DGGE-based detection method for Quahog Parasite Unknown (QPX)

Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.     

Molecular diagnostics,field validation,and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam Mercenaria mercenaria

Quahog Parasite Unknown (QPX) is a protistan parasite that causes disease and mortality in the hard clam Mercenaria mercenaria. PCR primers and DNA oligonucleotide probes were designed and evaluated for sensitivity and specificity for the QPX organism specifically and for the phylum Labyrinthulomycota in general. The best performing QPX-specific primer pair amplified a 665 bp region of the QPX small-subunit ribosomal DNA (SSU rDNA) and detected as little as 1 fg cloned QPX SSU rDNA and 20 fg QPX genomic DNA. The primers did not amplify DNA of uninfected hard clams M. mercenaria or of the thraustochytrids Schizochytrium aggregatum, Thraustochytrium aureum, and T. striatum. The general labyrinthulomycete primers, which were designed to offer broader specificity than the QPX primers, amplified a 435 bp region of SSU rDNA from QPX, and a 436 to 437 bp region of SSU rDNA from S. aggregatum, T. aureum, and T. striatum, but did not amplify that of the clam M. mercenaria. Field validation of the QPX-specific primer pair, through comparative sampling of 224 clams collected over a 16 mo period from a QPX endemic site in Virginia, USA, indicated that the PCR assay is equivalent to histological diagnosis if initially negative PCR products are reamplified. Oligonucleotide DNA probes specific for QPX and the phylum Labyrinthulomycota were evaluated for in situ hybridization assays of cell smears or paraffin-embedded tissues. Two DNA probes for QPX offered limited sensitivity when used independently; however, when used together as a probe cocktail, sensitivity was greatly enhanced. The probe cocktail hybridized to putative QPX organisms in tissues of hard clams collected from Virginia, New Jersey, Massachusetts and Canada, suggesting that the QPX organisms in these areas are either very closely related or the same species. The QPX probe cocktail did not hybridize with clam tissue or with the thraustochytrids S. aggregatum, T. aureum, and T. striatum. The labyrinthulomycete DNA probe hybridized with QPX and the 3 thraustochytrids, with no background hybridization to clam tissue. SSU rDNA sequences were obtained for the putative QPX organisms from geographically distinct sites. Phylogenetic analyses based on the QPX and Labyrinthulomycota sequences confirmed earlier reports that QPX is a member of this phylum, but could not definitively demonstrate that all of the QPX organisms were the same species.     

Environmental distribution and persistence of Quahog Parasite Unknown (QPX)

Quahog Parasite Unknown (QPX) is the cause of mass mortality events of hard clams Mercenaria mercenaria from Virginia, USA, to New Brunswick, Canada. Aquaculture areas in Massachusetts, USA, have been particularly hard hit. The parasite has been shown to be a directly infective organism, but it is unclear whether it could exist or persist outside of its clam host. We used molecular methods to examine water, sediment, seaweeds, seagrass and various invertebrates for the presence of QPX. Sites in Virginia and Massachusetts were selected based upon the incidence of QPX-induced clam die-offs, and they were monitored seasonally. QPX was detectable in almost all of our different sample types from Massachusetts, indicating that the parasite was widely distributed in the environment. Significantly more samples from Massachusetts were positive than from Virginia, and there was a seasonal pattern to the types of samples positive from Massachusetts. The data suggest that, although it may be difficult to completely eradicate QPX from the environment, it may be possible to keep the incidence of disease under control through good plot husbandry and the removal of infected and dying clams.     

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: I. Dynamics of QPX disease

Quahog Parasite Unknown (QPX) causes disease and mortality in hard clams, Mercenaria mercenaria. Seasonality of QPX disease prevalence in the field and changes in QPX growth and survival in vitro suggest a role of temperature in the hard clam-QPX interaction and disease development. This study specifically examined the effect of temperature on QPX disease development and dynamics. Naturally and experimentally infected clams were separately maintained in the laboratory at 13 °C, 21 °C, or 27 °C for 4 months. Following this initial treatment, temperature was adjusted to 21 °C for 5 additional months to simulate seasonal changes of temperature in the field and to investigate the effect of temperature variations on QPX disease dynamics. Mortality was continuously monitored during the experiment and clams were sampled at 2, 4 and 9 months for the assessment of QPX disease prevalence and intensity using our standard histological and quantitative PCR techniques. Results demonstrated significantly higher QPX disease prevalence and intensity, as well as higher mortality, in naturally-infected clams maintained at 13 °C as compared to those held at 21 °C or 27 °C. Similarly, disease development was significantly higher in experimentally infected clams maintained at the colder temperature (70% prevalence after 4 months) as compared to those maintained under warmer conditions (<10%). Additionally, our results demonstrated an improvement in the condition of clams initially maintained at 13 °C for 4 months after transfer to 21 °C for 5 additional months, with a significant reduction of QPX prevalence (down to 19%). Interestingly, disease development or healing in clams maintained at different temperatures exhibited a strong relationship with clam defense status (jointly submitted paper) and highlighted the impact of temperature on clam activity and QPX disease dynamics. These findings should be taken into account for the timing of activities involving the monitoring, movement (e.g. relays, transplants) or grow out (e.g. commercial culture, municipal enhancement) of hard clams in enzootic areas.     

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: II. Defense parameters

Quahog Parasite Unknown (QPX) is a protistan parasite affecting hard clams Mercenaria mercenaria along the Northeastern coast of the United States. The geographic distribution and occurrence of disease epizootics suggests a primary role of temperature in disease development. This study was designed to investigate the effect of temperature on constitutive and QPX-induced defense factors in M. mercenaria. Control and QPX-challenged (both experimentally and naturally) clams were maintained at 13, 21 and 27 °C for 4 months. Control and experimentally-infected clams originated from a southern broodstock (Florida, no prior reports of disease outbreak) while naturally-infected clams originated from a northern broodstock (Massachusetts, enzootic area). Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. Measured parameters included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated a strong influence of temperature on constitutive clam defense factors with significant modulation of cellular and humoral parameters of control clams maintained at 13 °C compared to 21 and 27 °C. Similarly, clam response to QPX challenge was also affected by temperature. Challenged clams exhibited no difference from controls at 27 °C whereas different responses were observed at 21 °C and 13 °C compared to controls. Despite differences in infection mode (experimentally or naturally infected) and clam origin (northern and southern broodstocks), similarities were observed at 13 °C and 21 °C between QPX infected clams from Florida and Massachusetts. Clam response to temperature and to QPX exhibited interesting relationship with QPX disease development highlighting major influence of temperature on disease development.     

Modulatory effects of hard clam (Mercenaria mercenaria) tissue extracts on the in vitro growth of its pathogen QPX

Quahog parasite unknown (QPX) is a fatal protistan parasite affecting cultured and wild hard clams Mercenaria mercenaria along the northeastern coasts of the USA and maritime Canada. Field investigations and laboratory transmission studies revealed some variations in the susceptibility of different hard clam stocks to QPX infection. In this study, we used in vitro QPX cultures to investigate the effect of plasma and tissue extracts from two different clam stocks on parasite survival and growth. Results demonstrated the presence of factors in clams that significantly modulate QPX growth. Extracts from gills and mantle tissues as well as plasma inhibited in vitro QPX growth, whereas foot and adductor muscle extracts enhanced parasite growth. Investigations of anti-QPX activities in plasma from two clam stocks displaying different susceptibility toward QPX disease in vivo demonstrated higher inhibition of QPX growth by plasma from New York (resistant) clams compared to Florida (susceptible) clams. Some clams appeared to be deficient in inhibitory factors, suggesting that such animals may become more easily infected by the parasite.     

Quantitative Real-Time PCR Assay for QPX (Thraustochytriidae), a Parasite of the Hard Clam (Mercenaria mercenaria)

We developed a real-time quantitative PCR (qPCR) assay targeting the rRNA internal transcribed spacer region of the hard clam pathogen QPX. The qPCR assay was more sensitive than was histology in detecting clams with light QPX infections. QPX was detected in 4 of 43 sediment samples but in none of 40 seawater samples.The thraustochytrid called QPX (for quahog parasite unknown) has caused high mortalities in hatchery-reared and wild hard clams (Mercenaria mercenaria, also known as quahogs) from Prince Edward Island (Canada) to Virginia (United States) since the late 1950s (17, 22, 25, 29). In the summer of 2002, QPX infections appeared in the previously healthy Raritan Bay (off the coast of Staten Island in New York) M. mercenaria population, causing significant clam mortality and closure of the fishery (6). Management of hard clam populations affected by QPX disease is hampered by an incomplete understanding of factors controlling the occurrence and severity of QPX infections. Environmental factors, such as salinity and temperature, appear to be important (22), as do clam population density and the planting of seed from nonlocal sources (7). More quantitative information about the occurrence and progression of QPX disease in relation to these and other variables would support better prediction of, and response to, QPX outbreaks. QPX is thought to be an opportunistic pathogen (4, 7, 11), capable of growing outside its host. However, there is very little known about substrates that might support QPX organisms outside of hard clams (4). The abilities to detect and enumerate QPX cells in potential reservoirs would allow the dynamics of the QPX organism in the environment to be related to the occurrence of QPX disease, offering new insight into fundamental questions about the natural transmission mechanisms of the infection.The 18S ribosomal DNA (rDNA) primer pair QPX-F and QPX-R2 can be used in a standard PCR assay to detect the presence of QPX DNA in clam tissue samples (26). Unfortunately, the products are too long (∼650 bp), and often include too much primer dimer, for use in a SYBR green real-time quantitative PCR (qPCR) assay. The low sequence variability in rRNA genes made it difficult to design other primers specific for QPX 18S rDNA. Instead, we used our previously reported rRNA internal transcribed spacer (ITS) region (including ITS1, the 5.8S rRNA gene, and ITS2) sequences for QPX isolates from Massachusetts and New York (20) to develop a qPCR assay targeting the more variable ITS region (1).     

Effects of salinity on hard clam (Mercenaria mercenaria) defense parameters and QPX disease dynamics

QPX (Quahog Parasite Unknown) is a protistan parasite affecting hard clams (Mercenaria mercenaria) along the Northeast coast of the United States. The fact that QPX disease epizootics are usually observed in field sites with high salinities led to the general assumption that salinity represents an important factor for disease distribution. This study was designed to investigate the effect of salinity on QPX disease development as well as constitutive and QPX-induced defense factors in M. mercenaria. Naïve and QPX-infected (both experimentally and naturally) clams were submitted to 17 and 30 psu for 4 months. Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. These included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated higher QPX-associated mortality in naturally infected clams maintained at high salinity compared to those held at 17 psu. Our findings also showed an increase in mortality following experimental challenge with QPX in clams submitted to 30 psu but not in those held at 17 psu. Constitutive clam defense factors and the response to QPX challenge were also affected by salinity. QPX challenge caused significant but transitory changes in hemolymph parameters that were obvious at 2 months but disappeared at 4 months. Overall, our results show that salinity modulates clam immunity and the progress of QPX disease although its impact appears secondary as compared to findings we reported earlier for temperature.     

Molecular genetic variation within and among isolates of QPX (Thraustochytridae), a parasite of the hard clam Mercenaria mercenaria

The thraustochytrid known as QPX (Quahog Parasite Unknown) has sporadically caused disease in the hard clam Mercenaria mercenaria along the east coast of North America since the 1960s. We hypothesized that genetically distinct QPX strains might be responsible for outbreaks of QPX disease in different areas and tested this hypothesis by comparing several QPX isolates recovered from the recent outbreak in Raritan Bay, New York with QPX strains isolated from 2 outbreaks in Massachusetts, USA. There was no variation in small subunit rDNA (SSU rDNA), 5.8S rDNA, or 4 mitochondrial gene sequences. In contrast, both of the ribosomal ribonucleic acid (rRNA) operon intergenic spacers, internal transcribed spacers 1 and 2 (ITS1 and ITS2), revealed substantial sequence variation. However, strain-specific sequences were not detected because the ITS sequence variation within QPX isolates was comparable to the variation between isolates. ITS1 sequences recovered from an infected clam by amplification with a QPX ITS2-specific primer were identical to those recovered from the QPX isolates.     

Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect <10 CFU per PCR reaction. To evaluate the effectiveness of the Arcobacter TaqMan assays with field isolates the assays were used to screen DNA samples prepared from faecal, hide and environmental samples obtained from two meat processing plants. In these studies, the TaqMan assays revealed that 2/150 (1.3%) samples were A. butzleri-positive, 11/150 (7.3%) were A. cryaerophilus-positive and the identity of generated amplicons was confirmed by DNA sequencing. Our results show that these TaqMan assays provide improvements in sensitivity and species-representation over other published Arcobacter PCR assays and they are compatible with detecting Arcobacters in sub-optimal matrices.     

Development of a TaqMan quantitative PCR assay specific for Cryptosporidium parvum

A rapid detection method that is both quantitative and specific for the water-borne human parasite Cryptosporidium parvum is reported. Real-time polymerase chain reaction (PCR) combined with fluorescent TaqMan technology was used to develop this sensitive and accurate assay. The selected primer-probe set identified a 138-bp section specific to a C. parvum genomic DNA sequence. The method was optimized on a cloned section of the target DNA sequence, then evaluated on C. parvum oocyst dilutions. Quantification was accomplished by comparing the fluorescence signals obtained from test samples of C. parvum oocysts with those obtained from standard dilutions of C. parvum oocysts. This real-time PCR assay allowed reliable quantification of C. parvum oocysts over six orders of magnitude with a baseline sensitivity of six oocysts in 2 h.     

Immunomagnetic bead-based recovery and real time quantitative PCR (RT iq-PCR) for sensitive quantification of aflatoxin B(1)

Aflatoxin B₁ is an unavoidable natural mycotoxin that enters the food chain by contamination of food grains and feedstuffs, potentially posing carcinogenic risks to animal and human health. Immuno-PCR methods have the potential to address the need of meeting the regulatory limits by detecting trace levels of toxins present in food and animal feeds. This paper describes a real-time immuno-quantitative PCR (RT-iqPCR) assay for quantification of aflatoxin B₁ suspended in methanol:water solution that can also serve as an extraction solvent. Immuno-PCR approaches were examined including direct vs. indirect sandwich assays using monoclonal vs. polyclonal antibodies. Our best approach was obtained using monoclonal antibodies to capture aflatoxin in solution prior to immobilizing the F_c portion of the capture antibodies onto to protein G magnetic beads. This was followed by the addition of a polyclonal ‘signal antibody’ tethered with an oligonucleotide template for a subsequent PCR assay. The RT-iqPCR assay described herein leads to the sensitive detection and quantification of aflatoxin B₁ from 10 ppb down to 0.1 ppb with high correlation (r² = 0.97) and efficiency (99.5%). The approach also detected the high-dose ‘hook effect’ phenomenon (excess antigen) which was overcome by the use of dilution protocols to eliminate false negatives that may occur at levels above quantification limits of the assay. The RT-iqPCR approach discussed here is presented as a model system that could easily be adapted for aflatoxin detection in a variety of food or animal feed samples using a simple methanol:water solution as an extraction solvent.     

Development and evaluation of a real-time quantitative PCR assay for Aspergillus flavus

Aspergillus flavus is a ubiquitous mold and the most common mold contaminating foodstuffs. Many strains of A. flavus produce aflatoxins. In addition it is an allergen and an opportunistic pathogen of animals and plants. A. flavus often is underestimated in traditional culture analyses due to the expertise required and the cost associated with speciating members of the genus Aspergillus. The goal of this study was to develop and validate a primer and probe set for the rapid detection and quantitation of A. flavus in pure culture using real-time quantitative polymerase chain reaction (QPCR) amplification. Unique DNA regions were located in the genome of the target organism by sequence comparison with the GenBank database, and several candidate oligonucleotides were identified from the scientific literature for potential use with the TaqMan QPCR technology. Three primer and probe sets were designed and validated for specificity and sensitivity in laboratory experiments. Initial screening to test for sensitivity was performed with seven A. flavus isolates and selected nontarget fungi. Specificity testing was conducted with the selected primer and probe set, which amplified all nine A. flavus isolates tested, including an aflatoxin producing strain. The primers did not amplify DNA extracted from 39 other fungal species (comprising 16 genera), including 18 other Aspergillus species and six Penicillium species. No amplification of human or bacterial DNA was observed; however cross-reactivity was observed with Aspergillus oryzae. PCR analysis of DNA dilutions and the use of an internal positive control demonstrated that 67% of the fungal DNA samples assayed contained PCR inhibitors. The assay validated for the target organism is capable of producing PCR results in less than 1 h after DNA extraction. The results of this research demonstrate the capabilities of QPCR for the enhanced detection and enumeration of fungi of significance to human health.     

Development and evaluation of real competitive PCR for high-throughput quantitative applications

Real competitive PCR (rcPCR) has been shown to have high sensitivity, reproducibility, and high-throughput potential. We describe further development and evaluation of this methodology as a tool for measuring nucleic acid abundance within a cell. Modifications to the original protocol allow analysis of gene expression levels using standard conditions regardless of mRNA abundance and assay type, thereby increasing throughput and ease of reaction setup while decreasing optimization time. In addition, we have developed a software package, TITAN, to automatically analyze the results. The details are relevant to researchers performing competitive PCR using any detection technique. The effectiveness of the described developments is demonstrated using 12 genes known to have differential expression in cell lines grown under normal and hypoxic conditions. Quantitative and qualitative comparisons to real-time PCR are presented. It is also demonstrated that the technique is capable of detecting submicroscopic chromosomal DNA deletions.     

Development and evaluation of a quantitative PCR assay targeting sandhill crane (Grus canadensis) fecal pollution

While the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous to Bacteroidetes and Clostridia. The Crane1 marker targeted a dominant clade of unclassified Lactobacillales sequences closely related to Catellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e., n = 553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n = 16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n = 165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics.     

腐皮镰刀菌SYBR Green实时荧光定量PCR快速检测方法的建立

目的建立一种能够快速、灵敏、特异的鉴定腐皮镰刀菌的SYBR Green实时荧光定量PCR。方法运用SYBR Green实时荧光定量PCR反应体系检测腐皮镰刀菌,并对此方法的特异性、灵敏度和稳定性进行评价。结果通过对45例样品的检测,结果显示SYBR Green实时荧光定量PCR特异性好,其检出率高于普通PCR;灵敏度高,对重组质粒标准品的检测灵敏度为1.0×10~2copies/μL;稳定性好,对质粒为1.0×10~7copies/μL、1.0×10~5copies/μL、1.0×10~3copies/μL的标准品重复检测10次,结果显示扩增反应Ct值的变异系数为0.96%~1.68%。结论SYBR Green实时荧光定量PCR检测腐皮镰刀菌,不仅特异性好,灵敏度高,稳定性好,而且简便、快速、易操作。