Sensitivity and permissivity of Cyprinus carpio to cyprinid herpesvirus 3 during the early stages of its development: importance of the epidermal mucus as an innate immune barrier
© Ronsmans et al.; licensee BioMed Central Ltd. 2014
Received: 9 August 2014
Accepted: 24 September 2014
Published: 4 October 2014
Cyprinid herpesvirus 3 (CyHV-3) causes a lethal disease in common and koi carp (Cyprinus carpio). The present study investigated the ability of CyHV-3 to infect common carp during the early stages of its development (from embryos to fingerlings) after inoculation by immersion in water containing the virus. Fish were inoculated at different times after hatching with a pathogenic recombinant CyHV-3 strain expressing luciferase. The sensitivity and permissivity of carp to CyHV-3 were investigated using in vivo bioluminescence imaging. The susceptibility of carp to CyHV-3 disease was investigated by measuring the survival rate. Carp were sensitive and permissive to CyHV-3 infection and susceptible to CyHV-3 disease at all stages of development, but the sensitivity of the two early developmental stages (embryo and larval stages) was limited compared to later stages. The lower sensitivity observed for the early developmental stages was due to stronger inhibition of viral entry into the host by epidermal mucus. In addition, independent of the developmental stage at which inoculation was performed, the localization of light emission suggested that the skin is the portal of CyHV-3 entry. Taken together, the results of the present study demonstrate that carp are sensitive and permissive to CyHV-3 at all stages of development and confirm that the skin is the major portal of entry after inoculation by immersion in infectious water. The results also stress the role of epidermal mucus as an innate immune barrier against pathogens even and especially at the early stages of development.
The common carp (Cyprinus carpio) is one of the oldest cultivated fish species. In China, the cultivation of carp dates back to at least the 5th century BC and in Europe, carp farming began during the Roman Empire . Common carp is currently one of the most economically valuable species in aquaculture, as it is one of the main cultivated fish for human consumption with worldwide production of 3.4 million tons per year . Common carp is also produced and stocked in fishing areas for angling purposes. Its colorful ornamental varieties (koi carp), grown for personal pleasure and competitive exhibitions, represent one of the most expensive markets for individual freshwater fish, with some prize-winners being sold for US $10 000–1 000 000 .
Carp development comprises successive stages associated with specific morphological and physiological characteristics. The incubation period of fertilized eggs varies between 48 and 72 h depending on water parameters ,. From hatching to 3–5 days post-hatching, fish are called embryos. The digestive tract is not fully developed so the fish rely entirely on the yolk sac as a source of nutrients ,. Because the gills are not yet formed, gas exchange occurs through the blood vessels of the yolk sac and the caudal fin. By 3 to 5 days post-hatching the yolk sac is entirely absorbed and most organs formed and functional. From this moment, the fish are referred to as larvae. In contrast to embryos, larvae rely on exogenous feeding and their respiration is mediated by the gills. Around 2–3 weeks post-hatching, larvae acquire an adult shape and are no longer transparent. When the metamorphosis is complete, the fish are referred to as juveniles. The fish then develop paired fins and maturation of the organs is finalized. Around 35 days post-hatching, the fish are called fingerlings. Fingerlings are fully developed, entirely covered with scales (with the exception of fish that have no scales), and appear just like adults ,. Despite their adult appearance, the immune system of fingerlings is not yet fully mature and functional.
Teleosts develop both innate and adaptive immune responses . During the early stages of life described above, the lymphomyeloid organs of teleosts are not yet fully mature and the fish are unable to develop an effective adaptive immune response. In carp, adaptive immune competency occurs roughly 2 months after hatching ,. Early in life, fish immunity relies on passively transferred maternal factors and on innate immune mechanisms which are activated just after egg fertilization and fully functional at hatching ,. Among the components of the innate immune system, the mucus covering the external and internal surfaces of the fish is thought to play a key role in the inhibition of pathogen entry into the host ,.
Koi herpesvirus (KHV), also known as cyprinid herpesvirus 3 (CyHV-3) (genus Cyprinivirus, family Alloherpesviridae, order Herpesvirales), is the etiological agent of an emerging and lethal disease in common and koi carp. Since its emergence in the late 1990s, this highly contagious and dreadful disease has caused severe economic losses in both the common and koi carp culture industries worldwide. Publication of the CyHV-3 sequence  and the cloning of its genome as an infectious bacterial artificial chromosome (BAC)  allowed the production of CyHV-3 recombinant strains. Recently, we took advantage of these advances to construct a luciferase (LUC)-expressing recombinant strain via intergenic insertion of a LUC expression cassette. Using this recombinant strain and in vivo imaging system (IVIS), we showed that the skin covering the fins and body of carp fingerlings, but not the gills, is the major portal of entry after inoculation by immersion in water containing the virus . Using similar approaches, epidermal mucus has been shown to act as an innate immune barrier, drastically reducing CyHV-3 binding to epidermal cells .
The epidermis of adult teleost fish is a living stratified squamous epithelium divided into three layers. The surface and basal layers are single-cell layers composed of keratinocytes and undifferentiated basal cells, respectively. The pluristratified intermediate layer is composed of unicellular glands (mucous cells and club cells), ionocytes and undifferentiated cells. All three layers contain cells capable of mitotic division. The scales are dermal structures covered by the epidermis . Compared to adults, the epidermis of embryos has a simplified structure consisting of surface and basal layers (single-cell layers) interrupted by unicellular glands and ionocytes.
The susceptibility of carp to CyHV-3 disease has been tested at different developmental stages after inoculation by immersion in water containing the virus. Though juveniles, fingerlings, and adults are susceptible to CyHV-3 disease -, larvae have been reported to be resistant to infection based on PCR analysis of experimentally inoculated subjects . These results suggest that the early stages of carp development are resistant to CyHV-3. Interestingly, this resistance could be due to a lack of expression of a component essential for completion of the viral infection (e.g., a cell surface binding receptor) by the early developmental stages and/or the expression of an innate immune mechanism capable of preventing the infection that is not expressed by later development stages.
In the present study, we investigated the ability of CyHV-3 to infect common carp during the early stages of its development (from embryos to fingerlings) after inoculation by immersion in water containing the virus. Fish were inoculated at different times post-hatching with a pathogenic recombinant CyHV-3 strain expressing LUC as a reporter gene. The sensitivity (ability to support viral entry into host cells) and permissivity (ability to support viral replication) of carp to CyHV-3 infection were investigated using IVIS. The susceptibility of carp to CyHV-3 disease was investigated by measuring the survival rate over a period of one month after inoculation. The results of the present study demonstrate that carp are sensitive and permissive to CyHV-3 infection and susceptible to CyHV-3 disease at all developmental stages. However, the fish express reduced sensitivity during the two earlier stages of development (embryo and larval stages) due to efficient inhibition of viral entry into the host by epidermal mucus. This study further supports the importance of the skin as the major portal of entry of CyHV-3 after inoculation by immersion in infectious water and the role of the epidermal mucus as an innate immune barrier against pathogens even and especially during the early developmental stages.
Materials and methods
Cells and virus
Common carp brain (CCB) cells  were cultured in minimum essential medium (Sigma) containing 4.5 g/L of glucose (D-glucose monohydrate; Merck) and 10% fetal calf serum. The cells were cultured at 25 °C in a humid atmosphere containing 5% CO2. CCB cells were used to produce and titrate CyHV-3. The FL BAC revertant ORF136 LUC strain of CyHV-3, hereafter referred to as the FL LUC strain, was described previously . This recombinant strain derived from the FL BAC clone of CyHV-3 encodes a wild type ORF55 locus (in which the BAC cassette was initially cloned) and a firefly (Photinus pyralis) luciferase expression cassette under control of the human cytomegalovirus immediate early promoter. The LUC cassette was inserted in the intergenic region between open reading frames ORF136 and ORF137.
Common carp (Cyprinus carpio) were produced by artificial reproduction. Breeders free of CyHV-3 based on PCR analysis of peripheral mononuclear blood cells and serological analysis that generated offspring sensitive to CyHV-3 disease were hosted in a recirculating aquaculture system at 23 °C (CEFRA, University of Liège, Belgium). Mature breeders were selected based on morphological characteristics. Ovulation and spawning were induced by two intraperitoneal (IP) injections of Ovaprim (Syndel) performed at an 8 h interval. The first injection was a dose of 0.05 mL/kg of body weight. The dose of the second injection differed between males and females: 0.25 and 0.5 mL/kg of body weight, respectively. Eggs were stripped 12 h after the second hormonal injection and fertilized with stripped sperm. All manipulations of the breeders were performed under anesthetic tranquilization with benzocaine (50 mg/L of water). Fertilized eggs were incubated in Zoug bottles until hatching. Embryos (mean body weight 1.2 mg) were transferred to a 50 L tank (2500 larvae per tank). The water temperature was regulated at 24 °C and the O2 concentration maintained above 6 ppm. Larvae were fed ad libitum with artificial larval food Gemma micro (Skretting). The food was distributed manually 6 times per day during the first 45 days post-hatching. Juveniles reached a mean body weight of 500 mg within their first month of life.
CyHV-3 inoculation of fish
For viral inoculation mimicking natural infection, fish were kept for 2 h in water containing the FL LUC strain of CyHV-3 (400 plaque-forming units (pfu)/mL). At the end of the incubation period, the fish were returned to 50 L tanks where they were kept in floating breeding nests (SERA). To avoid removal of the epidermal mucus, fish were caught using a container rather than a fish net. In some experiments, the skin mucus was partially removed before inoculation. Carp were caught with a fish net, placed on a plastic sheet, and the upper side of the body was gently rubbed with a floqswab ultrathin (Copan flock technologies, Brescia, Italy) to remove the epidermal mucus. Carp were also inoculated by IP injection as follows. Carp were anesthetized by immersion in water containing benzocaine (25 mg/L) and distributed individually to the wells of a macroscopic slide (12 cavity microscopic slide, VWR international) containing a viscous solution (NaCl 292 mg/L, KCl 12.6 mg/L, CaCl2.2H2O 48.3 mg/L, MgSO4.7H2O 81.6 mg/L, methylene blue 1 mg/L, carboxyl methyl cellulose high viscosity 20 g/L). Culture medium containing 3.6 × 106 pfu/mL of the FL LUC strain was injected intraperitoneally (corresponding to an approximate dose of 40 pfu per fish) using a microinjector (Femtojet, Eppendorf, VWR international) and tapered beveled pipettes (15–20 μm). Injection was performed using the following parameters, leading to the administration of an approximate volume of 0.01 μL: automatic mode, injection pressure of 200 hPa, compensation pressure of 0 hPa, and injection time of 0.2 - 0.8 s according to fish size. After injection, the fish were returned to 50 L tanks. This animal study was accredited by the local ethics committee of the University of Liège.
In vivo bioluminescence imaging
Imaging of firefly LUC expression was performed using IVIS (IVIS spectrum, Xenogen, Caliper LifeSciences) as described previously ,,. D?luciferin (Xenogen, Caliper LifeSciences) (15 mg/mL) in phosphate-buffered saline (PBS) was microinjected into the peritoneal cavity as described above. Efficient injection of D?luciferin into fish was controlled by examination of the fish using full field epifluorescent microscopy (Eclipse TE2000-S, Nikon). D?luciferin emits a green fluorescence when excited with a blue light (488 nm). After an incubation period of 10 min, the carp were analyzed by IVIS. Images were collected using the following settings: field of view A, binning on small, automatic exposure time with a maximum of 1 min, and a subject height of 0.35 cm. The relative intensities of transmitted light from bioluminescence were represented as a pseudocolor image ranging from violet (least intense) to red (most intense). For quantitative comparisons, regions of interest were manually drawn by surrounding the fish outline, and the Living Image software 3.2 (Caliper Life Sciences) was used to calculate the corrected average radiance (p/s/cm2/sr) by subtracting the average radiance of the background for each image.
Euthanized fish were fixed by immersion in Carnoy solution (ethanol:acetic acid:chloroform 6:1:3, v/v/v) for 24 h at 4 °C. After dehydration with ethanol, samples were embedded in paraffin blocks . Five micron sections were stained with a combined Periodic Acid-Schiff, Alcian Blue and hematoxylin-eosin staining prior to microscopic analysis using a Leica DM 2000 LED microscope equipped with a DFC 450C camera (Leica, Heerbrugg, Switzerland).
Significant differences in the log of the average radiance emitted by IVIS-positive fish, according to time of inoculation (days post-hatching) and time post-inoculation or according to time of inoculation and mode of inoculation, were assessed using two-way ANOVA with interactions. Significant differences in the number of IVIS-positive fish according to time of inoculation (days post-hatching), time post-inoculation and mode of inoculation were assessed using logistical analysis and the chi-squared test. Significant differences in survival rates according to time of inoculation (days post-hatching) were assessed using a contingency table and chi-squared test.
The goal of the present study was to investigate the ability of CyHV-3 to infect Cyprinus carpio during the early stages of its development. The data presented in this manuscript are representative of duplicate independent experiments. Importantly, though the different experiments were initially performed successively, the data set presented in this manuscript represents the parallel repetition of all experiments with the same batch of fish.
Sensitivity and permissivity of Cyprinus carpio to CyHV-3 during the early stages of development
Independent of the developmental stage at the time of inoculation and the time of analysis post-inoculation (24 or 72 hpi), at least one positive fish was detected (Figure 3), demonstrating that carp are sensitive to CyHV-3 infection at all stages of development. Photon emission was significantly higher 72 hpi compared to 24 hpi for the following times of inoculation (Figure 3A): 3 (p?<?0.05), 7 (p?<?0.05), 28 (p?<?0.0001), and 35 (p?<?0.0001) days post-hatching. These data suggest that carp are permissive to CyHV-3 at all stages of development, which was further supported by the IVIS images (Figure 4). Compared to images collected 24 hpi, those collected at 72 hpi systematically revealed more spots of light with a larger area and greater maximum radiance independent of the time of inoculation (Figure 4, second vs. third rows of panels).
These data demonstrate that all developmental stages of carp are sensitive and permissive to CyHV-3 infection. However, the percentage of positive fish was significantly lower after inoculation 0 – 14 days post-hatching compared to inoculation at later stages (Figure 3b, p?<?0.0001). No significant difference was found in the average radiance (p/sec/cm2/sr) of positive fish (independent of the stage at which inoculation was performed) (p?<?0.05), implying that, once infected, replication occurs similarly in fish, at least at the portal of entry.
Effect of epidermal mucus on the sensitivity of Cyprinus carpio to CyHV-3 during the early stages of development
Taken together, the results demonstrate that the lower sensitivity to CyHV-3 observed for the early developmental stages of carp is due at least in part to a strong inhibition of viral entry into the host by the epidermal mucus.
Susceptibility of Cyprinus carpio to CyHV-3 disease during the early stages of development
Carp were mock-inoculated or inoculated with the FL LUC strain at the indicated times post-hatching. Survival rates were measured over a period of 30 days post-inoculation (dpi). For most groups, surviving fish were counted on days 15 and 30 post-inoculation.
The goal of the present study was to investigate the ability of CyHV-3 to infect common carp during the early stages of its development (from embryos to fingerlings) after inoculation by immersion in infectious water. Using IVIS, we demonstrated that carp are sensitive and permissive to CyHV-3 infection at all stages of development. However, the sensitivity of the two early stages (embryo and larval stages) was limited compared to the older stages (juvenile and fingerling stages). Inoculation after removal of the epidermal mucus demonstrated that the reduced sensitivity of the early developmental stages was caused by a stronger inhibition of viral entry into the host by the epidermal mucus. Finally, the susceptibility of carp to CyHV-3 disease was tested by inoculating carp at different times after hatching and measuring the survival rate over a period of one month after inoculation. The data are consistent with the conclusion that carp are susceptible to CyHV-3 at all stages of development and that the sensitivity to CyHV-3 infection increases during ontogenesis.
Ito et al. reported that the larvae from two independent strains of common carp were resistant to CyHV-3 infection but juveniles from the same strains were highly susceptible . The results of the present study contradict this earlier report by demonstrating that carp are sensitive and permissive to CyHV-3 at all stages of development. Importantly, the results of the present study are representative of two experiments performed with unrelated breeders. All of the breeders used in this study were proved to be free of CyHV-3. In their study, Ito et al. did not mention whether they controlled the serological status of the carp used for reproduction. One interesting hypothesis that could explain the paradox between the two studies is that the previous study used seropositive genitors. These immune genitors could have transferred transient humoral immune protection against CyHV-3 to their offspring. Teleost fish lay telolecithal eggs in which passive transfer of maternal antibodies occurs ,. Whether embryos from immune versus naive breeders exhibit different sensitivity to CyHV-3 would be interesting to determine in the future, as well as how long this passive protection lasts. Experiments to test this hypothesis are currently in progress.
Epidermal mucus acts as an innate immune barrier against CyHV-3 entry into the host . The results of the present study suggest that the lower sensitivity to CyHV-3 of the early developmental stages is due to more efficient protection by the mucus, which is conferred through two types of complementary mechanisms. First, the mucus forms an efficient mechanical barrier that constantly moves downstream along the fish and off of the trailing edges. Similar to the muco-ciliary escalator of the respiratory tract of pulmonate animals, fish mucus reduces pathogen access to epithelial cells. Second, the mucus contains numerous proteins, such as immunoglobulins, enzymes, and lytic agents, capable of neutralizing microorganisms -. Several hypotheses, which are not mutually exclusive, could explain the more efficient inhibition of CyHV-3 entry by the epidermal mucus in the early developmental stages. One hypothesis is that the mucus layer of the early stages of development forms a more uniform mechanical barrier on the fish body, possibly due to its biochemical composition, the hydrodynamic parameters of the fish, or as a consequence of reduced physical interactions between the fish and an object or other fish . Another hypothesis is that the mucus produced by early stages of development contains a higher concentration of biologically active molecules capable of neutralizing CyHV-3. Even if technically difficult, it would be very interesting to compare the neutralization activity of soluble mucus extracts from larvae versus older stages that are more sensitive to CyHV-3. These experiments could reveal that the early stages of development express biologically active innate immune molecules in their mucus that are not expressed, or expressed at lower levels at later stages. In addition to their interest to fundamental science, such conclusions could be very interesting for application-oriented research, such as that trying to enhance fish resistance against pathogens by up-regulating innate immunity.
In conclusion, the present study demonstrates that carp are sensitive and permissive to CyHV-3 infection at all stages of development, even if the sensitivity of the early stages is reduced due to efficient inhibition of viral entry by the epidermal mucus. This study further supports the importance of the skin as the major portal of entry of CyHV-3 after inoculation by immersion in infectious water. It stresses the role of the epidermal mucus as an innate immune barrier against pathogen even and especially during the early stages of development.
MR did most of the experiments. MR, MB, KR, and AV contributed to the design of the study. MR and MB drafted the figures. DD performed the histological analysis of carp epidermis. MR, MV, and FE developed the procedure for microinjection into carp embryos and larvae. CM and FL performed carp reproduction. Statistical analyses were performed by FF. MR and AV conceived the study and drafted the manuscript. All authors read and approved the final manuscript.
Bacterial artificial chromosome
Common carp brain
Cyprinid herpesvirus 3
In vivo imaging system
MR is a research fellow of the “Fonds National Belge de la Recherche Scientifique” (FNRS). This work was supported by a grant from the University of Liège and by grants from the “Fonds National Belge de la Recherche Scientifique” (FNRS). KR and AV are members of the BELVIR consortium (IAP, phase VII) granted by the Belgian Science Policy Office (BELSPO) (Belgium).
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