Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs.

Osteosarcoma (OSA) is a malignant tumor of middle-aged dogs and adolescent humans. The clinical outcome of OSA has not improved over more than three decades, and dogs typically succumb to metastatic disease within 6 months despite tumor resection through limb amputation and adjuvant chemotherapy. Therefore, undetectable tumor cells with potential to form metastases are present at diagnosis. An assay to identify canine immortalized and primary OSA cells through flow cytometric detection of intracellular collagen 1 (Col I) and osteocalcin was optimized, and applied to blood samples from tumor-bearing dogs for detection of circulating tumor cells (CTCs). Spiking variable number of OSA cells into normal dog blood recovered 50-60% of Col I positive cells with high forward and variable side light scatter. An algorithm to exclude nonviable, doublet, and autofluorescent cells was applied to sequential blood samples from three dogs obtained prior to and after limb amputation, and at approximately, triweekly intervals over 121, 142, and 183 days of chemotherapy, respectively. Dogs had >100 CTC/106 leukocytes prior to amputation, variably frequent CTC during chemotherapy, and an increase up to 4,000 CTC/106 leukocytes within 4 weeks before overt metastases or death. Sorted CTCs were morphologically similar to direct tumor aspirates and positive for Col I. Although preliminary, findings suggest that CTCs are frequent in canine OSA, more numerous than carcinoma CTC in humans, and that an increase in CTC frequency may herald clinical deterioration. This assay may enable enumeration and isolation of OSA CTC for prognostic and functional studies, respectively. © 2019 International Society for Advancement of Cytometry.

Short communication: The effect of calcium supplementation at calving on neutrophil function.

Low total blood calcium concentration after calving has been demonstrated to be a risk factor for reduced neutrophil function. The objective of this study was to evaluate whether administration of an injectable calcium supplement product soon after calving increased neutrophil oxidative burst or phagocytosis capacity. Cows (n = 27) from 4 farms were blocked by parity and randomly assigned to receive either calcium gluconate (35% wt/vol) in combination with calcium glucoheptonate (10% wt/vol; Theracalcium, Vétoquinol Canada Inc., Lavaltrie, Quebec, Canada) or a placebo within 12 h after calving and again 24 h later. Each dose of 120 mL was injected subcutaneously over 2 sites. Total serum calcium concentration, neutrophil oxidative burst, and neutrophil phagocytosis capacity were measured from coccygeal blood samples before (time 0) and 72 h after first treatment. There was no difference between treatment groups in lactation number, total calcium concentration, oxidative burst, or phagocytosis at time of enrollment. There was no effect of treatment on oxidative burst or phagocytosis by neutrophils. This preliminary study does not support an effect of supplemental calcium to improve neutrophil oxidative burst or phagocytosis capacity of low-parity parturient cows.

Randomized clinical trial of a calcium supplement for improvement of health in dairy cows in early lactation.

Prophylactic Ca supplementation immediately after calving is a common strategy to prevent clinical and subclinical hypocalcemia in parturient dairy cows. The objective of this study was to evaluate the effect of prophylactic administration of an injected Ca supplement on blood Ca concentration at 24 and 48h after treatment, incidence risk of clinical disease and culling, milk production in early lactation, and probability of pregnancy at first insemination. Cows without signs of visible milk fever (n=984) from 7 farms were blocked by parity and randomly assigned to receive either Ca gluconate (35% wt/vol) in combination with Ca glucoheptonate (10% wt/vol; TheraCalcium, Vétoquinol Canada Inc., Lavaltrie, Quebec) or a placebo (medication vehicle solution with no Ca) at first contact with each cow after calving and again 12 to 24h later. Each dose was 120mL injected subcutaneously over 2 sites. Total serum Ca concentration (tCa) was measured from coccygeal blood samples before (time 0) and 24 and 48h after first treatment in a subsample of cows (n=129). Blood ß-hydroxybutyrate concentrations were measured from all cows twice between 3 and 16d in milk at weekly visits and cows were evaluated for vaginal discharge once between 28 and 42d in milk. Disease events, production data from the first 3 Dairy Herd Improvement milk tests, reproduction, and culling data were collected from each herd. For cows that had received 1 injection of Ca before the blood sample at 24h (n=95), tCa was significantly higher in the treated cows: mean ± standard error, 2.03±0.03 versus 1.90±0.03mmol/L, accounting for tCa at time of enrollment and a treatment by tCa at enrollment interaction. At 48h, no significant difference was found in tCa between treatment and control (mean ± SE, 2.12±0.02 and 2.10±0.03mmol/L, respectively). Cows treated with the Ca product were significantly less likely to have received intravenous, subcutaneous, or oral supplemental Ca for exhibiting clinical signs of hypocalcemia than control cows (5.0 vs. 8.4%). No effect was found of treatment on retained placenta, metritis, hyperketonemia, prevalence of purulent vaginal discharge, culling from the herd, early lactation production, probability of pregnancy to first artificial insemination, or time to pregnancy. With this subcutaneous prophylactic Ca treatment regimen, blood Ca levels were temporarily increased at 24h after treatment, but no effect was observed of supplemental Ca on the risk of disease or culling, milk production, or reproductive performance.

Expressed sequence tags from Madagascar periwinkle (Catharanthus roseus).

The Madagascar periwinkle (Catharanthus roseus) is well known to produce the chemotherapeutic anticancer agents, vinblastine and vincristine. In spite of its importance, no expressed sequence tag (EST) analysis of this plant has been reported. Two cDNA libraries were generated from RNA isolated from the base part of young leaves and from root tips to select 9,824 random clones for unidirectional sequencing, to yield 3,327 related sequences and 1,696 singletons by cluster analysis. Putative functions of 3,663 clones were assigned, from 5,023 non-redundant ESTs to establish a resource for transcriptome analysis and gene discovery in this medicinal plant.