Minnesota's Swine & U

Sarah Schieck Boelke:
Hello and welcome to Minnesota's Swine and U podcast series, a University of Minnesota Extension Swine Program. Today's podcast is a research update on the longitudinal evaluation of mycoplasma hyopneumoniae detected in processing fluids in swine breed-to-wean farms.

My name is Sarah Schieck Boelke, your host, and I'm a Swine Extension Educator with the University of Minnesota. Joining me today is Albert Canturri, who is a pathologist and assistant professor in the Department of Veterinary Population Medicine and the Veterinary Diagnostic Laboratory. To get us started today, Albert, will you share a little bit about yourself and including who did you work with to complete this project?

Albert Canturri:
Sure. Well, hello! Thank you for having me. My name is Albert Canturri. I'm a veterinary pathologist by formation and the research that I will be talking to you about today was included in my PhD thesis that was done here at the University of Minnesota and the mycoplasma research lab, and it was under the supervision of Dr. Maria Pieters. So, and since last year I have been working as a food animal diagnostician and pathologist here at the diagnostic lab.

Sarah Schieck Boelke:
Great, thank you for sharing that. And what research topic will you be sharing with us today? I guess you kind of alluded to it in your introduction as to working with the mycoplasma research lab. But can you explain a little bit more.

Albert Canturri:
Yeah. So today I would like to talk to you about the first study of my PhD thesis, and it was about the detection of mycoplasma hyopneumoniae, which is a bacterial pathogen of the respiratory tract of pigs in processing fluids. I would like to to share the lessons that we learned from that study and especially the implications that this has for diagnosis of mycoplasma hyopneumoniae in this case, but also for for other infectious agents in swine and in other species, in terms of sampling strategies, for example, or environmental contamination of samples or PCR testing. And among other topics. So yeah, we'll talk about these things today.

Sarah Schieck Boelke:
Great. I'm anxious to learn more. So, Albert, before we get too far in our discussion here, we should probably give some recognition to the funding source for this research project, because, as you and I know, but maybe our listeners are not that familiar with that research that happens at the University of Minnesota as well as other institutions, needs a funding source. So can you please share the funding sources for this project that we're talking about today?

Albert Canturri:
Sure. And again, as you said, this is very important for us, so it's always and good, and we need to acknowledge those funding agencies. In this case it was the Minnesota Pork Board and the Minnesota Agricultural Experiment Station through a Rapid Response Fund. So thank you both of them for that funding. And that opportunity to do this research.

Sarah Schieck Boelke:
So now let's talk a little bit more about your research and get into kind of the specifics can you give? A brief introduction to this research study explaining, why was it a valuable project to do?

Albert Canturri: Yeah. So the reason behind this project, I think it started around 2018, and it was based on a very interesting and highly unexpected finding. That again would could not be explained with the knowledge about the bacterium, about mycoplasma hyopneumoniae that we had at the moment, and and that we currently have.

So remember that, as I said, mycoplasma hyopneumoniae is a bacterium, and that affect pigs and causes respiratory disease, and very importantly, it is considered to be restricted to the respiratory tract. So the knowledge, the knowledge of these pathogen is that it doesn't travel outside the respiratory. That’s from one side, right. The other side is processing fluids alright. So processing fluids are a relatively new sample type that consist of the fluid collected from tail docking and castration of pigs, which is a process called processing. So processing pigs.

This, of course, when piglets are one to three days old, and with that fluid we have seen that this fluid that is, the translate of those testicles, and the tails can be used to detect pathogens. For example, PRRS, PCV 2 and 3, and see that mainly viral pathogens. So it's a good sample type to monitor the status of the sow farm for some of the pathogens right?

But again, this was, you know PRRS specific to viral pathogens. And then the surprise came when a swine practitioner from here from Minnesota found, or we detected Mycoplasma hyopneumoniae in processing. So this is this was very unprecedented, very surprising, and it raised the interest of here the researchers at the University of Minnesota to look forward to, to, you know, to investigate more this finding.

So later. We did some preliminary studies that were led by Carlos Vilada and Maria Pieters, and in those two studies mycoplasma was indeed detected by PCR in those processing fluids in different sow parities in different times of the year. So you know, that was not anecdotal. So that was real. Then there was mycoplasma, detection in processing fluids.

And in another study, and that was very interesting in those preliminary studies, the detection was observed at the same time as a mycoplasma outbreak in a previously negative farm.

Okay? So those two studies, or those two observation observations, raised an important question. And it was. you know, why are the processing fluids, a valuable sample to detect the infection of mycoplasma hyopneumoniae in sow farms?

You know what is the true value of those observations for disease diagnosis? What are we detecting? Right? We have a lot of questions. How does mycoplasma get into the processing fluids?

You know, if it appears that it does not travel outside the respiratory tract? So lots of questions that we needed to design a study to answer some of them, or at least to understand better what was happening. So the objective of of the present, the present study that II will talk about today was to expand the information and that could be considered, you know, very particular or very anecdotal, that was based on few observations and some very particular conditions, by involving more farms from different production systems, and to collect samples for a long period of time.

Sarah Schieck Boelke:
Interesting. So you had mentioned that it was kind of by pure chance originally that Mycoplasma was picked up by the processing fluids. Was that right?

Albert Canturri:
Yeah. Well, yeah, I don't know if it was chance, or there was something behind that. But yeah, that was maybe it can be called chance. But yeah, that that was unexpected for sure.

Sarah Schieck Boelke:
Interesting. So how do you and the others go about completing this study?

Albert Canturri:
Yeah. So this study this was a big study. So because again, for this study, we wanted to to expand the umbrella of observations. You know, I talked about those two preliminary studies. And we wanted to have much more information. To include more farms, more animals and to follow all those farms and animals. Not, for, you know, a short period of time, 1, 2 months. In this case we wanted to follow that for an entire year. You know, to have the to gather the most information as possible.

So in order to do that enrolled ten sow farms from three different production systems. Five were positive to mycoplasma hyopneumoniae and ten of those farms were negative to mycoplasma hyopneumoniae. And from each of these farms, we collected monthly. So each month we collected 30 individual litter processing fluid samples. So within each litter the processing fluids of that litter were collected in our back, and of those 30 in individual litter processing fluid samples, 10 were from gilt litters, 10 from we're from parity two gilts or sows, sorry, and 10 were from parity three and older litters.

And we did again. We did that monthly for an entire year. And we did this stratified sampling because we wanted to determine, or you know, we were curious about determining if there was an association between the detection of the Mycoplasma hyopneumoniae by PCRs and the parity of the sows right, because this is this was something that we saw in those preliminary studies that I mentioned earlier. We wanted to expand that information.
And also the fact of collecting samples monthly for an entire year was because we wanted to get numerous observations. But also because we wanted to assess if there was an association between the detection of Mycoplasma and the season of the year.

So at the end, when we finish the sampling step or the the sampling of this study, we obtained about actually a lot of samples, we obtain about 3,000 individual litter processing fluids. Again, lots of samples, and we put them in pools of five for PCR testing. So basically, we tested near 600 samples in this style. And I want to reinforce an important idea here. So again, testing was based on PCR, so detection of genetic material of Mycoplasma hyopneumoniae. So Mycoplasma hyopneumoniae DNA.

And this is the most common test for the detection of this bacterium, because for this bacterium bacterial culture, which is a test or an assay that, you know is very common, for other bacterial species does not work very well for Mycoplasma hyopneumoniae.

So remember, this is just PCR detection of DNA, and we don't have information about the viability of mycoplasma. There is no way to know with what we are detecting in those processing fluids is live with that mycoplasma. So keep that in mind for later.

Sarah Schieck Boelke:
So a couple of questions I thought of as you were going through this as you mentioned. You know the herds had either they were, you know, positive right for Mycoplasma or not. Can you just explain just briefly, Mycoplasma is something that commonly circulates in swine herds. Correct? That's, I shouldn't say like an underline. That's kind of always there. But right, it's fairly prevalent?

Albert Canturri:
Yeah. Well, I'm happy to say that I think we are changing that that was, you know, maybe 20 years ago, or or even 10 years ago, maybe the majority of the farms worldwide, or you know, here in the Upper Midwest were positive to Mycoplasma hyopneumoniae at the sow herd and but there has been, you know, for the last decade.

I'm led by Dr. Maria Pieters here at the University of Minnesota, an effort, and, you know, combined with practitioners and veterinarians and production systems here in Minnesota, especially to eliminate Mycoplasma hyopneumoniae from the farms through elimination strategies or allocation programs. And these have been very or you know, some of them the majority of them have been very successful. So I would say, you know, one of the problems that we had designing this study was to obtain positive mycoplasma, hyopneumoniae sow herds in Minnesota. Because, you know we are doing a great job eliminating these important disease from the source from the sow farm. So yeah, I think we are changing that trend. And that's very important.

Sarah Schieck Boelke:
Good. Thank you for further explaining that. And also pointing out that yeah, with Dr. Maria Pieters lab, yeah, that's her focus in trying to help not only the swine industry here in Minnesota, but also in other states, as well to try and eliminate that. So thank you for explaining that.

So what were the results of this research project?

Albert Canturri:
So the results were again another surprise, at least for me. At first, right? So the results of the PCR testing showed that the Mycoplasma hyopneumoniae was abundantly detected in positive farms. You know the positivity rate in those positive farms. Remember, we had five positive farms and five negative farms.

In the ones that were positive, the positivity rate was higher than 90 percent so that was, and that was very consistent throughout time with all months of the year, having similar detection rates and also the quantity of the Mycoplasma hyopneumoniae so very low value. So it seems evident from this results that the detection of mycoplasma genetic material by PCR in processing fluids might be a common finding rather than a sporadic event, as we thought previously. As a detection, again as I said, was consistent across various farms of different production systems, regardless of the sow parity.

On the contrary, in those negative farms, Mycoplasma was generally not detected so very, very low detection rates. So when we obtain these results we saw that Mycoplasma was so much present in processing fluids of those positive farms. And again, the ct values were very low in some samples. So remember, that the lower the ct value in a PCR the higher the more quantity of the target. In this case mycoplasma, hyopneumoniae is in that sample.

So lots of mycoplasma in terms of samples and in terms of quantity. So we wanted to, and to look more into it. And we run an analysis that was not an objective at the beginning of the study. But it made sense at that moment to perform and to better understand what those positive results were meaning and in order to do that we did a selection of the positive sample representing various farms from various systems. And we did DNA sequencing of them. We obtain 7 sequences from those samples, from the again different farms. And interestingly, they were identical between them.

So the mycoplasma obtained there was identical between farms, and it was highly homologous, almost 100 percent homologous to the J strain of mycoplasma hyopneumoniae, and this is a strain that has been historically used for vaccine production. So very important information there with, you know, lots of takeaways and implications that you know we would talk about later. Maybe.

Sarah Schieck Boelke:
So I do have a follow up question, and hopefully, I'm not stealing away from something that you're gonna explain later. But with you saying that it's historically used for vaccine production, could it be that that herd was vaccinated? For Mycoplasma? And that's how it got picked up?

Albert Canturri:
Umhm. Yeah, I will. You know this is one of the conclusions of this is one of the hypotheses that we have. At this point. So yeah, that this is one of the conclusions that that I would like to talk about this project and those positive farms were most likely vaccinating for mycoplasma hyopneumoniae.

And with this study, we don't have enough evidence in order to say that this is 100 percent the origin of what we detected. But the hypothesis at this point is, yes, that what we were detecting was vaccine products. So environmental contamination of those processing fluids by vaccine products. So if that were to be true, that again, with evidence that we have here. We can be, you know, very suspicious that this is the case. But we don't have enough evidence to just clearly say that. This is our hypothesis, again trying to give a mechanistic explanation to that right?

So our hypothesis is that when the workers perform processing in a given day maybe they have been previously vaccinating pigs at weaning, right? Piglets that get vaccinated with mycoplasma hyopneumoniae at weaning so they can be you know, might rest, or remnants of those that vaccine can get into your hands or your clothes, or, you know, coveralls, etc. And this can, you know environmentally, be a source of environmental contamination for those processing fluids that are later collected.

And that might be the source of the detection in processing of mycoplasma in processing fluid. This is one of our hypotheses. I mean this has great implications. For again, for sampling, for PCR testing that I would be, you know we can talk about later.

Sarah Schieck Boelke:
So you might have alluded to some of them already, but based off of your results, what conclusions can you make from this research project?

Albert Canturri:
Yeah. So lots of conclusions. Again. This was the first study of my PhD. And some of the things that we learned paved the way for further research and for expanding and for later other research opportunities that made up my whole PhD research. Right?

So first we saw that mycoplasma hyopneumoniae was frequently detected by PCR in positive farms, both in terms of quantity of genetic material and consistency of detection throughout time. While it was generally not detected in many farms, so I guess from now on it shouldn't surprise us that DNA from the bacterium is detected in processing fluids from those positive farms right? So at the beginning I thought it was very surprising to everybody. Now I wouldn't. It wouldn't surprise us right for everything that we have explained.

However and again this relates to what I was talking to you about earlier, the mechanisms by which this genetic material of mycoplasma reach those processing fluids is still unclear. Even though, as I said, the mycoplasma is considered to be restricted to the respiratory tract. And there are a few studies based on experimental conditions, or even in vitro studies that have suggested that Mycoplasma can disseminate outside the tracts, or, you know, for very at least transiently or for very short periods of time. So at this point we are not completely sure about the origin of the detected DNA in the processing fluids but you know, however, as I said this consistency of detection and the high bacterial loads that we obtain made us consider performing DNA sequencing, and just, you know, again, to try to characterize the nature and the possible origin of the genetic material.

As you said, we obtained a high homology with a strain that is used in commercial vaccine products. So again, our hypothesis is that the origin of the detected genetic material may be associated to environmental contamination of the processing fluids with mycoplasma, hyopneumoniae vaccine products rather than through presence of the bacterium in the testicles or in the associated fluid.

But again, this is just a hypothesis, and I think more research is needed in order to know better or to be 100% sure about the origin of the detection of mycoplasma in processing fluids.

Sarah Schieck Boelke:
So why are the results you shared important takeaways from your study?

Albert Canturri:
So I think this again, this study, there are many ramifications. You know many branches from the results of this study, and I would like to focus, perhaps, on two, the two most important ones, in my opinion.

So first, this study served, I think, to raise awareness on the importance of environmental contamination of samples and the prolonged stability of DNA in the environment. So we know that DNA in the environment can be there for long periods of time. So, and that can be a problem for testing and you know it's good to see that we were not the only ones that encountered detection issues with mycoplasma using PCR. So there are all other reports, at least two other reports out there around the same time that described diagnostic challenges associated with this environmental contamination of vaccine products, especially.

So this serves us, and this I think this should make us realize that for an appropriate interpretation of PCR results, especially for Mycoplasma. But I would say, for all other pathogens, efforts should be made to minimize the possibility of pre-analytical error. And you know this is a term that we use here in the lab and that is all the procedures that occur before the sample is analyzed in the lab.

So including sample everything that what happens in the outside, the lab, right from sample collection in the farm to handling that sample to transport in that sample everything that happens before that the sample is tested. It's extremely important for later, for interpreting the diagnostic, the results. So, for example, what can we do to in order to improve that, or to minimize that pre analytical error, right to improve the quality of the samples.
One thing easy things that we can do in the farm is determining a clean and dirty area when sampling as well as cleaning and disinfecting the sampling materials regularly. I think it would be beneficial in most cases, and for most pathogens. And all these measures, all these, you know, cleaning measures should be maximized in time with scenarios, such as, for example, when we monitor gilts for introduction to a naive herd, especially when these gilts have been vaccinated prior to sampling at the Gdu, for example, these are high risk scenarios that you know can give us a lot of trouble. If you know, diagnostic travel or diagnostic interpretation issues if it's not, you know, if they are not done correctly.

This was, first, the second takeaway from this study is to an, and this this was maybe the most important part of my of the PhD is how the lack of a test that would determine bacterial viability for mycoplasma hyopneumoniae have important implications in this case. So, for example, imagine that we had had a way to determine that if what we were detecting was dead or alive, right? If the Mycoplasma that was detected in those processing fluids was dead or alive, that perhaps would have helped us and don't get me wrong. PCR testing is a great diagnostic tool. It has high sensitivity, high specificity.

We use it every day here in the lab. Right? It's great. It's great. But the assay is not able to determine the viability of the genetic material. That we are detecting. So you know, differentiating live versus dead. So it might lead to interpretation issues. So in order to overcome this limitation. We have developed a viability PCR assay for mycoplasma an assay, that is able to tell us if the mycoplasma that we are detecting is dead or alive.

And we are using this assay in that different. We have use this assay and we are still, you know, developing this assay as a diagnostic tool in different scenarios with very promising results. So I'm sure we will learn a lot about mycoplasma in general, and we will solve some of the diagnostic problems with mycoplasma from with this new assay.

Sarah Schieck Boelke:
Very interesting. So in your role as a diagnostician, you're not necessarily using this type of assay, yet it's still in the developmental.

Albert Canturri:
Yes, yes, so we do not have it here set up in the diagnostic lab. So it has been developed in the lab already, you know, validated in the lab, in the mycoplasma research lab, and we are using it. Now, with the, you know, in special specific scenarios in the field. So you know, testing it outside the lab, you see. So, and we are collecting very good data from all those studies, and we hope that sooner rather than later can be added to the workflow of the diagnostic lab for you know, to try to help us in some of those very particular scenarios.

It won't replace the actual, the current PCR, of mycoplasma hyopneumoniae, you know. This, you know, detecting DNA is it's good, and it served us for the vast modality of what we are looking for. And you know it's great. It's a great diagnostic tool. But there's certain scenarios in which, you know, knowing the viability would be very important. And again at this point it is research. But yeah, I hope we can use it here soon.

Sarah Schieck Boelke:
Thanks for that further explanation. So to wrap up our discussion here. Is there any closing remarks that you would like to make at all?

Albert Canturri:
Well, I just want to take advantage of this opportunity. To reinforce the idea that diagnostics in general, the ways in which we detect pathogens, and and especially the ways in which we interpret laboratory results are fundamental for disease control. So it's my belief. And I think history has proven that without good diagnostics, there is no way to control or treat diseases in animals especially.

You know, we talk about pigs here. So pigs, of course. But you know this is animal diseases in general. So I think efforts at improving diagnostics. And you know, receiving good diagnostics tests or reports. It's either the veterinarian. It helps the veterinarian a lot. In all animal species, and I think diagnostics are very important for the veterinary profession in general. So I would encourage everybody that is listening to try to get the best quality samples and to contact the best quality labs in order to get very good diagnosis for the diseases that they are seeing clinically, because this will help us treat and control those diseases.

Sarah Schieck Boelke:
Well, thank you, Albert, for sharing this research with us on Mycoplasma hyopneumoniae diagnostics. And thank you to those listening to the University of Minnesota, swine, and you. Podcast this has been Sarah Schieck Boelke, Swine Extension Educator, along with Albert Canturri, a veterinary pathologist at the Veterinary Diagnostic Lab.

To further connect with the University of Minnesota Swine Extension, please visit the swine specific webpages on the University of Minnesota's website at www.extension.umn.edu/swine, and on those pages you will find connections to our blog and Facebook page. To learn about research being done by our swine faculty and veterinary medicine, please visit their Swine and Minnesota blog www.umnswinenews.com.