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  • Author or Editor: Dori L. Borjesson x
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Abstract

Objective—To develop a quantitative PCR assay for detection of Borrelia burgdorferi DNA in formalin-fixed, paraffin-embedded tissues; compare results of this assay with results of immunohistochemical staining of tissues from seropositive dogs; and determine whether B burgdorferi DNA could be detected in renal tissues from dogs with presumptive Lyme nephritis.

Design—Cohort study.

Sample Population—Archived tissue samples from 58 dogs.

Procedures—A quantitative PCR assay was performed on formalin-fixed, paraffin-embedded tissue sections from the dogs. Results were compared with results of immunohistochemical staining, B burgdorferi serostatus, clinical signs, and necropsy findings.

Results—38 dogs were classified as having positive or equivocal results for Lyme borreliosis, and 20 were classified as having negative results on the basis of clinical signs, serologic findings, and pathologic abnormalities. Borrelia burgdorferi DNA was amplified from tissue samples from only 4 (7%) dogs, all of which had been classified as having positive or equivocal results for Lyme borreliosis and had signs of presumptive Lyme nephritis. Results of PCR assays of renal tissue were positive for only 1 dog, and there was no agreement between results of immunohistochemical staining (ie, detection of B burgdorferi antigen) and results of the PCR assay (ie, detection of B burgdorferi DNA) for renal tissues.

Conclusions and Clinical Relevance—Results indicated that detection of B burgdorferi DNA in formalin-fixed, paraffin-embedded tissues is feasible, but that intact B burgdorferi DNA is rarely found in tissues from naturally infected dogs, even tissues from dogs with presumptive Lyme borreliosis. Further, findings support the contention that Lyme nephritis may be a sterile, immune complex disease.

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in Journal of the American Veterinary Medical Association

Abstract

Objective—To evaluate the clinical accuracy of reagent test strips used to estimate BUN concentration in dogs and cats.

Design—Prospective study.

Animals—116 dogs and 58 cats.

Procedure—Blood samples were collected at the time of admission to the hospital. Estimates of BUN concentration obtained with reagent test strips (category 1 [5 to 15 mg/dL], 2 [15 to 26 mg/dL], 3 [30 to 40 mg/dL], or 4 [50 to 80 mg/dL]) were compared with SUN concentrations measured with an automated analyzer. For dogs, category 1 and 2 test strip results were considered a negative result (nonazotemic) and category 3 and 4 test strip results were considered a positive result (azotemic). For cats, category 1, 2, and 3 test strip results were considered a negative result (nonazotemic) and category 4 test strip results were considered a positive result (azotemic).

Results—On the basis of SUN concentration, 40 of the 174 (23%) animals (20 dogs and 20 cats) were classified as azotemic. One dog and 2 cats had falsenegative test strip results, and 1 dog had a false-positive result. Sensitivity and specificity were 95% (20/21) and 99% (94/95), respectively, for dogs and 87% (13/15) and 100% (43/43), respectively, for cats.

Conclusions and Clinical Relevance—Results suggest that reagent test strips are a reliable method for rapidly estimating BUN concentrations in dogs and cats. Because test strip results are only semiquantitative and there remains a potential for misclassification, especially in cats, urea nitrogen concentration should ultimately be verified by means of standard chemistry techniques. (J Am Vet Med Assoc 2005;227:1253–1256)

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in Journal of the American Veterinary Medical Association

Abstract

Objective—To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days.

Animals—13 healthy adult Thoroughbreds.

Procedures—Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4°C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined.

Results—Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 μg of streptavidin-PE/1 × 106 RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively.

Conclusions and Clinical Relevance—Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.

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in American Journal of Veterinary Research

Abstract

Objective—To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells.

Animals—10 adult Quarter Horses and 8 newborn Thoroughbred foals.

Procedures—eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated.

Results—BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP activity and calcium deposition. There was no significant difference in BM-eMSC yield from the tuber coxae or sternum, but yield was significantly greater from the first 60-mL aspirate than from subsequent aspirates. The BM-eMSC expansion rate was significantly higher when cells were cultured in fetal bovine serum instead of autologous serum (AS).

Conclusions and Clinical Relevance—eMSCs from BM possessed the highest in vitro osteogenic potential; eMSCs from adipose tissue also had robust osteogenic potential. The tuber coxae and the sternum were viable sources of BM-eMSCs in yearlings, and 60 mL of BM aspirate was sufficient for culture and expansion. Expanding BM-eMSCs in AS to avoid potential immunologic reactions decreased the total yield because BM-eMSCs grew significantly slower in AS than in fetal bovine serum. Additional studies are needed to determine optimal ex vivo eMSC culture and expansion conditions, including the timing and use of growth factor—supplemented AS. (Am J Vet Res 2010;71:1237-1245)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To optimize the isolation and culture of mesenchymal stem cells (MSCs) from umbilical-cord blood (UCB), identify variables that predicted successful MSC isolation, and determine whether shipping, processing, and cryopreservation altered MSC viability, recovery rates, and expansion kinetics.

Sample Population—UCB samples from 79 Thoroughbred and Quarter Horse mares.

Procedures—UCB samples were processed to reduce volume and remove RBCs. Nucleated cells (NCs) were cryopreserved or grown in various culture conditions to optimize MSC monolayer expansion and proliferation. Donor and UCB-sample factors were analyzed to determine their influence on the success of MSC isolation and monolayer expansion.

Results—MSCs capable of multilineage in vitro differentiation were expanded from > 80% of UCB samples. Automated UCB processing and temperature-controlled shipping facilitated sterile and standardized RBC reduction and NC enrichment from UCB samples. The number of NCs after UCB samples were processed was the sole variable that predicted successful MSC expansion. The UCB-derived MSCs and NCs were successfully cryopreserved and thawed with no decrease in cell recovery, viability, or MSC proliferation. The use of fibronectin-coated culture plates and reduction of incubator oxygen tension from 20% to 5% improved the MSC isolation rate. Some UCB-derived MSC clones proliferated for > 20 passages before senescence. Onset of senescence was associated with specific immunocytochemical changes.

Conclusions and Clinical Relevance—Equine UCB samples appeared to be a rich source of readily obtainable, highly proliferative MSCs that could be banked for therapeutic use.

Full access
in American Journal of Veterinary Research