Advances in crustacean cell culture

In 2006, for the first time in history, more seafood was produced from aquaculture raised animals than wild caught fisheries. However, crustacean aquaculture continues to be plagued by disease, due to the lack of sensitive investigative methods that assist diagnostics and pathogen control. Prevention and control of diseases are an absolute priority for the durability of the crustacean aquaculture industry. \ud \ud The development of a permanent in vitro model for crustacean species is imperative. The research presented herein attempted to utilise novel modern technologies to develop the first continuous cell line from crustacea. The first investigation involved assessing methodologies that explored the existence of crustacean components in hybridised cell lines, which were developed by combining crustacean cells with immortal fish cells (Chapter 4). The methodologies involved cellular assessment from genomic and protein approaches, along with exploration of viral susceptibility of the cells. Using PCR, no crustacean 18S rRNA genes or haemocyanin genes were found in any of the seven hybrid cell lines. No crustacean proteins were detected, nor did any viral amplification occur when hybrid cells were inoculated with two crustacean viruses, indicating that these hybrid cell populations were not suitable for crustacean virological studies (Chapter 5). \ud \ud The second investigation involved optimisation of in vitro methods using the Australian freshwater crayfish, Cherax quadricarinatus (Chapters 6 and 7). The approach included scientifically optimising the culture medium by comparing cell proliferation with three different cell culture media and 25 different media supplements. Leibovitz-15 medium, along with supplemental iron, copper, foetal bovine serum, and non-essential amino acids, was found to significantly increase cell proliferation (F = 6.231, df = 1,19, p< 0.05) and also augment the longevity of cells in vitro.\ud \ud While spontaneous transformation of somatic cells can occur, transgenesis often expedites immortality. Therefore, the third investigation explored the transfection of C. quadricarinatus primary cells (Chapter 8). A lipofection reagent was used to introduce an observable green fluorescent protein vector into the cytoplasm of the cells. Transfection of the cells was then attempted using human oncogenes. \ud \ud Papillomaviruses are non-enveloped, double-stranded DNA tumour viruses that play a critical role in the formation of human anogenital cancer. Early studies have demonstrated that the human papillomavirus-expressed E6 and E7 proteins function concomitantly to disrupt the p53 and Rb tumour suppressor genes, regulators of the cell-cycle checkpoints at the first gap (G1) phase of the cell cycle. To help C. quadricarinatus cells pass through the G1 phase and enter the DNA synthesis stage of the cell cycle, HPV E6 and E7 genes were transfected into the C. quadricarinatus cells. Successful transfection was demonstrated by the presence of oncogene mRNA by RT-PCR. At day 150, transfected cells remained viable, although cell proliferation was stagnant. It may be that, although transfection of the oncogenes was successful, no proliferation of the C. quadricarinatus cells was evident, due to a lack of telomere maintenance. \ud \ud Overall, attempts to create a crustacean cell line remain elusive. Although the end product of a permanent cell line has not been forthcoming, this research made successful advancements in crustacean cell culture methodologies and explored new techniques and technologies that may assist eventual immortalisation

OIE white spot syndrome virus PCR gives false-positive results in Cherax quadricarinatus

White spot syndrome virus (WSSV) is an intranuclear bacilliform virus (IBV) that is a serious, notifiable crustacean pathogen. The Office International des Epizooties (OIE) PCR protocol for WSSV uses primer sets initially developed by Lo et al. (1996). It yields a first-step PCR amplicon of 1441 bp and a nested PCR amplicon of 941 bp. An amplicon (941 bp) purported to specifically detect WSSV was obtained when using template DNA extracted from Cherax quadricarinatus in a WSSV PCR\ud detection protocol recommended by the OIE. Sequencing and analysis of the 941 bp amplicon and an occasional 550 bp amplicon from C. quadricarinatus revealed no phylogenetic relationship with WSSV, and suggested a possible lack of sufficient primer specificity for WSSV in the OIE test. This suggestion was supported by the fact that the OIE outer primer sequence (146F1) was present in both the forward and reverse position of the 941 bp and the forward position of the 550 bp nested amplicons from C. quadricarinatus. As WSSV is a notifiable pathogen, the consequences of false-positive results are harsh in WSSV-free zones and can lead to incorrect quarantine and unnecessary destruction of animals. Therefore, urgent attention and revision is necessary for the current OIE PCR protocol for WSSV detection

Methods to enhance the intensity of intranuclear bacilliform virus infection in Cherax quadricarinatus

Many studies have examined the morphology, pathology and epizootiology of the intranuclear bacilliform virus (IBV) of Cherax quadricarinatus, but little research has been conducted to acquire specific knowledge of the virus. This is partly due to difficulties in detecting the virus and in obtaining sufficient material for viral isolation and purification. As quantified by light microscopy, we significantly (p < 0.01) enhanced IBV intensities from 10.56 to 16.67% in C. quadricarinatus by using salinity stress (12 ppt) and ingestion of infected hepatopancreatic tissue, which increased intensities from 4.33 to 10.77%. It was also found that phosphotungstic acid-eosin stain was superior to standard haematoxylin and eosin stain in visualizing IBV inclusion bodies. It is expected that these new techniques will enhance the detectability of the virus and provide sufficient viral material for viral purification, characterization and development of molecular tools for detection and phylogenetic analysis

Attempts at producing a hybridised Penaeus mondon cell line by cellular fusion

The lack of a standardised system for the isolation, identification and purification of prawn viruses, is a major obstacle to the control of viruses in penaeid aquaculture. To date, spontaneous and induced transformation of somatic penaeid cells has failed. Hybrid cells with the aim of supporting the growth of penaeid viruses were created using polyethylene glycol (PEG)-mediated fusion with two immortal cell lines, Epithelioma papulosum cyprinid (EPC) and Spodoptera frugiperda pupal ovarian cells (Sf9), fused with Penaeus monodon haemocytes. The immortal cell lines were biochemically blocked with actinomycin D and puromycin before fusion occurred. A total of 78 hybrid clones were created. The methods used to confirm the presence of P. monodon genes and proteins in the hybrid cells did not detect crustacean components, nor was any viral amplification detected by real-time PCR after hybrid cells were inoculated with two P. monodon parvoviruses, Penaeus merguiensis densovirus and infectious hypodermal and haematopoietic necrosis virus. These results suggest although the creation of the hybrid cells appeared successful, the cell lines lacked key crustacean cell components required for their use as an in vitro system for virus replication

Attempts at immortalization of crustacean primary cell cultures using human cancer genes

Primary cell cultures from crustacea have been initiated since the 1960s, yet no permanent cell line is available. Primary cells have a limited proliferative capacity in culture due to cellular senescence, which is regulated by a group of dominant senescence genes. The aim of this research was to manipulate cell cycle regulation by transfecting Cherax quadricarinatus primary cells with oncogenes, in an effort to induce a permanent cell line. Human papillomaviruses (HPV) play a critical role in the formation of anogenital cancer. Research has demonstrated that the HPV-expressed E6 and E7 proteins function concomitantly to disrupt the p53 and retinoblastoma (Rb) tumor suppressor genes, regulators of the cell-cycle checkpoints at the first gap (G1) phase. HPV E6 and E7 genes were transfected into the C. quadricarinatus cells by lipofection. Successful transfection was demonstrated by the presence of oncogene messenger RNA by reverse transciptase polymerase chain reaction. At day 150, transfected cells still remain viable, although cell proliferation was stagnant. It may be that while transfection of the oncogenes was successful, no proliferation of the C. quadricarinatus cells was evident due to a lack of telomere maintenance

Stakeholders’ perspectives on clinical trial acceptability and approach to consent within a limited timeframe: a mixed methods study

Objectives The Bronchiolitis Endotracheal Surfactant Study (BESS) is a randomised controlled trial to determine the efficacy of endo-tracheal surfactant therapy for critically ill infants with bronchiolitis. To explore acceptability of BESS, including approach to consent within a limited time frame, we explored parent and staff experiences of trial involvement in the first two bronchiolitis seasons to inform subsequent trial conduct.Design A mixed-method embedded study involving a site staff survey, questionnaires and interviews with parents approached about BESS.Setting Fourteen UK paediatric intensive care units.Participants Of the 179 parents of children approached to take part in BESS, 75 parents (of 69 children) took part in the embedded study. Of these, 55/69 (78%) completed a questionnaire, and 15/69 (21%) were interviewed. Thirty-eight staff completed a questionnaire.Results Parents and staff found the trial acceptable. All constructs of the Adapted Theoretical Framework of Acceptability were met. Parents viewed surfactant as being low risk and hoped their child’s participation would help others in the future. Although parents supported research without prior consent in studies of time critical interventions, they believed there was sufficient time to consider this trial. Parents recommended that prospective informed consent should continue to be sought for BESS. Many felt that the time between the consent process and intervention being administered took too long and should be ‘streamlined’ to avoid delays in administration of trial interventions. Staff described how the training and trial processes worked well, yet patients were missed due to lack of staff to deliver the intervention, particularly at weekends.Conclusion Parents and staff supported BESS trial and highlighted aspects of the protocol, which should be refined, including a streamlined informed consent process. Findings will be useful to inform proportionate approaches to consent in future paediatric trials where there is a short timeframe for consent discussions.Trial registration number ISRCTN11746266