Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish

Sepúlveda, Dagoberto; Bohle, H.; Labra, Álvaro; Grothusen, Horst; Marshall, Sergio H.

doi:10.1186/1746-6148-9-183

Cited by 20 publications

(18 citation statements)

References 29 publications

(37 reference statements)

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“…Relative mRNA expressions in the RTS11 cell line were evaluated using the 2 -ΔΔCt method [ 37 ]. Elongation factor 1α (ELF) was used as the housekeeping gene, using the forward and reverse GIM-ELF2 primers [ 38 ] and the same PCR conditions described above.…”

Section: Methodsmentioning

confidence: 99%

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model

et al. 2016

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Piscirickettsia salmonis is a facultative intracellular bacterium that causes the disease called “salmon rickettsial syndrome”. Attempts to control this disease have been unsuccessful, because existing vaccines have not achieved the expected effectiveness and the antibiotics used fail to completely eradicate the pathogen. This is in part the product of lack of scientific information that still lacks on the mechanisms used by this bacterium to overcome infected–cell responses and survive to induce a productive infection in macrophages. For that, this work was focused in determining if P. salmonis is able to modify the expression and the imbalance of IL-12 and IL-10 using an in vitro model. Additionally, we also evaluated the role the antimicrobial peptide hepcidin had in the control of this pathogen in infected cells. Therefore, the expression of IL-10 and IL-12 was evaluated at earlier stages of infection in the RTS11 cell line derived from Oncorhynchus mykiss macrophages. Simultaneously, the hepcidin expression and location was analyzed in the macrophages infected with the pathogen. Our results suggest that IL-10 is clearly induced at early stages of infection with values peaking at 36 hours post infection. Furthermore, infective P. salmonis downregulates the expression of antimicrobial peptide hepcidin and vesicles containing this peptide were unable to merge with the infective bacteria. Our results suggest that P. salmonis is able to manipulate the behavior of host cytokines and likely might constitute a virulence mechanism that promotes intracellular bacterial replication in leukocytes cells lines of trout and salmon. This mechanism involves the generation of an optimum environment for the microorganism and the downregulation of antimicrobial effectors like hepcidin.

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Section: Methodsmentioning

confidence: 99%

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model

et al. 2016

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“…The resulting RNA was recovered in a final volume of 30 l of nuclease-free deionized water. Triplicates of 5 l each were used to confirm ISAV by standard qRT-PCR procedures (19,22).…”

Section: Methodsmentioning

confidence: 99%

Bona Fide Evidence for Natural Vertical Transmission of Infectious Salmon Anemia Virus in Freshwater Brood Stocks of Farmed Atlantic Salmon (Salmo salar) in Southern Chile

Marshall

Ramírez

Labra

et al. 2014

J Virol

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Infectious salmon anemia (ISA) is a severe disease that affects farmed Atlantic salmon (Salmo salar), causing outbreaks in seawater in most salmon-producing countries worldwide, with particular aggressiveness in southern Chile. The etiological agent of this disease is a virus belonging to the Orthomyxoviridae family, named infectious salmon anemia virus (ISAV). Although it has been suggested that this virus can be vertically transmitted, even in freshwater, there is a lack of compelling experimental evidence to confirm this. Here we demonstrate significant putative viral loads in the ovarian fluid as well as in the eggs of two brood stock female adult specimens that harbored the virus systemically but without clinical signs. The target virus corresponded to a highly polymorphic region 3 (HPR-3) variant, which is known to be virulent in seawater and responsible for recent and past outbreaks of this disease in Chile. Additionally, the virus recovered from the fluid as well as from the interior of the eggs was fully infective to a susceptible fish cell line. To our knowledge, this is the first robust evidence demonstrating mother-to-offspring vertical transmission of the infective virus on the one hand and the asymptomatic transmission of a virulent form of the virus in freshwater fish on the other hand. IMPORTANCEThe robustness of the data presented here will contribute to a better understanding of the biology of the virus but most importantly will constitute a key management tool in the control of an aggressive agent constantly threatening the sustainability of the global salmon industry.

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“…25.2 ± 0.8 n.d. and can be transferred to other host-pathogen pairs by designing specific primer pairs. Such tools are already used routinely in terrestrial agriculture (Miller et al, 2009) and animal mariculture (Sep ulveda et al, 2013) and will facilitate a sustainable development of seaweed cultivation.…”

Section: Resultsmentioning

confidence: 99%

A highly prevalent filamentous algal endophyte in natural populations of the sugar kelp Saccharina latissima is not detected during cultivation in Northern Brittany

Bernard

Rousvoal

Collet

et al. 2019

Aquat. Living Resour.

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The sugar kelp Saccharina latissima is cultivated in Europe for food, feed and ultimately the production of chemical commodities and bioenergy. Being cultivated in the open sea, S. latissima is exposed to potentially harmful organisms, such as Laminarionema elsbetiae, a filamentous brown algal endophyte with a very high prevalence in wild populations of European S. latissima. As it was shown previously that S. latissima sporophytes get infected by L. elsbetiae very early in their life, seeding the spores on collectors and keeping them under controlled conditions during the critical time of a possible infection with filamentous endophytes could be advantageous over direct seeding techniques, where the ropes are deployed within days after seeding. We used a qPCR-assay to assess the prevalence of the endophyte L. elsbetiae in S. latissima cultivated during winter in Northern Brittany, comparing individuals from direct-seeded ropes and collector-seeded lines that were kept in laboratory conditions for different time spans. No DNA of the endophyte was detected in the samples, suggesting that either the kelps were not infected or the amount of endophytic filaments were below the detection rate of the qPCR assay. Furthermore, L. elsbetiae could not be detected in the seawater surrounding the kelp farm, indicating that L. elsbetiae is not fertile or disperses at a very small scale in Northern Brittany during the deployment time of young kelps. Our results suggest that infections of cultivated S. latissima with the endophyte L. elsbetiae might be a minor problem in kelp farms in Northern Brittany if the seeding production is kept under controlled conditions without external contamination.

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Design and evaluation of a unique RT-qPCR assay for diagnostic quality control assessment that is applicable to pathogen detection in three species of salmonid fish

Cited by 20 publications

References 29 publications

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model

Bona Fide Evidence for Natural Vertical Transmission of Infectious Salmon Anemia Virus in Freshwater Brood Stocks of Farmed Atlantic Salmon (Salmo salar) in Southern Chile

A highly prevalent filamentous algal endophyte in natural populations of the sugar kelp Saccharina latissima is not detected during cultivation in Northern Brittany

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