- Email: [email protected]
Enhanced bacterin permeability and side effects using low frequency sonophoresis at 37 kHz in rainbow trout
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Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
2 Department of Pathology and Microbiology, University of Prince Edward Island, Charlottetown, PEI, Canada; 3 Department of Biomedical Sciences, University of Prince Edward Island, Charlottetown, PEI, Canada
americanus to WSSV challenge than to either bacterial or parasitic pathogens. * Corresponding author. E-mail address: [email protected] (K.F. Clark)
Abstract This is the first high-throughput transcriptomic study investigating the immune response of the commercially valuable American lobster (Homarus americanus). An in vivo infection challenge was conducted using the naturally occurring lethal lobster pathogen Aerococcus viridans var. homari. This Gram-positive bacterium is able to multiply and spread systemically in lobsters when temperatures are above 10 oC. We used a novel H. americanus microarray to monitor the expression of over 14,000 genes in hepatopancreatic tissue. The expression of hundreds of new lobster immune genes was monitored for the first time to determine their role in response to this Gram-positive bacteria. Microarray analysis determined that 148 genes were differentially expressed in the lobster immune response. Verification of microarray results was undertaken by using RTqPCR to monitor the expression of: anti-lipopolysaccharide (ALFHa-1, ALFHa-2, ALFHa-4), a thioredoxin, acute phase serum amyloid protein A, hexokinase and two trypsin genes. There is good agreement between RTqPCR and microarray findings where RT-qPCR highlights the role of multiple ALFHa isoforms and SAA. The differential expression of ALFHa isoforms indicates that the immune system of H. americanus may have some pathogen specificity in that its response could be tailored to the class of pathogen causing disease. Additionally, the discovery of the significant increase in SAA could indicate a new biomarker for lobster or crustacean health and disease. SAA is a commonly used marker of innate immune activation in human and veterinary medicine that could potentially be applied to crustacean health studies as well. * Corresponding author. E-mail address: [email protected] (B. Novoa)
O-409. Response of the American lobster (Homarus americanus) to a white spot syndrome virus challenge trial K.F. Clark 1, 2, *, S.J. Greenwood 1, 3, A.R. Acorn 1,2, P.J. Byrne 4. 1 AVC Lobster Science Centre, University of Prince Edward Island, Charlottetown, PEI, Canada; 2 Department of Pathology and Microbiology, University of Prince Edward Island, Charlottetown, PEI, Canada; 3 Department of Biomedical Sciences, University of Prince Edward Island, Charlottetown, PEI, Canada; 4 Charlottetown Aquatic Animal Pathogen Biocontainment Laboratory, Department of Fisheries and Oceans, Charlottetown, PEI, Canada
Abstract High-throughput transcriptomics has the capacity to rapidly progress our understanding of the American lobster (Homarus americanus) immune response to pathogenic pressures. This technique has been applied to bacterial and parasitic models of lobster disease, but the absence of a known viral H. americanus pathogen has made it difficult to investigate the lobster's immune response to viral pathogens. White Spot Syndrome Virus is a lethal pathogen of shrimp and is the most significant impediment to growth and health in the global shrimp aquaculture industry. WSSV is capable of infecting scores of crustacean species so an in vivo challenge experiment was undertaken to determine if WSSV infected lobsters could be used as a model of H. americanus viral disease. Our studies have shown that WSSV is capable of infecting H. americanus and replicating in many of its tissues. Additionally, transcriptomic analysis of over 14,000 genes in hepatopancreatic tissue has found that 132 genes are differentially expressed during WSSV infection. RT-qPCR was conducted on a subset of these genes to verify their differential expression during WSSV challenge. We also performed RT-qPCR on some genes known to be differentially expressed during bacterial and parasitic infection in H. americanus to determine if they are involved in responding to WSSV infection. Our results present some very interesting differences in the response of H.
O-419. Enhanced bacterin permeability and side effects using low frequency sonophoresis at 37 kHz in rainbow trout C. Cobo 1, *, K. Makosch 1, R. Jung 2, K. Kohlmann 1, K. Knopf 1. 1
Department of Ecophysiology and Aquaculture, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany; 2 Bandelin GmbH & Co, Berlin, Germany Abstract Low frequency sonophoresis (LFS) has been categorized as one of the most advanced technologies in transdermal substances delivery (TSD), where focal skin applications in mammalians revealed important advances during the last decades. Based on these works, LFS has been suggested for fish as a potential technology to be used for enhancement in immersion vaccination. In contrast to mammalians where LFS is applied to discrete regions of the skin here the whole fish needs to be exposed for practical reasons. The present work evaluated the effect of LFS at 37 kHz on the uptake of an Aeromonas salmonicida bacterin and side effects of the treatment in rainbow trout. Quantitative PCR (qPCR) and immunohistochemistry were used to examine the bacterin uptake into skin and gill tissue. Side effects were assessed by histology and blood serum chemistry. A sonication intensity of 171 mW/cm2 was enough for increasing skin permeability, but caused damage to the gill epithelium. Sonication at 105 mW/cm2 or lower did not modify skin permeability, but enhanced the bacterin uptake into the gill tissue by factor 15 compared to conventional immersion. However, heavy erratic swimming of the fish gives rise to ethical concerns at this intensity. Following sonication, 20 and 120 min the bacterin uptake into the gill tissue was still enhanced by factor 3 and 2, respectively. Further reduction of the sonication intensity to 57 mW/cm2 did not induce erratic swimming, and the bacterin uptake into the gill tissue was still increased by three times. In addition, an increasing albumin-globulin ratio in the serum of the rainbow trout within 40 min revealed that LFS leads to an inflammatory response. Consequently, based on both, increased bacterin uptake and the inflammatory response, low intensity LFS has the potential to enhance vaccine immunity * Corresponding author. E-mail address: [email protected] (C. Cobo)
O-414. Analysis of Toll-like receptor 2 of antarctic teleosts M.R. Coscia 1, *, S. Varriale 1, S. Ferraresso 2, S. Giacomelli 1, L. Bargelloni 2, U. Oreste 1. 1
Institute of Protein Biochemistry, CNR, Via P. Castellino 111, Naples, Italy; 2 Department of Public Health, Comparative Pathology, and Veterinary Hygiene University of Padova, viale dell'Università, 16, Legnaro (PD), Italy Abstract Sixteen Toll-like receptors (TLRs) have been identified in teleosts, TLR2 being among the most studied ones. Its up-regulation following stimulation with lipopeptides from Gram-positive bacteria, have been demonstrated in many fish species. To study the cold-adaptive modifications of the protein structure, Antarctic teleosts are very interesting models to use, and, in this context, TLR are very intriguing molecules to study. We have investigated evolutionary selection on TLR2 from two Antarctic teleosts, Trematomus bernacchii (Nototheniidae), and Chionodraco hamatus (Channichtyidae). Full length cDNA sequences of both species were determined and the deduced amino acid sequences were divided into the extracellular region,
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
2 Department of Pathology and Microbiology, University of Prince Edward Island, Charlottetown, PEI, Canada; 3 Department of Biomedical Sciences, University of Prince Edward Island, Charlottetown, PEI, Canada
americanus to WSSV challenge than to either bacterial or parasitic pathogens. * Corresponding author. E-mail address: [email protected] (K.F. Clark)
Abstract This is the first high-throughput transcriptomic study investigating the immune response of the commercially valuable American lobster (Homarus americanus). An in vivo infection challenge was conducted using the naturally occurring lethal lobster pathogen Aerococcus viridans var. homari. This Gram-positive bacterium is able to multiply and spread systemically in lobsters when temperatures are above 10 oC. We used a novel H. americanus microarray to monitor the expression of over 14,000 genes in hepatopancreatic tissue. The expression of hundreds of new lobster immune genes was monitored for the first time to determine their role in response to this Gram-positive bacteria. Microarray analysis determined that 148 genes were differentially expressed in the lobster immune response. Verification of microarray results was undertaken by using RTqPCR to monitor the expression of: anti-lipopolysaccharide (ALFHa-1, ALFHa-2, ALFHa-4), a thioredoxin, acute phase serum amyloid protein A, hexokinase and two trypsin genes. There is good agreement between RTqPCR and microarray findings where RT-qPCR highlights the role of multiple ALFHa isoforms and SAA. The differential expression of ALFHa isoforms indicates that the immune system of H. americanus may have some pathogen specificity in that its response could be tailored to the class of pathogen causing disease. Additionally, the discovery of the significant increase in SAA could indicate a new biomarker for lobster or crustacean health and disease. SAA is a commonly used marker of innate immune activation in human and veterinary medicine that could potentially be applied to crustacean health studies as well. * Corresponding author. E-mail address: [email protected] (B. Novoa)
O-409. Response of the American lobster (Homarus americanus) to a white spot syndrome virus challenge trial K.F. Clark 1, 2, *, S.J. Greenwood 1, 3, A.R. Acorn 1,2, P.J. Byrne 4. 1 AVC Lobster Science Centre, University of Prince Edward Island, Charlottetown, PEI, Canada; 2 Department of Pathology and Microbiology, University of Prince Edward Island, Charlottetown, PEI, Canada; 3 Department of Biomedical Sciences, University of Prince Edward Island, Charlottetown, PEI, Canada; 4 Charlottetown Aquatic Animal Pathogen Biocontainment Laboratory, Department of Fisheries and Oceans, Charlottetown, PEI, Canada
Abstract High-throughput transcriptomics has the capacity to rapidly progress our understanding of the American lobster (Homarus americanus) immune response to pathogenic pressures. This technique has been applied to bacterial and parasitic models of lobster disease, but the absence of a known viral H. americanus pathogen has made it difficult to investigate the lobster's immune response to viral pathogens. White Spot Syndrome Virus is a lethal pathogen of shrimp and is the most significant impediment to growth and health in the global shrimp aquaculture industry. WSSV is capable of infecting scores of crustacean species so an in vivo challenge experiment was undertaken to determine if WSSV infected lobsters could be used as a model of H. americanus viral disease. Our studies have shown that WSSV is capable of infecting H. americanus and replicating in many of its tissues. Additionally, transcriptomic analysis of over 14,000 genes in hepatopancreatic tissue has found that 132 genes are differentially expressed during WSSV infection. RT-qPCR was conducted on a subset of these genes to verify their differential expression during WSSV challenge. We also performed RT-qPCR on some genes known to be differentially expressed during bacterial and parasitic infection in H. americanus to determine if they are involved in responding to WSSV infection. Our results present some very interesting differences in the response of H.
O-419. Enhanced bacterin permeability and side effects using low frequency sonophoresis at 37 kHz in rainbow trout C. Cobo 1, *, K. Makosch 1, R. Jung 2, K. Kohlmann 1, K. Knopf 1. 1
Department of Ecophysiology and Aquaculture, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany; 2 Bandelin GmbH & Co, Berlin, Germany Abstract Low frequency sonophoresis (LFS) has been categorized as one of the most advanced technologies in transdermal substances delivery (TSD), where focal skin applications in mammalians revealed important advances during the last decades. Based on these works, LFS has been suggested for fish as a potential technology to be used for enhancement in immersion vaccination. In contrast to mammalians where LFS is applied to discrete regions of the skin here the whole fish needs to be exposed for practical reasons. The present work evaluated the effect of LFS at 37 kHz on the uptake of an Aeromonas salmonicida bacterin and side effects of the treatment in rainbow trout. Quantitative PCR (qPCR) and immunohistochemistry were used to examine the bacterin uptake into skin and gill tissue. Side effects were assessed by histology and blood serum chemistry. A sonication intensity of 171 mW/cm2 was enough for increasing skin permeability, but caused damage to the gill epithelium. Sonication at 105 mW/cm2 or lower did not modify skin permeability, but enhanced the bacterin uptake into the gill tissue by factor 15 compared to conventional immersion. However, heavy erratic swimming of the fish gives rise to ethical concerns at this intensity. Following sonication, 20 and 120 min the bacterin uptake into the gill tissue was still enhanced by factor 3 and 2, respectively. Further reduction of the sonication intensity to 57 mW/cm2 did not induce erratic swimming, and the bacterin uptake into the gill tissue was still increased by three times. In addition, an increasing albumin-globulin ratio in the serum of the rainbow trout within 40 min revealed that LFS leads to an inflammatory response. Consequently, based on both, increased bacterin uptake and the inflammatory response, low intensity LFS has the potential to enhance vaccine immunity * Corresponding author. E-mail address: [email protected] (C. Cobo)
O-414. Analysis of Toll-like receptor 2 of antarctic teleosts M.R. Coscia 1, *, S. Varriale 1, S. Ferraresso 2, S. Giacomelli 1, L. Bargelloni 2, U. Oreste 1. 1
Institute of Protein Biochemistry, CNR, Via P. Castellino 111, Naples, Italy; 2 Department of Public Health, Comparative Pathology, and Veterinary Hygiene University of Padova, viale dell'Università, 16, Legnaro (PD), Italy Abstract Sixteen Toll-like receptors (TLRs) have been identified in teleosts, TLR2 being among the most studied ones. Its up-regulation following stimulation with lipopeptides from Gram-positive bacteria, have been demonstrated in many fish species. To study the cold-adaptive modifications of the protein structure, Antarctic teleosts are very interesting models to use, and, in this context, TLR are very intriguing molecules to study. We have investigated evolutionary selection on TLR2 from two Antarctic teleosts, Trematomus bernacchii (Nototheniidae), and Chionodraco hamatus (Channichtyidae). Full length cDNA sequences of both species were determined and the deduced amino acid sequences were divided into the extracellular region,