A diagnostic evaluation of real-time PCR, fluorescent antibody and microscopic agglutination tests in cases of equine leptospiral abortion.

REASONS FOR PERFORMING STUDY: A comprehensive evaluation of the real-time PCR assay for leptospirosis in comparison with other diagnostic assays on a large-scale basis is fundamental in validating the assay and determining the causes of equine abortions. OBJECTIVES: To compare and evaluate the diagnostic value of real-time PCR assay for leptospirosis with traditional methods in equine leptospiral abortions. STUDY DESIGN: Cross-sectional observational study. METHODS: A Leptospira spp. fluorescent antibody test (FAT), microscopic agglutination test (MAT) and real-time PCR (targeting the LipL32 gene) were compared and evaluated in equine fetal necropsy specimens (placenta, kidney, liver and heart blood) and maternal serum (when available) in 339 equine fetuses. RESULTS: From a total of 339 equine fetuses necropsied, 21 cases (6.19%) were diagnosed as leptospiral abortion. The majority of leptospiral abortions occurred in January (8 cases) and February (5 cases). Real-time PCR detected 21 of 21 cases, whereas MAT and FAT detected 19 and 18 (including 2 suspicious cases) cases, respectively. Comparing tissues, placenta yielded somewhat similar cycle of threshold values by real-time PCR compared with kidney, whereas kidney was the best specimen for the diagnosis of leptospirosis by the FAT test. In all MAT positive cases, the predominant titre in fetal heart blood was to serovar Pomona (ranging 1:100 to 1:204,800) with little or no cross-reaction to serovar Grippotyphosa. CONCLUSIONS: The results indicate that real-time PCR is an effective method for the diagnosis of leptospiral abortion in horses. However, MAT should continue to be used in clinical cases for serovar determination.

The growth response of BG-1 ovarian carcinoma cells to estradiol, 4OH-tamoxifen, and tamoxifen: evidence for intrinsic antiestrogen activation.

The influence of estrogen (E) and antiestrogen (AES) on the in vitro growth of BG-1 ovarian carcinoma cells, which express steroid receptors was examined (K. R. Geisinger, T. E. Kute, M. J. Pettenati, C. E. Welander, Y. Dennard, L. A. Collins, and M. E. Berens, Characterization of a human ovarian carcinoma cell line with estrogen and progesterone receptors, Cancer 63, 280-288, 1989). All determinations were simultaneously referenced under similar conditions to MCF-7 cells, a well-established cell line for modeling hormonal responses in breast cancer. In "complete" media containing fetal calf serum (FCS, 10%), MCF-7 cell numbers increased approximately 7 x in 7 days, remaining at this level Days 8-15. In contrast, BG-1 cells achieved similar numbers by Day 7, but showed apparent exponential growth over Days 8-15 to 15-20 x. Phenol red-free media containing 10% FCS (less than 20 pg estradiol (E2)/ml by RIA) was used to assess responses to E and AES. Growth of both MCF-7 and BG-1 cells slowed in E-free media. E2 (10 nM) stimulated the growth of both cell lines, yet was responsible for exponential increases during Days 8-15 only in BG-1 cell numbers (50-70 x). The metabolically active AES (4OH-tamoxifen, 50 nM) reduced E2-stimulated MCF-7 growth to 3-4 x, while tamoxifen (50 nM) had no effect. Rescue with 10 microM E2 fully overcame the AES inhibition of MCF-7 proliferation. In contrast, BG-1 cells experienced significant E2-stimulated growth reductions in the presence of either 4OH-tamoxifen or tamoxifen. E2 was observed to rescue BG-1 cells from both of these antagonists. We conclude that BG-1 ovarian carcinoma cells respond in vitro to E and AES. Moreover, by virtue of responses to tamoxifen, BG-1 cells may have an intrinsic capacity to hydroxylate tamoxifen to its active metabolite. This property of ovarian carcinoma cells might be worth exploiting in the design of more effective combination chemotherapy regimens.