Roles of phagocytosis activating protein (PAP) in Aeromonas hydrophila infected Cyprinus carpio

Accepted Manuscript Roles of Phagocytosis activating Protein (PAP) in Aeromonas hydrophila infected Cyprinus carpio Monwadee Wonglapsuwan, Pataraporn Kongmee, Naraid Suanyuk, Dr. Wilaiwan Chotigeat PII:

S0145-305X(15)30104-X

DOI:

10.1016/j.dci.2015.12.021

Reference:

DCI 2522

To appear in:

Developmental and Comparative Immunology

Received Date: 19 October 2015 Revised Date:

28 December 2015

Accepted Date: 29 December 2015

Please cite this article as: Wonglapsuwan, M., Kongmee, P., Suanyuk, N., Chotigeat, W., Roles of Phagocytosis activating Protein (PAP) in Aeromonas hydrophila infected Cyprinus carpio, Developmental and Comparative Immunology (2016), doi: 10.1016/j.dci.2015.12.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Roles of Phagocytosis activating Protein (PAP) in Aeromonas hydrophila

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infected Cyprinus carpio

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Monwadee Wonglapsuwanab, Pataraporn Kongmeeab, Naraid Suanyukc,

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Wilaiwan Chotigeat* ab a

Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand, bDept. of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand, c Dept. of Aquatic Science, Faculty of Natural Resources, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

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* Corresponding author: Dr. Wilaiwan Chotigeat, Department of Molecular Biotechnology

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and Bioinformatics, Faculty of Science, Prince of Songkla University, Hatyai, Songkhla,

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90112, Thailand. E-mail address: [email protected]

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Abstract Cyprinus carpio (koi) is one of the most popular ornamental fish. A major problem

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for C. carpio farming is bacterial infections especially by Aeromonas hydrophila. Previously

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studies had shown that the Phagocytosis Activating Protein (PAP) gene was involved in the

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innate immune response of animals. Therefore, we attempted to identify a role for the PAP

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gene in the immunology of C. carpio. The expression of the PAP was found in C. carpio

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whole blood and increased when the fish were stimulated by inactivated A. hydrophila.

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addition, PAP-phMGFP DNA was injected as an immunostimulant. The survival rate and the

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phagocytic index were significantly increased in the A. hydrophila infected fish that received

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the PAP-phMGFP DNA immunostimulant. A chitosan-PAP-phMGFP nanoparticle was then

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developed and feeded into fish which infected with A. hydrophila. These fish had a

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significantly lower mortality rate than the control. Therefore, this research confirmed a key

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role for PAP in protection fish from bacterial infection and the chitosan-PAP-phMGFP

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nanoparticle could be a good prototype for fish immunostimulant in the future.

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Keywords: PAP, Ribosomal protein L26, Carp, Phagocytosis, A. hydrophila, Chitosan

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1. Introduction One of the most popular ornamental fish is koi carp, Cyprinus carpio haematopterus,

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because of its fancy color and ability to survive and adapt to many climates and water

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conditions. Koi is one of subspecies of the common carp, Cyprinus carpio. Unlike another

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subspecies, koi shows variety color. Therefore, koi carp is a popular fish for ornamental.

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Recently, the hobby of keeping koi eventually spread worldwide. However, koi ornamental

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still has some problems. One of the major problems is infection by bacterium.

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Aeromonas hydrophila is one of the most bacterium infected in koi carp. A.

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hydrophila causes disease in fish known as “Motile Aeromonas Septicemia” (MAS),

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“Hemorrhagic Septicemia,” “Ulcer Disease,” or “Red-Sore Disease.” A. hydrophila has been

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categorized as an opportunistic pathogen. However, the term “opportunistic pathogen”

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conveys that A. hydrophila always is capable of producing disease if given the chance

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(Cipriano, 2001). Fish infected with A. hydrophila shown many different symptoms. These

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range from sudden death in otherwise healthy fish to lack of appetite, swimming

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abnormalities, pale gills, bloated appearance, and skin ulcerations. The skin ulcers may occur

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at any site on the fish and often are surrounded by a bright rim of red tissue. Infection by C.

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carpio results in a distended abdomen and the scales bristle out from the skin (Jagruthi et al.,

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2014).

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Ribosomal protein L26 (RPL26) is a ribosomal protein in the 60s subunit of the

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ribosome. The RPL26 gene has been named as a phagocytosis-activating protein (PAP)

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because there is some evidence that PAP is involved in an immune response. PAP has been

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found in the black tiger shrimp, Penaeus monodon infected with white spot syndrome virus

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(WSSV) (Deachamag et al., 2006). In addition, it was shown that PAP stimulated the immune

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response of the Pacific white shrimp, Lipopenaeus vannamei. PAP also protected the white

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shrimp against several pathogens such as WSSV, Yellow head virus (YHV) and Vibrio

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harveyi (Khimmakthong et al., 2011). There is also some evidence that the entry of PAP into

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phagocytic cells may be facilitated by the α2M protein (Chotigeat et al., 2007). Moreover, the

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PAP gene was activated in a mouse macrophage cell line when it was treated with silica, LPS

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and IFNγ (Segade et al., 1996). It was of interest, that in a previous study, we produced a

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complex of chitosan-PAP-phMGFP nanoparticles and this complex protected shrimp from

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pathogens (Khimmakthong et al., 2013).

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Chitosan is a copolymer of N-acetyl-D-glucosamine and D-glucosamine, obtained by

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the alkaline deacetylation of chitin. Various studies have referred to the use of chitosan as a

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gene carrier because of its low toxicity, low immunogenicity, excellent biocompatibility and

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high positive charge density (Shu and Zhu, 2012; Lee et al., 2015). A chitosan-based

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formulation for delivery of DNA has been published (Mao et al. 2010). There has been some

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research to indicate that a chitosan-DNA nanoparticle can also help to protect fish (Asian

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seabass, Lates calcarifer) from pathogens. A gene that encoded for an outer membrane

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protein (OMP) of Vibrio anguillarum was used to construct a DNA immunostimulant using

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pcDNA 3.1. The constructed plasmid was encapsulated in chitosan and orally vaccinated to

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L. calcarifer with a chitosan-OMP complex. This product showed a moderate protection

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against an experimental infection with V. anguillarum (Kumar et al., 2008).

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Therefore, in this study we attempted to investigate a role for the PAP gene in the

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immune response of C. carpio. Inactivated A. hydrophila was used to stimulate the immune

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response of C. carpio and then the PAP expression was measured. In addition we injected

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PAP to C. carpio with the PAP-phMGFP plasmid and fed the chitosan-PAP-phMGFP

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nanoparticles then checked the survival rate after challenging with A. hydrophila.

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2. Materials & Methods

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2.1 Animals Cyprinus carpio, average total length 13 cm and average total body mass 25 ± 3 g

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were purchased from a farm at Ratchaburi province. Fish were reared in a rectangular tank

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saturated with oxygen. Fish were fed with 3% commercial feed per body weight, twice a day.

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2.2 RT-PCR

Total RNA was extracted from fish blood using Trizol reagent (Invitrogen, Carlsbad,

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CA, USA) following the manufacturer’s protocol. Four hundred nanograms of total RNA was

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incubated with 200 ng of random primers at 70 oC for 5 min and cooled on ice for 5 min. This

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mixture was added to 1 x avian myeloblastosis virus (AMV) buffer, 1 mM deoxynucleotide

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triphosphate, and 10 U of AMV reverse transcriptase (Promega, Madison, WI, USA) in a 25

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µL reaction mixture and incubated at 48 oC for 2 h. The cDNA was used as a template. The

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primers for the PAP gene were designed from the conserved regions including the forward

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primer (PAP F: 5’ CAATGTCCGTGCCATGC 3’) and a reverse primer (PAP R: 5’

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CCGACCAGCAGCCTTGTT 3’). PCR was initiated with a first denaturation step of 5 min at

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95 oC, followed by 40 cycles of 94 oC 2 min, annealing at 50 oC for 1 min, and extension at

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72 oC for 1 min. Negative controls consisted of reactions with no template cDNA. The PCR

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products were detected after separation by electrophoresis on agarose gel.

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2.3 PAP sequence analysis

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The PAP gene was amplified from the blood of C. carpio which includes serum, red

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blood cell and white blood cell using PAP-F and PAP-R primers. The PCR product was

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cloned into the pGEM-T Easy (Promega, Madison, WI, USA) and sequence analysis was

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performed using the ABI prism 377 apparatus. The obtained nucleotide sequence was aligned

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with sequences in the GenBank databases using the BLASTx search program from NCBI

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(http://www.ncbi.nlm.nih.gov) to confirm the gene identity. PAP sequences from many

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organisms were obtained from GenBank. All sequences were aligned and analyzed using the

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CLUSTAL X version 2.7 to determine their similarity.

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2.4 Preparation of inactivated A. hydrophila

A. hydrophila was grown in Tryptic-Soy-Broth (TSB) at 28 oC until an OD of 0.8-1.0

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at 610 nm was reached. This culture was boiled at 100 oC for 30 min and centrifuged at 4000

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rpm for 15 min. The supernatant was discarded. The pellet, that contained inactivated A.

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hydrophila, was resuspensed in PBS until an OD of 0.8-1.0 was reached. The inactivation

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was confirmed by culturing the resuspened cells in Tryptic Soy Agar (TSA) at 28 oC for 48 h.

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The inactivated cells did not grow in the medium. The inactivated A. hydrophila was used as

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an immunostimulant.

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2.5 Stimulation of the fish immunity by inactivated A. hydrophila

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The 0.1 mL (1x109 CFU) of inactivated A. hydrophila was injected into the carp.

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After injection at 1, 2, 5 and 7 days, blood samples were collected. The total RNA was

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extracted and reversed to cDNA. The mRNA expression level was investigated by RT-PCR.

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The beta-actin gene was used as an internal control. The primers for the beta-actin gene were

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(5’

CAGATCATGTTYGAGACCTTC

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GATGTCCACGTCCACTTCAT 3’). The PCR products were separated by electrophoresis

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on a 1.5% agarose gel. The density of the DNA bands was calculated by the Scion image

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program. The expression levels for each group were compared statistically using a one-way

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ANOVA of the SPSS program at a 95% confidence level (p < 0.05).

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and

Beta-R:

(5’

2.6 PAP-phMGFP plasmid preparation

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The E. coli Top 10 that containing phMGFP or PAP-phMGFP plasmids were received

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from Khimmakthong et al., 2011. The plasmids were extracted following the protocol of

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Green & Sambook’s (Green and Sambrook, 2012). Briefly, the bacteria were culture in Luria

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Bertaini (LB) contained 80 µg/mL of ampicillin. The culture was incubated at 37 oC for 16-

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18 h and centrifuged at 10,000 xg for 1 min. The pellet was then resuspened with 100 µL of

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solution I (50 mM glucose, 25 mM Tris-HCl pH 8.0 and 10 mM EDTA) and incubated at

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room temperature for 5 min. Solution II (0.2 N NaOH and 1% SDS) was added and the tube

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was stored on ice for 5 min. The, solution III (50 M potassium acetate and glacial acetic acid)

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was added and then the tube was stored on ice for 30 min. The bacterial lysate was

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centrifuged at 12,000 xg for 15 min. The supernatant was transferred to a new tube. The

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plasmid was precipitated by adding one volume of ethanol at room temperature. The

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precipitated plasmid was harvested by centrifugation at 12,000 xg for 15 min. Then the pellet

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was washed with 70% ethanol, and sediment at 12,000 xg for 5 min. The pellet was dried by

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vacuum. Finally, the plasmid was dissolved in deionized water. The quality and quantity of

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the plasmid were checked by electrophoresis on an agarose gel and detected using a

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spectrophotometer at 260 and 280 nm.

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2.7 Protection efficiency C. carpio was divided into eight groups, and each group was consisted of 27 fish (the

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experiment was done in triplicate, 1N=9). Group 1, 2 and 3 were injected intraperitoneally

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with 200, 400 and 600 µg of PAP-phMGFP, respectively. Another three groups were injected

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intraperitoneally with 200, 400 and 600 µg of phMGFP, respectively. Group 7 was injected

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with PBS. After 7 days post injection, the fish were then challenged by A. hydrophila. The

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concentration of A. hydrophila was calculated from LD50 at day 7. Group 8 was injected with

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the PBS but was not injected with A. hydrophila for using as a negative control. Mortality

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was recorded twice a day for 28 days. The survival rate was recorded and relative survival

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rate (RSP) were calculated. The RPS was calculated as follows:

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Relative Percent Survival (RPS) = 1-

The percentage mortality of test group

X 100

The percentage mortality of positive group

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2.8 The distribution of the PAP-phMGFP expression in fish was determined by RT-PCR Fish were divided into 3 groups of 9 fish each. Group 1 and 2 were injected with 400

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µg of phMGFP and PAP-phMGFP, respectively. The last group was injected with PBS

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buffer. After 7 days post injection was found the highest expression of the PAP gene

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(Khimmakthong et al., 2011). So several organs including blood, kidney, liver, spleen, and

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heart were collected after 7 days post injection. Total RNAs were extracted from the organs.

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The mRNA expression of the PAP gene was investigated by RT-PCR using the PAP forward

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primer and the PAP reverse primer. Beta-actin primers were also used to produce the internal

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RT-PCR control.

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2.9 Phagocytosis activity of macrophage activated by PAP-phMGFP

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Fish were injected with 400 µg of PAP-phMGFP for 7 days. After that, fish were

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anesthetized by clove oil. Macrophages were obtained from the head kidney following

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Graham & Secombes protocol (Graham and Secombes, 1988). Briefly, the head kidney was

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dissected and transferred to L-15 medium. The kidney was homogenized gently and filtered

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through a 100 µm nylon mesh. The cells were centrifuged at 400 xg for 5 min at 4 oC. The

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supernatants were discarded. The pellets were washed by L-15 medium twice. The cell

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suspension was layered onto a 34 to 51% Percoll discontinuous density gradient and

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centrifuged at 400 xg for 30 min at 4 oC. Cells that were separated at the interface were

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collected, centrifuged at 400 xg for 5 min and resuspended in L-15 supplemented with 1%

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FCS. Viable cell concentrations were determined by trypan blue exclusion using a light

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microscope. About 98% or greater of these cells were used for the phagocytic activity

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experiment.

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The 400 µL of suspended macrophages, 2 x 106 cells/mL, were mixed with 0.1 mL of

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latex bead (2 x 108 /mL, particle diameter 2.0 µm), on a clean glass slide. The mixture was

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incubated at 25 oC for 30 min. The cells were fixed with 0.125% glutaraldehyde for 5 min.

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Then, the nonadherent cells were removed with PBS, followed by staining with Wright’s

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stain for 10 min. The number of ingested cells and ingesting cells were counted using a light

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microscope. The percentages of phagocytosis, phagocytic index (PI), the average number of

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the beads ingested per cell (ABPC) and the phagocytic index (PI) were calculated as follows:

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Percentage phagocytosis = (number of cells ingesting bead/number of cells observed) x 100

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ABPC = number of beads ingested*/number of cells ingesting bead

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Phagocytic index = (number of cells ingesting bead/number of cells observed) x (number of

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beads ingested*/number of cells observed) x 100

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*Beads ingested means the beads detected inside the cytoplasm of only the phagocytosed cells.

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2.10 Preparation of low molecular weight chitosan

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The chitosan-DNA nanoparticles were prepared following the method described by

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Mao et al, (2010). Briefly, high molecular weight chitosan (150 kDa) was dissolved in 1%

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acetic acid (w/v) and 0.1 M NaNO2 was dropped into it until the chitosan/NaNO2 ratio of

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0.01 was reached. The chitosan was precipitated by adjusting the pH and centrifuged at 7,500

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xg for 2 min and washed 10 times with deionized water. The pellet of chitosan was dialyzed

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and dried by lyophilization.

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2.11 Preparation of chitosan-PAP-phMGFP nanoparticles

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The chitosan-DNA nanoparticles were formed following the method described by

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Zheng et al., 2007. The chitosan was dissolved in 0.5% acetic acid with gently heating and

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the pH of the solution was adjusted to 5.6 – 6.9 with sodium hydroxide. The solution was

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filtered through the 0.2 µm membrane and diluted to 0.4 mg mL-1 of chitosan by 0.5% acetic

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acid, pH 6.9. Then, this chitosan was mixed with PAP-phMGFP. The ratio of the

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chitosan/PAP-phMGFP was 1:2. The mixture was vortexed intermittently for 30 sec and

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incubated at room temperature for 1 h before use. The chitosan-phMGFP nanoparticles were

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prepared by the same method. The chitosan-DNA nanoparticles were checked by

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electrophoresis on a 0.8% agarose gel.

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2.12 Survival rate of the fish treated orally with chitosan-PAP-phMGFP nanoparticles followed by a challenge with A. hydrophila.

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Chitosan-PAP-phMGFP nanoparticles were fed by mixing it with a fish diet to give

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200, 400 and 600 µg of chitosan-DNA. The mixture was fed once a day for 7 days. The fish

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were challenged by the intraperitoneal injection of A. hydrophila at 1 x 109 CFUmL-1. This A.

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hydrophila concentration resulted in a 50% death of the control fish in 7 days. Mortality was

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recorded for 28 days post A. hydrophila injection. The survival rate and RPS were calculated

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as previously described above.

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3. Results

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3.1 PAP in C. carpio

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Measurements of the PAP gene expression in C. carpio, RT-PCR was performed

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using the PAP -F: and PAP- R: primers. The PCR product was approximately 300 bps in size.

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The identity of the PCR product was confirmed by sequencing. The obtained sequence shared

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considerable identity with PAP genes from other organisms. The PAP of C. carpio shared a

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98% identity with the PAP of P. monodon, and a 66% similarity with the PAP of Salmo salar

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and Ictalurus punctatus. A comparison of the PAP sequences from various organisms is

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shown in figure 1. The results indicated that the PAP- F and PAP- R primers were specific to

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the PAP gene and could amplify PAP in C. carpio. Therefore, we used theses primers

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throughout the following experiments.

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3.2 PAP is activated by intraperitoneal immunostimulation

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To investigate a role for the PAP gene in the immune response, the C. carpio were

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injected with inactivated A. hydrophila to stimulate fish immunity. Then, blood was collected

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from the fish and the expression level of the PAP gene was investigated by RT-PCR (figure

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2 ). The PAP expression had significantly increased 2 days post A. hydrophila injection and

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the highest expression had occurred by day 7. These results indicated that PAP could play

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some role in the fishes immune response to infection by inactivated A. hydrophila.

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3.3 PAP-phMGFP is an immunostimulant for A. hydrophila infection.

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The efficiency of PAP itself to induce immune protection was then tested. To study

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whether PAP can act as an immunostimulant to protect the fish from bacterial infection, the

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fish were injected with PAP-phMGFP at various concentrations. After 7 days post injection,

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the vaccinated fish were challenged by A. hydrophila at its L50 dose. Mortality was

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investigated daily for 7 days. The highest relative percent survival (63.89 ±2.40) was found in

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the fish that received 400 µg of PAP-phMGFP (figure 3 and table 1). This highest survival

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rate was significantly different from the fish that received a lower concentration of PAP-

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phMGFP and the control group. These results indicated that PAP-phMGFP could be used as

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an immunostimulant to protect fish against A. hydrophila infections.

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Table 1. Relative Survival Rate (RSP) of fish injected with the immunostimulants and challenged by A. hydrophila.

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Concentration(µg/100µl)

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Relative Survival Rate (RSP) (%) 274

phMGFP

PAP-phMGFP

200

3.70±6.41

24.07±12.60

400

11.57±11.14

63.89±2.40*

600

8.33±7.22

12.04±0.80

(*) Asterisk indicates significantly different between groups. (N=27, p< 0.05).

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3.4 The distribution of PAP-phMGFP in organs after injection into the C. carpio. After injection of the PAP-phMGFP for 7 days, the distribution of PAP-phMGFP in

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various organs was investigated (figure 4). The PAP-GFP gene was only detected in the

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blood but no other organs of the fish that received 400 µg of PAP-phMGFP, and non was

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detected in any of the organs in the fish that received phMGFP or PBS.

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3.5 Phagocytic activity of macrophages can be activated by PAP-phMGFP

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PAP-phMGFP was tested for activation of macrophage phagocytic activity. The

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percentage phagocytosis of the PAP-phMGFP group was 55.77 ± 7.80 while the phMGFP

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and PBS groups were 46.14 ± 3.79 and 41.33 ± 4.16, respectively (figure 5A). However, the

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percentage phagocytic activity was not significantly different for any of the groups.

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The average number of the beads ingested per cell (ABPC) and the phagocytic index

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(PI) were also calculated. The PAP-phMGFP group showed a significantly higher ABPC

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(figure 5B). The ABPC of this group was 1.09 ± 0.03 whereas it was 1.01 ± 0.02 and 1.03 ±

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0.02, respectively for the controls.

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The results of the phagocytic index (PI) are shown in figure 5C. The PI of the PAP-

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phMGFP, phMFGP and PBS groups were 34.29 ± 9.30, 21.73 ± 3.41 and 17.67 ± 3.79,

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respectively. These results indicated that the PI of PAP-phMGFP group was significantly

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higher when compared to the control groups.

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3.6 PAP-phMGFP nanoparticles can protect fish from the A.hydrophila infection

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To try to enhance the efficiency of the transport of a DNA immunostimulant into a

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cell, PAP-phMGFP nanoparticles were produced and tested. Chitosan was selected as a

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vehicle to transport PAP-phMGFP into cells. The complex of PAP-phMGFP and chitosan

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was tested for its ability to migrate on an agarose gel subjected to electrophoresis (figure 6).

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The complex did not migrate in the gel because of its size and charge, while, the plasmid

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DNA itself did migrate easily in the gel.

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The ability of the chitosan-PAP-phMGFP nanoparticles was then tested for any

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protection against infection. The chitosan-PAP-phMGFP nanoparticles were mixed with the

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fish feed. The mixture was fed to the carp. The chitosan-phMFGP nanoparticles and chitosan

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only were also were fed as control groups. After feeding for 7 days, the carp were infected

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with A. hydrophila, and the survival rates and RPS were recorded and respectively (figure 7

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and table 2, respectively). There was a significantly higher survival rate in the group that

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received 600 µg of the chitosan-PAP-phMGFP nanoparticle compared to any of the others.

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There were no significant differences detected in the other groups when compared to the

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controls.

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Table 2. Relative Survival Rate (RSP) of fish fed with the DNA nanoparticles and challenged by A. hydrophila.

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Relative Survival Rate (RSP) (%)

Concentration (µg)

chitosan

phMGFP nanoparticles

PAP-phMGFP nanoparticles

200µg

5.56±6.41

7.87±0.80

11.57±0.80

400µg

8.68±6.85

11.57±6.05

11.57±0.80

600µg

11.11±11.11

15.28±6.05

34.72±2.41*

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(*) Asterisk indicates significantly different between groups. (N=27, p< 0.05).

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4. Discussion

There are several reports indicated that PAP is involved in immune response of

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organisms. The expression of PAP gene was up-regulated in response to macrophage

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activation in the mouse macrophage cell line when treated with silica, LPS and IFNγ (Segade

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et al., 1996). Moreover, the PAP has also been isolated from the hepatopancreas of a marine

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snail (Littorina littorea), which was up-regulated during anoxia exposure (Larade et al.,

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2001). In Penaeus japonicas, the PAP was constitutively expressed during the molt cycle

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(stage-specific transcripts) (Watanabe, 1999). We also demonstrated the expression of PAP

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was high when P. monodon was infected by WSSV. In addition, the expression of PAP in the

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haemolymph of P. monodon was induced via the intramuscular injection of the

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immunostimulants of inactivated WSSV, IVH and fucoidan, but it was not found in the

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hepatopancreas or lymphoid organ (Deachamag et al., 2006). These results show that PAP

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released from cells in the haemolymph could be one part of an innate immune response. This

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study showed that the koi carp’s PAP nucleotide sequence had a high identity to the P.

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monodon PAP and the PAP from other organisms. This result indicated that PAP gene is a

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conserve gene. Therefore, in this study, we investigated the role of PAP gene in innate

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immune response of the koi carp.

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Fish are known to have specific and non-specific immune responses to infection.

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Phagocytosis is one of the major gate-ways of a non-specific immune response in both

338

vertebrates and invertebrate. PAP is a macrophage activating protein and macrophage is one

339

of the non-specific immune responses in fish. PAP has been investigated in shrimp and has

340

been positively useful as a DNA immunostimulant to protect shrimp from viral infection as

341

well as bacterial infections (Khimmakthong et al., 2013). In the koi carp, The PAP was

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highly expressed in the carp after 7 days of activation by A. hydrophila, which is the same

343

pattern as for the PAP that was activated by WSSV in shrimp (Deachamag et al., 2006). In

344

addition, when the phMGFP-PAP was injected to express PAP. The expression was found

345

only in the blood where the phagocytosis occurs. These results suggested that PAP is also

346

involved in immune response of the fish. When the phMGFP-PAP was used as a

347

immunostimulant by injecting into carp and then challenged with A. hydrophila, it protected

348

the carp so that the RPS was 63.89 ±2.40. While the RPS of the PAP-phMGFP immunized

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shrimp after a challenge by WSSV, YHV and V. harveyi was 86.61%, 63.34% and 50%,

350

respectively. It was of interest that, although PAP activated phagocytosis, it also showed

351

protection against the WSSV and YHV viruses much better than against bacteria in shrimp

352

while the protection against bacteria in the carp (~64%) was slightly better than in shrimp

353

(50%). Several immunostimulants have been tried in fish for example, rainbow trout were

354

vaccinated with a formalin-killed, heated-killed and lipopolysaccharide from A. hydrophila

355

with RPS values of 84%, 67% and 34%, respectively, after challenging the fish with the live

356

A. hydrophila (Dehghani et al., 2012). In the case of a protein immunostimulant, the Indian

357

major carp that was vaccinated with a recombinant outer membrane protein (OMP) of A.

358

hydrophila had an RPS value of 56-87% after challenging the fish with various isolates of A.

359

hydrophila (Poobalanea et al., 2010), while, a 50% RPS was shown in Goldfish (Carassius

360

auratus) vaccinated with the same recombinant protein (Thangaviji et al., 2012).

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After injection of PAP-phMGFP, the average numbers of beads ingested per cell

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(ABPC) (Itami et al., 1994) and Phagocytic index (PI) were increased, although the percent

363

of phagocytosis had not been significantly changed. The ABPC indicates number of bead

364

engulfed by cell while the percent of phagocytosis indicates number of cell ingesting the

365

bead. The results indicated that the ability of ingestion the bead was increased in each cell.

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However, the number of the ingested cell was not increased. We suggested that PAP could

367

stimulate the activity of neutrophils, monocytes or both but not effect to the number.

In order to make the phMGFP-PAP more practical for use the chitosan-PAP-phMGFP was

369

tested by oral ingestion, Although the RPS (37.03% by oral or 63.89% by injection) was not

370

impressive but these results showed PAP involved in immune response of carp and there is the

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possibility of developing more efficient oral chitosan-DNA immunostimulant so further development

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of this immunostimulant should be investigated.

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Acknowledgements

This work was supported by National Research University Project of Thailand, Office

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of the Higher Education Commission and National Research Council of Thailand (NRCT).

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We thank Dr. Brian Hodgson, Prince of Songkla University, for assistance with the English

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and for valuable comments.

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Figure 1. Conservation of PAP sequence. Alignment of the nucleotide sequences of PAP from Carp with those of other PAP

3

homologues from other species. Species names are abbreviated at the left and shown as follows:

4

Macaca mulatta, GenBank accession number NM_001193566; Homo sapiens, GenBank

5

accession

6

NM_001304872; Mus musculus, GenBank accession number X80699; Salmo salar, GenBank

7

accession number NM_001245841; Ictalurus punctatus, GenBank accession number

8

NM_001200285; Penaeus monodon, GenBank accession number AY680836; Spodoptera

9

frugiperda, GenBank accession number AF400190. Sequence alignment was performed using

Ailuropoda

the CLUSTAL X program

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GenBank

accession

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Figure 2. Expression of PAP gene stimulated by inactivated A. hydrophila.

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melanoleuca,

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X69392.1;

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Fish were injected with inactivated A. hydrophila. After 1, 2, 5, and 7 days post infection,

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the blood was collected and the level of the PAP gene was investigated. The level of the β-actin

15

gene was also monitored to act as an internal control. PBS was also injected to the fish as another

16

control group. Each experimental group consisted of 3N. (1N consisted of 3 fish). (A) The

17

agarose gel electrophoresis has shown the expression of PAP and β-actin genes. (B) The relative

18

mRNA expression of PAP and β-actin genes measuring by semi-quantitative PCR. The white bar

19

represented the expression level of control groups whereas the black bar represented the

20

expression level of treatment groups. β-actin was used as the internal standard (Mean ± SD).

21

Asterisks indicated significant differences between control and treatment groups (p ≤ 0.05).

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Figure 3. Survival rate of fish injected intramuscularly with DNA immunostimulants at various doses after being exposed to A. hydrophila. The fish were injected with the PAP-phMGFP DNA at 200, 400 and 600 µg. Each

26

experimental group consisted of 27 fish. After 7 days post injection, the fish were challenged by

27

A. hydrophila and the mortality was observed for 28 days. The negative control was a group of

28

fish that was injected with PBS and was not injected with A. hydrophila. The positive control

29

was a group of fish was injected with PBS and injected with A. hydrophila. The chitosan and

30

chitosan-phMGFP-nanoparticle were also performed as the control groups.

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Figure 4. Expression levels of PAP and β-actin genes in organs after injected by DNA immunostimulants.

The fish were divided into 3 groups. PAP-phMGFP, phMGFP or PBS was injected into

35

each group. Each experimental group consisted of 3N (1N consisted of 3 fish). After 7 days post

36

injection, the organs including in blood (B), heart (H), kidney (K), liver (L) and spleen (S) were

37

collected. The expression level was investigated by PCR and 1.5% agarose gel electrophoresis.

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M; 100 bp DNA marker.

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Figure 5. Phagocytic activity of fish injected with the DNA immunostimulants

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Fish were injected with 400 µg of PAP-phMFGP. After 7 days pot injection, the kidneys

42

were collected and the macrophages were extracted. The phagocytic activity assay was

43

performed. (A) Percentage of phagocytosis. (B) Average number of the beads ingested per cell

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(ABPC). (C) Phagocytic index. Asterisk indicates a significant difference between the groups.

45

(N=3, p < 0.05).

47

Figure 6. Formation of the chitosan-PAP-phMGFP nanoparticle

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Electrophoretic migration of PAP-phMGFP before and after encapsulation with chitosan.

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Lane 1: phMGFP plasmid, 2: chitosan-phMGFP-nanoparticle, 3: chitosan-PAP-phMGFP-

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nanoparticle, 4: PAP-phMGFP plasmid.

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Figure 7. Effect of the chitosan-DNA-nanoparticle to the survival rate. Fish were fed with chitosan-DNA-nanoparticle at 200, 400 and 600 µg for 7 days and

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then challenged by A. hydrophila. Then, the mortality was observed for 28 days. The negative

55

control was a group of fish fed with normal feed and was not injected with A. hydrophila. The

56

positive control was a group of fish fed with normal feed and injected with A. hydrophila. The

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chitosan and chitosan-phMGFP-nanoparticle were also performed as the control groups. Each

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experimental group consisted of 27 fish.

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* 20 * 40 * 60 * 80 * 100 * ----------------------------------GGCCATCACCGAAGTGGGAGCGGCCAAAATGAAGTTTAATCCCTTTGTGACTTCCGACCGAAGCAAGAATCGCAAAAGGCATT --------------------------------------------------------GCCAAAATGAAGTTTAATCCCTTTGTGACTTCCGACCGAAGCAAGAATCGCAAAAGGCATT -----------------------------------GCCATCGCTGAAGTGCGAGCGGCCAAAATGAAGTCATATCCCTTTGTGACTTCTGACCGGAGCAAGAACCGTAAACGACATT --------------GTAGTTCTCTTTCCTTTTGCGGCCATCGGTGGATCGC-AGCCGCCAAAATGAAGTTCAATCCCTTCGTGACTTCTGACCGAAGCAAGAACCGCAAACGGCATT --------------------------------------AAGAGTTATCGGCAGGCCATCAACATGAAGCTGAATCCATTTGTGACATCCTCGCGGCGTAAGAACCGCAAGAGGCACT ---------------------------------------------AGCGAATAATCGCCATCATGAAGATCAATCGGTTTGTGACCTCCTCCCGGCGTAAGAACCGCAAGAGGCACT --------------------------------------------------------------ATGAGGATCCATAAGATGGTAACGGAGTCCCGGCGTAAAAACCGGCAACGATACT --------------------------------------------------------------------------------------------------------------------GGGGAGTATAATTTCTTTTTATTTCGGTTCTTTGCCGAGTTGTTTAGGTGAAGAGCGTCAGAATGAAGTACAATAAGCTCGTCACATCCTCCAGGAGGAAAAACAGGAAGAGGCACT ca atgaag at t gt ac tc ccg g aa aa cg aa g ca t

: 83 : 61 : 82 : 102 : 79 : 72 : 55 : : 117

M_mulatta H_sapiens A_melanole M_musculus S_salar I_punctatu P_monodon C_carpio S_frugiper

: : : : : : : : :

120 * 140 * 160 * 180 * 200 * 220 * TCAATGCACCTTCCCACATTCGCAGGAAGATTATGTCTTCCCCTCTTTCCAAAGAGCTGAGACAGAAGTACAACGTGCGATCCATGCCCATCCGAAAGGATGATGAAGTTCAGGTTG TCAATGCACCTTCCCACATTCGAAGGAAGATTATGTCTTCCCCTCTTTCCAAAGAGCTGAGACAGAAGTACAACGTGCGATCCATGCCCATCCGAAAGGATGATGAAGTTCAGGTTG TCAATGCACCTTCCCACATTCGCAGGAAGACATTGTCTTCCCCTCTTTCCAAAGAGCTGAGACAGAAGTACAATGTACGATCCATGCCCATCAGAAAGGATGATGAAGTGCAGGTTG TCAATGCGCCCTCTCACATTCGGAGGAAGATCATGTCTTCCCCTCTTTCCAAAGAGCTGAGACAGAAGTATAACGTTCGGTCTATGCCCATTCGGAAGGATGACGAAGTTCAGGTTG TCAATGCCCCCTCACACATCCGCAGGAAGATCATGTCCTCCCCCCTCTCCAAGGAGCTCCGTCAGAAGTACAATGCGAGGTCCATGCCTATCCGCAAGGACGACGAAGTCCAGGTTG TCAATGCCCCGTCACACATCCGCAGAAAGATAATGTCTTCACCTCTGTCCAAAGAGCTCCGCCAGAAGTACAACGTAAGGTCCATGCCCATCCGGAAGGACGATGAGGTCCAGGTGG TCAGCGCCCCTTCCCATATCAAGAGAAAGTTTATGTCTAGCCCCCTATCAAAGGAACTGCGTCAGAAGTACAATGTCCGTGCCATGCCAATTCGCAAAGATGACGAAGTACAGGTTG ----------------------------------------------------------------------CAATGTCCGTGCCATGCCA-TTCGCAA-GATGACGAAGTACAGGTTG TCAGCGCTCCTTCTCACATCAGACGAGTGCTTATGTCTGCGCCTCTGTCCAAGGAGTTGAGACAGAAGTTCAATGTAAAGTCTATGCCAATCCGCAGGGACGATGAAGTTCAGGTTG tca gc cc tc cacat g ag aag t atgtct c cc ct tccaa gagct g cagaagtacAA Gt g tCcATGCC aT cG AagGA GA GAaGT CAGGTtG

: : : : : : : : :

200 178 199 219 196 189 172 45 234

M_mulatta H_sapiens A_melanole M_musculus S_salar I_punctatu P_monodon C_carpio S_frugiper

: : : : : : : : :

240 * 260 * 280 * 300 * 320 * 340 * TACGAGGACACTATAAAGGTCAGCAAATTGGCAAAGTAGTCCAGGTTTACAGGAAGAAGTATGTTATCTACATTGAACGGGTGCAGCGGGAAAAGGCTAATGGCACAACGGTCCACG TACGTGGACACTATAAAGGTCAGCAAATTGGCAAAGTAGTCCAGGTTTACAGGAAGAAATATGTTATCTACATTGAACGGGTGCAGCGGGAAAAGGCTAATGGCACAACTGTCCACG TGCGAGGACACTACAAAGGTCAGCAGATTGGCAAAGTCGTCCAGGTTTACAGGAAGAAGTACGTTATCTACATAGAACGAGTGCAGCGAGAGAAGGCTAACGGCACAACCGTCCATG TTCGTGGACACTACAAAGGCCAGCAGATTGGCAAGGTGGTCCAAGTGTACAGGAAGAAGTACGTCATCTACATCGAACGAGTCCAGCGAGAGAAGGCTAATGGCACAACCGTCCACG TCCGTGGACACTACAAAGGCCAGCAGATTGGCAAGGTAGTGCAGGTCTACAGGAAGAAGTACGTCATCTACATTGAGCGCGTGCAGAGAGAGAAGGCCAACGGAACCACGGTGCACG TCCGGGGACACTACAAAGGCCAGCAGATTGGCAAAGTTGTCCAGGTATACAGGAAGAAATACGTCATTTACATTGAGCGTGTGCAGCGTGAGAAGGCCAATGGAACCACCGTTCATG TGCGCGGTCATTACGAAGGACAACAGGTTGGCAAAGTAGTCACTGTTTATCGCAAGAAGCTCTGCGTCTACATTGAGAGAATTCAGCGTGAAAAGGCCAACGGTGCATCAGTCTATG TGCGCGGTCATTAC-AAGGACAACAGGTTGGCAAAGTAGTCGCTGTTTATCGCAAGAAGCTCTGCATCTACATTGAGAGAATTCAGCGTGAAAAGGCCAACGGTGCATCAGTCTATG TCCGTGGTCACTACAAAGGCCAGCAAGTCGGCAAAGTAGTGCAGGTGTACCGTAAGAAGTTTGTCGTCTACATTGAGAGGATCCAGCGTGAGAAGGCTAACGGCGCCAGTGCATACG T CG GG CAcTAcaAAGG CAgCA TtGGCAAaGT GTcca GT TAc G AAGAAgt gt aTcTACATtGA G T CAGcG GA AAGGC AA GG C ac Gt A G

: : : : : : : : :

317 295 316 336 313 306 289 161 351

M_mulatta H_sapiens A_melanole M_musculus S_salar I_punctatu P_monodon C_carpio S_frugiper

: : : : : : : : :

360 * 380 * 400 * 420 * 440 * 460 TAGGCATTCACCCCAGCAAGGTGGTTATCACTAGGCTAAAACTGGACAAAGACCGCAAAAAGATCCTTGAACGGAAAGCCAAATCTCGCCAAG------TAGGAAAGGAAAAGGGCA TAGGCATTCACCCCAGCAAGGTGGTTATCACTAGGCTAAAACTGGACAAAGACCGCAAAAAGATCCTCGAACGGAAAGCCAAATCTCGCCAAG------TAGGAAAGGAAAAGGGCA TGGGCATTCACCCTAGCAAGGTGGTTATCACTAGACTAAAACTGGACAAAGACCGCAAAAAGATCCTTGAACGTAAAGCCAAATCTCGCCAAG------TAGGAAAGGAAAAGGGCA TGGGCATCCACCCCAGCAAGGTCGTTATCACCAGGCTAAAGCTGGACAAGGACCGCAAGAAGATCCTGGAGAGGAAAGCCAAGTCCCGACAAG------TAGGAAAGGAGAAGGGCA TCGGCATCCACCCCAGCAAGGTTGTGATCACCAGGCTAAAGCTGGACAAGGATCGCAAGAAGATCCTGGAGCGTAAGGCCAAGTCCCGCGCTG------ACGGAAAGGAGAAGGGCA TGGGCATCCACCCTAGCAAGGTTGTGATCACCAGGCTAAAGCTGGACAAGGATCGCAAGAAGATCCTGGAGCGCAAAGCCAAGTCACGGCAAG------AGGGCAAGGACAAGGGCA TTGGCATCCACCCTTCAAAAGTCTGTATTGTTAAGCTGAAGATGACAAAGTCCCGCAAGAGGATACTGGAAAACAAAGCTGCTGGTCGGGCTGCAGCTCAGGGCAAAGACAAGGAGA TTGGCATCCACCCTTCAAAAGTCTGTATTGTTAAGCTGAAGATGACAAAGTCCCGCAAGAGGATACTGGAAAACAAAGCTGCTGGTCGG---------------------------TCGGCATCCACCCTTCAAAGTGCGTGATTGTCAAACTAAAGATGAACAAGGACCGTAAATCGATCCTCGACCGCAGAGCGAAGGGCAGGTTGGCCGCCCTCGGCAAAGACAAGGGCA T GGCAT CACCC AAggt gt AT A gCTaAA TG acAA gacCGcAA a GATcCT GA g AaaGC aa cG g gg aa ga aagggca

: : : : : : : : :

428 406 427 447 424 417 406 250 468

M_mulatta H_sapiens A_melanole M_musculus S_salar I_punctatu P_monodon C_carpio S_frugiper

: : : : : : : : :

* 480 * 500 * 520 * 540 * 560 * 580 AATACAAGGAAGAAACAATTGAGAAGATGCAGGAATAAAGTAATCTTATATAAAAGCTTTGATTAAAACT--TGAAGCAAA-----------------------------------AATACAAGGAAGAAACCATTGAGAAGATGCAGGAATAAAGTAATCTTATATACAAGCTTTGATTAAAACT--TGAAACAAAAAAAAAGGGGAAGAAACGACAGCCTCACTTCTGTAT AATATAAGGAAGAAACAAACGAGAAAATGCAAGAGTAAAGTCATCTTA--------------------------------------------------------------------AATACAAGGAAGAAACGATCGAGAAGATGCAGGAGTAGAGACATCCCATGCACGGCTTTCATTAAAGACTGCTTAAGTAAAAAAAAAAAAAAAAAAAA------------------AATACAAGGAGGAAACCATTGAGAAGATGGCAGAGTGAA--AATC-----TATTGCTTACAATAAACTGCTGTACAAATCAAAAAAAAAAA-------------------------AATACAAGGAGGAGACCATTGAGAAAATGCAAGAGTGAA--CATTACCT-TTTTGCTAACAATAAAAGTCCACG------------------------------------------AGTTCACTGCTATGGACACCTAA-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AGTACACTGAGGAAACCGCCACCGCTATGGAGACCTCGTAAATATAGATTTTAAGACAAATAAAAAAGAAAAACTCCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA--------a taca ga ga ac a a atg t

: : : : : : : : :

507 521 475 545 508 488 429 576

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Highlights •

hydrophila.

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PAP gene was highly expressed in the C. carpio that were injected by inactivated A.

•

PAP-phMGFP DNA immunostimulant increased the phagocytic index and survival rate of A. hydrophila Infected fish.

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A chitosan-PAP-phMGFP nanoparticle was produced as a feed for fish.

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•

A chitosan-PAP-phMGFP nanoparticle can be an immunostimulant in fish for A.

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