Best of Leman 2018 series #3: J.Angulo – Understanding PRRSV infection dynamics in growing pigs in control and elimination programs

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:


Effects of Lawsonia intracellularis infection in the proliferation of different mammalian cell lines

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.

Highlights

  • 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

Arrows point towards cells highly infected by Lawsonia intracellularis.
Credit: Veterinary Mivrobiology

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.

Access to the entire paper

Abstract

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.

Science Page: Assessing the relative vulnerability of swine breeding herds to the introduction of PRRS virus

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. 

Key Points:

  • 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.

Study Summary:

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.

Follow-up study:

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)

Science Page: Protecting the Inevitable Risk; Biosecurity Evaluation at a Truck Wash

We hope you all had a great Thanksgiving! An ever increasing amount of you is visiting this blog every month so thank you, we appreciate your support!

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 Megan Bloemer on biosecurity at a truck wash. Megan, a 3rd-year veterinary student from the University of Illinois, presented this project at the Leman Conference this year and won the Morrison Swine Innovator Prize.

Key points:

  • Monitoring cab cleaning and hot shot handle cleaning via Glo Germ Gel is simple and cost-effective.
  • Wiping down the cab interior with intervention wipes only adds around 5 minutes. These minor cost and time additions to truck wash procedures can help to prevent a million-dollar PRRS break.
  • Truck wash crew and trailer washers are often overlooked but perform a job that is essential in maintaining biosecurity and disease outbreak and therefore herd health.

The objective of this study was to assess overall biosecurity at the truck wash and identify potential areas of concern, measure and evaluate these areas of concern, and suggest solutions.

Potential Areas of Concern Identified

Cab Cleaning

Glo Germ Gel under a UV light when the door handle was not cleaned (left) and was wiped down (right).

The areas observed for cleaning included: steering wheel, dash, handles, climate control buttons, and radio. These areas were not being focused on; but are critical areas touched each time a driver is in the cab. In addition, it was difficult for monitors to tell if a cab had been cleaned or not by visual inspection alone.

Equipment Movement

After the three-day observation period, it became apparent that all equipment besides hot shots stayed in the dryers. Thus, hot shots were identified as the main equipment of concern. They were not returning with each trailer load, leading to biosecurity concerns.

Monitor Movement

Monitors inspect both PRRS positive and PRRS negative trailers throughout the day, before the wash crew is allowed to disinfect each trailer. Although monitors change boots and put on Tyvek before inspecting negative trailers, there is no true clean / dirty line where they change shoes.

Evaluation

Cab Cleaning

Steering wheel, dash, door handle, climate control buttons, and radio control buttons were evaluated on how well they were cleaned with a Glo Germ Gel product. The Glo Germ Gel was applied while the trucks were waiting in line to be cleaned. The assessment was performed using an UV light for any trace of the Glo Germ, indicating whether the surface had or had not been cleaned. The interior of cabs were not being cleaned as well as possible as evidenced by the amount of fluorescence that was detected in those five critical areas.

Equipment Movement

All of the hot shot handles and prods were numbered in both the PRRS positive and PRRS negative equipment sheds on a Sunday. Every night for the next five days it was checked if each hot shot was present, which equipment shed it was in, and new ones were numbered as they appeared. Throughout the course of those five days hot shot handles and prods were not being returned on a consistent basis. However, the equipment was not switched between the PRRS positive and PRRS negative sheds.

Monitor Movement

Glo Germ Gel and Powder was applied to the shoes of monitors and on positive trailers before monitors inspected them. Although no Glo Germ was appreciated in the PRRS negative areas, it may still be a potential area of concern and should be further evaluated.

Interventions

Cab Cleaning

In order to ensure that the interior of cabs were being cleaned as well as possible,the truck wash crew was shown images of the cab interiors with the Glo Germ Gel comparing interiors that were wiped down and those that were not. Current protocols could be clarified, and the importance of cab cleaning should be emphasized. Glo Germ Gel also gives the monitors the ability to do random internal audits of cab cleaning.

Equipment Movement

In order to check hot shot handle and prod cleanliness Glo Germ can be applied at the same time monitors put Glo Germ in the cabs. To encourage returning hot shots the truck wash crew can continue to write down cull and gilt trailers that do not return with a hotshot. To stop any potential cross-contamination, the PRRS-positive hot shots could be painted red.

Monitor Movement

Although no Glo Germ was appreciated it is possible that monitor movement is still a potential biosecurity risk and should be further evaluated. It appears that the Glo Germ washed right off as the trailers were wet when the monitors inspected them.

Science Page: Emerging enrofloxacin and ceftiofur resistance in E. coli isolated from US swine clinical samples

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 from Dr. Shivdeep Singh Hayer from the STEMMA lab, on the emerging enrofloxacin and ceftiofur resistance in E.coli in swine.

Key points:

  • Nearly one-third of clinical E. coli isolates collected from swine samples were ceftiofur or enrofloxacin resistant
  • Genetic analysis revealed presence of rarely reported genes in antimicrobial resistant isolates
  • Most of the isolates were multi-drug resistant on both routine lab tests and genetic analysis

In a previous study, we analyzed the antimicrobial resistance in Escherichia coli isolates recovered from swine clinical samples from across USA during 2006-2016 at the University of Minnesota Veterinary Diagnostic Laboratory (UMN-VDL), and found a 47% annual increase in the prevalence of enrofloxacin resistance (from 1.5% in 2006 to 32% in 2016) while no trend was observed for the resistance to ceftiofur (that ranged between 32-39%). A follow-up study was conducted to evaluate the genetic basis of resistance against enrofloxacin and ceftiofur in E. coli isolates using whole genome sequencing (WGS).

153 swine clinical E. coli isolates collected in 2014-15 from 14 states across USA were selected and genes causing ceftiofur and enrofloxacin resistance were identified using WGS.

21 (out of 106) enrofloxacin-resistant isolates from 6 states harbored diverse plasmid mediated quinolone resistance (PMQR) genes (qnrB19, qnrB2, qnrS1, qnrS2 and qnrS15). The presence of PMQR genes alone was associated with clinical levels of resistance.

The most prevalent genes associated with ceftiofur resistance were blaCMY-2 (89/106, 84%). Moreover, 24 ceftiofur-resistant isolates harbored various blaCTX-M and blaSHV genes.

Additionally,  bacteria carrying blaCTX-M and qnr genes also contained genes coding for resistance mechanisms against other antimicrobial classes and were commonly resistant against ampicillin, tetracyclines, gentamycin, trimethoprim and sulfonamides.

These genes (blaCTX-M, qnr) have been rarely reported from farm animals in USA and have been implicated as important genetic mechanisms behind extended spectrum cephalosporin and fluoroquinolone resistance in human and animal populations in several countries. These genes are present on plasmids, making their dissemination across bacterial populations faster by horizontal transfer.

The presence of multiple antimicrobial resistance genes on the same plasmids also makes mitigation of this problem more difficult because of the possibility that using one antimicrobial class will exert positive selection pressure for resistance against other antimicrobial classes.

Use of processing fluids and serum samples to characterize PRRSv dynamics in 3 day-old pigs

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.

Key points

  • 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.

Methods

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.

Results

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.

ct value processing fluids versus serum samples PRRS
Cycle threshold (Ct) positive (≤35) and suspect (between >35 and 40) value distribution for serum (S, triangle) and processing fluid (P, circle) samples overtime (2, 5, 8, 11, 14, 17, 20 and 23 weeks post outbreak). Horizontal black lines indicate the mean Ct values for each week and sample type

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).

Abstract:

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.

Follow the link to read the entire article.

Science Page: Quarterly review of MSHMP reported PRRSv Restriction Fragment Length Polymorphism (RFLP) patterns

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 from the MSHMP team regarding reported PRRS RFLP patterns.

Keypoints:

  •  Recording PRRSv RFLP and sequences will provide better insights into the epidemiology of the disease at local, state and national level.
  • Building a RFLP database will allow us to assess which factors could be involved or related with the emergence of a new RFLP.
  • The predominant pattern RFLP in this quarterly review is the 1-7-4.

In the first quarter of the 2018/2019 incidence year, 20 breaks affecting 12 production systems were reported. Out of these, 4 occurred in July, 13 in August and 3 in September.

Of those 20 farms, three had a break while still being status 1, one was status 2 in the process of eliminating the disease (not using any immunization protocol at that point), 6 were using field virus as the acclimatization protocol (2fvi), 8 were using vaccine (2vx), one was provisionally negative (status 3) and one broke from a status 4 after being almost 4 years completely negative (see figure below).

RFLP patterns with status at break

The distribution of the breaks is wide and affects different states. Thus, we had 6, 1, 4, 1, 4, 2, 1 and 1 break in the states of IA, IN, MN, MO, NC, NE, OK and PA, respectively. The closest 2 farms that broke were 1.2 miles apart, belonged to the same company and had the break a week from each other (no sequences was provided).

Eight out of the 20 breaks reported were accompanied by the associated RFLP. The predominant (4 out of 8) RFLP pattern since July is 1-7-4. Iowa was the state with the highest number of 1-7-4 cases.