Growth enhancement and protective potential of feed

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(NaFish), Pahang, Malaysia. It was stored at – 80 °C in 15% glycerol stock culture in the. Molecular Pathology Laboratory, Faculty of Veterinary Medicine, ...
Growth enhancement and protective potential of feed-based outer membrane proteins against vibriosis in Macrobrachium rosenbergii Abdullateef Ajadi, M. Y. Sabri, A. B. Dauda, M. Y. Ina-Salwany & A. H. Hasliza Aquaculture International Journal of the European Aquaculture Society ISSN 0967-6120 Aquacult Int DOI 10.1007/s10499-018-0244-4

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Author's personal copy Aquacult Int https://doi.org/10.1007/s10499-018-0244-4

Growth enhancement and protective potential of feed-based outer membrane proteins against vibriosis in Macrobrachium rosenbergii Abdullateef Ajadi 1,2 & M. Y. Sabri 1 & A. B. Dauda 3,4 & M. Y. Ina-Salwany 3 & A. H. Hasliza 5

Received: 29 August 2017 / Accepted: 18 January 2018 # Springer International Publishing AG, part of Springer Nature 2018

Abstract The use of antibiotics to curtail vibriosis, which is a major infectious disease, plaguing shrimp and prawn is rather becoming less effective and the need for a better alternative is expedient. The outer membrane proteins (OMPs) of V. alginolyticus were extracted, mixed with powdered commercial feed and fed to the prawns to evaluate its effect on growth performance and protective potential. Sixty prawns were divided into groups A, B and C of 10 prawns each, with two replicates in six (150 L) glass aquaria. Groups A, B and C were fed with OMPs mixed diet, with OMPs-Freund’s incomplete adjuvant mixed diet and OMPs or adjuvant free diet (control diet) respectively. All the prawns were weighed weekly, and haemolymph was collected to determine the total haemocyte count (THC) and phenoloxidase (PO) activity. At the end of the feeding trial, prawns were intramuscularly challenged with 50 μL of 107 CFU V. alginolyticus. The treated groups were significantly higher in growth performance and THC than the control group, but no significant difference between the groups in terms of PO activity and mortality rate. The study, however, submitted that oral administration of OMPs with or without adjuvant is a good growth promoter and has

* Abdullateef Ajadi [email protected]

1

Department of Veterinary Pathology & Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

2

Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Ilorin, Ilorin PMB 1515, Nigeria

3

Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

4

Department of Fisheries and Aquaculture, Federal University, Dutsin-Ma, Dutsin-Ma, Katsina State PMB 5001, Nigeria

5

Department of Preclinical, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

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the potential for protection against vibriosis in giant freshwater prawn (Macrobrachium rosenbergii). Keywords Vibriosis . Macrobrachium rosenbergii . OMPs . Growth . Protection

Introduction The consistent global growing demand for prawns has brought about the increase in production of the aquatic species. Meanwhile, Macrobrachium rosenbergii is the world’s largest known palaemonid and the most popularly cultured freshwater prawns with significant contributions to the world aquaculture (Wowor and Ng 2007). The species is a suitable crustacean for adding significant value to the economy with an annual estimate of more than $1 billion (FAO 2011). The ability to breed easily, excellent meat quality, high hatching rate and market value are among the special features of M. rosenbergii that motivate its production (Keysami & Mohammadpour 2013). However, this increased production comes with its attendant challenges, and the major one is disease outbreak. Vibriosis caused by several pathogenic species of Vibrio is one of the major bacterial diseases plaguing the shellfish and finfish aquaculture. This could be attributed to the ubiquity of the pathogenic causative agents (Lavilla-Pitogo et al. 1998). The disease is characterised by high mortality rate and its devastating effect is a humongous economic loss (Lightner 2005). Antibiotics do not seem to be effective in the treatment and control of this disease. Meanwhile, the incessant use of antibiotics employed to control the disease has resulted to a more virulent pathogen, antibiotic resistant strains and accumulation of drug residues in the tissue of the host (Thakur et al. 2003). This deleterious effect is not restricted to the host only but secondarily affects man when antibiotic laden prawn products are consumed by the people. This has also led to several rejections at international market of shrimp and prawn products associated with the issue of drug residues (FAO 2011). However, for sustenance and increase in production of giant freshwater prawn in order to meet the growing demand, ensure a wholesome food fish for consumption and use of man, it is pertinent to seek better and safer methods of disease prevention and control. Although many studies have reported the protective role of components of microbial cell wall in aquatic crustaceans such as lipopolysaccharides (Barman et al. 2013), peptidoglycan (Purivirojkul et al. 2006), outer membrane proteins (Maftuch et al. 2013) and beta-glucan (Bai et al. 2014), injection route of substance administration appears to be the most efficacious; it is, however, costly, laborious and predisposes prawns to additional stress, whereas oral and immersion are the most preferred and practicable routes (Smith et al. 2003). Little information is available on the oral administration of outer membrane proteins of Vibrio spp. in prawns. This, if found effective, can be a cheaper and less laborious route of administration. This present study demonstrated the effect of oral administration of Vibrio’s outer membrane proteins on the growth of M. rosenbergii and protective potential against infection caused by Vibrio alginolyticus.

Materials and methods Culture and identification of putative Vibrio alginolyticus The V. alginolyticus (strain 2144) used for this study was obtained from diseased fish (Oreochromis niloticus) in an outbreak of vibriosis at National Fish Health Research Centre

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(NaFish), Pahang, Malaysia. It was stored at – 80 °C in 15% glycerol stock culture in the Molecular Pathology Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia. The organism was sub-cultured on thiosulfate citrate bile salts sucrose (TCBS) agar and incubated overnight at 30 °C. The identification was made using colony morphology, Gram stain, Analytical Profile Index (API) 20E and polymerase chain reaction (PCR).

Extraction of outer membrane proteins The outer membrane proteins (OMPs) of V. alginolyticus were extracted with little modification of Sabri et al. (2000). The putative bacteria were cultured on thiosulfate citrate bile salt sucrose (TCBS) agar for 24 h at 30 °C. The grown culture was inoculated in tryptic soy broth (TSB) and incubated at 30 °C with gentle shaking for 24 h. The bacterial cells were harvested by centrifugation using Avanti J-26S XPI centrifuge (Beckman Coulter, USA) at 3500×g for 10 min at 4 °C. The cells were subsequently washed three times with 25 mL sterile phosphate buffered saline (PBS) and resuspended in 10 mL sterile PBS before they were subjected to disruption by intermittent sonic oscillation (Ika Labortechnic Sonicator, USA). The bacterial cells were exposed to eight sonication treatments for 30 s in each exposure at 80% capacity. The debris of the cell and unbroken cells were removed by centrifugation at 10000×g for 20 min. The supernatant was obtained and subsequently centrifuged using Optima XPN-100 ultracentrifuge (Beckman Coulter, USA) at 100000×g for 2 h at 4 °C. The pellet was resuspended in 2 mL of 1% (w/v) sodium lauryl sarcosinate (Sigma, USA) and incubated at room temperature for 2 h. The suspension was further centrifuged at 100000×g for 2 h at 4 °C, the supernatant was discarded and the pellet was resuspended in 100 μL sterile PBS and stored at – 20 °C. The concentration of OMPs in the final preparation was determined using bicinchoninic acid (BCA) protein microassay kit (Sigma, USA).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The OMPs extracted from V. alginolyticus were separated by SDS-PAGE using 12% (w/v) resolving gel and 4% stacking gel, a method modification of Laemmli (1970). OMPs sample (9.54 μg total protein) was diluted in sample buffer containing 10% SDS, 0.5 M Tris-HCl pH 6.8, 20% glycerol, 0.55 (w/v) bromophenol blue and 5% (v/v) β-mercaptoethanol at the same ratio and heated for 5 min at 95%. The resultant sample was loaded into the wells of 0.75-mm thick gel and electrophoresis was run for 1 h at 140 V. Upon completion, the gel was stained with 0.025% Coomassie brilliant blue. The molecular weights of the outer membrane proteins were outlined with the Blueye pre-stained protein ladder (GeneDirex).

Immunoblotting According to the method of Towbin et al. (1979), with little modification, the polypeptides contained in the product of unstained SDS-PAGE gel were transferred onto 0.45 μm pore size nitrocellulose membrane (Sigma). The transfer was carried out in a buffer known as FLASHBlot™ transfer buffer (Advansta, USA) at 200 V for 20 min. Upon transfer, the nitrocellulose membrane was air dried and later submerged in deionized water for 5 min. The water was removed, and the membrane was covered with working solution of Ponceau S stain. The stained membrane was kept at room temperature for 15 min with gentle agitation until the protein bands were discernible before it was washed with copious deionized water. The membrane was allowed to dry and kept at – 20 °C until further use. The membrane with protein

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was cut into a strip and washed with PBS/0.5% Tween 20 (PBST) three times, 5 min each after which the membrane was incubated for 1 h at 37 °C with blocking buffer (PBST+1% Bovine Serum Albumin (BSA)). It was washed three times with PBST for 2 min each and incubated with hyperimmune serum diluted with PBST at 37 °C for 2 h with gentle shaking. The membrane was washed again as described earlier and incubated with goat anti-rabbit conjugate diluted with PBS only at 37 °C for one and a half h with a few time shaking and later washed. It was further washed for 5 min with Tris-buffer saline (TBS pH 7.5) and placed in the substrate (TMB, Promega) for 30 min. It was later washed with water, then allowed to dry and kept in the dark.

Preparation of OMPs incorporated feed Commercial feed by Dindings Aqua Feeds (Malaysia) (crude protein 32%, crude fat 10%, crude fibre 5% and crude ash 10%) and recommended for finfish and shellfish was obtained from a reliable aquatic feed shop; this was against the background knowledge that giant freshwater prawns are omnivorous. The feed was finely ground with the use of a mini blender (Waring commercial blender, USA) and mixed evenly with 30 μg/kg of earlier extracted OMPs, OMPs combined with Freund’s Incomplete Adjuvant (FIA) and Phosphate Buffered Solution (PBS) respectively. The resultant feed mixtures were passed through the pelletizer to form feed pellets, air dried, cut into smaller sizes and kept at 4 °C. Kjeldahl method of proximate analysis was used to determine the percentage of crude proteins of the treatment and control feeds. The formulation of the pellet contained 30 μg/kg of OMPs (Maftuch et al. 2013). The formulated feed containing 30 μg/kg of OMPs, the feed with FIA and PBS were fed to the respective treatments twice daily at 4% body weight.

Experimental prawns and design Apparently, healthy adult freshwater prawns with the average weight of 26 ± 2 g were obtained from a farm in Sungai Dulang, Bagan Datoh, Perak and transported to UPM. The prawns were acclimatised for 1 week prior to the commencement of the experiment and haemolymph was randomly obtained to check for the presence of Vibrio spp. The prawns were divided into three groups A, B and C of 10 prawns each with two replicates in 6 (150 l) glass aquaria and a blank control group of 10 prawns only (Table 1). Group A was treated with OMPs mixed diet, group B with OMPs-FIA mixed diet, while group C was fed with non-OMPs or adjuvant diet (control diet). Groups A and B were fed for 7 days, alternated for 7 days with control diet and a booster dose for another 7 days while group C was fed throughout with control diet. All prawns were weekly weighed; haemolymph was collected to determine the total haemocyte count (THC) and Table 1 Experimental design Groups A B C Blank control

1 2 1 2 1 2 –

No of prawn

Treatment

Bacterial challenge

10 10 10 10 10 10 10

OMPs mixed feed

IM injection of V. alginolyticus

OMPS +FIA mixed feed

IM injection of V. alginolyticus

Commercial feed only

IM injection of V. alginolyticus

Commercial feed only

IM injection of PBS

50 μL of 1 × 107 CFU of V. alginolyticus was injected

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phenoloxidase activity (PO). Water parameters such as temperature, dissolved oxygen, pH and salinity were measured every other day using YSI (Yellow Spring Inc. Ohio, USA). All experimental prawns in groups A, B and C, were injected intramuscularly between the second and third abdominal segments with 50 μL of 107 CFU of V. alginolyticus and blank control with 50 μL of PBS using a 1-mL sterile syringe (25 gauge). (The V. alginolyticus was first cultured in a tryptone soy broth (TSB) (prepared according to the manufacturer’s instruction) and left in an incubating shaker overnight at 30 °C. This was then centrifuged at 603×g for 20 min; the supernatant was removed and the pellet was resuspended in 0.85% NaCl. This process was repeated two other times. The prepared V. alginolyticus solution was diluted with 0.85% NaCl to obtain a concentration of 1 × 107 using 0.5 McFarland standard). The prawns were injected and observed for 24 h (Maftuch et al. 2013).

Measurement of body weight Treatment and control groups and their replicates were maintained and reared in continuously aerated and recirculating system. All prawns were drawn from each group weekly to record the average weight of the prawn in each of the treatment.

Total haemocyte count (THC) The THC was measured by the modified method of Chiu et al. (2010). One hundred microliters of haemolymph was withdrawn from the heart of each sampled prawn using a 1-mL sterile syringe (25 gauge) and mixed with 900 μL of anticoagulant solution. A drop of the diluted haemolymph (haemolymph-anticoagulant mixture) was placed on Neubauer haemocytometer, and the number of haemocytes was counted microscopically. THC was calculated using the formula adopted by Maftuch et al. (2013). THC ¼ total no of counted haemocytes  dilution factor  2  104 No of square counted where (2 × 104) includes the depth and number of chambers.

Phenoloxidase (PO) activity The PO activity was spectrophotometrically measured by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) following the modified procedure of Li et al. (2010). One thousand microliters of diluted haemolymph (1:9) was centrifuged at 800×g for 20 min at 4 °C. The supernatant was kept for another observation, and the pellet was washed with PBS and centrifuged. The resultant pellet was resuspended gently in the 1-mL cacodylate-citrate buffer and then centrifuged as above. The supernatant was discarded, the pellet was resuspended in 200 μL cacodylate buffer, and the aliquot (cell suspension) was equally split into two (100 μL each) tubes. One tube was incubated for 10 min at room temperature with 50 μL of trypsin (1 mg/ mL−1), which served as an elicitor. Fifty microliters of L-DOPA was subsequently added followed by 800 μL of cacodylate buffer 5 min later. Fifty microliters of cacodylate buffer and 50 μL of LDOPA were added to the other aliquot (100 μL) the same way, and this was measured as PO background activity in all test conditions. The PO activity of the prawns was measured at 490 nm optical density using a spectrophotometer, and the results were expressed as dopachrome formation of 50 μL of haemolymph.

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Statistical analysis The data obtained were recorded in Microsoft Excel 2013 and statistical analyses were carried out using Medcalc for windows, version 16.8.4 (Medcalc software, Mariakerke, Belgium). All data were presented as mean plus or minus standard error. Levene’s test was carried out for homogeneity of variance and one-way analysis of variance (ANOVA) was employed to determine whether significant differences existed between the different groups for each parameter tested. Significant differences were indicated at P < 0.05 and Duncan’s multiple range test was used as post-hoc test.

Results The outcome of the tested parameters including bacterial identification, SDS-PAGE analysis, average weight gain, total haemocyte count, phenoloxidase activity and bacterial challenge were presented in this section.

Bacterial identification The identity of the bacteria was phenotypically confirmed by colony morphological analysis, revealing colonies that were large (2–3 mm in diameter), round, yellow, smooth, raised centre, regular but translucent periphery and opaque. The bacteria turned the green colour agar yellow, indicating they are sucrose fermenter. The growth of the organism had covered the entire petri dish by 24 h post-inoculation. Gram staining microscopic examination showed that the organisms were Gram-negative (pink) and bacilli (rod shape) with a polar flagellum. They appeared in the cluster, pair and rarely single. The organism was confirmed to be Vibrio alginolyticus using the AP 20 E software. The API profile number was 4146124, and the percentage identification was 97.7%. The PCR showed positive bands (using V. alginolyticus specific primers) at 800 bp.

SDS-PAGE analysis The outer membrane proteins of V. alginolyticus, when resolved in 12% SDS-PAGE, showed several bands of molecular weights of polypeptides (Fig. 1) ranging from 11 kDa to above 75 kDa. The major bands were observed at 32 and 42 kDa. The other minor bands were seen at 25, 48, 70, 73 and 75 kDa.

Immunoblotting Immunoblotting results revealed three antigenic bands. Two major polypeptide bands with molecular sizes of 42 and 32 kDa are strongly immunogenic to hyperimmune serum against V. alginolyticus, and one minor polypeptide band of 20 kDa is lightly immunogenic (Fig. 2).

Average weight gain The average growth of each group was weekly obtained. There was statistically a significant difference between treatment groups and the control group (P < 0.05) but not between the treatment groups. Growths were enhanced in the treatment groups more than the control group in Fig. 3.

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kDa

M

1

75 63 48 42 kDa 32 kDa

35 25

20 17 11 Fig. 1 Coomassie brilliant blue stained SDS-PAGE profile of sonicated OMPs. Lane M molecular mass marker, lane 1 OMPs of V. alginolyticus

Total haemocyte count The THC in treatment groups increased significantly more than the control group as shown in Fig. 4. This observation occurred in week 1, 2 and 3. Although there was an increment in all

1

M

kDa

75 63

42 kDa 32 kDa

48 35 25 20 17 11

Fig. 2 Immunoblot analysis of OMPs of V. alginolyticus with immunoreactivity of the polypeptides intensely showing at the molecular mass of 42, and 32 kDa, light immunoreactivity reaction at 20 kDa. Line 1 immunoreactive bands, lane M molecular mass marker

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Weight (g)

25 Group Cx Omp Omp+FIA

20 15 10 5 0 0

1 2 3 Weeks post-stimulation

4

Fig. 3 Weekly (mean ± SE) growth pattern of Macrobrachium rosenbergii in the three different groups. (Cxcontrol group; OMP-group fed with OMP only diet; OMP + FIA-group fed with OMP + FIA diet)

groups in week 3, the increment was more significant in treatment groups than the control. However, there was a drop in THC (2.08 ± 2 × 106 cells/mL) in week 4 across all the groups to almost same level as in pre-experimental week (2.55 ± 0.40 × 106 cells/mL). No significant difference (P > 0.05) between the OMPs and OMP + FIA groups at each sampling. 50

Total Haemocyte Counts

40

30

Group

20

Cx OMP OMP+FIA

10

0 0

1

2

3

Weeks post-stimulation

4

Fig. 4 Weekly (mean ± SE) THC obtained from the three groups. (Cx-control group; OMP-group fed with OMP only diet; OMP + Adj-group fed with OMP + FIA diet)

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Phenoloxidase (PO) activity There was no significant difference among the groups (Fig. 5). The result showed a significant weekly decrease in the activity of phenoloxidase in all the groups, with only slight decrease in the control group from week 2 to week 3.

Experimental challenge All the prawns in each group that were intramuscularly injected with V. alginolyticus (50 μL of 1.0 × 107 CFU) died 24 h post-injection. No distinguishing clinical sign was noticed except body flexion of the dead prawns.

Discussion The SDS-PAGE analysis of the OMPs of V. alginolyticus in this study showed diverse bands consisting of few major and several minor bands. The polypeptides of different molecular mass and the immunogenic activity observed demonstrated that the OMPs had the ability to induce an immune response and might have the potential protective capability against pathogens of their stereotypes. This is in agreement with Li et al. (2010), who reported that OMPK was a good antigen to elicit an immune response and could possess the ability to mount protection against V. harveyi. Pati et al. (1996) earlier reported that OMPs were agent that could effectively evoke the production of antibodies in the vaccinated animals. The OMPs of many Gram-negative bacteria, especially the ones that are of aquatic importance such as Aeromonas species (Thangaviji et al. 2012) and Vibrio species (Qian et al. 2008) have been studied and reported for their suitability as vaccine candidates. Components of microbial cell wall such as

OD (490 nm)

0.3

0.2 Group

Cx OMP OMP+FIA

0.1

0.0 1

2

3

Weeks post-stimulation Fig. 5 Weekly (mean ± SE) phenoloxidase activity of the three groups. (Cx-control group; OMP-group fed with OMP only diet; OMP + FIA-group fed with OMP + FIA diet)

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lipopolysaccharides (Sung et al. 2000), peptidoglycan (Purivirojkul et al. 2006), β-glucan (Scholz et al. 1999) and outer membrane proteins (Maftuch et al. 2013) have been reported to enhance growth and serve as good immunostimulants. However, limited information is available on the effect of oral administration of outer membrane proteins in giant freshwater prawns. This study demonstrated the enhancement of growth of prawns fed with OMPs containing diets. This was one of the features of immunostimulants that initiated the interests of many researchers and the potential benefits of repeated immunostimulation in terms of weight gain had been reported by Azad et al. (2005). The mean weight gain of treated groups was significantly higher than the control group (P < 0.05). This might be connected with enhanced activities of proteases and other enzymes, which enabled a fast nutrient absorption from the gut and aided digestion (Keysami et al. 2007). It might also be associated with an increase in crude protein in treatment feeds due to the addition of OMPs. Although earlier studies have reported the effects of microbial immunostimulants such as bacterin (Sung 1990), β-glucan (Barman et al. 2013) and peptidoglycan (Itami et al. 1998) on the growth of crustaceans either in terms of weight gains or body length, we are not aware of any information about the effect of outer membrane proteins on the growth of giant freshwater prawn, therefore this study might be the first to report such. Haemocytes play significant roles in recognition of antigens, phagocytosis, encapsulation, cytotoxicity, melanization and cell to cell communication (Johansson et al. 2000). It was demonstrated in this study that the administration of OMPs led to increase in the total haemocyte count of the prawns. This agrees with Maftuch et al. (2013), who reported a significant increase in THC observed in the group of shrimps treated with OMPs more than the control group. The more the number of haemocytes in the haemolymph, the better the chance to enhance more protection against infectious challenge (Bai et al. 2014). At the end of the third week where the booster dose was administered, the value of THC got to a peak (37–40 × 106 cells/mL) in the treatment groups. However, by the end of the fourth week (a week after the booster dose), the THC fell drastically to almost below the baseline (2.08 × 106 cells/mL) in all the groups. This decrease could be associated with extrinsic or environmental factors (Tseng and Chen 2004; Cheng and Wang 2001). The decrease in the mean THC in this study 7 days after booster dose was an indication that the increased THC induced by the administration of OMPs was transient. This conforms with the submission of Smith et al. (2003) that most immunostimulants employed to enhance resistance to disease in crustaceans are shortlived in nature. This finding is also in agreement with Sung et al. (1996) who reported the apparently immunomodulating effect of glucan Vibrio bacterin administered by immersion, lasted only for a maximum of 24 h before declining to the initial control level. Therefore, it is pertinent that in order to achieve the effective role of immunostimulant in growth and protection, there must be prolonged or booster administration. This agrees with Azad et al. (2005) that the enhancement of growth and induction of protective response in shrimp is better attained with a booster dose of the immunostimulants. This study also demonstrated the insignificant difference in the value of phenoloxidase activity among all the groups. It was observed that PO is not best measured in the absence of polysaccharide which is essential for the activation of prophenoloxidase to its active form (phenoloxidase) under the action of serine protease. This agrees with Söderhäll and Cerenius (1998) and Smith et al. (1984) that described phenoloxidase as the terminal enzyme which derivation from its inactive prophenoloxidase form was activated by peptidoglycan or lipopolysaccharide from bacteria and β-glucan from fungi through a recognition of pathogenassociated molecule pattern. The alteration in the mean value of the activity of the

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phenoloxidase might be due to the physiological state of the prawn and environmental factors (Liu and Chen 2004). The total mortality in all the groups could primarily be due to external stressors and probably reaction to stress from the injection site, coupled with existing low level of circulating haemocytes at the time of challenge to combat the bacterial invasion (Sahoo et al. 2007), thus, causing further depletion of circulating haemocytes. This study demonstrated that oral administration OMPs of V. alginolyticus with or without Freund’s incomplete adjuvant can be used as a growth promoter due to their effect on weight gain and may have the potential to protect the giant freshwater prawn against vibriosis when administered with unique antigen protection vehicle at an optimal dose and regimen. Therefore, further study is required to establish the optimal dose and right duration of administration. It is also recommended that PO activity should be examined on a daily basis. Acknowledgements We would like to thank Mr. Alli, a farmer, and members of staff of Aquaculture Research Station UPM, Puchong, Selangor, for their excellent technical assistance. Funding information This project was funded by the Ministry of Science, Technology and Innovation (MOSTI), Malaysia, under the vote no. 6363400.

References Azad IS, Panigrahi A, Gopal C, Paulpandi S, Mahima C, Ravichandran P (2005) Routes of immunostimulation vis-à-vis survival and growth of Penaeus monodon postlarvae. Aquaculture 248(1–4):227–234. https://doi. org/10.1016/j.aquaculture.2005.04.011 Bai N, Gu M, Zhang W, Xu W, Mai K (2014) Effects of β-glucan derivatives on the immunity of white shrimp Litopenaeus vannamei and its resistance against white spot syndrome virus infection. Aquaculture 426-427: 66–73. https://doi.org/10.1016/j.aquaculture.2014.01.019 Barman D, Nen P, Mandal SC, Kumar V (2013) Immunostimulants for aquaculture health management. J Marine Sci Res Dev 3(3) Cheng W, Wang CH (2001) The susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae and its resistance to copper sulfate stress. Dis Aquat Org 47(2):137–144. https://doi. org/10.3354/dao047137 Chiu ST, Hsieh SL, Yeh SP, Jian SJ, Cheng W, Liu CH (2010) The increase of immunity and disease resistance of the giant freshwater prawn, Macrobrachium rosenbergii by feeding with selenium enriched-diet. Fish and Shellfish Immunology 29(4):623–629. https://doi.org/10.1016/j. fsi.2010.06.012 FAO. (2011). World aquaculture 2010. FAO Fisheries and Aquaculture Department. Technical paper (Vol. No. 500/1.). Retrieved from http://www.fao.org/docrep/014/ba0132e/ba0132e.pdf Itami T, Asano M, Tokushige K, Kubono K, Nakagawa A, Takeno N, Nishimura H, Maeda M, Kondo M, Takahashi Y (1998) Enhancement of disease resistance of kuruma shrimp, Penaeus japonicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilum. Aquaculture 164(1):277–288. https://doi.org/10.1016/S0044-8486(98)00193-8 Johansson MW, Keyser P, Sritunyalucksana K, Söderhäll K (2000) Crustacean haemocytes and haematopoiesis. Aquaculture 191(1–3):45–52. https://doi.org/10.1016/S0044-8486(00)00418-X Keysami MA, Mohammadpour M (2013) Effect of Bacillus subtilis on Aeromonas hydrophila infection resistance in juvenile freshwater prawn, Macrobrachium rosenbergii (de Man). Aquac Int 21(3):553–562 Keysami MA, Saad CR, Sijam K, Daud HM, Alimon AR (2007) Effect of Bacillus subtilis on growth development and survival of larvae Macrobrachium rosenbergii (de Man). Aquac Nutr 13(2):131–136. https://doi.org/10.1111/j.1365-2095.2007.00463.x Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. https://doi.org/10.1038/227680a0 Lavilla-Pitogo CR, Leaño EM, Paner MG (1998) Mortalities of pond-cultured juvenile shrimp, Penaeus monodon, associated with the dominance of luminescent vibrios in the rearing environment. Aquaculture 164(1–4):337–349. https://doi.org/10.1016/S0044-8486(98)00198-7

Author's personal copy Aquacult Int Li CC, Yeh ST, Chen JC (2010) Innate immunity of the white shrimp Litopenaeus vannamei weakened by the combination of a Vibrio alginolyticus injection and low-salinity stress. Fish Shellfish Immunol 28(1):121– 127. https://doi.org/10.1016/j.fsi.2009.10.003 Lightner, D. V. (2005). Biosecurity in shrimp farming: pathogen exclusion through use of SPF stock and routine surveillance. Journal of the World Aquaculture Society, 36(3), 229–248 Liu CH, Chen JC (2004) Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus. Fish Shellfish Immunol 16(3):321–334. https://doi.org/10.1016 /S1050-4648(03)00113-X Maftuch, Prasetio E, Sudianto A, Rozik M, Nurdiyani R, Sanusi E et al (2013) Improvement of innate immune responses and defense activity in tiger shrimp (Penaeus monodon Fabricius.) by intramuscular administration of the outer membrane protein Vibrio alginolyticus. SpringerPlus 2(1):432 Pati US, Srivastava SK, Roy SC, More T (1996) Immunogenicity of outer membrane protein of Pasteurella multocida in buffalo calves. Vet Microbiol 52(3–4):301–311. https://doi.org/10.1016/S0378-1135(96)00066-1 Purivirojkul W, Areechon N, Srisapoome P (2006) The effect of peptidoglycan on immune response in black tiger shrimp (Penaeus monodon Fabricius). Kasetsart J (Nat Sci) 187:181–187 Qian RH, Xiao ZH, Zhang CW, Chu WY, Wang LS, Zhou HH, Wei Yw, Yu L (2008) A conserved outer membrane protein as an effective vaccine candidate from Vibrio alginolyticus. Aquaculture 278(1–4):5–9. https://doi.org/10.1016/j.aquaculture.2008.03.010 Sabri MY, Zamri-Saad M, Mutalib a R, Israf D a, Muniandy N (2000) Efficacy of an outer membrane protein of Pasteurella haemolytica A2, A7 or A9-enriched vaccine against intratracheal challenge exposure in sheep. Vet Microbiol 73(1):13–23. https://doi.org/10.1016/S0378-1135(99)00205-9 Sahoo PK, Pillai BR, Mohanty J, Kumari J, Mohanty S, Mishra BK (2007) In vivo humoral and cellular reactions, and the fate of injected bacteria Aeromonas hydrophila in freshwater prawn Macrobrachium rosenbergii. Fish Shellfish Immunol 23(2):327–340. https://doi.org/10.1016/j.fsi.2006.11.006 Scholz U, Garcia Diaz G, Ricque D, Cruz Suarez LE, Vargas Albores F, Latchford J (1999) Enhancement of vibriosis resistance in juvenile Penaeus vannamei by supplementation of diets with different yeast products. Aquaculture 176(3-4):271–283. https://doi.org/10.1016/S0044-8486(99)00030-7 Smith VJ, Brown JH, Hauton C (2003) Immunostimulation in crustaceans: does it really protect against infection? Fish Shellfish Immunol 15(1):71–90. https://doi.org/10.1016/S1050-4648(02)00140-7 Smith VJ, Söderhäll K, Hamilton M (1984) β 1, 3-Glucan induced cellular defence reactions in the shore crab, Carcinus maenas. Comp Biochem Physiol A Physiol 77(4):635–639. https://doi.org/10.1016/0300-9629(84)90176-2 Söderhäll K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10(1):23–28. https://doi.org/10.1016/S0952-7915(98)80026-5 Sung HH (1990) Enhancement of growth in tiger shrimp (Penaeus monodon) by bacterin prepared from Vibrio vulnificus. Bull Eur Ass Fish Pathol 10(4):98–99 Sung HH, Yang YL, & Song YL (1996). Enhancement of microbicidal activity in the tiger shrimp Penaeus monodon via immunostimulation. J Crust Biol 16(2):278-284 Sung H-H, Kuo P-A, Kao W (2000) Effect of lipopolysaccharide on in vitro phagocytosis hemocytes from giant freshwater prawn (Macrobrachium rosenbergii). Jpn Soc Fish Pathol 3(35) Thakur AB, Vaidya RB, Suryawanshi SA (2003) Pathogenicity and antibiotic susceptibility of Vibrio species isolated from moribund shrimps. Indian J Marine Sci 32(1):71–75 Thangaviji V, Michaelbabu M, Anand SB, Gunasekaran P (2012) Microbial & Biochemical Technology Immunization with the Aeromonas OMP provides protection against Aeromonas hydrophila in goldfish (Carassius auratus). Microbial Biochem Technol 4(2):45–49 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76(9):4350–4354. https://doi. org/10.1073/pnas.76.9.4350 Tseng IT, Chen JC (2004) The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress. Fish Shellfish Immunol 17(4):325–333. https://doi.org/10.1016/j.fsi.2004.04.010 Wowor D, Ng PKL (2007) The giant freshwater prawns of the Macrobrachium rosenbergii species group (Crustacea: Decapoda: Caridea: Palaemonidae). Raffles Bull Zool 55(2):321–336