Science Page: Biosecurity screening tool; Benchmarking PRRSv biosecurity vulnerability using a short survey

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. Linhares’ lab at Iowa State University. In this Science Page are the results of a study looking at biosecurity aspects associated with PRRS frequency.

Key Points

  • New methods allow estimation of the overall PRRS-vulnerability risk score by asking 20 or less questions.
  • This can help producers and veterinarians to (a) measure and benchmark key biosecurity aspects, and (b) toidentify sites at relatively higher (or lower) risk of PRRSv introduction.

Study Summary: This study aimed to identify a small set of biosecurity aspects that, when combined, have a strong association with the frequency of PRRSv introduction into swine breeding herds.

Parameters included in the 2 models (A and B) to predict the number of PRRS outbreaks in farms for the past 5 years.

Preliminary Results: A cross-sectional study assessed biosecurity aspects in 84 breeding herds from 14 production systems in 2017. Models were trained to predict whether a farm had or not reported a PRRS outbreak in the past 5 years, given a set of biosecurity aspects. Two methods were used, and both models were able to classify the herds with a great overall performance based on few biosecurity aspects (See figure). The variables used by both methods were related to the frequency of risk events in the farm, swine density around the farm, farm characteristics/ requirements to visitors, and operational connections to other sites.

Note: The Gini coefficient (or index) is a single number aimed at measuring the degree of inequality in a distribution. (Source: Wikipedia) The higher the number, the less equally distributed the farms will be.

When comparing the predicted positive value obtained by the models, they showed a strong positive correlation (0.7 and 0.76, respectively) with the frequency of past outbreaks.

Enroll on our follow-up study: Study farms will be asked to fill a short survey. Using the methods above, the PRRS-vulnerability risk score will be generated for each farm enrolled. The information will be collected via an Excel file and the name of the farms and production systems will be kept confidential.

To enroll or request additional information please contact: Gustavo Silva (gustavos-at-iastate.edu) or Daniel Linhares (linhares-at-iastate.edu) at Iowa State University.

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.

ASF threat: 3 swine vets share insights from the frontline

The rapid spread of African swine fever (ASF) throughout China and other regions of the world has raised concerns the disease will ultimately make its way to the US — a development that could cripple the nation’s pork industry if it doesn’t adequately prepare.

That was the ominous warning of three US swine veterinarians who came together for a roundtable discussion on ASF following their recent trip to China. Among them was Dr. John Deen from the University of Minnesota, coming back from the Leman China conference.

Follow the link to listen to their podcast.

The informative session was organized by the editors of Pig Health Today and sponsored by Zoetis.

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.

Keeping up with a changing world: new challenges, new technologies, new people

From hog cholera and pseudorabies to dendograms and microbiome

Things looked very different in the world of swine health and production when the Leman Swine Conference was inaugurated in 1974. It would be another four years until hog cholera would be officially eradicated from the USA, and 13 years until the appearance of PRRS. The US was a net importer of pork and pseudorabies was an emerging disease. Artificial insemination was virtually unheard of on commercial farms, and biosecurity, as we know it, was in its infancy.  The personal computer was about to make its debut in 1975, but it would be more than a decade (most notably with the development of PigCHAMP at the University of Minnesota), until computerized herd management software would evolve to become a mainstay of managing herds. Back then, access to data and information was at a premium, and for the 44 years since The Leman Swine Conference has provided a vibrant venue to exchange and discuss ideas and experiences, both practical and scientific. However, in contrast to 1974, our challenge is no longer how to access information, but how to digest and make sense of the deluge of information coming at us from endless sources.

While the transformation of swine production in the field has been stunning, it has been no less so in the scientific realm. Words such as PCR, sequencing, dendrogram, and microbiome, flow easily from the tongues of veterinarians today, but were not in the lexicon in 1974. We all know we are now in the era of “Big Data” where advancements in computer science and computational analysis have endowed us with tools to perform complex analyses at unprecedented speeds. The time-honored goal and purpose of the Leman Swine Conference, namely to foster the cross-fertilization of ideas between the science and practice of swine health and production, must find its footing in this new world of near real-time information acquisition, analysis and reporting. For those of us who were weaned on to traditional diets of veterinary medicine and animal sciences, this is not a trivial challenge, and more than ever there is a need for us to work across disciplines with people who have the relevant skill sets. We are fortunate that the state of Minnesota and its university have been proactive in recognizing and responding to these new opportunities.

New researchers to address these new challenges

In the 2015 legislative session, the Minnesota state legislature authorized a multi-year investment known as the Agricultural Research, Education, Extension and Technology Transfer Program (AGREETT).  The vision of the AGREETT program was to support positions for new faculty, technicians and graduate students to work in seven key areas to support agriculture in Minnesota:

  • Crop and livestock productivity
  • Microbial science
  • Advancing soil fertility and water quality
  • Agricultural technology and decision-making
  • Nutrient recycling and management
  • Agro-ecological innovation
  • Technologies aimed at managing pest resistance and climate change

Many of these new positions at the University have recently been filled, including six faculty hires at the College of Veterinary Medicine, three of whom were featured in the 2018 Leman Conference program.

Dr. Kim VanderWaal

Kim VanderWaal, PhD is a native Minnesotan with degrees from the University of Minnesota and University of California-Davis.Kim was recruited for the “Big Data” AGREETT position in the Department of Veterinary Population Medicine (VPM) where she was already working with the swine group on disease modeling projects and is involved in data analysis with the Morrison Swine Health Monitoring Project. Kim’s interests surround the use of large data sets to better understand pathogen movements within agricultural production systems, and other complex problems including aspects of food safety and antibiotic resistance. Kim lead the pre-conference workshop titled “Geeks to Geeks: A practitioner’s guide to designing research studies” involving several speakers addressing issues of study design and analysis, including case studies. As you are all aware, the growth of applied research conducted in industry makes this an important area for today’s veterinarians to build their skill base.

Dr. Noelle Noyes

Noelle Noyes, DVM, PhD, was recruited for the AGREETT position in antimicrobial resistance in the VPM department. She is a native of New York who did her undergraduate studies at Amherst, Massachusetts, then completed a joint DVM/PhD program at Colorado State University. Her doctoral research focused on antimicrobial use and resistance in feedlot cattle. Noelle brings state-of-the-art expertise in bioinformatics and shotgun sequencing of the microbiome, and is eager to apply her skills for the benefit of the swine industry in Minnesota.  At the Leman Conference, Noelle spoke in the break-out session titled “New Directions in Antibiotic Use and Resistance”. Her talk was titled “Antimicrobial use and Antimicrobial Resistance – How Will We Ever Understand It?” where she presented her perspectives on how new tools and approaches can help us address this important challenge.

Dr. Declan Schroeder

VPM also gained an AGREETT position in Pathogen Discovery and Surveillance, and successfully recruited Declan Schroeder, PhD to this position. Declan is an experienced molecular virologist who holds an honorary Chair in Viral Metagenomics in the School of Biological Sciences at the University of Reading, United Kingdom. He has over 20 years of research experience as a molecular biologist in the areas of virology, biodiversity, pathology, and genomics – in particular, the use of genomic tools to study key biological processes. His research focuses on a diverse array of host-virus systems, including the honeybee. He was the former Director of the Marine Biological Association of the UK Culture Collection where he was also a Senior Research Fellow in Viral and Molecular Ecology (2001-2018). Declan made a presentation titled ‘Molecular diagnostics: Present and future, in the Disease Diagnosis and Research break out session. His move to Minnesota means that the Schroeder lab will continue to develop molecular tools to enhance detection and surveillance to secure and improve agricultural productivity.        

Space does not permit detailed introduction of another dozen AGREETT hires in other Departments and Colleges across the University, but several of them have roles that will support the swine industry. These include Erin Cortus (agricultural engineer focused on manure and odor management, CFANS); Andres Gomez (microbiome, CFANS); Melissa Wilson (manure management and soil science, CFANS); Diane DeWitte (swine extension educator, CFANS); and Peter Larsen (host-pathogen interactions) and Mathew Aliota (vector-borne diseases) in the Department of Veterinary and Biomedical Sciences, CVM.

This group of talented researchers complements a well-established group of swine researchers,including our new Leman Chair Cesar Corzo, to strengthen our capacity to work with stakeholders to address the daily and emerging challenges of the swine industry.

Please keep a look out for Kim, Noelle, Declan, Erin and Andres, and help welcome them to our community. It is important to let them know or what you are seeing and doing in the field to help bring science and practice into alignment,and honor the tradition of Al Leman and Bob Morrison.

Cold plasma technology to clean swine barn air

Porcine reproductive and respiratory syndrome virus (PRRSv) costs the US swine industry more than $580 million each year. First described in North Carolina, Iowa, and Minnesota in the late 1980s, the virus rapidly spreads through swine barns and is one of the industry’s biggest game changers. Additionally, pigs infected with virulent strains exhales aerosols containing a large quantity of the virus.

Today, researchers in the Veterinary Diagnostic Lab at the CVM are looking to apply research they are doing on decontaminating foods in collaboration with the University of Minnesota College of Science and Engineering (CSE) to swine barn air filtration in an effort to further promote swine health and safety in the food industry at large.

Plasma, served cold

Plasma is defined as partially or fully ionized gases with neutral net charge. It consists of a cocktail of photons, ions, free radicals, molecules, and atoms—many of which are highly reactive, which allows for many applications, including water decontamination. Plasma sources can also be engineered to produce plasma at close to room temperature—often referred to as cold plasma—enabling the treatment of highly heat-sensitive surfaces, such as some foods.

2D- integrated coaxial micro hollow dielectric barrier plasma discharge array
Plasma (purple) is produced inside the holes of the array, through which air is blown. Pathogens are inactivated when they come into contact with the air coming through the holes in the array.

The United States Department of Agriculture is supporting Sagar Goyal, PhD, professor in the Department of Veterinary Population Medicine at the CVM; Peter Bruggeman, PhD, professor of Mechanical Engineering at the CSE; and their team of researchers in pursuing the use of cold atmospheric gaseous plasma technology for decontaminating food and food-processing surfaces.

The team is seeing success in the lab—bacteria and viruses stand little chance against the cold plasma they are making.

According to Goyal, the laboratory results look extremely promising. “If a surface is contaminated with viruses or bacteria, we can kill them,” says Goyal. “If food is contaminated—as early as during harvest by food handlers—our goal is to use cold plasma to kill the contaminants.”

A pig impact

“Meanwhile,” says Goyal, “swine farmers are already using air filtration systems to mitigate disease. But these are not foolproof, so if we can combine them with this cold plasma, it would be helpful in getting rid of any disease affecting swine that can be transferred by air.” This includes, but is not limited to, PRRSv. So, cold plasma could positively impact the food and agricultural industry in more ways than one.

Follow the link to read more about how cold plasma could be used in swine barns.

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.