Monoclonal antibodies against loggerhead sea turtle ...

Developmental and Comparative Immunology 87 (2018) 12–15

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Monoclonal antibodies against loggerhead sea turtle, Caretta caretta, IgY isoforms reveal differential contributions to antibody titers and relatedness among other sea turtles

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Maria L. Rodgers1, Charles D. Rice∗ Department of Biological Sciences, Clemson University, Clemson, SC, 29634, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Sea turtle IgY Sea turtle IgY-specific mAbs Caretta caretta loggerhead sea turtles mAb LH12 mAb LH1 mAb LH9

Serum from loggerhead sea turtles, Caretta caretta, was collected from the southeast Atlantic Ocean during routine summer monitoring studies in 2017. Serum immunoglobulin IgY was purified and used to develop IgY isoform-specific monoclonal antibodies (mAb). mAb LH12 was developed against the 66 kDa heavy chain of IgY, mAb LH1 was developed against the truncated heavy chain of approximately 37 kDA, and mAb LH9 was developed against the 23 kDa light chains. mAb LH9 reacts with the light chains of all sea turtles, mAb LH12 reacts with the long heavy chain of all sea turtles within the family Cheloniidae, and mAb LH1 reacts with the truncated form of IgY in both olive and Kemp's ridley turtles. Circulating IgY antibodies against three different marine bacterial pathogens were determined in 16 loggerhead samples using these mAbs. mAb LH12 detects higher titers than mAb LH1, and mAb LH9 detects the highest titers.

1. Introduction Sea turtles, like all reptiles, produce immunoglobulin Y (IgY) as their mature antibody response to antigens (Leslie and Clem, 1972; Warr et al., 1995; Wei et al., 2009; Work et al., 2015a). Current knowledge suggests that IgY is phylogenetically derived from IgM and IgA, and subsequently gave rise to mammalian immunoglobulin G (IgG) and immunoglobulin E (IgE) (Zhang et al., 2017). Typical IgY has two heavy and two light chains with a total molecular weight of approximately 180 kDa (Taylor et al., 2009; Zhang et al., 2017). Another common isoform of IgY exists as a truncated form of approximately 120 kDa, known as IgY (ΔFc). Reptiles and birds may produce the 180 kDa IgY isoform, the 120 kDa IgY (ΔFc) isoform (Fab), or both (Leslie and Clem, 1969). Based on what is known about avian IgY expression, it can be assumed that the Fab product in turtles is the product of alternative splicing of heavy chain mRNA at specific switch regions (Munhoz et al., 2014). The larger isoform of IgY consists of two heavy chains, known as 7S IgY, each having a mass of approximately 63 kDa, with one variable and four constant regions. The smaller Fab isoform consists of two heavy chains, known as 5.7S IgY, each having a mass of approximately 33.8 kDa, with one variable and two constant regions. The light chain for IgY has a mass of approximately 23.1 kDa (Muñoz et al., 2013).

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Due to its presence in diverse taxa, little is known about the function of IgY across vertebrate phyla that produce this immunoglobulin (Zhang et al., 2017). In addition, the two isoforms of IgY do not always possess the same functional abilities. For example, in animals that produced both IgY and Fab, or solely IgY, IgY may be transferred to the embryo thorough yolk, but in animals solely producing the Fab, it is transmitted to the embryo via the yolk-sac (Warr et al., 1995). Moreover, IgY, but not the Fab, may contribute to anaphylaxis, and thus evolutionary pressure may have selected for the production of a certain amount non-anaphylaxis inducing isoform in circulation. In ducks the approximate ratio of IgY to Fab in serum is 3:5, but this ratio may vary when examined in other species, including sea turtles (Lundqvist et al., 2006). The ability to measure pathogen-specific IgY responses would enable investigators to determine which kinds of pathogens sea turtles respond to immunologically, and to determine the relative contribution of the two isoforms to immunity. Previous attempts by others were made to quantify IgY in loggerhead sea turtles using a monoclonal antibody (HL673) against the light chains of desert tortoise immunoglobulin (Kaplan et al., 2016; Schumacher et al., 1993). However, at this time it is not known if that antibody is specific for the light chains of IgY or IgM, and it is not species-specific like monoclonal antibodies generated against green sea turtle IgY (Herbst and Klein, 1995;

Corresponding author. Department of Biological Sciences, 132 Long Hal, Clemson, SC 29634, USA. E-mail address: [email protected] (C.D. Rice). Current address: Division of Coastal Sciences, School of Ocean Science and Technology, University of Southern Mississippi, Ocean Springs, Mississippi, 39564, USA.

https://doi.org/10.1016/j.dci.2018.05.015 Received 7 May 2018; Received in revised form 18 May 2018; Accepted 18 May 2018 Available online 19 May 2018 0145-305X/ © 2018 Elsevier Ltd. All rights reserved.


M.L. Rodgers, C.D. Rice

2.4. Development of enzyme-linked immunosorbent assays (ELISAs) for quantifying bacteria-specific IgY titers in individual turtles

Work et al., 2015b), and loggerhead IgY described herein. Previous work in our lab generated mouse polyclonal antisera against loggerhead IgY, but polyclonal antisera does not allow us to determine the relative contribution of each IgY isoform in various experimental inquiries (Rodgers et al., 2018). In the study described herein, loggerhead turtle serum samples were collected during routine monitoring efforts over the summer of 2017, and used to purify serum IgY and produce highly specific monoclonal antibodies (mAbs) against the full length heavy chain, the truncated Fab heavy chain, and light chains. These mAbs were then characterized as to their isotypes, cross reactivity with other in-hand sea turtle serum samples, and to determine the contribution of IgY isoforms to total circulating IgY titers against select marine bacteria. To our knowledge, this is the first study to develop mAbs against both IgY isoforms in loggerhead sea turtles, and the first to examine the relative contributions of the different IgY isoforms to specific antigens.

Recent studies demonstrated that loggerhead turtle produce IgY that recognizes several marine bacterial pathogens (Rodgers et al., 2018). In this study, Mycobacterium marinum, Vibrio parahaemolyticus, and Escherichia coli were obtained from ATCC, and grown using recommended materials and methods. Background on each bacteria and rationale for querying sea turtle antibody responses to these pathogens is provided in previously published work as a supplemental file (Rodgers et al., 2018). To perform enzyme-linked immunosorbent assays (ELISAs), the above three marine bacteria were again grown under their specific conditions and coated onto ELISA plates as previously described. Loggerhead serum suspensions were made by adding 10 μL of serum from each individual turtle into 990 μL of PBS in 1.5 mL snap cap tubes. Forty μL of diluted serum solution from 16 individuals were added to 6 wells containing 40 μL PBS, and incubated at 4 °C overnight. The next day, plates were washed three times with PBS-TW20, then 75 μL of a 5 μg/ml solution of the three different mAbs were added to the wells. Plates were washed three times with PBS-TW20 after 2 h of room temperature incubation. Goat anti-mouse IgG AP (1:1000) was added in 75 μL volumes to each well and incubated at room temperature for 1.5 h before being washed four times with PBS-TW20. Finally, 100 μL of 1 mg/mL p-nitrophenol phosphate (ThermoFisher, Waltham, MA, USA) in AP buffer were added to each well, and the plates were read for optical densities at 405 nm after 30 min and the data were recorded.

2. Materials and methods 2.1. Animals and serum collection Serum samples from individual loggerhead turtles and Kemp's ridley turtles were collected in the summer of 2017 during routine monitoring efforts by the South Carolina Department of Natural Resources and the Southern Atlantic Fish Management Council, and shipped overnight to Clemson University. Serum samples for leatherback, green, olive ridley, and hawksbill turtles were kindly provided by Dr. Theirry Work, USGS HI. All serum samples were maintained at −80 °C until further analysis.

2.5. Statistical analysis Titers were compared to one another by a one-way analysis of variance (ANOVA) with a Tukey's multiple contrast post-hoc test whenever differences were noted by the ANOVA. GraphPad Prism's version 7 statistical software (GraphPad Software, La Jolla, CA, USA) was used for all statistical analysis, and the α-value was set at 0.05 prior to the study. Statistical summaries of all comparisons are provided as a supplemental file (Supplemental file 1).

2.2. Monoclonal antibody production and characterization Previous work in our lab and by other investigators shows that sea turtle IgY can be purified using the immunoglobulin-binding Protein-G from Streptococcal bacteria (Rodgers et al., 2018), and these same procedures were used in this study. Six-week old female Balb/c mice were housed at the Godley-Snell Animal Facility at Clemson University under IACUC approved protocols. Mice were given a sub-cutaneous (s.c.) injection of 50 μg purified IgY in 0.9% saline containing TiterMax Gold adjuvant on day 1. Fourteen days later mice received a second s.c. immunization using Freund's incomplete adjuvant. Subsequent boosters at 21 day intervals were given in saline via s.c. immunizations, and the final booster was given intraperitoneally. Five days after the last booster immunization, mice were sacrificed using slow lethal CO2 hypoxia, and their spleens removed using aseptic methods. Procedures for splenic plasma cell fusion with Sp02-14 myelomas, and for screening, cloning, purification, and isotyping of the resulting antibodies have been described elsewhere (Margiotta et al., 2017; Rice et al., 1998).

3. Results and discussion 3.1. Reactivity of mAbs against loggerhead IgY and in other turtles As shown in previous work, loggerhead and green turtle IgY is easily purified using protein G columns, and contains heavy chains, light chains, and the truncated IgY heavy chain (Fig. 1A). Attempts to develop mAbs against all three chains of loggerhead IgY were successful, and after further characterizations, mAb LH1 (IgG2a, κ1) was found to be specific for the truncated IgY heavy chain, mAb LH9 (IgG1, κ1) to be specific for IgY light chains, and mAb LH12 (IgG1, κ1) to be specific for the heavy chains of IgY (Fig. 1B). In this study we found that mAb LH12 recognizes the heavy chain of not only loggerhead, but green, olive ridley, hawksbill, and Kemp's ridley IgY, yet not leatherback turtles (Fig. 1C). Leatherback sea turtles are within the family Dermochelyidae, while all others are within the family Cheloniidae, and thus the phylogenetic distance probably precludes reactivity of mAb LH12 with leatherback IgY heavy chains. In general, antibodies generated against immunoglobulins of a particular species are not reactive with other species, even of the same genera. However, our observations may indicate that sea turtles within the family Cheloniidae are more closely related than previously assumed, or that the constant domain recognized by mAb LH12 is highly conserved across the family. At the time of study, serum samples from the Australian flatback sea turtle, Natator depressus, were not available, but we predict that mAb LH12 would recognize the same protein in these turtles. Previous work shows that loggerhead sea turtles are more closely related to Kemp's ridley and olive ridley than other sea turtles, and this can be visualized immunologically in our study using mAb LH1 against the truncated form

2.3. Gel electrophoresis and immunoblotting To determine the reactivity of mAbs with purified IgY, 5 μg of purified loggerhead IgY or serum diluted 1:20 in PBS were subjected to SDS-PAGE and transferred to PVDF membranes. After blocking with 10% FBS in PBS, the membranes were probed with 5 μg/ml of each mAb for 1 h at room temperature, washed 3x with PBS-Tween 20 (PBSTW20), then probed again with goat anti-mouse IgG secondary antibody conjugated with alkaline phosphatase (1:1500 in PBS) (ThermoFisher, Waltham, MA, USA) for 1 h at room temperature. Following three washings in PBS-TW20, alkaline phosphatase activity was visualized with substrate BCIP/NBT. We also determined the reactivity of anti-loggerhead IgY mAbs with serum from leatherback (Dermochelys coriacea), green (Chelonia mydas), olive ridley (Lepidochelys olivacea), hawksbill, (Eretmochelys imbricate), and Kemp's ridley (Lepidochelys kempii) sea turtles. 13



Fig. 1. (A). SDS-PAGE and Coomassie blue staining of protein-G purified IgY products. Lane 1: molecular weight marker; Lane 2: loggerhead (LH) IgY. Strong bands appear at approximately 63 kDa, 34 kDa, and 25 kDa, corresponding to the heavy chains, a truncated form, and the light chains of IgY, respectively. (B). Immunoreactivity of mAb LH12, mAb LH1, and mAb LH9 against purified loggerhead sea turtle IgY. Lanes 1, 4, and 7 contain molecular weight marker; lanes 2, 5, and 8 contain purified IgY. (C). Immunoreactivity of mAb LH12, mAb LH1, and mAb LH9 against whole serum from loggerhead (LH), leatherback (LB), green (G), olive ridley (OR), hawksbill (HB), and Kemp's ridley (KR). Blots were ultimately probed with goat-anti-mouse IgG to target mAbs.

antibody shows promise for exploring antibody responses in all sea turtles to a variety of potential pathogens. Sea turtles spanning the oceans of tropical and temperate regions across the globe experience threats from multiple abiotic and biotic sources, including contaminants, climate change, sea level rise, and emerging diseases (Christianen et al., 2012; Perrault et al., 2014; Pike et al., 2015; Pilcher et al., 2014; Rodgers et al., 2018). Very little is known about diseases of sea turtles beyond fibropapillomatosis (Jones et al., 2016), though there have been routine isolation events of Escherichia coli (Al-Bahry et al., 2009; Foti et al., 2009; Raidal et al., 1998; Santoro et al., 2006, 2008) and Vibrio parahaemolyticus (Chuen-Im et al., 2010) from various individual sea turtles. An important component to understanding overall sea turtle health and disease is the ability to measure acquired immune responses to marine pathogens in the form of antibody responses. To that end, we herein present the development, characterization, and some technical applications of monoclonal antibodies against both isoforms of loggerhead sea turtle IgY with applications in other species of sea turtles. Going forward, these new additions to the limited tool box of sea turtle-specific reagents are now available to the scientific community examining the health of these animals in their natural habitat, as well as animals in recovery centers and aquariums.

of IgY heavy chain (Fig. 1C). One important application of mAb LH1 going forward is in the accurate identification of serums samples derived from these three species compared to the other species of sea turtles. Of particular note, mAb LH9 recognizes IgY light chains in all of the turtle species examined. The observation that mAb LH9 recognizes leatherback proteins as well indicates that the constant region of the light chains in sea turtles are highly conserved. Moreover, this may also indicate that sea turtles, like some birds and microbats, have only one light chain isotype (λ), compared to amphibians who have three (κ, λ, and σ), and most other tetrapods, including mammals, that have two (κ, λ) (Das et al., 2008). To our knowledge, the only other mAbs against sea turtle IgY developed to date are for green sea turtles (Herbst and Klein, 1995; Work et al., 2015a). The cross reactivity of some green sea turtle-specific mAbs against IgY light chains produced by Herbst and Klein was similar to mAb LH9 in our study, but the availability of those reagents is unknown at this time. The cross reactivity of mAbs against IgY light chains produced by Work et al. is also unknown at this time. The panel of mAbs generated herein may be very useful for researchers in developing sensitive ELISA systems to quantify IgY (total and pathogen specific) in all species of sea turtles, especially within the family Cheloniidae, whether as standalone reagents, or in combination with these aforementioned mAbs against green turtle IgY. 3.2. ELISA results

Acknowledgements

In our previous work, polyclonal antibodies against loggerhead and Kemp's ridley turtle IgY were developed and shown to recognize all three chains (Rodgers et al., 2018). In that study, circulating IgY titers against select marine bacteria were determined, revealing that responses were different between the two species of turtles, even though it was concluded that all individuals were healthy. By developing mAbs specific to the different chains of IgY, we now have the means to determine the relative contribution of the two isoforms to circulating antibody titers. Using 16 different loggerhead serum samples and three representative marine bacteria, we found that relative titers against Mycobacterium marinum were higher than for Vibrio parahaemolyticus, with lower responses to Escherichia coli (Fig. 2). By comparing the relative contribution of IgY chains to recognize each bacterium, we can see that the full length heavy chain contributes more to total activity than the fragmented isoform in recognizing each. The differences in titers between the two isoforms is more pronounced against M. marinum than the other two bacterial species examined. Since mAb LH9 recognizes light chains from both IgY isoforms, the total measured activity is higher when using it as the primary antibody in the ELISA system. With the observation that mAb LH9 recognizes light chains in all sea turtle species examined, including leatherback sea turtles, this

Sincere thanks are extended to Dr. Mike Arendt and Jeffery Schwenter (SC DNR), and Julia Byrd (SAFMC) for access to loggerhead and Kemp's ridley serum samples collected in 2017 under NOAA permit # Section 10(A)(1)(a) permits 15566 and 19621. Special thanks are also extended to Dr. Thierry Work, USGS, HI for access to serum samples from green, leatherback, hawksbill, and olive ridley turtles under NOAA permit 15685. This work was funded by the National Park Service through the Piedmont-South Atlantic Cooperative Ecosystems Unit (#H5000-08-5050).

Appendix B. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.dci.2018.05.015.

Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.dci.2018.05.015. 14



Fig. 2. Relative IgY antibody titers by ELISA against three marine bacteria in loggerhead turtles using mAb LH12 against heavy chains, mAb LH1 against the truncated heavy chain isoform, and mAb LH9 against light chains. Overall, antibody titers against M. marinum (A) were higher than that of V. parahaemolyticus (B) and E. coli (C). The contribution of the heavy chains to antibody responses were higher than that of the truncated isoform of IgY. The highest antibody titers against each bacterium were determined using the IgY light chain-specific monoclonal antibody. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Data represent the mean ± standard error of the mean (n = 16 loggerheads sampled in 2017).

References

exposure, superoxide dismutase activity and plasma protein electrophoretic profiles in wild-caught Kemp's ridley sea turtles (Lepidochelys kempii) in southwest Florida. Harmful Algae 37, 194–202. Pike, D.A., Roznik, E.A., Bell, I., 2015. Nest inundation from sea-level rise threatens sea turtle population viability. Royal Society Open Science 2, 150127. Pilcher, N.J., Perry, L., Antonopoulou, M., Abdel-Moati, M.A., Al Abdessalaam, T.Z., Albeldawi, M., Al Ansi, M., Al-Mohannadi, S.F., Baldwin, R., Chikhi, A., Das, H.S., Hamza, S., Kerr, O.J., Al Kiyumi, A., Mobaraki, A., Al Suwaidi, H.S., Al Suweidi, A.S., Sawaf, M., Tourenq, C., Williams, J., Willson, A., 2014. Short-term behavioural responses to thermal stress by hawksbill turtles in the Arabian region. J. Exp. Mar. Biol. Ecol. 457, 190–198. Raidal, S.R., Ohara, M., Hobbs, R.P., Prince, R.I.T., 1998. Gram-negative bacterial infections and cardiovascular parasitism in green sea turtles (Chelonia mydas). Aust. Vet. J. 76, 415–417. Rice, C.D., Schlenk, D., Ainsworth, J., Goksøyr, A., 1998. Cross-reactivity of monoclonal antibodies against peptide 277–294 of rainbow trout CYP1A1 with hepatic CYP1A among fish. Mar. Environ. Res. 46, 87–91. Rodgers, M.L., Toline, C.A., Rice, C.D., 2018. Humoral immune responses to select marine bacteria in loggerhead sea turtles Caretta caretta and Kemp's ridley sea turtles Lepidochelys kempii from the southeastern United States. J. Aquat. Anim. Health 1, 20–30. Santoro, M., Hernandéz, G., Caballero, M., García, F., 2008. Potential bacterial pathogens carried by nesting leatherback turtles (Dermochelys coriacea) in Costa Rica. Chelonian Conserv. Biol. 7, 104–108. Santoro, M., Hernández, G., Caballero, M., García, F., 2006. Aerobic bacterial flora of nesting green turtles (Chelonia mydas) from Tortuguero National Park, Costa Rica. J. Zoo Wildl. Med. 37, 549–552. Schumacher, I.M., Brown, M.B., Jacobson, E.R., Collins, B.R., Klein, P.A., 1993. Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease. J. Clin. Microbiol. 31, 1454–1460. Taylor, A.I., Fabiane, S.M., Sutton, B.J., Calvert, R.A., 2009. The crystal structure of an avian IgY-Fc fragment reveals conservation with both mammalian IgG and IgE. Biochemistry 48, 558–562. Warr, G.W., Magor, K.E., Higgins, D.A., 1995. IgY: clues to the origins of modern antibodies. Immunology today 16, 392–398. Wei, Z., Wu, Q., Ren, L., Hu, X., Guo, Y., Warr, G.W., Hammarström, L., Li, N., Zhao, Y., 2009. Expression of IgM, IgD, and IgY in a reptile, Anolis carolinensis. J. Immunol. 183, 3858–3864. Work, T.M., Dagenais, J., Breeden, R., Schneemann, A., Sung, J., Hew, B., Balazs, G.H., Berestecky, J.M., 2015a. Green turtles (Chelonia mydas) have novel asymmetrical antibodies. J. Immunol. 195, 5452–5460. Work, T.M., Dagenais, J., Breeden, R., Schneemann, A., Sung, J., Hew, B., Balazs, G.H., Berestecky, J.M., 2015b. Green turtles (Chelonia mydas) have novel asymmetrical antibodies. J. Immunol. 195, 5452–5460. Zhang, X., Calvert, R.A., Sutton, B.J., Doré, K.A., 2017. IgY: a key isotype in antibody evolution. Biol. Rev. 92, 2144–2156.

Al-Bahry, S., Mahmoud, I., Elshafie, A., Al-Harthy, A., Al-Ghafri, S., Al-Amri, I., Alkindi, A., 2009. Bacterial flora and antibiotic resistance from eggs of green turtles Chelonia mydas: an indication of polluted effluents. Mar. Pollut. Bull. 58, 720–725. Christianen, M.J.A., Govers, L.L., Bouma, T.J., Kiswara, W., Roelofs, J.G.M., Lamers, L.P.M., van Katwijk, M.M., 2012. Marine megaherbivore grazing may increase seagrass tolerance to high nutrient loads. J. Ecol. 100, 546–560. Chuen-Im, T., Areekijseree, M., Chongthammakun, S., Graham, S.V., 2010. Aerobic bacterial infections in captive juvenile green turtles (Chelonia mydas) and hawksbill turtles (Eretmochelys imbricata) from Thailand. Chelonian Conserv. Biol. 9, 135–142. Das, S., Nikolaidis, N., Klein, J., Nei, M., 2008. Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers. Proc. Natl. Acad. Sci. Unit. States Am. 105, 16647–16652. Foti, M., Giacopello, C., Bottari, T., Fisichella, V., Rinaldo, D., Mammina, C., 2009. Antibiotic resistance of Gram negatives isolates from Loggerhead sea turtles (Carettacaretta) in the central mediterranean sea. Mar. Pollut. Bull. 58, 1363–1366. Herbst, L.H., Klein, P.A., 1995. Monoclonal antibodies for the measurement of classspecific antibody responses in the green turtle, Chelonia mydas. Vet. Immunol. Immunopathol. 46, 317–335. Jones, K., Ariel, E., Burgess, G., Read, M., 2016. A review of fibropapillomatosis in Green turtles (Chelonia mydas). Vet. J. 212, 48–57. Kaplan, A.J., Stacy, N.I., Jacobson, E., Le-Bert, C.R., Nollens, H.H., Origgi, F.C., Green, L.G., Bootorabi, S., Bolten, A., Hernandez, J.A., 2016. Development and validation of a competitive enzyme-linked immunosorbent assay for the measurement of total plasma immunoglobulins in healthy loggerhead sea (Caretta caretta) and green turtles (Chelonia mydas). J. Vet. Diagn. Invest. 28, 5–11. Leslie, G.A., Clem, L.W., 1969. Phylogeny of immunoglobulin structure and function III. Immunoglobulins of the chicken. J. Exp. Med. 130, 1337–1352. Leslie, G.A., Clem, L.W., 1972. Phylogeny of immunoglobulin structure and function VI. 17S, 7.5S and 5.7S anti-DNP of the turtle., Pseudamys scripta. J. Immunol. 108, 1656–1664. Lundqvist, M.L., Middleton, D.L., Radford, C., Warr, G.W., Magor, K.E., 2006. Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck. Dev. Comp. Immunol. 30, 93–100. Margiotta, A.L., Bain, L.J., Rice, C.D., 2017. Expression of the major vault protein (MVP) and cellular vault Particles in fish. Anat. Rec. 300, 1981–1992. Munhoz, L.S., Vargas, G.D.Á., Fischer, G., Lima, M.D., Esteves, P.A., Hübner, S.D.O., 2014. Avian IgY antibodies: characteristics and applications in immunodiagnostic. Ciência Rural. 44, 153–160. Muñoz, F.A., Estrada-Parra, S., Romero-Rojas, A., Gonzalez-Ballesteros, E., Work, T.M., Villaseñor-Gaona, H., Estrada-Garcia, I., 2013. Immunological evaluation of captive green sea turtle (Chelonia mydas) with ulcerative dermatitis. J. Zoo Wildl. Med. 44, 837–844. Perrault, J.R., Schmid, J.R., Walsh, C.J., Yordy, J.E., Tucker, A.D., 2014. Brevetoxin

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