Occupancy Modeling for Improved Accuracy and Understanding of Pathogen Prevalence and Dynamics

Most pathogen detection tests are imperfect, with a sensitivity < 100%, thereby resulting in the potential for a false negative, where a pathogen is present but not detected. False negatives in a sample inflate the number of non-detections, negatively biasing estimates of pathogen prevalence. Histological examination of tissues as a diagnostic test can be advantageous as multiple pathogens can be examined and providing important information on associated pathological changes to the host. However, it is usually less sensitive than molecular or microbiological tests for specific pathogens. Our study objectives were to 1) develop a hierarchical occupancy model to examine pathogen prevalence in spring Chinook salmon Oncorhynchus tshawytscha and their distribution among host tissues 2) use the model to estimate pathogen-specific test sensitivities and infection rates, and 3) illustrate the effect of using replicate within host sampling on sample sizes required to detect a pathogen. We examined histological sections of replicate tissue samples from spring Chinook salmon O. tshawytscha collected after spawning for common pathogens seen in this population: Apophallus/echinostome metacercariae, Parvicapsula minibicornis, Nanophyetus salmincola/ metacercariae, and Renibacterium salmoninarum. A hierarchical occupancy model was developed to estimate pathogen and tissue-specific test sensitivities and unbiased estimation of host- and organ-level infection rates. Model estimated sensitivities and host- and organ-level infections rates varied among pathogens and model estimated infection rate was higher than prevalence unadjusted for test sensitivity, confirming that prevalence unadjusted for test sensitivity was negatively biased. The modeling approach provided an analytical approach for using hierarchically structured pathogen detection data from lower sensitivity diagnostic tests, such as histology, to obtain unbiased pathogen prevalence estimates with associated uncertainties. Accounting for test sensitivity using within host replicate samples also required fewer individual fish to be sampled. This approach is useful for evaluating pathogen or microbe community dynamics when test sensitivity is <100%.     

High-frequency transformation of human repair-deficient cell lines by an Epstein-Barr virus-based cDNA expression vector.

We constructed a human cDNA expression vector by combining an episomal Epstein-Barr virus (EBV) vector with the expression cassette from the transient-expression vector, pCDM8. This new vector, designated pEBS7, exhibited high-level expression of reporter genes in normal and repair-deficient xeroderma pigmentosum cell lines. Reconstruction experiments indicated that marker genes diluted to a frequency of 10(-5) can be rescued on a single transfection dish. Moreover, derivative cell lines that constitutively express the gene encoding EBV nuclear antigen 1 exhibited a tenfold enhancement in the frequency of rescue of marker genes. The feasibility of preparing large-scale directional or nondirectional cDNA libraries in pEBS7 was demonstrated and reconstruction experiments indicated that marker genes could be rescued from either library with equal efficiency. These results establish a high-efficiency system for the isolation of genes by direct phenotypic selection in human mutant cell lines.     

l-cystathionine and its selenium analogue in Neptunia amplexicaulis

Both l-cystathionine and l-selenocystathionine have been isolated from the selenium-accumulating legume Neptunia amplexicaulis.     

Molecular cloning of the murine substance K and substance P receptor genes.

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.     

Direct evidence for formation of hybrid vesicles by fusion of plasma and outer acrosomal membranes during the acrosome reaction in boar spermatozoa.

A variety of treatment procedures was utilized to identify the origin and composition of the vesicles formed during the acrosome reaction of boar spermatozoa. Whether the acrosome reaction occurred spontaneously or was induced chemically the vesicles were hybrid vesicles composed of roughly equal proportions of plasma and outer acrosomal membranes.     

O-alkyl lipid synthesis: the mechanism of the acyl dihydroxyacetone phosphate fatty acid exchange reaction

We have previously provided evidence for a mechanism by which acyl DHAP is converted enzymatically to O-alkyl DHAP. This mechanism involves, in part, the formation of an endiol of acyl DHAP, loss of the fatty acid by splitting of the DHAP carbon-1 to oxygen bond and the gain of a long chain fatty alcohol. It has been shown that acyl DHAP can exchange its fatty acid for one in the medium, presumably by the mediation of O-alkyl DHAP synthase. In the present investigation we have shown that the fatty acid which is gained by acyl DHAP in the exchange process retains both carboxyl oxygens, as predicted by our postulated mechanism. This reaction is exceptional because the usual action of acyl hydrolases is to cleave at the oxygen to acyl bond.     

Class II genes of the human major histocompatibility complex. The DO beta gene is a divergent member of the class II beta gene family

A novel class II beta chain gene is described. This gene, tentatively called DO beta, displays considerably less polymorphism than beta genes of the DP, DQ, and DR loci. The nucleotide sequence of the DO beta gene is strikingly similar to that of the previously identified murine A beta 2 gene. The DO beta gene displays the same exon/intron organization as other beta genes although the fifth exon and the translated portion of the sixth exon are longer than in other genes. A striking feature of the amino acid sequence deduced from the DO beta gene sequence is the pronounced hydrophobicity of the NH2-terminal region. This feature distinguishes the putative DO beta chain from other class II beta chains and raises the possibility that DO beta chains may interact with an alpha chain that is structurally different from those of the DP, DQ, and DR loci. It further suggests that the putative DO molecule may have a function different from those of other class II antigens.     

Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.