New Method for Lawsonia intracelullaris Quantification Based on Optical Density by Spectrophotometry

Studies investigating the pathogenesis of Lawsonia intracellularis often require bacterial quantification in suspension. However, due to the organism's fastidious growth requirements-being both intracellular and microaerophilic-traditional quantification methods, such as colony-forming unit counting, are not feasible. Currently, the only widely available method for quantifying L. intracellularis is real-time quantitative PCR (RT-qPCR). Unfortunately, the time required to perform RT-qPCR is incompatible with the bacterium's limited survival outside its intracellular and microaerophilic environment. As a result, bacterial suspensions are typically quantified subjectively, based on the researcher's experience for immediate use, with RT-qPCR conducted afterward. Optical density (OD) spectrophotometry is a rapid, although indirect, method of estimating bacterial concentrations in suspension, and it has been applied successfully to fast-growing prokaryotic species. Therefore, the objective of this study was to determine the correlation between RT-qPCR results and the optical density of L. intracellularis suspensions, with the goal of enabling the use of spectrophotometry for immediate bacterial quantification in experimental settings. Optical densities (ODs) were measured at 405 nm and 450 nm, using either a cuvette or microplate, while RT-qPCR was employed to establish a standard curve from samples of known concentration and to quantify the concentration of L. intracellularis in the test suspensions. Four comparison variations between OD and RT-qPCR were evaluated: (1) spectrophotometry at 405 nm using a cuvette vs. RT-qPCR; (2) spectrophotometry at 405 nm using a microplate vs. RT-qPCR; (3) spectrophotometry at 450 nm using a cuvette vs. RT-qPCR; and (4) spectrophotometry at 450 nm using a microplate vs. RT-qPCR. The tests were conducted in two independent replications, with each sample analyzed in duplicate. In all variations, the correlation between the bacterial concentrations determined by RT-qPCR and those estimated by OD was greater than 80%, with a statistical significance of p < 0.05. The following OD conversion equations for determining the number of microorganisms/mL were obtained: (1) f(x) = -7.438 × 10⁸ + 1.797 × 10¹⁰. x; (2) f(x) = 3.255 × 10⁸ + 3.003 × 10⁹. x; (3) f(x) = -8.006 × 10⁸ + 2.169 × 10¹⁰. x; (4) f(x) = 3.107 × 10⁸ + 3.758 × 10⁹. x. Here, "X" is the Ct value obtained by RT-qPCR. These findings enable researchers to improve the accuracy of their L. intracellularis experiments by utilizing optical spectrometry-a straightforward method that provides immediate results for determining bacterial concentration in suspensions.