Mirtha E. Suarez-Duarte

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.

Proliferative enteropathy is an enteric disease caused by the bacterium Lawsonia intracellularis, which affects several species of domestic and wild animals. The mechanisms underlying the mechanisms employed by L. intracellularis to cause host cell proliferation are poorly understood, mostly because this bacterium is extremely difficult to isolate and propagate in vitro. Comparative genomics methods for searching for genes orthologous to genes known to be associated with pathogenesis allow identification of genes potentially involved in pathogenesis by the pathogen of interest. The goal of this study was to carry out in silico research on L. intracellularis genes orthologous to genes required for intracellular invasion and survival present in other pathogenic bacteria, particularly Brucella abortus, B. melitensis, B. suis, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium subspecies paratuberculosis, Salmonella enterica, Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis. A total of 127 genes associated with invasion and intracellular survival from five known intracellular bacteria were mapped against the predicted proteomes of all L. intracellularis strains publicly available on GenBank, using the OrthoFinder program. A total of 45 L. intracellularis genes were orthologous to genes associated with pathogenesis of other intracellular bacteria. Genes putatively associated with signal the transduction of chemotaxis and cell motility were identified. Genes related to DNA binding and repair were also identified, with some of them supporting a possible association of bacteria with macrophages or inducing pro-inflammatory responses. The homology-based identification of these genes suggests their potential involvement in the virulence and pathogenicity of L. intracellularis, opening avenues for future research and insights into the molecular mechanisms of Lawsonia-elicited proliferative enteropathy.