Author: marina02

Microbial communities of farmed mussel Mytilus galloprovincialis and seawater in the estuary of Krka River in Croatia

Mytilus galloprovincialis, commonly known as the Mediterranean mussel, is significant shellfish species in Croatian marine aquaculture, with a total production of 926 tonnes live weight averaged across 2021, 2022 and 2023. The investigation of the mussel microbiome has gained significant attention due to its critical role in mussel physiology, health, and aquaculture productivity. Mussels serve not only as a food source but also as biofilters, efficiently removing particulate matter and potentially harmful microbes from the water, thus improving water quality and ecosystem health. In this study, the entire microbial community present in the tissues (gills and hepatopancreas) of farmed mussels, as well as in the marine water of the Krka River estuary was examined on a monthly scale during 2022 and 2023 using DNA metabarcoding. The analysis targeted the V4-V5 variable region of the 16S rRNA gene. Bioinformatic evaluation of the data revealed that Proteobacteria, Bacteroidota, and Firmicutes were the most abundant bacterial phyla in the mussel tissues, with notable seasonal variations. Among the 648 genera identified, 522 were found in gills, 516 in hepatopancreas, and 498 in seawater samples, with 331 genera common to all three sample types. Potentially pathogenic genera and coliforms such as Vibrio, Clostridium, and a group classified as Escherichia-Shigella were detected, albeit at very low abundances and exclusively in the hepatopancreas. Alpha diversity was highest in the seawater samples and lowest in the gills and hepatopancreas. The results provide insights into mussel host-microbe-environment interactions that can enhance aquaculture practices and support sustainable mussel farming. This research was supported by the Ministry of Agriculture of the Republic of Croatia through Operational Program for Maritime Affairs and Fisheries for the period 2014-2020 and the European Union through the European Fund for Maritime Affairs and Fisheries, under project INNODAGNJA.

1. TRUMBIĆ, ŽELJKA, University of Split, Department of Marine Studies, Croatia, Presenter
2. ĆURKO, TENA, University of Split, Croatia, Author
3. HRABAR, JERKO, Institute of Oceanography and Fisheries Split, Croatia, Author
4. BOGDANOV, LARISA, University of Split, Department of Marine Studies, Croatia, Author

Development of a recombinant S4 protein and nanoparticle-based vaccine for Tilapia Lake virus

Tilapia lake virus (TiLV) disease is a highly contagious infection causing significant mortality in tilapia, with no effective treatments or commercial vaccines currently available. This study aimed to develop a recombinant protein-based approach for vaccine development. TiLV segment 4 (S4) was cloned into the pET28a(+) vector and expressed in Escherichia coli BL21(DE3) with codon optimization to enhance protein production. Optimal expression conditions were identified using 0.1 mM galactose induction at 37°C for 1 hour, yielding an insoluble protein fraction that was denatured with 6 M urea and purified to a concentration of 250 mg/L. Dot blot immunodetection assays confirmed the immunoreactivity of the purified protein. Building on this, a chitosan nanoparticle-based immersion vaccine (CNS4) incorporating the recombinant S4 protein was developed and tested. The nanoTiLV-S4 vaccine had an average size of 284 ± 9.2 nm and was rapidly absorbed through fish gills and intestines, with epithelial uptake occurring within 30 minutes. Under laboratory and field conditions, the CNS4 vaccine achieved relative percent survival (RPS) rates of 25% and 31.88%, respectively, compared to unvaccinated controls. These findings provide a foundation for large-scale recombinant TiLV S4 protein production and highlight the potential of nanoparticle-based delivery systems in TiLV vaccine development, though further optimization and field trials are required to improve efficacy and assess performance under diverse aquaculture conditions.

1. LERTWANAKARN, TUCHAKORN, Department of Physiology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand, Presenter
2. SURACHETPONG, WIN, Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand, Author

Development and Evaluation of a Recombinant Protective Antigen-Based Vaccine Against Bacillus cereus Infection in Asian Sea Bass (Lates calcarifer)

This study aimed to develop a vaccine for Asian sea bass (Lates calcarifer) infected with Bacillus cereus group (Bcg), a pathogenic bacterium known to cause severe septicemia, ultimately leading to significant economic losses in commercial aquaculture systems. The clinical signs of infected fish included multifocal petechiae on the skin, and excessive accumulation of coelomic fluid in the body cavity, all of which indicate systemic infection and potential organ dysfunction. We identified a plasmid, pBS01, in the virulent B. cereus strain, which shares notable similarities with the Bacillus anthracis virulence plasmid pXO1. Further genomic and proteomic analyses revealed the presence of a protective antigen (PA) within this strain, analogous to the protective antigen found in B. anthracis. Given the established role of the protective antigen in anthrax pathogenesis and its importance as a vaccine target, we hypothesized that the PA from B. cereus might serve as an effective immunogen for vaccine development. To investigate the potential interaction between the protective antigen and host immune receptors, we conducted molecular docking studies between PA and anthrax toxin receptor 1 (ANTXR1). The results demonstrated that domain 4 (PD4) of the protective antigen has a binding affinity to ANTXR1, suggesting that this domain may play a critical role in host-pathogen interactions. Based on these findings, we proceeded to express and purify recombinant PD4 as a subunit vaccine candidate. For the vaccination trial, Asian sea bass were immunized at two time points: an initial dose at day 0, followed by a booster at day 14. Each fish received a 50 µg dose of the recombinant PD4 protein. After the immunization period, fish were challenged with B. cereus to assess vaccine efficacy. The results indicated that the vaccinated group exhibited a relative percentage survival (RPS) of 100%, demonstrating complete protection against B. cereus infection. In contrast, the control group, which did not receive the vaccine, exhibited a cumulative mortality rate of 72.5%, confirming the high virulence of the pathogen and underscoring the vaccine’s protective potential.

1. CHEN, SHIH-CHU, NATIONAL PINGTUNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, Presenter
2. CHENG, LI-WU, NATIONAL PINGTUNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, Author
3. WANG, PEI-CHI, NATIONAL PINGTUNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, Author

Cross protection efficacy test of an inactivated multivalent immersion vaccine against an intraperitoneal experimental challenge with Yersinia Ruckeri of different serotypes in Salmo salar

Introduction

Since the introduction and dissemination of Yersinia ruckeri into the Atlantic salmon, vaccines have been traditionally based on antigens from O1 serotype. However, since the discovery that a wider range of serotypes is responsible for infection in this fish species, concerns have been raised about vaccine cross-protecting efficacy. The objective of this study was to evaluate the efficacy of an inactivated vaccine administered by dip immersion against an intraperitoneal experimental challenge with Yersinia ruckeri (YR) of serotypes O1, O5 and O8 in Salmo salar in fresh water.

Material and Methods

Atlantic salmon fingerlings were divided into two groups and immunized either with ICTHIOVAC ERM or with placebo (water). Two doses of the vaccine were administered by immersions at approximately 3 g and 8 g. Then the fish of each group were redistributed into 3 subgroups of 50 fish and challenged by intraperitoneal injection with O1, O5 or O8 YR at 784 degree days post vaccination (since the first dose). Mortalities were monitored over 16 days.

Results and Conclusions

The challenges produced a mortality in the placebo group between 42 % and 76 % depending on the strain used. The Relative Percent Survival (RPS) observed in vaccinated groups were 100 %, 97.20 % and 90.48 % for the O1, O5 and O8 YR challenge, respectively.

The results demonstrated that ICTHIOVAC ERM was able to reduce and even prevent the mortality caused by O5, O8 and O1 YR strains. Considering that the vaccine contains YR serotypes O1 and O2, this finding suggest that the vaccine confer cross protection through the mentioned serotypes.

These results highlight the potential for broader vaccine strategies to combat diverse YR serotypes.

1. ALVAREZ DE HARO, NEILA, HIPRA SCIENTIFIC, Presenter
2. Parra, David, HIPRA SCIENTIFIC, Author
3. Baratelli, Massimiliano, HIPRA SCIENTIFIC, Author
4. Verdaguer Sentmartí, Joel, HIPRA SCIENTIFIC, Author
5. Callol, Agnès, HIPRA SCIENTIFIC, Author
6. Merino Pérez, Rosa María, HIPRA SCIENTIFIC, Author
7. Vicente Rubiano, Marina, HIPRA SCIENTIFIC, Author

Orally delivered OMVs from Photobacterium damselae subsp. picicida are efficiently taken up in the intestine of European sea bass and trout, highlighting their potential for oral fish vaccination

Outer membrane vesicles (OMVs) are nano-sized particles naturally released from the outer membrane of Gram-negative bacteria during growth. These vesicles carry a wide array of bioactive molecules, including outer membrane proteins, lipids, toxins, and pathogen-associated molecular patterns (PAMPs), making them highly effective at stimulating immune responses. Although OMVs have been successfully used as vaccine platforms in humans, including in vaccines against Neisseria meningitidis, their potential for fish vaccination remains largely underexplored.

Infectious diseases are a major challenge in aquaculture, driving the need for effective vaccination strategies that reduce mortality and antibiotic use. Oral vaccines are particularly advantageous due to ease of administration, reduced animal stress, and suitability for large-scale application. However, few oral vaccines are currently available for fish, and their protective efficacy remains limited and inconsistent. OMVs, due to their natural immunogenicity, intrinsic adjuvanticity and capability of targeting mucosal surfaces, represent a promising alternative for oral immunization in fish. Interestingly, it is possible to manipulate the antigen content of OMVs, to remove toxic and immunosupressive components and incorporate heterologous antigens from viral, bacterial and parasitic pathogens, opening doors to the development of OMVs-based multivalent vaccine formulations.

Recently, it was shown that Photobacterium damselae subsp. piscicida (Phdp), an important fish pathogen, secretes large amounts of OMVs in vitro and in vivo. These OMVs contain virulence factors, including the lethal toxin AIP56, hindering their direct use for vaccination. However, OMVs devoid of AIP56 administered intraperitoneally to European sea bass (Dicentrarchus labrax), without adjuvants, conferred partial protection against experimental Phdp infection, highlighting their vaccine potential.

In this study, we evaluated the intestinal uptake of detoxified Phdp OMVs following oral administration to European sea bass and rainbow trout (Oncorhynchus mykiss). Fish were inoculated with OMVs by oral gavage, and intestines were collected at 2, 6, 24, and 48 hours post-inoculation in both species, with an additional time point at 4 days included for sea bass. After controlled fixations, samples were histologically processed for paraffin embedding. The presence of OMVs in the intestinal tissues was evaluated by immunohistochemistry. The results revealed efficient uptake in the most distal region of the intestine in both species.

The demonstration that orally delivered Phdp OMVs are able to reach and interact with the intestinal mucosa of European sea bass and rainbow trout is a promising step toward developing an OMVs- based platform for producing effective, adjuvant-free oral vaccines for aquaculture.

1. Loureiro, Inês, I3S-INSTITUTO DE INVESTIGAÇÃO E INOVAÇÃO EM SAÚDE, UNIVERSIDADE DO PORTO, Author
2. Mendes, Nuno, I3S-INSTITUTO DE INVESTIGAÇÃO E INOVAÇÃO EM SAÚDE, UNIVERSIDADE DO PORTO, Author
3. dos Santos, Nuno MS, I3S-INSTITUTO DE INVESTIGAÇÃO E INOVAÇÃO EM SAÚDE, UNIVERSIDADE DO PORTO, Author
4. do Vale, Ana, I3S-INSTITUTO DE INVESTIGAÇÃO E INOVAÇÃO EM SAÚDE, UNIVERSIDADE DO PORTO, Presenter

Orally delivered OMVs from Photobacterium damselae subsp. picicida are efficiently taken up in the intestine of European sea bass and trout, highlighting their potential for oral fish vaccination

Outer membrane vesicles (OMVs) are nano-sized particles naturally released from the outer membrane of Gram-negative bacteria during growth. These vesicles carry a wide array of bioactive molecules, including outer membrane proteins, lipids, toxins, and pathogen-associated molecular patterns (PAMPs), making them highly effective at stimulating immune responses. Although OMVs have been successfully used as vaccine platforms in humans, including in vaccines against Neisseria meningitidis, their potential for fish vaccination remains largely underexplored.

Infectious diseases are a major challenge in aquaculture, driving the need for effective vaccination strategies that reduce mortality and antibiotic use. Oral vaccines are particularly advantageous due to ease of administration, reduced animal stress, and suitability for large-scale application. However, few oral vaccines are currently available for fish, and their protective efficacy remains limited and inconsistent. OMVs, due to their natural immunogenicity, intrinsic adjuvanticity and capability of targeting mucosal surfaces, represent a promising alternative for oral immunization in fish. Interestingly, it is possible to manipulate the antigen content of OMVs, to remove toxic and immunosupressive components and incorporate heterologous antigens from viral, bacterial and parasitic pathogens, opening doors to the development of OMVs-based multivalent vaccine formulations.

Recently, it was shown that Photobacterium damselae subsp. piscicida (Phdp), an important fish pathogen, secretes large amounts of OMVs in vitro and in vivo. These OMVs contain virulence factors, including the lethal toxin AIP56, hindering their direct use for vaccination. However, OMVs devoid of AIP56 administered intraperitoneally to European sea bass (Dicentrarchus labrax), without adjuvants, conferred partial protection against experimental Phdp infection, highlighting their vaccine potential.

In this study, we evaluated the intestinal uptake of detoxified Phdp OMVs following oral administration to European sea bass and rainbow trout (Oncorhynchus mykiss). Fish were inoculated with OMVs by oral gavage, and intestines were collected at 2, 6, 24, and 48 hours post-inoculation in both species, with an additional time point at 4 days included for sea bass. After controlled fixations, samples were histologically processed for paraffin embedding. The presence of OMVs in the intestinal tissues was evaluated by immunohistochemistry. The results revealed efficient uptake in the most distal region of the intestine in both species.

The demonstration that orally delivered Phdp OMVs are able to reach and interact with the intestinal mucosa of European sea bass and rainbow trout is a promising step toward developing an OMVs- based platform for producing effective, adjuvant-free oral vaccines for aquaculture.

1. Loureiro, Inês, I3S-INSTITUTO DE INVESTIGAÇÃO E INOVAÇÃO EM SAÚDE, UNIVERSIDADE DO PORTO, Author
2. Mendes, Nuno, I3S-INSTITUTO DE INVESTIGAÇÃO E INOVAÇÃO EM SAÚDE, UNIVERSIDADE DO PORTO, Author
3. dos Santos, Nuno MS, I3S-INSTITUTO DE INVESTIGAÇÃO E INOVAÇÃO EM SAÚDE, UNIVERSIDADE DO PORTO, Author
4. do Vale, Ana, I3S-INSTITUTO DE INVESTIGAÇÃO E INOVAÇÃO EM SAÚDE, UNIVERSIDADE DO PORTO, Presenter

Developing mRNA vaccines for Atlantic salmon

Introduction

Infectious diseases are a major problem in the fish farming industry. As over 400 million salmon are vaccinated annually in Norway, the high mortality rates indicate that the current vaccines are not efficient enough, and vaccines have not been developed for some diseases. A new and improved vaccine strategy is therefore necessary. mRNA vaccines are emerging as a powerful candidate to conventional vaccines, but the mRNA vaccine technology for fish is just in its beginning. In this study, we have tailored mRNA to salmon and studied the effect of poly(A) tail length and the use of modified nucleotides on protein expression in CHSE-214 cells.

Methodology

EGFP-encoding mRNA was made by in vitro transcription (IVT) and transfected into CHSE-214 cells. Protein expression was observed with fluorescence microscopy and flow cytometry 1-10 days post transfection.

Results

The protein expression by in vitro transcribed mRNA was strongly influenced by the length of the tail, as mRNA with a long tail yielded a significantly higher percentage of EGFP positive cells compared to mRNA with shorter tails. The use of modified nucleotides increased the percentage of EGFP positive cells compared to mRNA without modified nucleotides, in addition to influencing the stability of the mRNA.

Conclusion

Our findings show that mRNA can be used to express an antigen of interest in salmonid cells, and that optimization of the mRNA structure can have a great impact on the development of a vaccine for Atlantic salmon.

1. Fossum Krog, Thea, UNIVERSITY OF BERGEN, Presenter
2. Soo Haukland, Ida, UNIVERSITY OF BERGEN, Author
3. Teien Haugland, Gyri, UNIVERSITY OF BERGEN, Author

Enhancing sole larvae immunity: Artemia nauplii as NNV oral vaccine carriers

Senegalese sole (Solea senegalensis) is a marine flatfish with high commercial value in the Southern European aquaculture. However, its production is severely threatened by seasonal outbreaks of viral encephalopathy and retinopathy caused by the Nervous Necrosis Virus (NNV), which affects mainly larvae and juvenile fish. Nowadays, there is no effective treatment available, and all efforts are focused in developing a vaccine to protect sole production in hatcheries.  Given that traditional vaccination methods involving handling and stress are not suitable for the early developmental stages of fish, oral vaccination through live feed may represent a viable alternative for immunization. Brine shrimp (Artemia salina) nauplii are routinely used in fish farms as live feed for larval developmental stages. One of their notable biological traits is the ability to act as bioacumulators, due to its filter-feeding behaviour. In this study we have explored the potential of Artemia salina nauplii as carriers of an NNV-protein based subunit vaccine towards Senegalese sole larvae for its implementation in hatcheries. For this purpose, Artemia nauplii (100 nauplii/ml) were incubated for 24 hours with different concentrations of an NNV-protein based subunit vaccine or nanospheres loaded with NanoLuc luciferase as reporter protein. Protein uptake and retention within nauplii were demonstrated using immunofluorescence (IFA) and chemiluminescence assays. Subsequently, sole larvae were fed ad libitum with the recombinant protein-enriched Artemia for 4 days. Delivery of the recombinant protein to the larvae was tested through immunohistochemistry. Our results showed the effective internalization and retention of the recombinant protein within Artemia nauplii, as well as the subsequent delivery of the antigen to sole larvae. Further assays should be conducted to assess the specific immune stimulation in larvae and the level of protection conferred.

This work was funded by the European Union under the Horizon Europe Programme, Grant Agreement Nº 101084204 (Cure4Aqua). SS: Grant RYC2023-045820-I funded by MCIN/AEI/ 10.13039/501100011033 and FSE+.

1. Vazquez-Salgado, Lucía, ARCUS, Universidade de Santiago de Compostela, Author
2. Jadhav, Swapnil, ARCUS, Universidade de Santiago de Compostela, Author
3. Barreiro-Piñeiro, Natalia, CIQUS, Universidade de Stiago de Compostela, Author
4. López-Teijeiro, Adrián, CIQUS, Universidade de Stiago de Compostela, Author
5. Martinez-Costas, Jose, CIQUS, Universidade de Stiago de Compostela, Author
6. Bandín, Isabel, ARCUS, Universidade de Santiago de Compostela, Author
7. Souto, Sandra, ARCUS, Universidade de Santiago de Compostela, Presenter

Immune response of Senegalese sole to a NNV protein-based vaccine

Vaccination is the preferred strategy for preventing viral diseases in aquaculture. However, developing effective vaccines for aquatic environments presents several challenges. Key factors such as antigen presentation, delivery method, and antigen concentration must be carefully considered. Additionally, due to the unique characteristics of the fish immune system, vaccines must be specifically designed and tested for each target species.
Nervous necrosis virus (NNV) is among the most threatening viral pathogens in aquaculture, prompting significant efforts to protect economically important species such as sea bass, gilthead sea bream, and Senegalese sole. Vaccination against NNV is particularly challenging, as it requires immunization at the earliest developmental stages.
In this study, we aimed to advance immunization strategies against NNV in Senegalese sole. We evaluated the immune responses of sole post-larvae (0.5 g) and early juveniles (15 g) to experimental vaccination using two NNV antigens based on the viral coat protein. These antigens were produced in E. coli and encapsulated into nanospheres using an innovative IC-tagging system. Vaccines were administered via intraperitoneal injection in early juveniles and by a 10-minute immersion in post-larvae. In early juveniles, blood samples were collected after a prime and booster dose to assess specific antibody production. Whole post-larvae were homogenized for RNA-seq analysis.
Results demonstrated the production of specific antibodies in early juveniles after the booster dose. RNA-seq analysis of post-larvae revealed differential gene expression patterns and antigen-dependent immune responses, highlighting the capacity of the sole immune system to be stimulated through vaccination.

Funded by the European Union under the Horizon Europe Programme, Grant Agreement Nº 101084204 (Cure4Aqua). SS: Grant RYC2023-045820-I funded by MCIN/AEI/ 10.13039/501100011033 and FSE+.

1. Souto, Sandra, ARCUS, Universidade de Santiago de Compostela, Presenter
2. Vazquez-Salgado, Lucía, ARCUS, Universidade de Santiago de Compostela, Author
3. Salgueiro-Lago, Ana, ARCUS, Universidade de Santiago de Compostela, Author
4. Barreiro-Iglesias, Antón, ARCUS, Universidade de Santiago de Compostela, Author
5. Barreiro-Piñeiro, Natalia, CIQUS, Universidade de Stiago de Compostela, Author
6. Martinez-Costas, Jose, CIQUS, Universidade de Stiago de Compostela, Author
7. Bandín, Isabel, ARCUS, Universidade de Santiago de Compostela, Author

Cetyltrimethylammonium bromide-based mucoadhesive nano vaccine delivered via immersion enhances mucoadhesion and skin penetration in Striped catfish (Pangasianodon Hypophthalmus) against Edwardsiella Ictaluri

Edwardsiella ictaluriis a major bacterial pathogen responsible for severe disease outbreaks in striped catfish (Pangasianodon hypophthalmus), resulting in substantial economic losses in aquaculture. Traditional vaccine delivery methods, such as injection and oral administration, are limited by issues including fish stress, antigen instability, and inconsistent immune responses. Immersion vaccination offers a practical, non-invasive alternative by enabling antigen uptake through mucosal surfaces, making it especially suitable for mass immunization in aquaculture. Although chitosan-based nanovaccines have demonstrated promise in enhancing mucosal delivery, their performance can be inconsistent due to variations in physicochemical stability under different conditions. To address this, cetyltrimethylammonium bromide (CTAB), a cationic surfactant, was selected for its strong electrostatic interaction with negatively charged mucosal surfaces and its ability to stabilize nanoparticles. This study aimed to evaluate the effectiveness of immersion delivery of a CTAB-based mucoadhesive nanovaccine by assessing its physicochemical characteristics, mucoadhesive behavior, and skin penetration capacity in striped catfish. A formalin-inactivatedE. ictaluristrain (ED-INK04) was used to formulate two vaccine types: a conventional formalin-killed control and a CTAB-based nanoemulsion (CTAB-NE). The CTAB-NE was prepared by combining sonicated bacterial antigens with lipid carriers and CTAB to enhance surface charge and adhesion. The zeta potential of the vaccine particles was measured to determine surface charge. Striped catfish were vaccinated via immersion for 1–5 minutes, after which gill and skin tissues were collected for analysis. Nile Red fluorescent dye was used to evaluate mucoadhesion and tissue penetration. Imaging was conducted using fluorescence microscopy, spectrophotometry, and confocal laser scanning microscopy to assess penetration depth up to 100 µm. The CTAB-NE formulation exhibited a positive surface charge (+20.41 ± 1.29 mV), in contrast to the negatively charged control (−28.38 ± 0.22 mV), which enhanced its affinity for mucosal surfaces. Strong fluorescence signals in gill tissues confirmed superior mucoadhesion in the CTAB-NE group. Moreover, confocal imaging revealed a time-dependent increase in skin penetration, with CTAB-NE reaching depths of up to 100 µm within 5 minutes of immersion, while the control showed minimal uptake. In conclusion, immersion delivery of the CTAB-based nanovaccine significantly improved mucosal adhesion and transdermal penetration compared to the conventional formulation. These findings highlight the potential of CTAB-NE as an effective, non-invasive vaccine delivery system for striped catfish. Furtherin vivostudies are needed to evaluate its immunogenicity and protective efficacy againstE. ictaluri.

1. Yostawonkul, Jakarwan, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani 12120 Thailand, Presenter
2. Kamble, Manoj T., Center of Excellence in Wildlife, Exotic, and Aquatic Animal Pathology, Faculty of Veterinary Science, Chulalongkorn University,, Author
3. Sukkarun, Pimwarang, Faculty of Veterinary Science, Rajamangala University of Technology Srivijaya, Nakhon Si Thammarat, 80240, Thailand, Author
4. Thompson, Kim D., Moredun Research Institute, Pentlands Science Park, Penicuik, EH26 0PZ, UK, Author
5. Pirarat, Nopadon, Center of Excellence in Wildlife, Exotic, and Aquatic Animal Pathology, Faculty of Veterinary Science, Chulalongkorn University,, Author