Improved method and test strategy for recovery of enteric viruses from shellfish

An improved recovery method and testing strategy were devised for recovery of low numbers of enteric viruses from each of three commercially important shellfish species. Effective recovery of virus depended as much upon details of the test strategy adopted for use of the improved method with each species as on the method itself. The most important test details involved sample composition, pool size, and method of use of cell cultures. Recovery sensitivity measured permitted detection of 25 to 3 plaque-forming units of enteroviruses and 100 to 27 plaque-forming units of reovirus through their recovery in cell culture, with effectivenesses averaging 64 and 46%, respectively. Test samples prepared by the improved recovery method were virtually cytotoxicity free. Optimal recovery of virus on 45-cm2 cell culture monolayers was obtained with 1-ml inocula adsorbed for 2 h. The most effective recovery of virus from shellfish samples was made by a sequential adsorption procedure which allowed equal exposure of an entire sample to each of two or more cell cultures. Removal of nonviral contaminants from test samples by antibiotic treatment was preferable to the use of ether or membrane filtration procedures.     

Methods for recovering poliovirus and rotavirus from oysters

Polioviruses and rotaviruses are potential indicators of sewage pollution of water and shellfish. Several methods for detecting these viruses in oysters were assessed. Elution-precipitation involving Catfloc for clarification and skim milk for subsequent flocculation resulted in the recovery of an average of 79% of poliovirus type 1 and 37% of rotavirus SA-11 from oyster homogenates inoculated with low numbers of these viruses. Adsorption-elution-precipitation did not improve the recovery of poliovirus and was detrimental to the recovery of rotavirus. Ultrafiltration or ultracentrifugation resulted in improved recovery of rotavirus but also in higher toxicity of oyster extracts to cell cultures. We recommend the use of the described elution-precipitation method for detecting viral pollutants in sample of oysters.     

Methods for recovering poliovirus and rotavirus from oysters.

Polioviruses and rotaviruses are potential indicators of sewage pollution of water and shellfish. Several methods for detecting these viruses in oysters were assessed. Elution-precipitation involving Catfloc for clarification and skim milk for subsequent flocculation resulted in the recovery of an average of 79% of poliovirus type 1 and 37% of rotavirus SA-11 from oyster homogenates inoculated with low numbers of these viruses. Adsorption-elution-precipitation did not improve the recovery of poliovirus and was detrimental to the recovery of rotavirus. Ultrafiltration or ultracentrifugation resulted in improved recovery of rotavirus but also in higher toxicity of oyster extracts to cell cultures. We recommend the use of the described elution-precipitation method for detecting viral pollutants in sample of oysters.     

单管半套式RT-PCR法检测贝类中轮状病毒的研究

轮状病毒(Rotavirus)是一种以贝类为载体的重要食源性病毒,目前来说,RT_PCR是贝类中轮状病毒最有效的检测方法,但通常由于病毒含量较低以及贝类中存在大量的PCR抑制物,致使将其运用于实际样本的检测时灵敏度仍较低。在实验室模拟自然环境,以人工播毒的方式使贝类富集轮状病毒,运用单管半套式RT_PCR法进行检测,并将单管半套式RT_PCR与ELISA、RT_PCR的检测灵敏度做了比较,表明单管半套式RT_PCR比ELISA的灵敏度高10 0 0倍,是传统RT_PCR的10倍,有时甚至10 0倍。另外,单管半套式RT_PCR法降低了实验过程中的内源性和外源性污染,使检测所需时间从6h缩短至4 5h ,大大改进和完善了食源性病毒检测方法。并同时以整只贝和仅以贝的消化道为样检测表明,以消化道为样时的病毒检出率和检测灵敏度较高。     

Human enteroviruses in oysters and their overlying waters.

The presence of enteroviruses in oysters and oyster-harvesting waters of the Texas Gulf coast was monitored over a period of 10 months. Viruses were detected in water and oyster samples obtained from areas both open and closed to shellfish harvesting. Viruses were detected periodically in waters that met current bacteriological standards for shellfish harvesting. No significant statistical relationship was demonstrated between virus concentration in oysters and the bacteriological and physiochemical quality of water and shellfish. Viruses in water were, however, moderately correlated with total coliforms in water and oysters and with fecal coliforms in oysters. Total coliforms in water were realted to total coliforms in sediment were related only to total coliforms in sediment. Among the physiochemical characteristics of water, turbidity was related statistically to the organic matter content of water and to fecal coliforms in water. There was a marked effect of rainfall on the bacteriological quality of water. Of a total of 44 water samples, 26 yielded virus in concentrations from 4 to 167 plaque-forming units per 100-gallon (ca. 378.5-liter) sample. Of a total of 40 pools of 10 to 12 oysters each, virus was found in 14 pools at a concentration of 6 to 224 plaque-forming units per 100 g of oyster meat. On five occasions, virus was found in water samples when no virus could be detected in oysters harvested from the same sites. This study indicates that current bacteriological standards for determining the safety of shellfish and shellfish-growing waters do no reflect the occurrence of enteroviruses.     

Human enteroviruses in oysters and their overlying waters.

The presence of enteroviruses in oysters and oyster-harvesting waters of the Texas Gulf coast was monitored over a period of 10 months. Viruses were detected in water and oyster samples obtained from areas both open and closed to shellfish harvesting. Viruses were detected periodically in waters that met current bacteriological standards for shellfish harvesting. No significant statistical relationship was demonstrated between virus concentration in oysters and the bacteriological and physiochemical quality of water and shellfish. Viruses in water were, however, moderately correlated with total coliforms in water and oysters and with fecal coliforms in oysters. Total coliforms in water were realted to total coliforms in sediment were related only to total coliforms in sediment. Among the physiochemical characteristics of water, turbidity was related statistically to the organic matter content of water and to fecal coliforms in water. There was a marked effect of rainfall on the bacteriological quality of water. Of a total of 44 water samples, 26 yielded virus in concentrations from 4 to 167 plaque-forming units per 100-gallon (ca. 378.5-liter) sample. Of a total of 40 pools of 10 to 12 oysters each, virus was found in 14 pools at a concentration of 6 to 224 plaque-forming units per 100 g of oyster meat. On five occasions, virus was found in water samples when no virus could be detected in oysters harvested from the same sites. This study indicates that current bacteriological standards for determining the safety of shellfish and shellfish-growing waters do no reflect the occurrence of enteroviruses.     

Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and fecal samples.

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO(2) incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.     

Assessment of enterovirus contamination in mussel samples from Morocco

Summary In Morocco, shellfish sanitary quality analysis does not currently include enteric virus detection. Enteroviruses are classically detected by cell culture, but molecular methods such as RT-PCR are now broadly used alone or associated to cell culture. RT-PCR has the advantage of requiring less time and budget than cell culture. Bivalve mussels, being filter-feeders tend to accumulate viruses in contaminated seawater. In order to assess mussel contamination by enteroviruses, we screened samples of two origins, an aquaculture system and an area where wild mussels grow. Domestic sewage samples from an outlet near the above wild mussels growing area were also analysed. Viruses were concentrated from mussel meat by direct glycine elution and PEG 8000 precipitation. Total RNA was then extracted from the PEG precipitate by the guanidine thiocyanate method and used in RT-PCR. Enterovirus genomes were detected in 10% of wild shellfish samples, whilst none was present in the aquaculture samples. Since organisms harvested in both growing areas are used for human consumption, the enterovirus contamination observed in this study may highlight a potential public health risk and illustrate the importance of including routine virological analysis of shellfish in Morocco.     

Comparative Sensitivity of Various Cell Culture Systems for Isolation of Viruses from Wastewater and Fecal Samples

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO₂ incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.     

Maximizing recovery and detection of Cryptosporidium parvum oocysts from spiked eastern oyster (Crassostrea virginica) tissue samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Influence of environmental factors on virus detection by RT-PCR and cell culture

The effects of clay, humic acid, u.v. light and shellfish tissue residues on the detection of poliovirus type 2 from environmental samples by culture and RT-PCR were investigated. RT-PCR showed 10-100 times greater sensitivity for PV2 detection in the absence of sample contaminants than did culture by plaque assay in BGM cell monolayers. Bentonite clay (100-1000 mg l-1) and shellfish tissue residues reduced virus detection by plaque assay, but the effect of bentonite was mitigated by simple elution procedures. Bentonite clay, humic acid (5-150 mg l-1) and mussel tissue reduced virus detection by RT-PCR by between 1 and 8 logs, although this was mitigated in part by elution and Sephadex filtration of extracts. Sephadex filtration of samples reduced culturable PV2 by 32-50%. Exposure of PV2 in water to u.v. light reduced culturability of PV2 but not detection by RT-PCR. This study demonstrates that virus detection in environmental samples is strongly influenced by naturally occurring substances and disinfection approaches. The accuracy of results of viral analyses of this nature should be carefully scrutinized with respect to sample constituents.     

Medium for development of bee cell cultures (<Emphasis Type="Italic">Apis mellifera</Emphasis>: Hymenoptera: Apidae)

A media for the production of cell cultures from hymenopteran species such as honey bee, Apis mellifera L. (Hymenoptera: Apidae) was developed. Multiple bee cell cultures were produced when using bee larvae and pupae as starting material and modified Hert–Hunter 70 media. Cell culture systems for bees solves an impasse that has hindered efforts to isolate and screen pathogens which may be influencing or causing colony collapse disorder of bees. Multiple life stages of maturing larvae to early pupae were used to successfully establish cell cultures from the tissues of the head, thorax, and abdomen. Multiple cell types were observed which included free-floating suspensions, fibroblast-like, and epithelia-like monolayers. The final culture medium, WH2, was originally developed for hemipterans, Asian citrus psyllid, Diaphorina citri, and leafhopper, Homalodisca vitripennis cell cultures but has been shown to work for a diverse range of insect species such as bees. Bee cell cultures had various doubling times at 21–23°C ranging from 9–15 d. Deformed wing virus was detected in the primary explanted tissues, which tested negative by rt-PCR for Israeli acute paralysis virus (IAPV), Kashmir bee virus, acute bee paralysis virus, and black queen cell virus. Culture inoculation with IAPV from an isolate from Florida field samples, was detectable in cell cultures after two subcultures. Cell culture from hymenoptera species, such as bees, greatly advances the approaches available to the field of study on colony collapse disorders.     

Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster (Crassostrea virginica) Tissue Samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Investigating the establishment of primary cell culture from different abalone (<Emphasis Type="Italic">Haliotis midae</Emphasis>) tissues

The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.     

Infectivity of the DNA from four isolates of JC virus.

The infectivity of JC virus DNA was demonstrated in its most permissive cell culture, primary human fetal glial cells. The amount of infectivity observed in these heterogeneous cultures varied considerably between batches of cells. Contrary to results obtained with the papovaviruses simian virus 40 and BK virus, the calcium technique (F. L. Graham and A. J. van der Eb, Virology 52:456--467, 1973) was found to be more efficient at promoting JC virus DNA infectivity than the DEAE-dextran method (J. H. McCutchan and J. S. Pagano, J. Natl. Cancer Inst. 41:351--357, 1968): maximum infectivity titers of 4 x 10-(4) and 6 x 10(3) fluorescent cell units per microgram of DNA, respectively. These values represent an approximate recovery of infectivity from virus of between 0.02 and 0.14%. Comparisons of infectivity of DNAs obtained from four isolates of JC virus and which differed in their degrees of heterogeneity did not reveal significant differences. The JC virus DNA was not infectious in primary human fetal lung and kidney cells.     

Quantitative PCR Method for Enumeration of Cells of Cryptic Species of the Toxic Marine Dinoflagellate Ostreopsis spp. in Coastal Waters of Japan

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.     

Diagnosis of bovine respiratory syncytial virus infection by complement fixation test.

The complement fixation test by the microtiter method was applied to the serological diagnosis of bovine respiratory syncytial (RS) virus infection. When used as complement fixing antigens, untreated infected cell culture fluid, fluorocarbon-treated, and ether-treated materials showed no differences in antigenicity among them. The complement fixing antigenicity of bovine RS virus appeared in bovine kidney and Vero cell cultures for the first time 4 days after inoculation. Both the infectivity and complement fixing antigenicity reached a maximum 6 days after inoculation. In detecting complement fixing antibody from infected cattle, the most outstanding specific reaction was obtained when 5% fresh normal calf serum had been added to the diluent of complement. Neutralizing and complement fixing antibodies were examined in serum samples from two cattle in the course of experimental infection. It was found that both antibodies turned to be positive 2 weeks after inoculation. There was a linear correlation between neutralizing and complement fixing antibody titers, when serum samples from 40 natural cases were tested in the acute and convalescent stages. In addition, common antigenicity was demonstrated between the virus of bovine origin and the Long strain of human RS virus by complement fixation test.     

Impact of Procedural Steps and Cryopreservation Agents in the Cryopreservation of Chlorophyte Microalgae

The maintenance of traditional microalgae collections based on liquid and solid media is labour intensive, costly and subject to contamination and genetic drift. Cryopreservation is therefore the method of choice for the maintenance of microalgae culture collections, but success is limited for many species. Although the mechanisms underlying cryopreservation are understood in general, many technical variations are present in the literature and the impact of these are not always elaborated. This study describes two-step cryopreservation processes in which 3 microalgae strains representing different cell sizes were subjected to various experimental approaches to cryopreservation, the aim being to investigate mechanistic factors affecting cell viability. Sucrose and dimethyl sulfoxide (DMSO) were used as cryoprotectants. They were found to have a synergistic effect in the recovery of cryopreserved samples of many algal strains, with 6.5% being the optimum DMSO concentration. The effect of sucrose was shown to be due to improved cell survival and recovery after thawing by comparing the effect of sucrose on cell viability before or after cryopreservation. Additional factors with a beneficial effect on recovery were the elimination of centrifugation steps (minimizing cell damage), the reduction of cell concentration (which is proposed to reduce the generation of toxic cell wall components) and the use of low light levels during the recovery phase (proposed to reduce photooxidative damage). The use of the best conditions for each of these variables yielded an improved protocol which allowed the recovery and subsequent improved culture viability of a further 16 randomly chosen microalgae strains. These isolates included species from Chlorellaceae, Palmellaceae, Tetrasporaceae, Palmellopsis, Scenedesmaceae and Chlamydomonadaceae that differed greatly in cell diameter (3–50 µm), a variable that can affect cryopreservation success. The collective improvement of each of these parameters yielded a cryopreservation protocol that can be applied to a broad range of microalgae.     

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two‐dimensional difference gel electrophoresis (2D‐DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D‐DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D‐DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample. Biotechnol. Bioeng. 2010; 105: 306–316. © 2009 Wiley Periodicals, Inc.