Measurement of the viability of Lawsonia intracellularis.
The objective of this study was to develop and test both a flow cytometry method (FCM) and a direct count method (DCM) that both use fluorescent stains to determine the viability of Lawsonia intracellularis (LI), an obligate intracellular bacterium and the cause of proliferative enteropathy (PE) in pigs and other animal species. Live LI were passaged in cell culture and harvested from infected McCoy cells. Dead LI were prepared by exposing live LI to 70% isopropyl alcohol for 30 min. Seven samples with dead:live ratios of 0:100 (live control), 10:90, 30:70, 50:50, 70:30, 90:10, and 100:0 (dead control) were prepared for testing by both the FCM and the DCM. For the FCM, TO-PRO-3 iodine was applied to the samples, and viable LI were counted. For the DCM, the samples were stained with LIVE/DEAD BacLight, which contains SYTO 9 and propidium iodine, then filtered through 0.2-microm Nuclepore black polycarbonate filters, viewed, and counted with the use of an epifluorescence microscope. Data were evaluated by estimating 95% limits of agreement and the concordance correlation coefficient (CCC). The limits of agreement between the FCM and the DCM versus the standard ratio of added LI showed mean differences not equal to zero, suggesting that systematic bias was introduced. The CCC showed almost perfect agreement (r = 0.9898). With a specific fluorescent probe, the FCM is useful and as good as the DCM for determining LI viability.
Department of Veterinary Population Medicine, College of Veterinary Medicine, 385 Animal Science/Veterinary Medicine, University of Minnesota, 1988 Fitch Avenue, St. Paul, Minnesota 55108, USA. watta004@umn.edu
Suphot
S
Wattanaphansak
Connie
C
Gebhart
Michael
M
Olin
John
J
Deen
D000818:Animals; D002470:Cell Survival; D015169:Colony Count, Microbial / Q000379:methods / Q000662:veterinary*; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D007202:Indicators and Reagents; D020639:Lawsonia Bacteria / Q000254:growth & development*; D009930:Organic Chemicals; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods / Q000662:veterinary*; D013552:Swine; D013997:Time Factors
D007202:Indicators and Reagents; D009930:Organic Chemicals; C103389:SYTO 9