Mycoplasma hyorhinis and Mycoplasma hyosynoviae dual detection patterns in dams and piglets

Today, we are sharing an original research article published by the MycoLab and Dr. Maria Pieters in PLOS One regarding detection patterns for 2 species of mycoplasmas in sows and piglets.

The objectives of this study were to:

  • describe when Mycoplasma hyorhinis and Mycoplasma hyosynoviae can be detected in piglets and is sows,
  • assess if there was a correlation between detection of the mycoplasmas in the sow and in the piglet, and
  • assess if there was a correlation between lameness and mycoplasma detection.

Conclusions

Under the conditions of this investigation, dams appeared to be consistently positive for both M. hyorhinis and M. hyosynoviae prior to weaning.

In contrast, higher detection was observed in piglets at week 3, in comparison to week 1 post-farrowing, with M. hyorhinis, while detection of M. hyosynoviae was remarkably minimal.

The relative risk of developing lameness in postweaning piglets was highly associated with the detection of M. hyorhinis at 3 weeks of age

This research article is available in open-access on the PlOS One website.

Methods

To answer their questions, researchers selected a 2,000 farrow-to-wean farm located in the Midwest with an unknown prevalence of the 2 mycoplasmas. 128 piglets were randomly selected from 30 sows, representative of the parity structure of the farm.

Swabs of the piglets and sows tonsils were taken 1 and 3 weeks post-farrowing. Starting at 5 weeks of age, piglets were evaluated for lameness every 2 weeks until they reached 22 weeks.

Tonsillar swabs were sent to the UMN VDL and were tested by PCR for Mycoplasma hyorhinis and Mycoplasma hyosynoviae.

Experimental design

Lameness scores were determined as follows: Score of 0) pig gets up immediately from a lying position and moves freely in the pen with balanced weight on all four limbs. Score of 1) pig rises immediately but a reluctant movement is observed, with short steps and uneven distribution of body weight. Score of 2) pig moves slowly in the pen with short steps and reduced weight in the sore limb, or pig rises slowly and the affected limb was not weight bearing. Score of 3) pig is reluctant to rise, with muscle shivering when standing and lifts the sore limb from the floor, or pig refuses to walk or walks on three limbs only and Score of 4) pig only rises when forced and when standing has marked signs of pain (e.g. reluctance to move, limping and vocalization).

Results

Dams appeared to be consistently positive for both M. hyorhinis (72% positive) and M. hyosynoviae (72% and 55% of positive sows respectively at week one; 65% and 48.3% positive at week 3). On the other hand, M. hyorhinis and M. hyosynoviae were detected in a small proportion of piglets in week one (8.3% and 0% of piglets positive respectively). However, M. hyorhinis was detected in half of the sampled piglet population just prior to weaning whereas only 0.9% of them were positive for M. hyosynoviae.

Mycoplasmas detection percentages in tonsillar swabs

M. hyorhinis was detected in a higher proportion of first parity dams than in multiple parity dams in both weeks of sampling, although this difference was only significant on week 3 of sampling. Detection of M. hyosynoviae, however, was higher in multiple parity dams in the first week of sampling, yet an increase in PCR detection was observed in first parity dams in week 3. The pattern of increasing detection between weeks one and three post-farrowing observed for both microorganisms in first parity dams may reflect a more recent transmission event and consequent colonization.

PCR detection of mycoplasmas in parity 0 and older sows

The risk of developing lameness at least once during post-weaning was higher if the piglets were detected positive for M. hyorhinis at week three. Additionally, there was a significant association between positive detection of M. hyorhinis at week 3 and a positive lameness score during its post-weaning age.
However, the association between positive detection of M. hyosynoviae and lameness score in post-weaning was not established due to fewer numbers of positive cases in week three.

Abstract

Mycoplasma hyorhinis and M. hyosynoviae are agents associated with arthritis in pigs. This study investigated the tonsillar detection patterns of M. hyorhinis and M. hyosynoviae in a swine population with a history of lameness. The plausibility of dual PCR detection of these agents in dams at one and three weeks post-farrowing and their offspring at the same time was determined. The association between M. hyorhinis and M. hyosynoviae detection in piglets and potential development of lameness in wean-to-finish stages was evaluated by correlating individual piglet lameness scores and PCR detection in tonsils. Approximately 40% of dams were detected positive for M. hyorhinis and M. hyosynoviae at both one and three weeks post-farrowing. In first parity dams, M. hyorhinis was detected in higher proportions (57.1% and 73.7%) at both weeks of sampling compared to multi-parity dams. A lower proportion of first parity dams (37.5%) were detected positive at week one with M. hyosynoviae and an increase in this proportion to 50% was identified in week three. Only 8.3% of piglets were detected positive for M. hyorhinis in week one compared to week three (50%; p<0.05). The detection of M. hyosynoviae was minimal in piglets at both weeks of sampling (0% and 0.9%). Lameness was scored in pigs 5–22 weeks of age, with the highest score observed at week 5. The correlation between PCR detection and lameness scores revealed that the relative risk of developing lameness post-weaning was significantly associated with detection of M. hyorhinis in piglets at three weeks of age (r = 0.44; p<0.05).The detection pattern of M. hyorhinis and M. hyosynoviae in dams did not reflect the detection pattern in piglets. Results of this study suggest that positive detection of M. hyorhinis in piglets preweaning could act as a predictor for lameness development at later production stages.

Antimicrobial Resistance Projects: Towards Antimicrobial Stewardship

This article was written by Drs. CJ Gebhart, KE Olsen and JL Torrison from the Veterinary Diagnostic Laboratory, University of Minnesota.

The emergence of antimicrobial resistance in humans, animals and the environment is a major global public health threat to both human and veterinary medicine.  Efforts to address this important issue involve government, industry, academia, and most notably, veterinary diagnostic laboratories (VDLs).  These efforts include surveillance to assess the extent resistance in human and animal pathogens and the development of policies to monitor and control antimicrobial resistance.

A collaborative effort involving the stakeholders listed above is the key to addressing this emerging threat of antimicrobial resistance and VDLs play major roles in these collaborative efforts.  As reported in a Commentary by GK Hendrix in the Journal of Veterinary Diagnostic Investigation in 2018, VDLs are the “nexus in the battle against antimicrobial resistance” (1). The University of Minnesota VDL Bacteriology Section performs almost 30,000 bacterial cultures annually, and most of the pathogenic isolates are archived for future use.  These uses include further testing (subtyping, virulence gene assays, serotyping, etc.), use in disease control efforts (autogenous vaccines, etc.), various research projects, and surveillance studies.  Almost 5,000 of these pathogenic bacteria are subjected to antimicrobial resistance testing annually, and these antimicrobial minimum inhibitory concentration data are archived for decades for further use.

Performing one of many antimicrobial susceptibility tests in the University of Minnesota Veterinary Diagnostic Laboratory, Bacteriology Section.

For our part in this aforementioned collaborative effort in antimicrobial stewardship, the University of Minnesota VDL is actively involved in two collaborative government-organized antimicrobial resistance projects as well as several collaborative academic research projects on antimicrobial resistance.  The common goal of the collaborative government projects is to determine the population and distribution of resistant bacteria in the U.S. 

The first of these projects is the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service National Animal Health Laboratory Network (NAHLN) project (2).  This project has 19 AAVLD-accredited laboratories throughout the U.S. and Canada participating with the objective of monitoring antimicrobial resistance profiles in animal pathogens routinely isolated from VDLs.  Ultimately, this project will result in a national centralized data collection and reporting process, using harmonized methods and antimicrobial resistance interpretation and reporting standards.  It aims to monitor data for trends in antimicrobial resistance phenotypes (and eventually genotypes) by identifying new or emerging resistance profiles, monitoring usefulness of antimicrobials over time, and reporting these trends to facilitate antimicrobial stewardship efforts. 

This USDA project began in January, 2018, and initially involved collection of isolates and antimicrobial resistance data from Escherischia coli (all species), Salmonella enterica (all species), Mannheimia haemolytica (cattle) and Staphylococcus intermedius group (companion animals) from routine VDL submissions.  A target of about 3,000 isolates will be collected from the participating VDLs annually and archived for further testing.  The antimicrobial testing data will be tracked and stored by USDA for each isolate and an annual report will be prepared for stakeholders.  This report will include antimicrobial resistance trends for antibiotics important for human and animal health and the distribution of minimum inhibitory concentrations for each antimicrobial monitored for each bacterial pathogen for each animal species included in the study.

The second of these collaborative antimicrobial resistance projects is the Food and Drug Administration (FDA), Center for Veterinary Medicine, Veterinary-Laboratory Investigation and Response Network (Vet-LIRN) project (3). This project has 21 AAVLD-accredited laboratories participating with the objective of performing surveillance of antimicrobial susceptibility testing results and whole genome sequencing of pathogens from the National Antimicrobial Resistance Monitoring System scope of interest (4). 

This FDA project began in January, 2017, and initially involved collection of isolates and data for three zoonotic bacterial pathogens, with several other bacterial species added to the project in July, 2018.  About 2,000 isolates have been collected since project inception, and the FDA has randomly selected about 200 of these isolates for whole genome sequencing.  The remaining isolates have been archived for future studies.  As an additional benefit related to this project, the University of Minnesota VDL received funds from FDA to purchase an Illumina iSeq Sequencer and participate in a collaborative project designed to increase the number and capabilities of network laboratories involved in the whole genome sequencing portion of this FDA project.  Standardization and harmonization of these bacterial genome sequencing abilities among participating laboratories is further designed to increase the network capacity and facilitate future outbreak investigations.

In summary, in support of antimicrobial stewardship efforts, the University of Minnesota VDL Bacteriology Section provides clinical isolates and antimicrobial susceptibility testing data for two collaborative government-initiated projects, one in collaboration with the USDA and the other with the FDA.  Further, the VDL as a whole provides leadership in antimicrobial stewardship on a daily basis, cooperating with disease outbreak investigations, collaborating with academic and industrial researchers, and educating veterinarians, clients and the public on issues of antimicrobial stewardship (1).

References

1.  Hendrix, GK. 2018.  The Role of Veterinary Diagnostic Laboratories in the Fight Against Antimicrobial Resistance

2.  USDA. 2018. USDA’s Role in Combatting Antimicrobial Resistance. 

3.  FDA. 2018.  Veterinary Laboratory Investigation and Response Network

UMN swine groups meet with the Minnesota Pork Board members

On December 17th, swine-focused faculty members from the College of Veterinary Medicine and the College of Food, Agricultural and Natural Resource Sciences met with the members of the Minnesota Pork Board research committee. 

At the Christensen Farms’ truck wash

This year was the third iteration of this meeting, hosted for the first time by Christensen Farms. Guests were invited to tour either the feed mill or the truck wash located in Sleepy Eye before sitting down for a day of productive exchange. 

After a short presentation by the host company, Deans Brian Buhr and Trevor Ames gave an update on the main initiatives from their respective Colleges. Dean Ames introduced the latest AGREETT faculty hires, Noelle Noyes and Declan Schroeder, as well as the anticipated arrival of Dr. Matheus Costa in the new year. The 2+2 program in collaboration with South Dakota State University was received with enthusiasm by the swine producers.

After lunch, attendees were divided into groups to brainstorm research ideas in four main areas: swine nutrition, swine health, manure management, and swine production and housing. After two hours of spirited discussion, 15 researchable questions were identified as the most pressing problematics faced by the Minnesotan pork industry. The meeting left the researchers inspired to keep offering science-driven solutions, and all agreed to meet again in 2019.

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.

Recordings from the ASF session at the 2018 Leman conference are available

African Swine Fever is a topic of concern for the industry. The organizing committee of the 2018 Allen D. Leman Swine conference recognized that and responded by dedicating an entire session of the conference on this topic. Below are the recordings from that session.

Dr. Chris Oura: African Swine Fever – a real and present global threat

Dr. Gustavo Lopez: ASF experience in a large commercial system

Dr. Patrick Webb – ASF awareness, prevention & response efforts at the national level

Dr. Scott Dee – Risk of African Swine Fever (ASF) virus in feed and mitigation strategies

 

African Swine Fever in China: a Swine Disease Global Surveillance Report

This report was published from the Swine Health Information Center and prepared by the University of Minnesota.

Thursday, August 16, 2018

There is now a second case of African swine fever (ASF) in China.  It was found in a slaughterhouse where 30 pigs died of ASF. The slaughterhouse is located in the city of Zhengzhou in the Henan province. The pigs came from Tangyuan district of the city of Jiamusi, in the Heilongjiang province, over 500 miles to northeast of the first reported herd, which was detected approximately two weeks ago.

By road, the distance from the farm to the slaughterhouse is approximately 1,400 miles, travelling through areas with high pig density. The slaughterhouse is a large commercial facility, owned by Shuanghui, part of the WH Group, the world’s largest pork producer.

china-asf-second-case-e1534428267522.png

Government officials are moving quickly to try to isolate the disease. The slaughterhouse has been closed with a no movement zone within a radius of approximately of 6 miles and a zone of 2 miles for destroying all pigs.

This news outbreak signals that there may be a number of concerns about the status of ASF in China.  The distance between the original detection in Shenyang and this newly identified farm, as well as slaughterhouse, shows that the disease is being transported widely. The areas of concern now involve multiple Chinese provinces and heighten the likelihood of further cases.

china-4-locations-interest-asf.png

4 key locations: Red dot, new ASF outbreak reported in slaughterhouse is located in the city of Zhengzhou in the Henan province; blue: first ASF outbreak reported in a small pig farm in district of Shenbei New, in the province of Liaoning; green dot: farm (owner Wang) located in the Hunnan District, Shenyang city, Liaoning province; purple: Tangyuan district of the city of Jiamusi, in the Heilongjiang province.

The Chinese press is reporting that the outbreak started as early as April of this year.  (http://www.chinanews.com/sh/2018/08-15/8600530.shtml) A partial translation is below.

The first ASF case was officially confirmed on Aug 3, 2018 on a small farm (owner Zhang) located in the Shenbei District, Shenyang city, Liaoning province.  Further investigations indicated that the owner of the ASF index farm purchased 45 pigs on July 5, 2018 from a farm (owner Wang) located in the Hunnan District, Shenyang city, Liaoning province. Fecal samples collected from Wang’s farm were confirmed ASFV RNA positive by PCR.

Wang said that the last entry of pigs onto the farm occurred on March 24, 2018. There were 100 piglets purchased from Chuanying District, Jilin city, Jilin province.  In April, some pigs started to become sick and then died on the Wang farm. Wang did not report the abnormal pig death; instead the remaining live pigs were sold to the Zhang farm in Shenyang and other people. So far, all of the pigs initially from the Wang farm have been tracked and culled.  Wang was held in a detention center due to violation of the laws.