We launched a new series on the blog last year. Once a month, we are sharing with you a presentation given at the Allen D. Leman swine conference, on topics that the swine group found interesting, innovative or that lead to great discussions.
We can find all of the presentations selected from last year’s conference on the blog here.
Our third presentation for this year is from Dr. Jose Angulo from Zoetis and Dr. Paul Yeske from Swine Vet Center regarding PRRS infection dynamics in growing pigs.
Click on the image below to see his presentation at the conference:
Today, we are sharing a publication by Dr. Talita Resende, a phD candidate working with Drs. Gebhart and Vannucci. Dr. Resende’s research focuses on the mechanisms enabling Lawsonia intracellularis’ infectivity and pathogenesis. Her latest paper, available in open access from Veterinary Microbiology, looks at the effects of Lawsonia intracellularis on different cell lines.
Effects of L. intracellularis on intestinal cell lines in vitro is unknown.
Impact of nutrient deprivation on cell proliferation was cell line dependent.
L. intracellularis did not lead to proliferation of the cell lines tested.
L. intracellularis and Ki-67 were co-localized in all cell lines tested.
Single cell cultures are not a suitable model for L. intracellularis pathogenesis.
Material and Methods
4 different intestinal epithelial cells lines were compared in this study: IPEC-J2 , IEC-18, Caco-2, and McCoy cells. McCoy were used as a reference since previous publications have shown that Lawsonia intracellularis can grow in this cell type.
Each cell line was infected with 2 types of Lawsonia intracellularis: low and high passage. Infected cell lines were used as control during the experiment. At days 1, 4, and 7 post-infection, the number of cells highly infected by Lawsonia (i.e. that had more than 30 organisms in their cytoplasm) was counted. To estimate cell proliferation, the amount of DNA in each cell line was evaluated. Additionally, a fluoerescence marker called Ki-67 was used to identified eukaryotic cells undergoing division. Lastly, a wound closure assay was done by scraping infected cell lines with a pipette and measure the width of the “wound” over time.
Results and Discussion
All cell lines tested were susceptible to L. intracellularis infection with typical intracellular bacterial growth of about 30–100 per cell in the cytoplasm of infected cells.
There was no statistical difference in cellular proliferation within or among groups at 0 and 1 dpi. Additionally, no increased proliferation in any cell line infected by L. intracellularis was noted, regardless of the bacterial passage status.
To verify whether cells infected by L. intracellularis would proliferate and migrate faster than non-infected cells through a scratched monolayer, a wound closure assay was executed. There were no differences among treatment groups for wound closure at any time point (0 to 24h and 24h to 48h)
It is suggested that L. intracellularis preferentially infects actively proliferating cells in intestinal crypts. By looking at both Lawsonia and Ki-67 markers, it was noted that in the majority of treatment groups and with the exception of the IPEC-J2 cell line, the proportion of cells that were double positive (L. intracellularis was co-localized with Ki-67) was higher than cells that were L. intracellularisinfected, but negative for Ki-67.
Taken together, these findings have decisively shown that two-dimensional intestinal epithelial in vitro cultures do not reproduce the characteristic proliferative effect of L. intracellularis infection in vivo.
Lawsonia intracellularis is an obligate intracellular bacterium that causes proliferative enteropathy in various animal species. While cellular proliferation of intestinal cells is recognized as the hallmark of L. intracellularis infection in vivo, it has not been demonstrated in in vitromodels. In order to assay the effect of L. intracellularis, various cell lines were infected with pathogenic and non-pathogenic passages of the bacterium. Because of the high proliferative rate of these cell lines, serum deprivation, which is known to reduce proliferation, was applied to each of the cell lines to allow the observation of proliferation induced by L. intracellularis. Using antibodies for Ki-67 and L. intracellularis in dual immunofluorescence staining, we observed that L. intracellularis was more frequently observed in proliferating cells. Based on wound closure assays and on the amount of eukaryotic DNA content measured over time, we found no indication that cell lines infected with L. intracellularis increased proliferation and migration when compared to non-infected cells (p > 0.05). Cell arrest due to decreased serum in the culture media was cell-line dependent. Taken together, our findings provide data to support and expand previous subjective observations of the absence of in vitro proliferation caused by L. intracellularis in cell cultures and confirm that cell lines infected by L. intracellularis fail to serve as adequate models for understanding the cellular changes observed in proliferative enteropathy-affected intestines.
This is our Friday rubric: every week a new Science Page from the Bob Morrison’s Swine Health Monitoring Project. The previous editions of the science page are available on our website.
This week we are sharing a report by Dr. Daniel Linhares’ lab at Iowa State University. The report summarizes the findings of his study regarding the factors making a sow farm vulnerable for PPRS introduction.
A model to quantify and identify biosecurity vulnerability in breeding herds is now available.
Events related to swine movements, transmission by air and water, and people movements were the variables most associated with PRRS outbreak.
Biosecurity vulnerability scores may help producers/veterinarians prioritize biosecurity investments.
Herd-specific biosecurity assessments are needed to determine herd-specific risk for PRRS outbreaks. Thus, we developed and validated a biosecurity vulnerability score (BVS) that measures the relative vulnerability of swine breeding herds to PRRSv introduction. The BVS was based on a multi-criteria decision algorithm that ranked risk events associated with outbreaks. A comprehensive biosecurity assessment was used to obtain information of the biosecurity practices from each participating herd. The practices performed in each herd were weighted by the relative importance of each event obtained from an expert opinion panel resulting in a score that identifies the events that should be prioritized. In two independent data sets, the scores consistently revealed that farms with higher scores had a higher frequency of PRRS outbreaks. In addition, results suggest that events related to swine movements,transmission by air and water, and people movements should be prioritized.
We are developing a new screening tool to validate the minimum number of questions associated with frequency of PRRS outbreak. Study farms will be asked to fill out a short survey. This can help producers and veterinarians to identify sites at relatively higher risk of PRRSv introduction.
To enroll or to request additional clarification please contact: Gustavo Silva at Iowa State University (gustavos-at-iastate.edu)