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.

Morrison Swine Health Monitoring Project 2018 Summary

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, Dr Cesar Corzo shares the summary of the year 2018 for the Morrison Swine Health Monitoring Project.

During 2018 the MSHMP continued to make progress in different areas related to our main objective of developing the capacity to support the industry respond to emerging pathogens.

1) Database – Database has been structured to be able to capture a larger volume of data. This is a major step forward as we continue to work towards building the capacity of adding more sites and disease entities if needed.

2) Prospective PRRS sequence monitoring – The process of capturing diagnostic data continues, although not yet automated it is still adding sequences to the database. The database currently has 23,414 PRRS sequences from 20 systems and 21 states. Analyses of the database have begun with a subset but ultimately, we will be exploring trends and seasonal relationships involving spatialͲtemporal approaches. The database continues to provide a resource for MSHMP participants when conducting outbreak investigations.

3) Transport data capture and analysis – After a challenging year with our transport App we have decided to go back to basics and try a new approach to capturing transport data. The new approach which involves technology already validated in the trucking industry is currently being tested; we will follow up on this objective later this year.

4) Expansion – MSHMP continues to grow through three different ways:

  • 1) Current MSHMP participants continue to add new established farms,
  • 2) New participants have joined the project, two new production systems are already reporting and 2 more are in the process of providing data and
  • 3) Growing herd data inclusion into MSHMP has begun and is in the early stages as we learn how to link it with the breeding herd.

We have also continued our commitment with creating value to our producers through specific research projects that have been shared through conferences, MSHMP participant meeting during AASV and Leman Conference.

Peer Reviewed Publications

1. Vilalta C, Sanhueza J, Alvarez J, Murray D, Torremorell M, Corzo C, Morrison R. Use of processing fluids to determine porcine reproductive and respiratory syndrome virus infection status in pig litters. Vet Microbiol. 2018. 225:149Ͳ156. DOI: 10.1016/j.vetmic.2018.09.006

2. Machado, G., C. Vilalta, A.M. Corzo, C., Torremorrell, M., Perez, K. VanderWaal. Predicting outbreaks of Porcine Epidemic Diarrhea virus through animal movements and spatial neighborhoods. Nature Scientific Reports. Accepted.

3. Kinsley, A.C., A. Perez, M.E. Craft, K. VanderWaal. Characterization of swine movements in the United States and implications for disease control. Preventive Veterinary Medicine. Submitted.

4. Sanhueza JM, Vilalta C, Corzo C, Arruda AG. Factors affecting Porcine Reproductive and Respiratory Syndrome virus time-to-stability in breeding herds in the Midwestern United States. Transbound Emerg Dis. 2018. Dec 6. Doi: 10.11111/tbed.13091.

5. Arruda AG, Sanhueza J, Corzo C, Vilalta C. Assessment of area spread of porcine reproductive and respiratory syndrome (PRRS) virus in three clusters of swine farms. Transbound Emerg Dis. 2018. DOI: 10.1111/tbed.12875.

6. Arruda AG, Vilalta C, Puig P, Perez A, Alba A. Time-series analysis for porcine reproductive and respiratory syndrome in the United States. PLoS One. 2018. 13(4):e0195282. DOI: 10.1371/journal.pone.0195282. eCollection.

7. VanderWaal, K, Perez A, Torremorrell A, Morrison R, Craft M. Role of animal movement and indirect contact among farms in transmission of porcine epidemic diarrhea virus. Epidemics. 2018. 24:67-75. DOI: 10.1016/j.epidemic.2018.04.001.

Acknowledgements

We would like to acknowledge the strong team of faculty members, post-docs, students and staff that make this project possible. Additionally, this project would not be possible without the commitment of participants and practitioners and funding from the Swine Health Information Center.

Faculty: B. Morrison, C. Corzo, A. Perez, M. Torremorell, K. VanderWaal, J. Torrison and D. Linhares (ISU), D. Holtkamp (ISU), A. Arruda (OSU), and G. Machado (NCSU)

Post-Docs and Students: Carles Vilalta (Data visualization, PRRS testing), Juan Sanhueza (TTS, spatialͲtemporal analysis), Mariana Kikuti (PRRS sequence trends), Paulo Fioravante (IT Director), Emily Geary (Data manager), Kaushi Kanankege (Spatial analysis), Igor Paploski (Regional PRRS sequence analysis), Belinda Befort (Diagnostic trends)

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:


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.

NHF: What does livestock-associated MRSA mean for the neighborhood?

Our monthly collaboration with the National Hog Farmer continues; this month Dr Peter Davies from the University of Minnesota, College of Veterinary Medicine,  explains what is livestock-associated MRSA and if it affects people living near pig farms.

“Livestock-associated” MRSA first isolated in 2004

Methicillin Resistant Staphylococcus aureus (MRSA) Source: Wikimedia Commons

Most people are likely familiar with MRSA (methicillin resistant Staphylococcus aureus), a flagship “superbug” that is a major concern to human medicine. And just about everybody in the pig industry has heard that certain variants of MRSA are very common in some livestock populations (including pigs), and these are referred to as “livestock-associated” MRSA (LA-MRSA).

A novel variant of MRSA (labelled ST398 using a DNA typing method) was first found in pigs in the Netherlands in 2004. Subsequently, ST398 MRSA and several other types (e.g., ST9 in Asia, ST5 in North America) were reported in pigs in numerous countries, and often in their caretakers as well. The discovery that pigs may be a large MRSA reservoir created some justifiable panic and confusion, raising questions about the implications for human health, particularly for industry workers (e.g., farmers, veterinarians, processing plant workers), pork consumers and, last but not least, people living in the neighborhood of pig farms.

Generally, LA-MRSA  lack most of the key “virulence factors” that enable the bacteria to cause clinical infections in people.

Human clinical infections by “livestock-associated” MRSA are rare

Although workers on MRSA-positive farms often harbor LA-MRSA in their nose, significant clinical infections in healthy workers have been rare. Human clinical infections with LA-MRSA do occur, but most cases tend to be of relatively mild disease (such as skin infections), with more severe infections typically limited to elderly and medically compromised patients.

Remembering that about 2% of healthy U.S. citizens carry human adapted variants of MRSA, the relative clinical importance of LA-MRSA appears to be minimal in most countries. Globally since 2004, there have been around 10 fatal cases of LA-MRSA infections reported, compared with about 50 fatal MRSA cases per day (18,650 per year in 2005) in the United States alone.

2016 study of Iowa hospitals found probable livestock variants in only 0.24% of MRSA cases, and 1% of S. aureus infections. In North Carolina, another leading swine-producing state, there were no LA-MRSA variants among more than 1,200 MRSA isolated from human bloodstream infections between 1995 and 2015 (Dr. Vance Fowler, Duke University, personal communication).

Living next to pig farms does not increase the risk of exposure

Although MRSA can be isolated from meat products, there is little evidence to suggest cause for concern about food-borne transmission. In contrast, conclusions of studies looking at the neighborhood risk of exposure to LA-MRSA from pig farms are conflicting. We will focus on the findings of studies that have compared pig workers and neighbors directly, measured the distance from pig barns to residences directly; and used laboratory testing to confirm the presence of LA-MRSA in the study populations. 

Across three early studies in Europe, LA-MRSA prevalence (nasal carriage) was greater than 180 times higher in 352 pig industry workers (44%) than in 2,094 rural residents without farm exposure (0.24%).

  • A similar study in Holland in 2017 showed similar prevalence of nasal colonization (0.56%) in people without livestock contact, but also found the positive people on average lived closer to farms (of any type). Importantly, the authors noted that routes of transmission underlying this were not known.
  • A very detailed study of cases of MRSA infection in Denmark showed that overall MRSA risk did not differ between pig-dense regions versus other regions. However, the likelihood that a MRSA infection would be a LA-MRSA type was higher in the pig-dense regions, confirming some “spillover” from the industry to the community.
  • Notably, a follow-up study published in 2018 found that, within pig-dense areas, the patients with LA-MRSA infections did not live closer to pig farms than population controls. The authors conclude that direct environmental spread from neighboring pig farms was unlikely and suggested that community spread through contact with people working with livestock,  might be the predominant mechanism.

In summary, the overall impact of LA-MRSA relative to human variants remains very small in most countries including the United States. There is no evidence that residence in rural areas increases overall MRSA risk.

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.