Detection and quantification by molecular techniques of early infection by Lawsonia intracellularis in suckling piglets

Abstract

Background: Lawsonia intracellularis is the causative agent of Porcine Proliferative Enteropathy (PPE), one of the most prevalent pig enteric diseases worldwide, but with sparse information about early infections in suckling piglets in the epidemiology of PPE. With that aim, this study evaluates the prevalence of L. intracellularis in 3-week-old piglets by analysing ileal digesta content and mucosal scrapings from 383 pigs from 16 farms (aprox., 25 pigs/batch) by real-time qPCR and droplet digital PCR (ddPCR).

Results: Forty-nine samples yielded a qPCR positive result. Eleven samples from eight farms were confirmed as positive with concentrations of L. intracellularis from 3.5 log₁₀ to 4.5 log₁₀ bacteria/g of sample. Another 16 samples, eight farms, were classified as low positive (2.07-2.38 log₁₀ bacteria/g) and 22 provided an uncertain result. Finally, 334 samples tested negative for L. intracellularis. At batch level, half of the farms included in the study had at least one positive sample and in 10 farms (62.5%) there was at least one low positive sample. The ddPCR was run in 50 of the 383 samples based on their PCR output (including low positive, uncertain and negative samples). Correlation analyses revealed a strong association between qPCR and the ddPCR results (ρ = 0.75; p < 0.001). The ddPCR allowed us to detect and confirm a positive result in the 19 samples classified as uncertain by the qPCR and detect L. intracellularis in 8 of 15 negatives by qPCR.

Conclusions: The results of the study demonstrate that a number of piglets are already infected with L. intracellularis during the suckling period evidencing early infection in certain animals, adding information of PPE epidemiology and opening new research topics such as sow-piglet transmission. Study results also evidence the usefulness of a combination of qPCR and ddPCR to improve qPCR sensitivity but assuring high specificity.

Keywords: Enteric infection; Ileitis; Pathogen; Porcine proliferative enteropathy; Swine; Transmission.

Fig. 1

Concentration of Lawsonia intracellularis estimated by gram of sample (digesta and/or ileum mucosa) in positive piglets from the 15 farms tested. Figure 1A shows results from positive samples in eight farms and Fig. 1B shows summarizes the data from low positive samples detected in piglets from 12 farms

Fig. 2

Lawsonia intracellularis droplet digital PCR results obtained in 50 samples collected from piglets. Figure 2A droplet counts in samples categorized as negative, uncertain or low positive by qPCR. Whiskers show significant differences among categories. Figure 2B correlation analysis between qPCR quantification and droplet counts. Pearson correlation ^® and P-value estimated for the data analysed