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.
Dr. Kim VanderWaal and Dr. John Deen from the University of Minnesota co-authored a new publication available now in the Proceedings of the National Academy of Sciences of the United States of America.
The objectives of this study were to identify priority swine pathogens, characterize temporal and geographic trends in research priorities.
57,471 publications covering 40 swine pathogens, compiled from 3 major database searches and dating from 1966 to 2016 were included in this analysis.
The top 10 pathogens published on were:
Foot and Mouth Disease
Porcine Reproductive and Respiratory Syndrome
Classical Swine Fever
African Swine Fever
The number of publications on swine infectious diseases increased over time as the hog production intensified. However, 8 pathogens increased faster than expected, particularly in the past 15 years: hepatitis E virus, Nipah virus, influenza, Streptococcus suis, Lawsonia intracellularis, porcine circovirus 2, PRRS, and PED.
On the contrary, some diseases had a slower growth in number of publications than expected. These included pseudorabies, Pasteurella multocida, Actinobacillus pleuropneumoniae, Brachyspira hyodysenteriae, and transmissible gastroenteritis virus. All of these pathogens were production diseases whose importance to the industry had declined in recent decades due to better control or even regional eradication.
Differences among world regions were identified except for influenza virus which appeared in the top 5 in most regions of the world. Southern regions where extensive hog production may still be the norm, tended to focus more on parasitic infections compared to Northern areas. Western Europe centered more on pathogens related to zoonotic and foodborne concerns compared to Northern America.
Pork accounts for more than one-third of meat produced worldwide and is an important component of global food security, agricultural economies, and trade. Infectious diseases are among the primary constraints to swine production, and the globalization of the swine industry has contributed to the emergence and spread of pathogens. Despite the importance of infectious diseases to animal health and the stability and productivity of the global swine industry, pathogens of swine have never been reviewed at a global scale. Here, we build a holistic global picture of research on swine pathogens to enhance preparedness and understand patterns of emergence and spread. By conducting a scoping review of more than 57,000 publications across 50 years, we identify priority pathogens globally and regionally, and characterize geographic and temporal trends in research priorities. Of the 40 identified pathogens, publication rates for eight pathogens increased faster than overall trends, suggesting that these pathogens may be emerging or constitute an increasing threat. We also compared regional patterns of pathogen prioritization in the context of policy differences, history of outbreaks, and differing swine health challenges faced in regions where swine production has become more industrialized. We documented a general increasing trend in importance of zoonotic pathogens and show that structural changes in the industry related to intensive swine production shift pathogen prioritization. Multinational collaboration networks were strongly shaped by region, colonial ties, and pig trade networks. This review represents the most comprehensive overview of research on swine infectious diseases to date.
Sow vaccination decreased influenza infections in piglets at weaning.
Influenza positive gilts at entry were associated with positive piglets at weaning.
More work is needed to assess herd closure, gilt isolation and gilt vaccination.
83 farms from 2 different pig production companies and located in Iowa, Minnesota and South Dakota were enrolled in this study. Samples were collected at weaning on a monthly basic for a little less than 6 years as part of routine surveillance programs. The majority of farms submitted 4 oral fluid samples per month but some collected nasal swabs or oro-pharyngeal swabs.
23% of the samples tested positive for influenza allowing the collection of 173 hemagglutinin sequences. In the H1 hemagglutinin subtype, isolates were 93.8% to 99% similar between each other and 94.3% to 97.4% similar to the vaccine strains. The largest discrepancy was found in the delta 1 clade. In the H3 hemagglutinin subtype, isolates were 95.9 to 99.7% similar among each other and 997.3% to 97.5% similar to the vaccine strains.
The influenza status of the piglets at weaning was influenced by several factors.
Seasons and vaccination status of the sows against influenza influenced piglet infection status at weaning. Indeed, sow influenza vaccination was significantly associated with a decreased probability of piglets testing influenza positive at weaning. Both whole-herd and pre-farrow vaccination protocols were better compared to no vaccination and there were no differences between both protocols. Additionally, having influenza positive gilts at entry increased the probability of detecting positive piglets at weaning.
Among all the factors evaluated, sow influenza vaccination and gilt influenza status at entry were the only factors associated with influenza in piglets at weaning in Midwestern breed-to-wean farms.
Breed-to-wean pig farms play an important role in spreading influenza A virus (IAV) because suckling piglets maintain, diversify and transmit IAV at weaning to other farms. Understanding the nature and extent of which farm factors drive IAV infection in piglets is a prerequisite to reduce the burden of influenza in swine. We evaluated the association between IAV infection in piglets at weaning and farm factors including farm features, herd management practices and gilt- and piglet-specific management procedures performed at the farm. Voluntarily enrolled breed-to-wean farms (n = 83) agreed to share IAV diagnostic testing and farm data from July 2011 through March 2017 including data obtained via the administration of a survey. There were 23% IAV RT-PCR positive samples of the 12,814 samples submitted for IAV testing within 2989 diagnostic submissions with 30% positive submissions. Among all the factors evaluated (n = 24), and considering the season-adjusted multivariable analysis, only sow IAV vaccination and gilt IAV status at entry significantly reduced (p-value<0.05) IAV infections in piglets at weaning. Results from this study indicate that veterinarians and producers could manage these identified factors to reduce the burden of influenza in piglets prior to wean and perhaps, reduce the spread of IAV to other farms and people.
This new publication in Veterinary Microbiology describes the best methodology to monitor 3-day-old piglets for PRRS, using both serum and processing fluid samples. The first author of the publication is Dr. Carles Vilalta, member of the Morrison Swine Health Monitoring Program (MSHMP) team.
Processing fluids (PF) constitute a useful sample to detect PRRSV infections at processing.
PRRSV can circulate in the farm at a low prevalence, increasing the chances of a re-break.
Young parity female litters should be targeted for PRRSV detection.
Current practice to bleed 30 pigs could be underestimating PRRSV prevalence in the herd.
The decrease in sensitivity at the litter level can be compensated by sampling more litters to detect PRRSV at the herd level.
The study was conducted in a 6,000 sow farm with a PRRS stable status. Every 3 weeks, serum samples and processing fluids were collected from all piglets in 10 randomly chosen litters. This process was then repeated 8 times, meaning that the farm was monitored for a total of 24 weeks. All samples were tested via PCR. 3 samples with the lowest Ct value were tested by virus isolation and sequencing of the ORF5 gene was performed.
10.6% of the piglets tested positive for PRRSv via serum PCR, representing 29.8% of the litters. The same number of litters tested positive via processing fluid PCR testing.
The percentage of processing fluid positive samples was significantly higher is parity 1 and 2 sows compared to parity 3 and older sows. Additionally, a significant association between parity and probability of detecting a positive pig was observed.
A significant higher proportion of positive serum samples was observed in males compared to females. A similar trend was obtained when comparing positive Ct values by gender with values from males being lower (i.e., higher viral load) than those from females.
Using a Ct value of 37, processing fluid samples had a Se and Sp of 87% (95% CI: 66%–97%) and 94% (95% CI: 85%–99%), respectively when compared with litter RT-PCR results obtained from individual serum samples. The total agreement between both tests was 92.2% and the positive and negative predictive values were 87% (95% CI: 66%–97%) and 94% (95% CI: 85%–99%), respectively. False negative processing fluids were identified in litters having 2 or less PRRSV positive piglets
The agreement between the PF and serum results was kappa = 0.81 (95% CI: 0.59–1.00). The difference in the proportion of positive samples between both types of sample was not statistically significant (McNemar test, p = 1).
Collection of serum samples of pigs at weaning to monitor for porcine reproductive and respiratory syndrome virus (PRRSV) has become a common practice to determine PRRSV herd infection status. Diagnostic sensitivity of this practice is low in herds undergoing PRRSV elimination once prevalence of infection is near zero. Thus, the goal of this study was to characterize the dynamics of PRRSV infection in 3 day-old pigs overtime using serum and serosanguineous fluids obtained as part of castration and tail docking practices (processing fluids (PF)). Secondary goal was to estimate sensitivity and specificity of PF in the 3 day old population. A 6000 breed-to-wean sow herd was monitored every three weeks for 23 weeks after a PRRSV outbreak by collecting both PF and individual serum samples from all pigs in the selected litters. Out of the 77 litters tested, 23 (29.8%) were identified as positive using the PF and the serum samples, with a Cohen’s kappa statistic of 0.81 (95% CI: 0.59–1) between the results obtained in each sample type. The sensitivity and specificity of the PF relative to the results in serum was 87% (95% CI: 66%–97%) and 94% (95% CI: 85%–99%) respectively. The percentage of PRRSV positive litters decreased over time and litters from gilts were more likely to test positive than those from older sows. Overall, the study demonstrates that PF can be a convenient and reliable specimen to monitor PRRSV infection in breeding herds.