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Vaccination of fish against Aeromonas hydrophila infections using the novel approach of transcutaneous immunization with dissolving microneedle patches in aquaculture
Journal Pre-proof Vaccination of fish against Aeromonas hydrophila infections using the novel approach of transcutaneous immunization with dissolving microneedle patches in aquaculture Saekil Yun, Seung-Jun Lee, Sib Sankar Giri, Hyoun Joong Kim, Sang Geun Kim, Sang Wha Kim, Se Jin Han, Jun Kwon, Woo Taek Oh, Se Chang Park PII:
S1050-4648(19)31154-4
DOI:
https://doi.org/10.1016/j.fsi.2019.12.026
Reference:
YFSIM 6668
To appear in:
Fish and Shellfish Immunology
Received Date: 16 September 2019 Revised Date:
29 November 2019
Accepted Date: 9 December 2019
Please cite this article as: Yun S, Lee S-J, Giri SS, Kim HJ, Kim SG, Kim SW, Han SJ, Kwon J, Oh WT, Chang Park S, Vaccination of fish against Aeromonas hydrophila infections using the novel approach of transcutaneous immunization with dissolving microneedle patches in aquaculture, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2019.12.026. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Abstract The aim of this study was to develop and evaluate a novel route of administration for vaccinating fish against Aeromonas hydrophila infection using a dissolving microneedles (MNs) patch. The A. hydrophila JUNAH strain was inactivated with formalin and used as a vaccine antigen. It was mixed with dissolvable carboxymethyl cellulose (CMC) as the matrix material to produce the MNs patches. When examined with a scanning electron microscope, each patch has 282 uniformly distributed, pyramid-shaped needles on a circular base. In the skin insertion experiment, the MNs patches were confirmed to be capable of penetrating the skin of the fish. Through agglutination assay and analysis of non-specific parameters like lysozyme and superoxide dismutase, it was verified that the antigen embedded into the patch induced adaptive and innate immune responses in the fish. In the challenge experiment, the group inoculated with the MNs patch and the group injected with formalin killed cells (FKC) showed a similar survival rate. Our results suggest that the FKC-loaded MNs patch is a wholly viable method alternative to injection for the vaccination of fish.
Keywords: Aeromonas hydrophila, carboxymethyl cellulose, cyprinid loach, dissolving microneedles, transcutaneous immunization.
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Vaccination of fish against Aeromonas hydrophila infections using the novel approach of
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transcutaneous immunization with dissolving microneedle patches in aquaculture
3
Saekil Yuna, Seung-Jun Leeb, Sib Sankar Giria, Hyoun Joong Kima, Sang Geun Kima, Sang Wha Kima,
4
Se Jin Hana, Jun Kwona, Woo Taek Oha, Se Chang Parka, *
5 6
a
7
Veterinary Science, Seoul National University, Seoul 08826, Republic of Korea
8
b
9
Technology, Seowon University, Cheongju 28674, Republic of Korea
Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for
Department of Pharmaceutical Science and Engineering, School of Convergence Bioscience and
10 11
* Corresponding author: Se Chang Park, DVM, Ph.D.
12
Mailing address: College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul
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National University, 81-417, 1 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea 08826
14
Phone: +82-2-880-1282, Fax: +82-2-880-1213. E-mail: [email protected]
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
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1. Introduction
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Aeromonas hydrophila is a gram-negative, rod-shaped bacterium [1] and it is a significant pathogen
35
affecting the aquaculture industry. This pathogen can cause hemorrhagic septicemia in many species
36
of fish, resulting in massive mortalities in freshwater fish, as well as in higher vertebrates [2–4]. This
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is important from the point of view of public health as there is possibility of human infection [5–7].
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Vaccination of the fish is gradually gaining popularity as a cost-effective method of combating this
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disease [8]. There have been many achievements while researching the vaccine against A. hydrophila
40
including DNA, recombinant protein, and live attenuated vaccines [9–11].
41
There are several routes of administration to deliver the vaccine antigen to the fish. One route is
42
injection; this method is reliable as there is direct delivery of small amounts of antigen, and generally
43
results in effective protection from the disease [12]. However, this procedure should be performed
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only by an expert, and also requires the fish to be anesthetized; as such, it is labor-intensive and
45
requires a greater financial investment. The second route is by immersion, where the fish are placed in
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tanks containing the vaccine; this method is simple, but cannot be adopted by all aquaculture farms.
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Oral administration is the third method used by the aquaculture industry. It can be performed by
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coating the feed with the antigen or by mixing the antigen with the feed. This route is advantageous as
49
it can be easily administered to a large number of fish at once. However, there is no uniformity in the
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amount of antigen ingested by each fish and this method may be ineffective in some species where the
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antigen is damaged in the stomach [13]. In this study, we are studying transcutaneous immunization
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(TCI) which is being used in fish for the first time. This route has the advantage of being easily
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administrable and being reliably effective, thus overcoming the difficulties faced by the methods
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currently in use.
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The administration of vaccines through the skin (using its large surface area) is called TCI. The skin is
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abundant in trained antigen presenting cells (APCs), there are dendritic cells (DCs) in the dermis and
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Langerhans cells (LCs) in the epidermis; these cells can activate primary immune responses [14, 15].
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This immunization method is gaining recognition as the skin is very easily accessible and has various
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immunological features [16]. Permeation of antigen through the skin is interrupted by stratum
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corneum, the outermost layer of the epidermis [17]. In the case of fish, the skin is composed of two
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layers, the epidermis and the dermis. The thickness of the epidermis varies depending on factors like
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part of the body, age, sex, stage of reproductive cycle, and environmental stresses [18]. However, the
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biggest obstacle to using TCI in fish is the presence of scales. Most fish are covered with these
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protective scales, which grow out of their skin.
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TCI using dissolving microneedles (MNs) has received great attention; it has already been studied for
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a variety of human diseases such as tetanus, diphtheria, malaria, and influenza [19, 20]. However,
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there has been no research into the use of MNs for the development of fish vaccines. The aim of this
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study was to develop and evaluate a dissolving MNs patch with formalin killed A. hydrophila cells
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(FKCs) The efficacy of this novel method was evaluated in a cyprinid loach model by comparing one
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group vaccinated with the MNs patch and another group vaccinated by intraperitoneal injection.
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2. Materials and methods
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2.1. Animals
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A total of 400 cyprinid loaches (Misgurnus anguillicaudatus), with a mean body weight ± standard
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deviation of 9.12 ± 2.91 g, were purchased from commercial fish farms in the Gyeonggi province,
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Republic of Korea. The fish were brought to the laboratory of the College of Veterinary Medicine of
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Seoul National University (SNU), Seoul, Republic of Korea. They were acclimatized for 30 days
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before commencing the experiment. The fish were kept in 200 liter fiberglass tanks at 25 ± 2 °C and
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fed twice a day with the commercial feed: TetraBits Complete (Tetra; USA). Approximately 30 % of
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the water in each tank was changed daily.
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All the protocols in this experiment were designed and performed in accordance with the Guidelines
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on the Regulation of Scientific Experiments on Animals and this study was approved by the
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Institutional Animal Care and Use Committee of the Seoul National University (Approval No: SNU-
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190611-5).
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2.2. Preparation of bacteria and antigen
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In 2009, there was a disease outbreak at a loach farm in the Republic of Korea that killed large
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numbers of fish; the A. hydrophila JUNAH strain was isolated from the infected animals. This
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bacterial strain was preserved in a lyophilized condition in our laboratory, and was used in the course
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of this study [21]. The preserved bacteria were cultured on tryptic soy agar (TSA) (Difco Laboratories
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Inc; Detroit, Michigan, USA) at 25 °C for 24 h, and then a single colony was cultured in tryptic soy
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broth (TSB) (Difco Laboratories Inc; Detroit, Michigan, USA) at 25 °C for 24 h. The A. hydrophila
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bacteria in culture medium were then inactivated by a 0.5 % final concentration of formalin for 48 h at
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25 °C and concentrated by centrifugation at 10,000×g and 4 °C for 10 min. The inactivated bacteria
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were washed twice in sterile phosphate-buffered saline (PBS), and resuspended in sterile PBS.
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2.3. Fabrication of molds
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The metal master was manufactured using a micromilling technique. The master for the MN tips
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contained 282 square pyramids (base width: 300 µm; height: 900 µm; needle spacing: 900 µm) on a
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circular disk. Using the master mold, female molds of polydimethylsiloxane (PDMS) were made. A
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PDMS monomer and a curing agent (SYLGARDTM 184 Silicone Elastomer, 10:1 w/w) were mixed
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well and poured into the master mold. These PDMS molds were detached from the master structure
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after 24 h at 25 °C. They were used to produce the FKC-loaded MNs patches for the study.
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2.4. Fabrication of FKC-loaded dissolving MNs patches
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Carboxymethyl cellulose (CMC) was used as the matrix material for the MNs patches as it has good
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mechanical strength and easily dissolved in water. The FKC-loaded MNs were fabricated using a
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simple molding process, casting a CMC solution containing 10% (w/v) CMC in distilled water. The
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CMC powder was completely dissolved in distilled water at 70 °C using a vortex for 30 min. The
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bubbles produced by this procedure were removed by centrifugation at 2,700×g for 30 min. The 10 %
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CMC solution was stored at 4 °C. The inactivated A. hydrophila cells prepared with PBS solution were
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centrifuged at 10,000×g for 10 min, and then the supernatant was decanted. The 10 % CMC solution
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was poured onto the inactivated cells and fully mixed with the antigen using a vortex for 10 min. The
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mixture of 10 % CMC and formalin killed cells was stored at 4 °C and used in the study.
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To fabricate the patch, 200 µl of the FKC-loaded solution was poured into the PDMS mold. The
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amount of antigen in the needle part of the patch was adjusted to 2 × 108 colony-forming units (CFU).
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The mold was placed in the desiccator for 10 min and then centrifuged at 2,700×g for 10 min. This
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procedure was repeated twice to fill the micro-cavities of the PDMS mold, to form the tips of MNs as
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well as to promote its compaction. The molds were then dried in an oven at 50 °C for 4 h. Eventually,
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the FKC-loaded MNs patches were detached from the molds and stored in the desiccator (containing
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silica gel) until further use. The morphology of the MNs was characterized using an optical
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microscope (Stereo Discovery V12) (Zeiss; Oberkochen, Germany) and a scanning electron
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microscope (JSM-6700F) (JEOL Ltd; Akishima, Tokyo, Japan).
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2.5. Skin insertion experiment
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The skin insertion experiment was performed to confirm that the MNs patches were capable of
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penetrating the skin. Crystal violet dye (1% w/v) was added to the mixture containing 10 % CMC
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solution and inactivated bacterial cells. The MNs patch was pressed onto the caudal skin of an
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anesthetized loach on a flat surface using thumb pressure for 10 s. Then, the MNs patch was removed
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and the insertion sites were observed using an optical microscope.
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2.6. Dissolving ability of the patches in water
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Following the MNs insertion test, the dissolving ability in water was simply evaluated on the skin of
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an anesthetized loach in vivo. The MNs patch was pressed onto the caudal skin of the loach on a flat
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surface using thumb pressure for 10 s; the loach was subsequently returned to its rearing tank. The
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inoculation sites were visually examined at predetermined time intervals. The dissolving ability of the
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patches was determined by measuring the time taken to dissolve completely in water.
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2.7. Vaccination and sample collection
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The 400 loaches were divided randomly into 4 experimental groups. The fish in the FKC group were
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administered 0.1 ml of formalin killed A. hydrophila cells via intraperitoneal injection; and for the fish
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in the MNsP group, transcutaneous inoculation with FKC-loaded MNs patches was performed. The
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MNs patches were inoculated onto the caudal skin of the fish by pressing with the thumb for 10 s. The
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total antigen content both in 0.1 ml of the vaccine for the FKC group and in one patch for the MNsP
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group was adjusted to 2 × 108 CFU. The fish in the control (CON) groups, CON-PBS and CON-MNs,
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were administered 0.1 ml of sterile PBS via intraperitoneal injection, or inoculated with MNs patches
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without antigen at the same site (caudal skin) respectively. Blood samples were collected from fish in
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each group following anesthetization with tricaine methanesulfonate, 300 ppm (MS-222). For
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antibody titer assays, the samples were collected at 0, 2, 4, 6, 8, 10, and 12 weeks post-vaccination
150
(wpv). To analyze the activities of lysozyme (LZM) and superoxide dismutase (SOD), the blood
151
samples were collected at 0, 1, 2, 3, and 4 wpv. The blood samples were centrifuged at 6,500×g for
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10 min at 4 °C, and then the serum was stored at −20 °C until use.
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2.8. Agglutination titer assay
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For the experiment, microtiter plates with U-shaped wells were used. Serum samples underwent serial
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two-fold dilutions in PBS and homologous inactivated A. hydrophila (107 CFU/ml) was added. The
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plates were kept overnight at 25 °C. Agglutination activity was determined by visual observation, and
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the endpoint titer was defined as the reciprocal of the highest dilution.
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2.9. Non-specific immune parameters
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The analysis of the LZM (Fish lysozyme-renal amyloidosis ELISA kit, CUSABIO, Houston, USA)
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and SOD (Fish Superoxide Dismutase ELISA kit, CUSABIO, Houston, USA) activities was
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performed using commercial kits according to the manufacturer's instructions.
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2.10. Challenge experiment
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The A. hydrophila JUNAH strain was administered as a challenge to the experimental groups (n = 30)
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at 4 wpv, i.e. the median lethal dose (LD50), by intraperitoneal injection. The clinical signs were
168
monitored and cumulative mortality was measured twice a day for 2 weeks until the end of the
169
experiment. The internal organs of the fish that died during this experiment were streaked onto TSA
170
medium and incubated at 25 °C for 24 h to identify the infective bacteria, and polymerase chain
171
reaction was performed on the isolates, as described in previous studies [23]. The challenge
172
experiment was repeated thrice, and all fish were anesthetized using MS-222 (300 ppm) prior to the
173
challenge. Vaccine efficacy was assessed by relative percent survival (RPS) using the following
174
formula:
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RPS = [1–(cumulative mortality of vaccinated group/cumulative mortality of control group)] × 100 %
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2.11. Statistical analysis
178
All data was analyzed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). A one-way analysis
179
of variance (ANOVA) was used to analyze the data, followed by Duncan's multiple range test, to
180
compare variations in immune parameters for differences at a significance level of 0.05. The
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mean ± standard error of the mean of assayed parameters was calculated for each group. The mean ±
182
standard error of the mean of the assayed parameters was calculated for each group.
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3. Results
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3.1. Fabrication of FKC-loaded MNs patches
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The FKC-loaded MNs patches were well fabricated; scanning electron microscope images of the MNs
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patches showed 282 uniformly distributed, pyramid-shaped needles on the circular base. The tips of
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these patches were well aligned with the base, and there were no damaged tips observed during the
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demolding process. The shape of the MNs without antigen loading was sharper than the FKC-loaded
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MNs. The textures on the surface of both types of MNs were, therefore, different. While the surface of
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the MNs without antigen loading looked very smooth, the surface of the FKC-loaded MNs showed a
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rich texture because of the antigen addition (Figure 1).
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3.2. Skin insertion
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Penetration of the skin is essential for transdermal delivery of an antigen using MNs patches. Loaches,
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which were used in this study, have scales on the outermost layer of their skin that need to be
197
penetrated. The skin penetration capability of the FKC-loaded MNs patches was investigated by
198
inoculating the patches for 10 s using thumb pressure, then removing them and visually observing the
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inoculation sites. Most of the tips went through the skin and had an insertion rate of 90.35% in the
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inoculated loaches (n = 9). The insertion sites were easily observed as they were dyed with crystal
201
violet (Figure 2).
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3.3. Dissolving ability in water
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The ability of the patch to dissolve in water was simply evaluated using the skin of loach in vivo. For
205
the experiment, the FKC-loaded MNs patches were inoculated onto the caudal skin of the loaches
206
using thumb pressure for 10 s. After that, the loach was left out of water for a minute and then moved
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back into the rearing tank. After treatment with the MNs patches, the insertion sites were visually
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observed at 0, 1, 5, 10, 15, and 20 mins. Following the experiments, the loaches slowly recovered from
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the anesthesia and showed no abnormalities during the 2-week observation. The patches inoculated
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onto the loaches, began to dissolve rapidly soon after replacing them in the water. The observation in
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this experiment was that the patch was not able to maintain its shape after 10 min in the water. After
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15 min, it became completely transparent except one small part. After 20 min, it was difficult to even
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observe the shape of the MNs patch on the skin of the fish (Figure 3).
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3.4. Antibody titers
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The antibody titers of all the vaccinated groups were measured for 12 wpv. The antibody titers were
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raised at 2 wpv in the group that had been inoculated with FKC-loaded MNs patches and also in the
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group that had been injected with formalin killed vaccine, titers showed a tendency to peak at 4 wpv
219
followed by a gradual decline. The antibody titers of the FKC group were significantly higher than that
220
of the MNsP group. However, there were no statistically significant differences between the 2
221
vaccinated groups from 4 wpv to the end of the experiment (Figure 4). Agglutination titers in the
222
control groups, which were administered with sterile PBS or MNs patches, remained at 0 throughout
223
the experiment. Serum titers indicated no detectable antibodies prior to vaccination in all groups.
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3.5. Non-specific immune response
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In the analysis of LZM activity, the MNsP group showed a significantly higher activity than the FKC
227
group at 1 and 2 wpv. The activity of the MNsP group peaked at 2 wpv and then declined.
228
Subsequently, there were no significant differences between the 2 groups after 3 wpv. Similar patterns
229
were seen on analyzing the SOD activity. The MNsP group showed significantly higher activity than
230
the FKC group at 1 and 2 wpv. The activity of the group peaked at 1 wpv and then gradually declined.
231
Again, there were no significant differences between the vaccinated groups at 3 and 4 wpv (Figure 5).
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3.6. RPS and mortality
234
The experimental challenge test was performed at 4 wpv to compare the efficacy of the vaccines. The
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survival rate was calculated after each group was intraperitoneally administered with the A. hydrophila
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JUNAH strain (Figure 6). The 2 vaccinated groups (MNsP and FKC) showed similar data in the
237
challenge experiment, and the survival rates of both these groups were predictably higher than those of
238
the 2 control groups (CON-PBS and CON-MNs). The survival rate of the MNsP group (53.3%) was
239
slightly higher than that of the FKC group (46.7%) for the challenge experiment. However, there were
240
no significant differences between the 2 groups. In all the groups, mortality began at 12 h post-
241
infection and continued to occur up to 60 h after the challenge. The subjects were observed for 2
242
weeks following the challenge, and the surviving fish remained alive for the rest of the experiment.
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4. Discussion
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The purpose of this study was to develop and evaluate a novel transdermal vaccine delivery system
246
using MNs, as an alternative to the injection method commonly used in the aquaculture industry. MNs
247
are considered a synergistic combination of transdermal patches and hypodermic needles. These
248
patches can penetrate the protective barrier of the skin, and enhance transdermal antigen delivery [24,
249
25]. Antigen-loaded dissolving MNs patches can induce effective immune responses. This TCI
250
method, when applied in aquaculture, has some advantages over the injection method. Vaccination
251
using injections requires the use of needles, which pose the risk of needle pricks during the
252
vaccination process [26, 27]. Moreover, if these injections are not performed by an expert, there is a
253
risk of organ damage that may result in mortality in the fish. However, this requirement for a skilled
254
professional increases the cost of the vaccination. On the other hand, dissolving MNs patches can be
255
easily and safely administered even by nonprofessionals.
256
The fundamental barrier formed by the skin of the fish prevents the entry of most exogenous
257
substances including the beneficial vaccine antigens. Transdermal inoculation is especially difficult in
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fish owing to the scales present on the skin. The thickness of the skin (consisting of the epidermal and
259
the dermal layer), and the strength and pattern of the scales vary widely among teleosts. Burrowing or
260
benthic species have characteristics such as thick epidermis containing large numbers, or large sized
261
mucous cells, and particularly reduced or absent scales [28, 29]. For this study, a species with thin and
262
soft scales would the best option for successful transdermal penetration of the vaccine. Therefore, the
263
loach was selected as the experimental animal.
264
Various substances were considered as matrix materials for the manufacture of MNs patches such as
265
CMC, hyaluronic acid, polyvinyl alcohol, and chitosan [30]. Finally, CMC, was selected as it is strong
266
enough to pierce the skin barrier, dissolves well in water, is easily produced, and can be used for a fish
267
vaccine as it is an inexpensive substance. All the above-mentioned characteristics made it the ideal
268
substance to use for this study. The MNs patches produced using CMC sufficiently penetrated the skin
269
of the fish, as shown in Figure 2, and also enhanced the innate and adaptive immunity of the fish. This
270
study used the one-step molding process for the fabrication of the MNs patches rather the than two-
271
step process. The two-step method divides the steps of making the tips and the base, such that the drug
272
is only included in the tip [31, 32]; this method is more suited for expensive substances such as
273
hormones. However, because the additives used in this study are cheap, the inactivated antigen was
274
added to both the tip and the base to simplify the fabrication process.
275
During the skin insertion tests, it was observed that some of the CMC tips were broken at the skin
276
surface. This incomplete tip insertion could result in inaccurate dosing of the transported antigen. To
277
ensure antigen delivery, the MNs should be strong enough to penetrate the skin without being broken
278
or bent when applied. A patch with enough strength to completely penetrate the skin was not produced
279
in the study. Therefore, further research may be required to find other materials that satisfy these
280
conditions.
281
In the dissolving capability test, the patches were found to remain in the skin of the fish for over 20
282
min, and this feature may serve as an advantage. Microchannels are created after the insertion of the
283
MNs patches; these microchannels might allow external pathogens to invade the fish from the
284
surrounding water [33, 34]. However, these channels are isolated from the external environment as the
285
MNs patches in this study remain on the skin for about 20 min. Blood spills were observed in the skin
286
of fish that were immediately removed from the patch after inoculation to the skin. This protects the
287
skin from invasion by external pathogens before it can recover using its natural rejuvenation process.
288
The antibody titers following vaccination showed a similar pattern in both the MNsP and the FKC
289
groups. The FKC group had a significantly higher titer at 2 wpv, but there was no statistical difference
290
between them at 4 wpv. The non-specific immune response was assessed by LZM and SOD analysis.
291
These enzymes play an important role in natural defense mechanisms [35]. LZM and SOD analysis
292
also showed no significant differences between the two vaccinated groups at 3 and 4 wpv respectively.
293
These results are also reflected in the survival rates following the challenge experiment, with no
294
significant differences in the survival rates of the 2 vaccinated groups. Various immunological
295
assessments confirmed that the efficacy of the vaccines was almost similar in both the vaccinated
296
groups. The purpose of this study is not to propose a vaccine that is more effective than the FKC
297
vaccine, but to suggest that TCI using MNs patches is an alternative to injection as a route of
298
administration. These results show that the TCI through MNs patches is a fully viable vaccination
299
method for fish.
300 301
5. Conclusions
302
In this study, vaccination by TCI using FKC-loaded MNs patches was evaluated by comparing it with
303
the FKC vaccine administered by injection in cyprinid loaches. The patch penetrated the skin of the
304
fish and dissolved well in water. The MNsP group inoculated with the MNs patches showed similar
305
adaptive and innate immune responses when compared to the loaches that were injected with the
306
vaccine. In addition, the 2 groups showed no difference in survival rates following the challenge
307
experiment. The results of this study showed that FKC-loaded MNs patches are wholly viable for the
308
vaccination of fish against A. hydrophila infections.
309 310 311
Conflicts of interest statement
312
The research was conducted in the absence of any commercial or financial relationships that could be
313
construed as a potential conflict of interest.
314 315
Acknowledgements
316
This research was supported by Cooperative Research Program for Agriculture Science and
317
Technology Development (Supportive managing project of Center for Companion Animals Research)
318
by Rural Development Administration (PJ0139852019), and by Global Ph.D Fellowship Program
319
through the National Research Foundation of Korea (NRF) funded by the Ministry of Education
320
(NRF-2015H1A2A1029732).
321 322 323 324 325 326 327
328
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1 2
Figure 1. Scanning electron microscope images of a microneedle with no antigen (A) and a
3
microneedle loaded with formalin-killed Aeromonas hydrophila (B).
4 5 6 7 8 9 10 11 12 13
14 15
Figure 2. Optical microscope images of the skin of a loach with no treatment (A) and the skin of a
16
loach inoculated with the microneedle patch (B). The microneedle patch was made evident by adding
17
crystal violet dye.
18 19 20 21 22
23 24
Figure 3. The dissolution of the microneedle patch in water following inoculation. The treated fish
25
were returned to their rearing tanks after inoculation. The inoculation site was photographed at 0, 1, 5,
26
10, 15, and 20 min post-treatment.
27
28 29
Figure 4. Serum agglutination titers in cyprinid loaches vaccinated with formalin-killed cells of
30
Aeromonas hydrophila JUNAH strain (FKC) by intraperitoneal injection and inoculated with the
31
microneedle patch (MNsP) loaded with formalin-killed cells. The control groups were administered
32
0.1 ml of sterile phosphate-buffered saline by intraperitoneal injection (CON-PBS), or inoculated with
33
MNs patches without the antigen (CON-MNs). The bars represent mean ± standard error of the mean
34
(n = 3). Different letters above the bars represent the statistical significance (p < 0.05) between the
35
different groups at the same time point.
36 37
38
39 40
Figure 5. The activity of lysozyme (A) and superoxide dismutase (B) in the blood of cyprinid loaches
41
vaccinated with formalin killed cells of Aeromonas hydrophila JUNAH strain (FKC) by
42
intraperitoneal injection and inoculation with the microneedle patch (MNsP) loaded
43
killed cells. The control groups were administered 0.1 ml of sterile phosphate-buffered saline by
44
intraperitoneal injection (CON-PBS), or inoculated with microneedle patches without antigen (CON-
45
MNs). The bars represent mean ± standard error of the mean (n = 5). Different letters above the bars
46
represent the statistical significance (p < 0.05) between different groups at the same time point.
47
[LZM: lysozyme; SOD: superoxide dismutase]
48 49 50 51 52 53 54 55
with formalin
56 57
Figure 6. The cumulative survival rate curve of the challenge experiment on cyprinid loaches.
58
Intraperitoneally administered formalin-killed cells of Aeromonas hydrophila JUNAH strain (FKC),
59
and inoculated with the microneedle patch (MNsP) loaded with formalin-killed cells. The control
60
groups were administered 0.1 ml of sterile phosphate-buffered saline by intraperitoneal injection
61
(CON-PBS), or inoculated with microneedle patches without antigen (CON-MNs). The bars represent
62
mean ± standard error of the mean (n = 30).
63
Highlights • • •
The administration for vaccinating fish using a dissolving microneedles (MNs) patch was developed and evaluated. The MNs patches were fabricated using a simple molding process, casting a carboxymethyl cellulose (CMC) solution. The patch induced adaptive and innate immune responses similar to the formalin-killed vaccine.
S1050-4648(19)31154-4
DOI:
https://doi.org/10.1016/j.fsi.2019.12.026
Reference:
YFSIM 6668
To appear in:
Fish and Shellfish Immunology
Received Date: 16 September 2019 Revised Date:
29 November 2019
Accepted Date: 9 December 2019
Please cite this article as: Yun S, Lee S-J, Giri SS, Kim HJ, Kim SG, Kim SW, Han SJ, Kwon J, Oh WT, Chang Park S, Vaccination of fish against Aeromonas hydrophila infections using the novel approach of transcutaneous immunization with dissolving microneedle patches in aquaculture, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2019.12.026. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Abstract The aim of this study was to develop and evaluate a novel route of administration for vaccinating fish against Aeromonas hydrophila infection using a dissolving microneedles (MNs) patch. The A. hydrophila JUNAH strain was inactivated with formalin and used as a vaccine antigen. It was mixed with dissolvable carboxymethyl cellulose (CMC) as the matrix material to produce the MNs patches. When examined with a scanning electron microscope, each patch has 282 uniformly distributed, pyramid-shaped needles on a circular base. In the skin insertion experiment, the MNs patches were confirmed to be capable of penetrating the skin of the fish. Through agglutination assay and analysis of non-specific parameters like lysozyme and superoxide dismutase, it was verified that the antigen embedded into the patch induced adaptive and innate immune responses in the fish. In the challenge experiment, the group inoculated with the MNs patch and the group injected with formalin killed cells (FKC) showed a similar survival rate. Our results suggest that the FKC-loaded MNs patch is a wholly viable method alternative to injection for the vaccination of fish.
Keywords: Aeromonas hydrophila, carboxymethyl cellulose, cyprinid loach, dissolving microneedles, transcutaneous immunization.
1
Vaccination of fish against Aeromonas hydrophila infections using the novel approach of
2
transcutaneous immunization with dissolving microneedle patches in aquaculture
3
Saekil Yuna, Seung-Jun Leeb, Sib Sankar Giria, Hyoun Joong Kima, Sang Geun Kima, Sang Wha Kima,
4
Se Jin Hana, Jun Kwona, Woo Taek Oha, Se Chang Parka, *
5 6
a
7
Veterinary Science, Seoul National University, Seoul 08826, Republic of Korea
8
b
9
Technology, Seowon University, Cheongju 28674, Republic of Korea
Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for
Department of Pharmaceutical Science and Engineering, School of Convergence Bioscience and
10 11
* Corresponding author: Se Chang Park, DVM, Ph.D.
12
Mailing address: College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul
13
National University, 81-417, 1 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea 08826
14
Phone: +82-2-880-1282, Fax: +82-2-880-1213. E-mail: [email protected]
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
33
1. Introduction
34
Aeromonas hydrophila is a gram-negative, rod-shaped bacterium [1] and it is a significant pathogen
35
affecting the aquaculture industry. This pathogen can cause hemorrhagic septicemia in many species
36
of fish, resulting in massive mortalities in freshwater fish, as well as in higher vertebrates [2–4]. This
37
is important from the point of view of public health as there is possibility of human infection [5–7].
38
Vaccination of the fish is gradually gaining popularity as a cost-effective method of combating this
39
disease [8]. There have been many achievements while researching the vaccine against A. hydrophila
40
including DNA, recombinant protein, and live attenuated vaccines [9–11].
41
There are several routes of administration to deliver the vaccine antigen to the fish. One route is
42
injection; this method is reliable as there is direct delivery of small amounts of antigen, and generally
43
results in effective protection from the disease [12]. However, this procedure should be performed
44
only by an expert, and also requires the fish to be anesthetized; as such, it is labor-intensive and
45
requires a greater financial investment. The second route is by immersion, where the fish are placed in
46
tanks containing the vaccine; this method is simple, but cannot be adopted by all aquaculture farms.
47
Oral administration is the third method used by the aquaculture industry. It can be performed by
48
coating the feed with the antigen or by mixing the antigen with the feed. This route is advantageous as
49
it can be easily administered to a large number of fish at once. However, there is no uniformity in the
50
amount of antigen ingested by each fish and this method may be ineffective in some species where the
51
antigen is damaged in the stomach [13]. In this study, we are studying transcutaneous immunization
52
(TCI) which is being used in fish for the first time. This route has the advantage of being easily
53
administrable and being reliably effective, thus overcoming the difficulties faced by the methods
54
currently in use.
55
The administration of vaccines through the skin (using its large surface area) is called TCI. The skin is
56
abundant in trained antigen presenting cells (APCs), there are dendritic cells (DCs) in the dermis and
57
Langerhans cells (LCs) in the epidermis; these cells can activate primary immune responses [14, 15].
58
This immunization method is gaining recognition as the skin is very easily accessible and has various
59
immunological features [16]. Permeation of antigen through the skin is interrupted by stratum
60
corneum, the outermost layer of the epidermis [17]. In the case of fish, the skin is composed of two
61
layers, the epidermis and the dermis. The thickness of the epidermis varies depending on factors like
62
part of the body, age, sex, stage of reproductive cycle, and environmental stresses [18]. However, the
63
biggest obstacle to using TCI in fish is the presence of scales. Most fish are covered with these
64
protective scales, which grow out of their skin.
65
TCI using dissolving microneedles (MNs) has received great attention; it has already been studied for
66
a variety of human diseases such as tetanus, diphtheria, malaria, and influenza [19, 20]. However,
67
there has been no research into the use of MNs for the development of fish vaccines. The aim of this
68
study was to develop and evaluate a dissolving MNs patch with formalin killed A. hydrophila cells
69
(FKCs) The efficacy of this novel method was evaluated in a cyprinid loach model by comparing one
70
group vaccinated with the MNs patch and another group vaccinated by intraperitoneal injection.
71 72
2. Materials and methods
73
2.1. Animals
74
A total of 400 cyprinid loaches (Misgurnus anguillicaudatus), with a mean body weight ± standard
75
deviation of 9.12 ± 2.91 g, were purchased from commercial fish farms in the Gyeonggi province,
76
Republic of Korea. The fish were brought to the laboratory of the College of Veterinary Medicine of
77
Seoul National University (SNU), Seoul, Republic of Korea. They were acclimatized for 30 days
78
before commencing the experiment. The fish were kept in 200 liter fiberglass tanks at 25 ± 2 °C and
79
fed twice a day with the commercial feed: TetraBits Complete (Tetra; USA). Approximately 30 % of
80
the water in each tank was changed daily.
81
All the protocols in this experiment were designed and performed in accordance with the Guidelines
82
on the Regulation of Scientific Experiments on Animals and this study was approved by the
83
Institutional Animal Care and Use Committee of the Seoul National University (Approval No: SNU-
84
190611-5).
85 86
2.2. Preparation of bacteria and antigen
87
In 2009, there was a disease outbreak at a loach farm in the Republic of Korea that killed large
88
numbers of fish; the A. hydrophila JUNAH strain was isolated from the infected animals. This
89
bacterial strain was preserved in a lyophilized condition in our laboratory, and was used in the course
90
of this study [21]. The preserved bacteria were cultured on tryptic soy agar (TSA) (Difco Laboratories
91
Inc; Detroit, Michigan, USA) at 25 °C for 24 h, and then a single colony was cultured in tryptic soy
92
broth (TSB) (Difco Laboratories Inc; Detroit, Michigan, USA) at 25 °C for 24 h. The A. hydrophila
93
bacteria in culture medium were then inactivated by a 0.5 % final concentration of formalin for 48 h at
94
25 °C and concentrated by centrifugation at 10,000×g and 4 °C for 10 min. The inactivated bacteria
95
were washed twice in sterile phosphate-buffered saline (PBS), and resuspended in sterile PBS.
96 97
2.3. Fabrication of molds
98
The metal master was manufactured using a micromilling technique. The master for the MN tips
99
contained 282 square pyramids (base width: 300 µm; height: 900 µm; needle spacing: 900 µm) on a
100
circular disk. Using the master mold, female molds of polydimethylsiloxane (PDMS) were made. A
101
PDMS monomer and a curing agent (SYLGARDTM 184 Silicone Elastomer, 10:1 w/w) were mixed
102
well and poured into the master mold. These PDMS molds were detached from the master structure
103
after 24 h at 25 °C. They were used to produce the FKC-loaded MNs patches for the study.
104 105
2.4. Fabrication of FKC-loaded dissolving MNs patches
106
Carboxymethyl cellulose (CMC) was used as the matrix material for the MNs patches as it has good
107
mechanical strength and easily dissolved in water. The FKC-loaded MNs were fabricated using a
108
simple molding process, casting a CMC solution containing 10% (w/v) CMC in distilled water. The
109
CMC powder was completely dissolved in distilled water at 70 °C using a vortex for 30 min. The
110
bubbles produced by this procedure were removed by centrifugation at 2,700×g for 30 min. The 10 %
111
CMC solution was stored at 4 °C. The inactivated A. hydrophila cells prepared with PBS solution were
112
centrifuged at 10,000×g for 10 min, and then the supernatant was decanted. The 10 % CMC solution
113
was poured onto the inactivated cells and fully mixed with the antigen using a vortex for 10 min. The
114
mixture of 10 % CMC and formalin killed cells was stored at 4 °C and used in the study.
115
To fabricate the patch, 200 µl of the FKC-loaded solution was poured into the PDMS mold. The
116
amount of antigen in the needle part of the patch was adjusted to 2 × 108 colony-forming units (CFU).
117
The mold was placed in the desiccator for 10 min and then centrifuged at 2,700×g for 10 min. This
118
procedure was repeated twice to fill the micro-cavities of the PDMS mold, to form the tips of MNs as
119
well as to promote its compaction. The molds were then dried in an oven at 50 °C for 4 h. Eventually,
120
the FKC-loaded MNs patches were detached from the molds and stored in the desiccator (containing
121
silica gel) until further use. The morphology of the MNs was characterized using an optical
122
microscope (Stereo Discovery V12) (Zeiss; Oberkochen, Germany) and a scanning electron
123
microscope (JSM-6700F) (JEOL Ltd; Akishima, Tokyo, Japan).
124 125
2.5. Skin insertion experiment
126
The skin insertion experiment was performed to confirm that the MNs patches were capable of
127
penetrating the skin. Crystal violet dye (1% w/v) was added to the mixture containing 10 % CMC
128
solution and inactivated bacterial cells. The MNs patch was pressed onto the caudal skin of an
129
anesthetized loach on a flat surface using thumb pressure for 10 s. Then, the MNs patch was removed
130
and the insertion sites were observed using an optical microscope.
131 132
2.6. Dissolving ability of the patches in water
133
Following the MNs insertion test, the dissolving ability in water was simply evaluated on the skin of
134
an anesthetized loach in vivo. The MNs patch was pressed onto the caudal skin of the loach on a flat
135
surface using thumb pressure for 10 s; the loach was subsequently returned to its rearing tank. The
136
inoculation sites were visually examined at predetermined time intervals. The dissolving ability of the
137
patches was determined by measuring the time taken to dissolve completely in water.
138 139
2.7. Vaccination and sample collection
140
The 400 loaches were divided randomly into 4 experimental groups. The fish in the FKC group were
141
administered 0.1 ml of formalin killed A. hydrophila cells via intraperitoneal injection; and for the fish
142
in the MNsP group, transcutaneous inoculation with FKC-loaded MNs patches was performed. The
143
MNs patches were inoculated onto the caudal skin of the fish by pressing with the thumb for 10 s. The
144
total antigen content both in 0.1 ml of the vaccine for the FKC group and in one patch for the MNsP
145
group was adjusted to 2 × 108 CFU. The fish in the control (CON) groups, CON-PBS and CON-MNs,
146
were administered 0.1 ml of sterile PBS via intraperitoneal injection, or inoculated with MNs patches
147
without antigen at the same site (caudal skin) respectively. Blood samples were collected from fish in
148
each group following anesthetization with tricaine methanesulfonate, 300 ppm (MS-222). For
149
antibody titer assays, the samples were collected at 0, 2, 4, 6, 8, 10, and 12 weeks post-vaccination
150
(wpv). To analyze the activities of lysozyme (LZM) and superoxide dismutase (SOD), the blood
151
samples were collected at 0, 1, 2, 3, and 4 wpv. The blood samples were centrifuged at 6,500×g for
152
10 min at 4 °C, and then the serum was stored at −20 °C until use.
153 154
2.8. Agglutination titer assay
155
For the experiment, microtiter plates with U-shaped wells were used. Serum samples underwent serial
156
two-fold dilutions in PBS and homologous inactivated A. hydrophila (107 CFU/ml) was added. The
157
plates were kept overnight at 25 °C. Agglutination activity was determined by visual observation, and
158
the endpoint titer was defined as the reciprocal of the highest dilution.
159 160
2.9. Non-specific immune parameters
161
The analysis of the LZM (Fish lysozyme-renal amyloidosis ELISA kit, CUSABIO, Houston, USA)
162
and SOD (Fish Superoxide Dismutase ELISA kit, CUSABIO, Houston, USA) activities was
163
performed using commercial kits according to the manufacturer's instructions.
164 165
2.10. Challenge experiment
166
The A. hydrophila JUNAH strain was administered as a challenge to the experimental groups (n = 30)
167
at 4 wpv, i.e. the median lethal dose (LD50), by intraperitoneal injection. The clinical signs were
168
monitored and cumulative mortality was measured twice a day for 2 weeks until the end of the
169
experiment. The internal organs of the fish that died during this experiment were streaked onto TSA
170
medium and incubated at 25 °C for 24 h to identify the infective bacteria, and polymerase chain
171
reaction was performed on the isolates, as described in previous studies [23]. The challenge
172
experiment was repeated thrice, and all fish were anesthetized using MS-222 (300 ppm) prior to the
173
challenge. Vaccine efficacy was assessed by relative percent survival (RPS) using the following
174
formula:
175
RPS = [1–(cumulative mortality of vaccinated group/cumulative mortality of control group)] × 100 %
176 177
2.11. Statistical analysis
178
All data was analyzed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). A one-way analysis
179
of variance (ANOVA) was used to analyze the data, followed by Duncan's multiple range test, to
180
compare variations in immune parameters for differences at a significance level of 0.05. The
181
mean ± standard error of the mean of assayed parameters was calculated for each group. The mean ±
182
standard error of the mean of the assayed parameters was calculated for each group.
183 184
3. Results
185
3.1. Fabrication of FKC-loaded MNs patches
186
The FKC-loaded MNs patches were well fabricated; scanning electron microscope images of the MNs
187
patches showed 282 uniformly distributed, pyramid-shaped needles on the circular base. The tips of
188
these patches were well aligned with the base, and there were no damaged tips observed during the
189
demolding process. The shape of the MNs without antigen loading was sharper than the FKC-loaded
190
MNs. The textures on the surface of both types of MNs were, therefore, different. While the surface of
191
the MNs without antigen loading looked very smooth, the surface of the FKC-loaded MNs showed a
192
rich texture because of the antigen addition (Figure 1).
193 194
3.2. Skin insertion
195
Penetration of the skin is essential for transdermal delivery of an antigen using MNs patches. Loaches,
196
which were used in this study, have scales on the outermost layer of their skin that need to be
197
penetrated. The skin penetration capability of the FKC-loaded MNs patches was investigated by
198
inoculating the patches for 10 s using thumb pressure, then removing them and visually observing the
199
inoculation sites. Most of the tips went through the skin and had an insertion rate of 90.35% in the
200
inoculated loaches (n = 9). The insertion sites were easily observed as they were dyed with crystal
201
violet (Figure 2).
202 203
3.3. Dissolving ability in water
204
The ability of the patch to dissolve in water was simply evaluated using the skin of loach in vivo. For
205
the experiment, the FKC-loaded MNs patches were inoculated onto the caudal skin of the loaches
206
using thumb pressure for 10 s. After that, the loach was left out of water for a minute and then moved
207
back into the rearing tank. After treatment with the MNs patches, the insertion sites were visually
208
observed at 0, 1, 5, 10, 15, and 20 mins. Following the experiments, the loaches slowly recovered from
209
the anesthesia and showed no abnormalities during the 2-week observation. The patches inoculated
210
onto the loaches, began to dissolve rapidly soon after replacing them in the water. The observation in
211
this experiment was that the patch was not able to maintain its shape after 10 min in the water. After
212
15 min, it became completely transparent except one small part. After 20 min, it was difficult to even
213
observe the shape of the MNs patch on the skin of the fish (Figure 3).
214 215
3.4. Antibody titers
216
The antibody titers of all the vaccinated groups were measured for 12 wpv. The antibody titers were
217
raised at 2 wpv in the group that had been inoculated with FKC-loaded MNs patches and also in the
218
group that had been injected with formalin killed vaccine, titers showed a tendency to peak at 4 wpv
219
followed by a gradual decline. The antibody titers of the FKC group were significantly higher than that
220
of the MNsP group. However, there were no statistically significant differences between the 2
221
vaccinated groups from 4 wpv to the end of the experiment (Figure 4). Agglutination titers in the
222
control groups, which were administered with sterile PBS or MNs patches, remained at 0 throughout
223
the experiment. Serum titers indicated no detectable antibodies prior to vaccination in all groups.
224 225
3.5. Non-specific immune response
226
In the analysis of LZM activity, the MNsP group showed a significantly higher activity than the FKC
227
group at 1 and 2 wpv. The activity of the MNsP group peaked at 2 wpv and then declined.
228
Subsequently, there were no significant differences between the 2 groups after 3 wpv. Similar patterns
229
were seen on analyzing the SOD activity. The MNsP group showed significantly higher activity than
230
the FKC group at 1 and 2 wpv. The activity of the group peaked at 1 wpv and then gradually declined.
231
Again, there were no significant differences between the vaccinated groups at 3 and 4 wpv (Figure 5).
232 233
3.6. RPS and mortality
234
The experimental challenge test was performed at 4 wpv to compare the efficacy of the vaccines. The
235
survival rate was calculated after each group was intraperitoneally administered with the A. hydrophila
236
JUNAH strain (Figure 6). The 2 vaccinated groups (MNsP and FKC) showed similar data in the
237
challenge experiment, and the survival rates of both these groups were predictably higher than those of
238
the 2 control groups (CON-PBS and CON-MNs). The survival rate of the MNsP group (53.3%) was
239
slightly higher than that of the FKC group (46.7%) for the challenge experiment. However, there were
240
no significant differences between the 2 groups. In all the groups, mortality began at 12 h post-
241
infection and continued to occur up to 60 h after the challenge. The subjects were observed for 2
242
weeks following the challenge, and the surviving fish remained alive for the rest of the experiment.
243 244
4. Discussion
245
The purpose of this study was to develop and evaluate a novel transdermal vaccine delivery system
246
using MNs, as an alternative to the injection method commonly used in the aquaculture industry. MNs
247
are considered a synergistic combination of transdermal patches and hypodermic needles. These
248
patches can penetrate the protective barrier of the skin, and enhance transdermal antigen delivery [24,
249
25]. Antigen-loaded dissolving MNs patches can induce effective immune responses. This TCI
250
method, when applied in aquaculture, has some advantages over the injection method. Vaccination
251
using injections requires the use of needles, which pose the risk of needle pricks during the
252
vaccination process [26, 27]. Moreover, if these injections are not performed by an expert, there is a
253
risk of organ damage that may result in mortality in the fish. However, this requirement for a skilled
254
professional increases the cost of the vaccination. On the other hand, dissolving MNs patches can be
255
easily and safely administered even by nonprofessionals.
256
The fundamental barrier formed by the skin of the fish prevents the entry of most exogenous
257
substances including the beneficial vaccine antigens. Transdermal inoculation is especially difficult in
258
fish owing to the scales present on the skin. The thickness of the skin (consisting of the epidermal and
259
the dermal layer), and the strength and pattern of the scales vary widely among teleosts. Burrowing or
260
benthic species have characteristics such as thick epidermis containing large numbers, or large sized
261
mucous cells, and particularly reduced or absent scales [28, 29]. For this study, a species with thin and
262
soft scales would the best option for successful transdermal penetration of the vaccine. Therefore, the
263
loach was selected as the experimental animal.
264
Various substances were considered as matrix materials for the manufacture of MNs patches such as
265
CMC, hyaluronic acid, polyvinyl alcohol, and chitosan [30]. Finally, CMC, was selected as it is strong
266
enough to pierce the skin barrier, dissolves well in water, is easily produced, and can be used for a fish
267
vaccine as it is an inexpensive substance. All the above-mentioned characteristics made it the ideal
268
substance to use for this study. The MNs patches produced using CMC sufficiently penetrated the skin
269
of the fish, as shown in Figure 2, and also enhanced the innate and adaptive immunity of the fish. This
270
study used the one-step molding process for the fabrication of the MNs patches rather the than two-
271
step process. The two-step method divides the steps of making the tips and the base, such that the drug
272
is only included in the tip [31, 32]; this method is more suited for expensive substances such as
273
hormones. However, because the additives used in this study are cheap, the inactivated antigen was
274
added to both the tip and the base to simplify the fabrication process.
275
During the skin insertion tests, it was observed that some of the CMC tips were broken at the skin
276
surface. This incomplete tip insertion could result in inaccurate dosing of the transported antigen. To
277
ensure antigen delivery, the MNs should be strong enough to penetrate the skin without being broken
278
or bent when applied. A patch with enough strength to completely penetrate the skin was not produced
279
in the study. Therefore, further research may be required to find other materials that satisfy these
280
conditions.
281
In the dissolving capability test, the patches were found to remain in the skin of the fish for over 20
282
min, and this feature may serve as an advantage. Microchannels are created after the insertion of the
283
MNs patches; these microchannels might allow external pathogens to invade the fish from the
284
surrounding water [33, 34]. However, these channels are isolated from the external environment as the
285
MNs patches in this study remain on the skin for about 20 min. Blood spills were observed in the skin
286
of fish that were immediately removed from the patch after inoculation to the skin. This protects the
287
skin from invasion by external pathogens before it can recover using its natural rejuvenation process.
288
The antibody titers following vaccination showed a similar pattern in both the MNsP and the FKC
289
groups. The FKC group had a significantly higher titer at 2 wpv, but there was no statistical difference
290
between them at 4 wpv. The non-specific immune response was assessed by LZM and SOD analysis.
291
These enzymes play an important role in natural defense mechanisms [35]. LZM and SOD analysis
292
also showed no significant differences between the two vaccinated groups at 3 and 4 wpv respectively.
293
These results are also reflected in the survival rates following the challenge experiment, with no
294
significant differences in the survival rates of the 2 vaccinated groups. Various immunological
295
assessments confirmed that the efficacy of the vaccines was almost similar in both the vaccinated
296
groups. The purpose of this study is not to propose a vaccine that is more effective than the FKC
297
vaccine, but to suggest that TCI using MNs patches is an alternative to injection as a route of
298
administration. These results show that the TCI through MNs patches is a fully viable vaccination
299
method for fish.
300 301
5. Conclusions
302
In this study, vaccination by TCI using FKC-loaded MNs patches was evaluated by comparing it with
303
the FKC vaccine administered by injection in cyprinid loaches. The patch penetrated the skin of the
304
fish and dissolved well in water. The MNsP group inoculated with the MNs patches showed similar
305
adaptive and innate immune responses when compared to the loaches that were injected with the
306
vaccine. In addition, the 2 groups showed no difference in survival rates following the challenge
307
experiment. The results of this study showed that FKC-loaded MNs patches are wholly viable for the
308
vaccination of fish against A. hydrophila infections.
309 310 311
Conflicts of interest statement
312
The research was conducted in the absence of any commercial or financial relationships that could be
313
construed as a potential conflict of interest.
314 315
Acknowledgements
316
This research was supported by Cooperative Research Program for Agriculture Science and
317
Technology Development (Supportive managing project of Center for Companion Animals Research)
318
by Rural Development Administration (PJ0139852019), and by Global Ph.D Fellowship Program
319
through the National Research Foundation of Korea (NRF) funded by the Ministry of Education
320
(NRF-2015H1A2A1029732).
321 322 323 324 325 326 327
328
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1 2
Figure 1. Scanning electron microscope images of a microneedle with no antigen (A) and a
3
microneedle loaded with formalin-killed Aeromonas hydrophila (B).
4 5 6 7 8 9 10 11 12 13
14 15
Figure 2. Optical microscope images of the skin of a loach with no treatment (A) and the skin of a
16
loach inoculated with the microneedle patch (B). The microneedle patch was made evident by adding
17
crystal violet dye.
18 19 20 21 22
23 24
Figure 3. The dissolution of the microneedle patch in water following inoculation. The treated fish
25
were returned to their rearing tanks after inoculation. The inoculation site was photographed at 0, 1, 5,
26
10, 15, and 20 min post-treatment.
27
28 29
Figure 4. Serum agglutination titers in cyprinid loaches vaccinated with formalin-killed cells of
30
Aeromonas hydrophila JUNAH strain (FKC) by intraperitoneal injection and inoculated with the
31
microneedle patch (MNsP) loaded with formalin-killed cells. The control groups were administered
32
0.1 ml of sterile phosphate-buffered saline by intraperitoneal injection (CON-PBS), or inoculated with
33
MNs patches without the antigen (CON-MNs). The bars represent mean ± standard error of the mean
34
(n = 3). Different letters above the bars represent the statistical significance (p < 0.05) between the
35
different groups at the same time point.
36 37
38
39 40
Figure 5. The activity of lysozyme (A) and superoxide dismutase (B) in the blood of cyprinid loaches
41
vaccinated with formalin killed cells of Aeromonas hydrophila JUNAH strain (FKC) by
42
intraperitoneal injection and inoculation with the microneedle patch (MNsP) loaded
43
killed cells. The control groups were administered 0.1 ml of sterile phosphate-buffered saline by
44
intraperitoneal injection (CON-PBS), or inoculated with microneedle patches without antigen (CON-
45
MNs). The bars represent mean ± standard error of the mean (n = 5). Different letters above the bars
46
represent the statistical significance (p < 0.05) between different groups at the same time point.
47
[LZM: lysozyme; SOD: superoxide dismutase]
48 49 50 51 52 53 54 55
with formalin
56 57
Figure 6. The cumulative survival rate curve of the challenge experiment on cyprinid loaches.
58
Intraperitoneally administered formalin-killed cells of Aeromonas hydrophila JUNAH strain (FKC),
59
and inoculated with the microneedle patch (MNsP) loaded with formalin-killed cells. The control
60
groups were administered 0.1 ml of sterile phosphate-buffered saline by intraperitoneal injection
61
(CON-PBS), or inoculated with microneedle patches without antigen (CON-MNs). The bars represent
62
mean ± standard error of the mean (n = 30).
63
Highlights • • •
The administration for vaccinating fish using a dissolving microneedles (MNs) patch was developed and evaluated. The MNs patches were fabricated using a simple molding process, casting a carboxymethyl cellulose (CMC) solution. The patch induced adaptive and innate immune responses similar to the formalin-killed vaccine.