A quantitative PCR method for assessing the presence of Pasteurella testudinis DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii).

Pasteurella testudinis has been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Our goal was to develop a sensitive and specific qPCR method for detecting DNA from P. testudinis in nasal lavage fluid collected from desert tortoises in the field. Probes for 16S ribosomal RNA and RNA polymerase ß-subunit (rpoB) genes were designed. A standard curve generated with DNA extracted from known numbers of bacterial cells determined by flow cytometry revealed a lower detection limit of 50 fg/ml (10 bacteria/ml). The nasal lavage fluid contained no interfering substances, and the qPCR method did not recognize normal flora DNA. The nasal lavage samples from 20 desert tortoises captured in Clark County, Nevada, USA in 2007 and housed at the Desert Tortoise Conservation Center, were all positive for P. testudinis DNA by qPCR. Another set of 19 lavage samples collected in 2010 from wild desert tortoises in the Mojave Desert were tested and 84% were positive for P. testudinis DNA. Fully validated, this qPCR method will provide a means of determining colonization rate. When used in conjunction with serological methods and clinical evaluations, both infection rate and disease rate can be determined for this potential URTD pathogen. This new assay provides an important tool for managing the threatened populations of the Mojave Desert tortoise.

Quantitative PCR method for detection of mycoplasma spp. DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii).

Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6pg ml(-1) to a high of 72,962pg ml(-1). Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise.

Flow cytometric method for quantifying viable Mycoplasma agassizii, an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii).

AIMS: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. METHODS AND RESULTS: Here, we demonstrate that the nonfluorescent molecule 5-carboxyfluorescein (5-CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5-CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml(-1) can be labelled and counted in less than 1 h. Experiments using temperature-induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. CONCLUSIONS: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.

Germline polymorphisms are potential metastasis risk and prognosis markers in breast cancer.

A number of theories have been proposed to account for the origins of metastasis, although none as yet have adequately explained all of its characteristics. With approximately 90% of cancer-related deaths due to the effects of disseminated tumors, improved understanding of this process is critical for reducing cancer-associated morbidity and mortality. Extensive research to investigate the molecular basis of this process has been conducted, and our lab has focused on the role of germline polymorphism in this complex process. Simple breeding experiments using a highly metastatic mouse model showed that germline polymorphisms significantly contribute to metastasis susceptibility. Genetic mapping studies revealed that a number of genomic regions are linked to metastasis susceptibility, including a metastasis modifier on mouse chromosome 19. Subsequent analysis identified Sipa1 as the most likely candidate for the observed linkage on Chr 19. Evaluation of SNPs in SIPA1 in a pilot association study in a human breast cancer cohort supported this possibility and demonstrated that SIPA1 polymorphisms are associated with various markers of poor prognosis including differential sentinel lymph node status. Taken together, these data suggest that germline polymorphism is an important modulating component in metastatic progression that needs to be investigated if we are to fully understand the metastatic process.

Host genetics and tumour metastasis.

Metastasis, the spread and growth of tumours at secondary sites, is an extremely important clinical event, since a majority of cancer mortality is associated with the metastatic tumours, rather than the primary tumour. In spite of the importance of metastasis in the clinical setting, the actual process is extremely inefficient. Millions of tumour cells can be shed into the vasculature daily; yet, few secondary tumours are formed. The classical hypothesis explaining the inefficiency was a series of secondary events occurring in the tumour, resulting in a small subpopulation of cells capable of completing all of the steps required to successfully colonise a distant site. However, recent discoveries demonstrating the ability to predict metastatic propensity from gene expression profiles in bulk tumour tissue are not consistent with only a small subpopulation of cells in the primary tumour acquiring metastatic ability, suggesting that metastatic ability might be pre-programmed in tumours by the initiating oncogenic mutations. Data supporting both of these seemingly incompatible theories exist. Therefore, to reconcile the observed results, additional variables need to be added to the model of metastatic inefficiency. One possible variable that might explain the discrepancies is genetic background effects. Studies have demonstrated that the genetic background on which a tumour arises on can have significant affects on the ability of the tumour to metastasise and on gene expression profiles. Thus, the observations could be reconciled by combining the theories, with genetic background influencing both metastatic efficiency and predictive gene expression profiles, upon which, subsequently, metastasis-promoting mutational and epigenetic events occur. If the genetic background is an important determinant of metastatic efficiency, it would have significant implications for the clinical prediction and treatment of metastatic disease, as well as for the design of potential prevention strategies.

Preparation of microparticulate beta-glucan from Saccharomyces cerevisiae for use in immune potentiation.

AIMS: To develop a method for the preparation of an immunologically active, homogeneous, nonaggregated, microparticulate beta-glucan-containing material from the budding yeast Saccharomyces cerevisiae. METHODS AND RESULTS: Using a combination of sonication and spray-drying, a homogeneous preparation of 1-2-mu diameter beta-glucan-containing particles was made from alkali- and acid-insoluble yeast cell wall material. This microparticulate beta-glucan remained in suspension longer and, following oral administration at 0.1 mg kg(-1) for 14 d, enhanced phagocytosis of mouse peritoneal macrophages significantly better than did aggregated beta-glucan particles. CONCLUSIONS: A new sonication and spray-drying method can be employed to overcome the problem of aggregation of beta-glucan microparticles in aqueous media. SIGNIFICANCE AND IMPACT OF THE STUDY: A microparticulate form of beta-glucan that remains in suspension longer for pharmaceutical applications and has superior immune potentiation characteristics has been developed.

Predisposition to efficient mammary tumor metastatic progression is linked to the breast cancer metastasis suppressor gene Brms1.

Tumor metastasis is one of the most important clinical aspects of neoplastic disease because patient mortality is frequently attributable to disseminated rather than primary tumors. However, it still is not possible to definitively distinguish those individuals at high risk for disseminated disease, who would benefit from aggressive adjuvant therapy, from the low-risk patients who might be spared the side effects of additional anticancer therapy. To identify factors that predispose toward metastatic disease, we have used a genetic approach. Using a highly metastatic model of mammary cancer, we identified previously inbred mouse strains (DBA/2J, NZB/B1NJ, and I/LnJ) that harbor genetic factors that significantly suppress metastatic efficiency. In this study, we report the results of four experiments to localize the genetic map locations of the metastasis efficiency modifier genes. One statistically significant locus was identified on proximal Chr 19 designated Mtes1. Secondary candidate intervals were detected on Chrs 6, 9, 13, and 17. Interestingly, Mtes1 colocalizes with the murine orthologue of the human breast cancer metastasis suppressor gene Brms1, suggesting that allelic variants of Brms1 might be responsible for the metastasis suppression observed.

Three loci modify growth of a transgene-induced mammary tumor: suppression of proliferation associated with decreased microvessel density.

In earlier studies it was observed that the genetic background significantly affected the phenotype of a transgene-induced mammary tumor. Tumors arising in an (I/LnJ x PyMT) F1 hybrid background appeared earlier than in the FVB/N-TgN(MMTV-PyVT)(634Mul) parent, but accumulated less tumor mass, indicating a net decrease in tumor growth. Quantitative genetic mapping in a backcross identified three loci that were associated with the decreased proliferative capacity of the I/LnJ F1 tumors. Molecular analysis of the tumors suggests that these loci may act by restricting the tumor's ability to recruit microvessels. The three loci, designated Mmtg1-3, are unlinked to the angiogenic genes Fgf2, Flt1, Flk4, Flk1, Vegf, and Vegfc, as well as the precursors of the endogenous antiangiogenic molecules angiostatin and endostatin. The Mmtg loci may therefore provide novel targets for antiangiogenic therapeutic strategies.