Differential Diagnosis Of Mixed Haplosporidium Costale And Haplosporidium Nelsoni Infections In The Eastern Oyster, Crassostrea Virginica, Using Dna Probes

Haplosporidium costale and Haplosporidium nelsoni are morphologically similar pathogens of the eastern oyster Crassostrea virginica. In the absence of the spore stage, infections of the two species are extremely difficult, if not impossible, to distinguish using traditional light microscopy of stained tissue sections. Species-specific molecular diagnostics were developed for H. costale from the small subunit ribosomal DNA (SSU rDNA) sequence. The polymerase chain reaction (PCR) primers amplified a 557 base pair (bp) region of the H. costale SSU rDNA, but did not amplify DNA from oyster (C. virginica) or from six other haplosporidans (H. nelsoni, H. louisiana, H. lusitanicum, Minchinia teredinis, M. chitonis, or M. tapetis). The DNA probe was used with in sim hybridizations of oyster tissue sections to visualize H. costale plasmodia and prespore stages; it did not hybridize with oyster (C. virginica) or other haplosporidans (H. nelsoni, H. louisiana, or Minchinia teredinis). DNA-based diagnostics for H. costale, in conjunction with molecular tools previously developed for H. nelsoni, have overcome limitations of histological examination. From in situ hybridizations using both probes, some Virginia oysters previously diagnosed with H. costale were found to have mixed infections consisting of approximately 80 to 90% H. costale plasmodia and 10 to 20% H. nelsoni plasmodia, Plasmodia of H. costale were not found in epithelial tissue, only in connective tissue. In addition, use of the DNA probe confirmed the presence of H. costale plasmodia in Virginia oysters collected in the fall, an unprecedented seasonality for an advanced H. costale infection

Detection Of Haplosporidium-nelsoni (Haplosporidia, Haplosporidiidae) In Oysters By PCR Amplification

Haplosporidium nelsoni is a protistan pathogen of the eastern oyster Crassostrea virginica, and has contributed to the decline of the oyster population in the Chesapeake Bay. From comparison of the sequence data of the 16S-like rDNA of H. nelsoni with those of Minchinia teredinis and other related organisms, 2 oligonucleotides which were specific to H. nelsoni and suitable for use as PCR primers were identified. These primers amplified a 564 base pair fragment of the small subunit (SSU) rRNA gene of H. nelsoni, but did not amplify genomic oyster DNA or the SSU rRNA genes of the haplosporidians Haplosporidium costale, Haplosporidium louisiana, or M. teredinis. The PCR primers were able to detect the H. nelsoni SSU rDNA from 50 ng of infected oyster genomic DNA or from 10 fg of cloned H. nelsoni SSU rDNA. The ability of the PCR primers to diagnose H. nelsoni-infected oysters was compared to the established techniques of hemolymph settlement analysis in Farley chambers and histological examination from a sample of 20 oysters. Hemolymph settlement analysis detected infection in 10 oysters and histology revealed infections in 11 oysters. PCR amplification of DNA from hemolymph initially detected infections in 15 oysters and reamplification of the PCR products detected an additional 4 infections. PCR amplification is a more sensitive diagnostic assay for H. nelsoni than traditional techniques

Investigating The Life Cycle Of Haplosporidium nelsoni (MSX)

Attempts to decipher the life cycle of Haplosporidium nelsoni began almost immediately after it was identified as the pathogen causing MSX disease in eastern oysters, Crassostrea virginica. But transmission experiments failed and the spore stage, characteristic of haplosporidans, was extremely rare. Researchers concluded that another host was involved: an intermediate host in which part of the life cycle was produced, or-if the oyster was an accidental host-an alternate host that produces infective elements. A later finding that spores were found more often in spat (\u3c 1 y old) than in adults revived the idea of direct transmission between oysters. The new findings and the availability of molecular diagnostics led us to revive life cycle investigations. Over several years, oyster spat were examined for spores and searched for H. nelsoni in potential non-oyster hosts using both histological and polymerase chain reaction (PCR) methodologies. Although spores occurred in a high proportion of spat with advanced infections, it was concluded that they were unlikely to be a principal source of infective elements because naive oysters used as sentinels to assess infection pressure became highly infected even after native oysters developed resistance, and infected spat could no longer be found. A histological survey of zooplankton and small bivalves in Delaware Bay found few recognizable parasites and nothing resembling a haplosporidan. A subsequent PCR study of water, sediment, and macro-invertebrates from Chesapeake, Delaware, and Oyster bays resulted in many positive samples, but in situ hybridization failed to identify any recognizable structures. PCR analysis of potential intermediate hosts for other molluscan pathogens has also resulted in many species yielding positive results but required in situ hybridization to verify infections. It is suggested that any future search for a nonoyster host of H. nelsoni be conducted in a relatively confined system and/or target specific phyla, strategies that have been successful in other life cycle studies. It is noted that candidate phyla could include those known to host haplosporidans and species whose abundance or distribution may have changed in concert with outbreaks of MSX disease in the northeastern United States in recent years

Resolving confusions about jarrah dieback - don’t forget the plants

The name jarrah dieback has been used for two different disorders, leading to considerable confusion. It was coined in the 1940s to describe the sudden death of groups of jarrah (Eucalyptus marginata) trees in south western Western Australia, which occurred on poorly drained sites, following exceptionally heavy rainfall. In the 1960s these sites were shown to be infested by Phytophthora cinnamomi and jarrah deaths were attributed to it, even though it was only isolated from 5 % of sampled trees. Also the definition of jarrah dieback was expanded to include deaths of many other plants on infested sites, from which P. cinnamomi was more readily isolated. Jarrah trees die from severe water deficiency, indicating problems with water conduction through roots. Xylem vessel diameters vary along roots, being narrow at the root collar, while distally they are larger, providing water storage. Jarrah transpires vigorously during summer, accessing water at depth on sites with deep soil, but being more dependent on internally stored water when root systems are shallower. Following waterlogging, sapwood vessels become blocked with tyloses, reducing both conductivity and potential water storage; such trees may have insufficient water reserves for summer survival. In jarrah P. cinnamomi is unlikely to cause water deficiency because sapwood invasion is rapidly contained in healthy roots. Recent investigations into P. cinnamomi invasion and host responses in other plants show that it can potentially cause a vascular wilt in Banksia spp. and chronic, symptomless infections in herbaceous plants. Susceptibility to waterlogging damage, and/or mortality resulting from infection by P. cinnamomi can only be clarified by detailed knowledge of the hosts and their vulnerabilities. This is essential for making diagnoses, devising management strategies, and avoiding the confusions of the past

Transformation Pathways of Silica under High Pressure

Concurrent molecular dynamics simulations and ab initio calculations show that densification of silica under pressure follows a ubiquitous two-stage mechanism. First, anions form a close-packed sub-lattice, governed by the strong repulsion between them. Next, cations redistribute onto the interstices. In cristobalite silica, the first stage is manifest by the formation of a metastable phase, which was observed experimentally a decade ago, but never indexed due to ambiguous diffraction patterns. Our simulations conclusively reveal its structure and its role in the densification of silica.Comment: 14 pages, 4 figure

An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification

Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability

To respond or not to respond - a personal perspective of intestinal tolerance

For many years, the intestine was one of the poor relations of the immunology world, being a realm inhabited mostly by specialists and those interested in unusual phenomena. However, this has changed dramatically in recent years with the realization of how important the microbiota is in shaping immune function throughout the body, and almost every major immunology institution now includes the intestine as an area of interest. One of the most important aspects of the intestinal immune system is how it discriminates carefully between harmless and harmful antigens, in particular, its ability to generate active tolerance to materials such as commensal bacteria and food proteins. This phenomenon has been recognized for more than 100 years, and it is essential for preventing inflammatory disease in the intestine, but its basis remains enigmatic. Here, I discuss the progress that has been made in understanding oral tolerance during my 40 years in the field and highlight the topics that will be the focus of future research

Accurate and Rapid Estimation of Phosphene Thresholds (REPT)

To calibrate the intensity of transcranial magnetic stimulation (TMS) at the occipital pole, the phosphene threshold is used as a measure of cortical excitability. The phosphene threshold (PT) refers to the intensity of magnetic stimulation that induces illusory flashes of light (phosphenes) on a proportion of trials. The existing PT estimation procedures lack the accuracy and mathematical rigour of modern threshold estimation methods. We present an improved and automatic procedure for estimating the PT which is based on the well-established Ψ Bayesian adaptive staircase approach. To validate the new procedure, we compared it with another commonly used procedure for estimating the PT. We found that our procedure is more accurate, reliable, and rapid when compared with an existing PT measurement procedure. The new procedure is implemented in Matlab and works automatically with the Magstim Rapid2 stimulator using a convenient graphical user interface. The Matlab program is freely available for download