Vibrio harveyi infections in Penaeus vannamei larvae. Dis Aquat Organ 32:151–155 Ro´zalski A, Sidorczyk Z, Kotełko K (1997) Potential virulence factors of Proteus bacilli. Microbiol Mol Biol Rev 61(1):65–89 Ro´zalski A, Kwil I, Torzewska A, Baranowska M, Staczek P (2007) Proteus bacilli: features and virulence factors. Postepy Hig Med Dosw 61:204–219 Shu XH, Xiao KY, Jin XL, Chen KY, Huang ZJ (1997) Studies on the pathogenic bacteria of the rotten-skin disease of bullfrog. J Fish China 21(suppl):71–76 Snyder AR, Williams HN, Baer ML, Walker KE, Stine OS (2002) 16S rDNA sequence analysis of environmental Bdellovibrio-and-like organisms (BALO) reveals extensive diversity. Int J Syst Evol Microbiol 52:2089–2094 Stock I (2003) Natural antibiotic susceptibility of Proteus spp., with special reference to P. mirabilis and P. penneri strains. J Chemother 15(1):12–26 Stolp H, Starr MP (1963) Bdellovibrio bacteriovorus gen. et sp. n., a predatory, ectoparasitic and bacteriolytic microorganism. Antonie Van Leeuwenhoek 29:217–248 Wakida-Kusunoki AT, Amador-del Angel LE, Alejandro PC, Brahms CQ (2011) Presence of Pacific white shrimp
123
Antonie van Leeuwenhoek (2014) 105:423–430 Litopenaeus vannamei (Boone, 1931) in the Southern Gulf of Mexico. Aquat Invasions 6(Suppl):S139–S142 Wang G, Wang Y (1998) Pathology of the caverned disease in soft-shelled turtle (Trionyx sinensis). Acta Univ. Agric 26(4):82–86 Boreali-occidentalis Wang L, Chen Y, Huang H, Huang Z, Chen H, Shao Z (2013) Isolation and identification of Vibrio campbellii as a bacterial pathogen for luminous vibriosis of Litopenaeus vannamei. Aquac Res. doi:10.1111/are.12191 Wen C, Lai X, Xue M, Huang Y, Li H, Zhou S (2009) Molecular typing and identification of Bdellovibrio-and-like organisms isolated from seawater shrimp ponds and adjacent coastal waters. J Appl Microbiol 4:1154–1162 Xu B, Ji WH, Xu H (1992) Proteus vulgaris: a new pathogen in Penaeus orientalis. J Fish China 16(2):130–136 Xu Q, Li J, Liu Q, Wang Q (2007) Effects of Bdellovibrio bacteriovorus and Rhodotorula glutinis on growth and non-specific immune competence of Fenneropenaeus chinensis. Mar Fish Res 28(5):42–47 Yair S, Yaacov D, Susan K, Jurkevitch E (2003) Small eats big: ecology and diversity of Bdellovibrio and like organisms, and their dynamics in predator-prey interactions. Agronomie 23:433–439 Zhang Q, Xiong Q, Xiao L, Yang H, Yang X (2005) A pathogen isolated from skin-ulcer Pseudosciaena crocea-Proteus mirabilis ZXS02 strain. J Fish China 29(6):824–830 Zhang X, Chen C, Yan B, Fang H, Qin G, Xu J (2009) Phenotypic and molecular characterization of pathogenic Vibrio parahaemolyticus isolated from Penaeus vannamei. Oceanologia et Limnologia Sini 5:654–662 Zheng T, Kong L, Zhu N (2011) Epidemiology and control strategy of red body disease of Penaeus vannamei in Zhejiang, China. Sci Fish Farm 12:48–49 Zhou Y, Zhang B, Chen X, Qian J (2003) Preliminary studies on the red body disease in Penaeus vannamei. Mar Sci 27(5):61–65 Zhou Q, Li K, Xie J, Liu B (2009) Role and functions of beneficial microorganisms in sustainable aquaculture. Bioresour Technol 100:3780–3786 Zhou J, Fang W, Yang X, Zhou S, Hu L, Li X, Qi X, Su H, Xie L (2012) A nonluminescent and highly virulent Vibrio harveyi strain is associated with ‘‘bacterial white tail disease’’ of Litopenaeus vannamei Shrimp. PLoS ONE 7(2):e29961
