Shrimp is one of the most preferred seafood around the world, increasing the tremendous demand leads to a spatial increase of shrimp farming worldwide. Along with these continuing trends, some new and renewed shrimp diseases have arisen. There have been several major disease outbreaks within shrimp ...
Shrimp is one of the most preferred seafood around the world, increasing the tremendous demand leads to a spatial increase of shrimp farming worldwide. Along with these continuing trends, some new and renewed shrimp diseases have arisen. There have been several major disease outbreaks within shrimp farming in various countries, including white spot syndrome (WSS), yellow head disease (YHD) and infectious myonecrosis (IMN) and more recently acute hepatopancreatic necrosis disease (AHPND). However, Outbreaks of WSS and AHPND were reported globally and the epidemiology of particular diseases has studied intensively while giving less concern on YHD and IMNV as they were affected only some Asian shrimp farming countries and Brazil. Moreover, most of the pathogenicity and immune response against YHD and IMN are largely unknown due to the lack of scientific studies. Therefore, the present study aimed to the evaluate diagnosis method, immune response and gut microbiome of yellow head disease (YHD) and infectious myonecrosis (IMN) in white leg shrimp (Litopenaeus vannamei).
In the first chapter, a novel real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay based on the TaqMan probe was developed for the detection and quantification of YHV-1 in shrimp. The present qRT-PCR assay was further validated using the shrimps obtained from shrimp farms, bioassay and, experimentally infection by cohabitation and intramuscular injection (IM) methods. Further, we aimed to evaluate the expression of antimicrobial peptide genes (AMPs) in the white leg shrimp experimentally infected with the YHV-1 by the RT-PCR assays. The results of the novel TaqMan probe-based qRT-PCR assay showed a strong linear correlation (r2=0.991) between threshold cycles (CT) and RNA quantities. The present qRT-PCR assay showed high reproducibility with the CVs < 5% and 100% specificity by giving negative results for other common shrimp pathogens, including YHV-8, WSSV, IMNV and Vp AHPND. The qRT-PCR assay could detect up to 10 copies of RNA from the infected shrimp tissues and it also showed 100% diagnosis sensitivity compared to OIE recommended nested RT-PCR assay. The relative expression of penaeidin (PEN-2a, PEN-3a and PEN-4a) and Crustin were more prominently expressed during the early stage of YHV-1 infection while the transcript of AMPs was highly expressed in the hemolymph and gill than the muscle and hepatopancreas. Based on the present results, the novel qRT-PCR assay can be used as a rapid and sensitive diagnostic method for the detection and quantification of YHV-1 from the infected shrimp tissues. The AMPs expression was revealed that hemolymph and gill are the main target organs of the YHV-1 infection and act a key role against YHV-1 infection in shrimp by expressing the various AMPs at the early stage of infection.
In the second chapter, IMNV experimental infection was performed to evaluate the distribution of the IMNV in skeletal muscle obtained from different body segments and the response of immune-related genes (AMPs, Hsp70 and proPO), pro-inflammatory cytokines (TNF-ά and IL1-β), antioxidant enzyme (MnSOD, CAT, GPx and TRx) and arginine kinase (AK) genes in muscle and hemolymph of IMNV infected shrimp. The main visible clinical sign appeared as opaque skeletal muscle and it was observed in most of the challenged shrimp after 5 dpi. There was no significant difference among the CT values for the detection of IMNV from skeletal muscle obtained from the different abdominal segments of the shrimp. The qRT-PCR assay for the detection of IMNV from hemolymph and skeletal muscle was observed a high viral load in hemolymph until the first two days of post-infection and later it was shown to be high viral load in the skeletal muscle. In the AMPs, the PEN-3a was shown the highest relative expression in the hemolymph and expression of AMPs were increased in muscle and hemolymph at the early stage of infection. The expression of proPO and Hsp70 in the hemolymph and muscle was increased at the early stage of IMNV infection. The relative expression of both pro-inflammatory cytokine genes was rapidly increased in muscle from the 5 dpi showing the clinical symptoms of white skeletal muscle. The antioxidant enzyme genes were expressed in muscle than the hemolymph under the IMNV infection. Moreover, the relative expression MnSOD and CAT genes were elevated at the early stage of IMNV infection while elevated relative expression of GPx and TRx genes were associated with the middle and late stage of IMNV infection. The elevated relative expression of AK in hemolymph was observed at the early stage of IMNV infection and the relative expression in muscle was increased at the late stage of IMNV infection. Collectively these results revealed that the rapid distribution of the IMNV to all segments through hemolymph after initial contact of IMNV. The upregulation of the inflammation, oxidative stress-related and arginine kinase genes in muscles at the time of clinical symptoms appeared could be related to muscle necrosis.
In the third chapter, the microbiota from the gut of healthy (n=6) and, YHV-1 (n=6) and IMNV (n=7) infected shrimps was characterized using the sequencing of the V4 hypervariable region of the 16S rRNA gene. The sequence data were further analyzed by using several bioinformatics databases and pipelines. Initially, high-quality reads were used for further analysis in the Ezbiocloud 16S based Microbiome Taxonomic Profiling (MTP) database. The YHV-1 infected group was shown a relatively high number of operational taxonomic units (OTUs) compared to the healthy and IMNV infected groups. The diversity and richness of gut microbiota were barely affected by YHV-1 and IMNV infection and only the gut microbiome of the IMNV infected group were shown significantly higher phylogenetic diversity. The abundance of predominant phyla such as Proteobacteria and Actinobacteria were upregulated significantly under YHV-1 and IMNV infection while Bacteroidetes were significantly decreased in the gut microbiome of both YHV-1 and IMNV infected shrimps. At the genus level, Photobacterium was increased significantly and Spongimonas and Litorilituus were decreased significantly compared to the healthy shrimps Moreover, the PCoA plot showed the independently clustered healthy, YHV-1 and IMNV infected groups. The UPGMA clustering could be differentiated YHV-1 infected shrimps independently. Although some shrimps of IMNV infected and healthy shrimps were clustered together, the rest of the shrimps were clustered separately in each group. Metagenomics predictions by PICRUSt showed slight variations in several KEGG pathways metabolism and biological processes but it was not significant. Based on the present results, the gut microbiome composition between healthy and infected shrimps indicates that both YHV-1 and IMNV infections could impact gut microbiome composition and not for the function in white leg shrimp. Moreover, further study with the increased sample size is needed for understanding more insight into gut microbiome dynamics under YHV-1 and IMNV infections.
Collectively, the availability of the novel qRT-PCR assay for the detection and quantification of the YHV-1 can be useful in future YHV-1 outbreaks. Elevated transcript levels in pro-inflammatory cytokines and oxidative stress were related to clinical signs of IMNV infection. Moreover, IMNV-causing white muscle might be due to inflammation by hydrogen peroxidase related to oxidative stress. The composition of the gut microbiome altered under YHV-1 and IMNV infection and no significant functional dynamics under infections might be due to the gut as an ectodermal origin tissue not infected by both YHV-1 and IMNV.
Shrimp is one of the most preferred seafood around the world, increasing the tremendous demand leads to a spatial increase of shrimp farming worldwide. Along with these continuing trends, some new and renewed shrimp diseases have arisen. There have been several major disease outbreaks within shrimp farming in various countries, including white spot syndrome (WSS), yellow head disease (YHD) and infectious myonecrosis (IMN) and more recently acute hepatopancreatic necrosis disease (AHPND). However, Outbreaks of WSS and AHPND were reported globally and the epidemiology of particular diseases has studied intensively while giving less concern on YHD and IMNV as they were affected only some Asian shrimp farming countries and Brazil. Moreover, most of the pathogenicity and immune response against YHD and IMN are largely unknown due to the lack of scientific studies. Therefore, the present study aimed to the evaluate diagnosis method, immune response and gut microbiome of yellow head disease (YHD) and infectious myonecrosis (IMN) in white leg shrimp (Litopenaeus vannamei).
In the first chapter, a novel real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay based on the TaqMan probe was developed for the detection and quantification of YHV-1 in shrimp. The present qRT-PCR assay was further validated using the shrimps obtained from shrimp farms, bioassay and, experimentally infection by cohabitation and intramuscular injection (IM) methods. Further, we aimed to evaluate the expression of antimicrobial peptide genes (AMPs) in the white leg shrimp experimentally infected with the YHV-1 by the RT-PCR assays. The results of the novel TaqMan probe-based qRT-PCR assay showed a strong linear correlation (r2=0.991) between threshold cycles (CT) and RNA quantities. The present qRT-PCR assay showed high reproducibility with the CVs < 5% and 100% specificity by giving negative results for other common shrimp pathogens, including YHV-8, WSSV, IMNV and Vp AHPND. The qRT-PCR assay could detect up to 10 copies of RNA from the infected shrimp tissues and it also showed 100% diagnosis sensitivity compared to OIE recommended nested RT-PCR assay. The relative expression of penaeidin (PEN-2a, PEN-3a and PEN-4a) and Crustin were more prominently expressed during the early stage of YHV-1 infection while the transcript of AMPs was highly expressed in the hemolymph and gill than the muscle and hepatopancreas. Based on the present results, the novel qRT-PCR assay can be used as a rapid and sensitive diagnostic method for the detection and quantification of YHV-1 from the infected shrimp tissues. The AMPs expression was revealed that hemolymph and gill are the main target organs of the YHV-1 infection and act a key role against YHV-1 infection in shrimp by expressing the various AMPs at the early stage of infection.
In the second chapter, IMNV experimental infection was performed to evaluate the distribution of the IMNV in skeletal muscle obtained from different body segments and the response of immune-related genes (AMPs, Hsp70 and proPO), pro-inflammatory cytokines (TNF-ά and IL1-β), antioxidant enzyme (MnSOD, CAT, GPx and TRx) and arginine kinase (AK) genes in muscle and hemolymph of IMNV infected shrimp. The main visible clinical sign appeared as opaque skeletal muscle and it was observed in most of the challenged shrimp after 5 dpi. There was no significant difference among the CT values for the detection of IMNV from skeletal muscle obtained from the different abdominal segments of the shrimp. The qRT-PCR assay for the detection of IMNV from hemolymph and skeletal muscle was observed a high viral load in hemolymph until the first two days of post-infection and later it was shown to be high viral load in the skeletal muscle. In the AMPs, the PEN-3a was shown the highest relative expression in the hemolymph and expression of AMPs were increased in muscle and hemolymph at the early stage of infection. The expression of proPO and Hsp70 in the hemolymph and muscle was increased at the early stage of IMNV infection. The relative expression of both pro-inflammatory cytokine genes was rapidly increased in muscle from the 5 dpi showing the clinical symptoms of white skeletal muscle. The antioxidant enzyme genes were expressed in muscle than the hemolymph under the IMNV infection. Moreover, the relative expression MnSOD and CAT genes were elevated at the early stage of IMNV infection while elevated relative expression of GPx and TRx genes were associated with the middle and late stage of IMNV infection. The elevated relative expression of AK in hemolymph was observed at the early stage of IMNV infection and the relative expression in muscle was increased at the late stage of IMNV infection. Collectively these results revealed that the rapid distribution of the IMNV to all segments through hemolymph after initial contact of IMNV. The upregulation of the inflammation, oxidative stress-related and arginine kinase genes in muscles at the time of clinical symptoms appeared could be related to muscle necrosis.
In the third chapter, the microbiota from the gut of healthy (n=6) and, YHV-1 (n=6) and IMNV (n=7) infected shrimps was characterized using the sequencing of the V4 hypervariable region of the 16S rRNA gene. The sequence data were further analyzed by using several bioinformatics databases and pipelines. Initially, high-quality reads were used for further analysis in the Ezbiocloud 16S based Microbiome Taxonomic Profiling (MTP) database. The YHV-1 infected group was shown a relatively high number of operational taxonomic units (OTUs) compared to the healthy and IMNV infected groups. The diversity and richness of gut microbiota were barely affected by YHV-1 and IMNV infection and only the gut microbiome of the IMNV infected group were shown significantly higher phylogenetic diversity. The abundance of predominant phyla such as Proteobacteria and Actinobacteria were upregulated significantly under YHV-1 and IMNV infection while Bacteroidetes were significantly decreased in the gut microbiome of both YHV-1 and IMNV infected shrimps. At the genus level, Photobacterium was increased significantly and Spongimonas and Litorilituus were decreased significantly compared to the healthy shrimps Moreover, the PCoA plot showed the independently clustered healthy, YHV-1 and IMNV infected groups. The UPGMA clustering could be differentiated YHV-1 infected shrimps independently. Although some shrimps of IMNV infected and healthy shrimps were clustered together, the rest of the shrimps were clustered separately in each group. Metagenomics predictions by PICRUSt showed slight variations in several KEGG pathways metabolism and biological processes but it was not significant. Based on the present results, the gut microbiome composition between healthy and infected shrimps indicates that both YHV-1 and IMNV infections could impact gut microbiome composition and not for the function in white leg shrimp. Moreover, further study with the increased sample size is needed for understanding more insight into gut microbiome dynamics under YHV-1 and IMNV infections.
Collectively, the availability of the novel qRT-PCR assay for the detection and quantification of the YHV-1 can be useful in future YHV-1 outbreaks. Elevated transcript levels in pro-inflammatory cytokines and oxidative stress were related to clinical signs of IMNV infection. Moreover, IMNV-causing white muscle might be due to inflammation by hydrogen peroxidase related to oxidative stress. The composition of the gut microbiome altered under YHV-1 and IMNV infection and no significant functional dynamics under infections might be due to the gut as an ectodermal origin tissue not infected by both YHV-1 and IMNV.
주제어
#White leg shrimp Yellow head disease Infectious myonecrosis Experimental infection Gut microbiome
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