Hello, I'm John House, and in this talk I was gonna provide some Perspectives on her biosecurity in reference to mycoplasma, I just want to acknowledge Mike Hazleton, Mark and I worked on preparing this talk. I'd also just like to acknowledge that, in this talk, I'll be presenting some different studies or results of different studies that have involved lots of different people. Some from the University of Sydney, John Morton from Jamorra, or Nen from Pure Source Dairy Farm Company in China, Fiona Monsall from the University of Florida, Craig Dwyer from Smith and Veterinary Services in Tasmania, and and Kitten Branco from the National Vet Institute Technical University of Denmark, and we were supported with this work through the Gardner Foundation.
And Dairy Australia. So, It's often useful to actually know the characteristics of pathogens because when you investigating the I guess the pathophysiology and the dissemination, if you, their capacity to survive and replicate influences outcomes. So mycoplasma are the smallest on self-replicating per carrier, the laxa cell wall, that's relevant when you're trying to treat it because things like penicillins and the B lactams.
Function by breaking down cell wall, but mycoplasma doesn't have one, so that's why, you know, a number of antimicrobials don't work. It's nutritionally fastidious and slow growing, requiring 3 to 7 days of growth before colonies become apparent. That actually you end up being important in Australia when mycoplasma sort of re-emerged because people sometimes knew they had a problem when they were submitting things for culture, but they, Weren't requesting a mycoplasma culture, and it wasn't showing up on regular blood agar because it was growing too slowly, and therefore, the diagnosis wasn't made.
It's capable of surviving in cool moist environments for weeks to months, so, and it has actually been shown that it can survive in, composted manure piles for a little while. That's relevant to, I guess, just environmental management. It's a facultative intracellular pathogen, which is relevant to, you know, treatment options, which aren't very good.
It colonises mucosal sites, plus and minus clinical disease. This is an important one because, mycoplasma can hang out in mucosal sites like upper nose, in vaginas, in around mucous membranes, around the eyes, without causing disease, so that's one of the tricky parts about identifying. Animals that are infected or colonised, depends on which way you want to call it.
And it's also capable of hematogenous spread, which means getting the bloodstream and spreading throughout the host. You'll see that because, for example, when calves are fed mycoplasmic contaminated milk, it will disseminate through the body and you'll see infections of joints, tendons, she, middle ears, lungs, even can cause, meningitis. So it does spread around the body.
There is a bunch of different species of Mycoplasma, there's over 100 species, there's less that have been associated with disease in cattle, the big one historically was Mycoplasma mycoide subspecies, Mycoidia small colony type or contagious bovine pleura pneumonia, the big one at the that's actually been eradicated from Australia and New Zealand, mycoplasma bobus I guess is the topical one at the moment, it's quite pathogenic, but there are several others and others have. Associated with disease, in Australia, there is a related organism called achola plasma, which are important because if you're doing culture as your diagnostic, they will grow on the same media and could be considered, you know, be read as a false positive. This is a perspective and my, my experience comes from working with herds in North America and in Australia, and I'm going to, I guess draw on that to provide that perspective.
Gave you the history that myco mycoides has been eradicated. In 1970, there is a, a report in the Australian literature of seven sew groups identified. In this report, one of those isolets from milk was mycoplasma bovis.
It sort of disappears off the radar for a long time. There is a report of mycoplasma alkaline in 1980s, and then mycoplasma Group 7, which is now called Mycoplasma lii, in the 1980s. And then about 2006, we were involved or saw a number of herds with mycoplasma fivers, and we also, some of the herds had dual infections with Californicum, and then we did a series of studies where we found a few other things like Boviginan, Canadans, Arginnini and Boba Rhinos.
So what's the sort of scenario that I'm referring to? Well, if you look at the literate in Australia in 1999, there was a PCR that reported a high prevalence of mycoplasma bus. Subsequently, there's this, this, PCR was questioned that it was actually amplifying, genes, well, genes that weren't mycoplasma and so it's sort of I guess been discounted.
We saw clinical disease outbreaks in 2006 and then there was a couple of surveys using PCR in 2010 and 2012, which suggested the prevalence was pretty low, down between 0.1 and 3.5.
These are probably underestimates because we know now that the frequency of shedding, In the bulk tank milk is actually relatively low, even in infected herds, and so this will probably, well, it will be an underestimation of the true prevalence, which hasn't been sort of re-evaluated now with Other tools yet in Australia. So what happened when this emergence in 2006-2007 occurred? So the disease outbreak in the first identified herds started in heifers that were purchased from a dispersal sale.
Five of the first seven herds identified 1000 plus cows, which was a lot larger than the herd average. So initially, the presentation was more skewed towards larger herds. 3 of the herds that were initially affected were expanding and had recently purchased cows, and I believe some of them actually might have purchased cows from the first herd that we referred to there, .
Two of the 1st 5 herds shared the same service providers, that was a veterinary service which was involved me, an AI technician who was, breeding across the farms and milking machine technicians, and I guess this raised a question in my mind that, of the possibility that service providers could play a role, In transmitting between herds because the, the second herd had not purchased any cattle, and at that time, and, and so the question was where, where did it come from and, and that was the only thing I could see that was common to the, to those two herds. And, and it was a freak like every day, breeding, for example, were there going between herds, so that was a possibility, but yeah, it's not proven. 3 of the 1st 5 affected herds experienced 10+% loss of milking cows.
So just to give you a sense of magnitude, And there were some herds early on that actually lost more than that, like 20+%. And one of the issues that occurred early on was that There wasn't much experience in Australia with mycoplasma. It really hadn't been an issue, and people, well, the vet profession wasn't aware of it and wasn't tuned into diagnosing it.
So, there was a number of these early herds that, were experiencing significant mastitis outbreaks, and they were accumulating infected cows in their hospital herds, and they were doing a bunch of cultures, coming back with no growth, and And struggling. And, and then they would end up putting cows on a truck because they wouldn't respond to treatment. And, this went on for, I think for one of them, it was actually 6 months.
And, and then they got a diagnosis. Once a diagnosis is in play, things come around relatively quickly. So clinical presentations, so a mastitis with increased incidence of clinical mastitis, often multiple quarters affected, non-responsive to treatment, and others appear full of milk, very little is produced.
This sort of pattern is, is really, you know, if you have multiple quarters not responding to treatment, others are, are feeling appear full, that that really needs to ring some alarm bells that or or certainly, Have you thinking of the post of mycoplasma, because as I said, you really need to have that consideration because if you just do a regular culture, it won't show up, you need to specifically ask for mycoplasma to be cultured for. So the magnitude of outbreaks are exacerbated by diagnostic failure, poor milking hygiene's a risk, poor treatment hygiene's a risk, and then management of fresh and hospital cows as a single group is a risk. So if you milk your fresh cows with your mastitis cows, you're basically putting the most vulnerable animal with the highest, you know, risk of infection animal, and it's a good recipe to have a, a problem.
The clinical cases in this scenario were cold. Another presentation that wasn't as common, on most farms, but then occasionally you'd have it as, as the problem was lameness. So this was a primary presentation less commonly, but on some farms, and animals would often present with multiple joints affected, and you could have three legs affected, and multiple joints on the same leg affected.
It causes an arthritisinosynovitis, so a lot of the inflammation. Is actually in the tendencies around the joint, so in this picture here we can see You get this yellow gelatinous material around the the tendons, the cartilage doesn't look too bad, and the whole, limbs inflamed, and it tracks up and down those tendencies, so you sort of get this diffuse swelling. These, these animals are often very painful.
Sometimes they'll actually get to the point where they'll be recumbent and they have to be shot. And people would, you know, they, they generally would be cold, but sometimes what they would do is treat them with like a macrolide and then withhold them, and they become a beef cow because you can't use the macrolides in milking cows. More common in heifers than cows, that was, that still holds true.
Often observed shortly after calving. And outbreaks often appeared to be associated with environmental stresses, so mud, crowding, excessive time standing, and I wonder if they had issues with heifers, they had slippery concrete, and the heifers were slipping and dropping on their knees, in the holding yard, and, every time they did that, then they'd end up blowing up a joint, the next thing you know it would have mic, so trauma seemed to be a, a risk as well. Again, clinical cases cold.
There are some less common clinical presentations. There is, this is infrequent, this facial swelling, but it's been about 3 areas I'm aware of that are reported this phenomena of, swelling of the face, and, that you, it may, usually it's not the only sign you see on the farm. You might have big legs, mastitis, and this, but it's a little bit of an odd one, but, that's.
Appears to occur in some herds. And then abortion can also be observed. And this seems to be more of an issue when, you have an outbreak in dry cows.
So that's a fairly rare event to have an outbreak in dry cows, but it, we have seen it in Australia and there are reports out of the US and, in the Australian scenario, what happened was, The herd was milking their cows. One of the neighbour cows got into the herd. They ended up milking it for a little while, and then they dried off their cows, because the cow got into the herd just before dry off.
And then shortly after dry off, they had cows with bad mastitis and going down, aborting. And it was a bit of a train wreck for them, and some of the cows died. And, and it ended up being mycoplasma.
And The thought that's come out of the US was that that actually the process of administering intramammary tubes in an infected herd when you've got mycoplasma around. Is that you actually can end up transmitting it between cows, and I've seen sort of how this could occur, when if you go to a dairy in the morning when it's, you know, the light's just coming in and people strip the cows onto the platform. If you've got the light in the right angle, you'll see a plume of, of sort of mists come up where the milk hits the platform and then there's a little bit of plume of aerosol that comes up.
So, what, what people sometimes do is they go, OK, we're going to dry off these cows, and they'll put all the tubes on the platform, and then they go ahead and strip the cows. And if you've got a microplasma amongst that group, you can get actually contamination all over everything. So it's really associated with actually an introduction at the time of dry off.
And one of the sort of, control methods that I was advised of when I was working in the States was actually to discontinue intramammary therapy in the midst of the outbreak because of concern that you in you effectively transmit mycoplasma from a mycoplasma cow to a cow that might be presenting for strep humerus or some other type of mastitis through basically inserting it when you put the tubes in. The other group that's often commonly affected are calves. And, the issue here is you've got mycoplasma in the milk.
And if you're feeding mascidic milk to calves, well, then you feed it to the calf and it can invade the tonsils and disseminate through the calf, so you will have animals die. You'll see pneumonia, tino synovitis, that's said, arthritis, tinosynovitis. Oitis Mur in turner, which middle ear infections, and then They can get decubital ulcers.
That's less common. So what's the risk? The risk is feeding unpasteurized colostrum and waste milk.
The waste milk would be a bigger risk than, the colostrum, but if you pull colostrum, that increases the risk a lot. So if you have, a cow that's infected at the time that she calves, and that does occur occasionally, And she's shedding mycoplasma in her colostrum. If you feed that colostrum to 1 or 2 calves, while those, you know, 1 or 2 calves get infected.
If you pull the colostrum from 5 cows and then feed it to, you know, say 10 cows or more, at 10 calves, then you're going to infect more calves. So it's recommended you don't pull colostrum. Waste milk obviously can be highly contaminated.
Infected cows can shed up to 106 per mL. So, even if you dilute it, you know, 10-fold, it's still 10 per mL, . Mixing infected and uninfected stock, so it, it does colonise the tonsils, and if, one of the things that's been associated with increased risk of pneumonia and feedlots is common access to water troughs, and I have, I can't prove it, but I suspect that from mycoplasma contents, if it, if animals slobber in the water and that's contaminated the mycoplasma, then potentially you could see how it could move across from things.
So aerosol, there is respiratory spreading calf bars, there's fun mite, so you get it on equipment like tits and so forth, water and feed troughs. So when, when this sort of all blew up, a lot of questions, you know, were asked by farmers and, you know, where did it come from? How do I control the disease?
How do I treat affected cows and cows? What should I do with affected animals? And that was a particular reference to he replacement.
Should I cull them or, you know, or should I keep them as replacements? And, and often, when it's things are not going well, it was a very keen interest to eradicate the disease. All good questions, but some of them are a bit tricky.
So, detection of infected animals and herds. So what tools do we have available? There's culture, there's PCR and serology.
So we can either try to grow the organism, we can look for, DNA from the organism, or we can look for the animal's response. To the organism, that's effectively what we're trying to do there. And when we started this, in Australia we were using culture, we, did use Paterproof, which is a commercial PCR and we also developed some, species-specific PCRs to look at, you know, the, the strains that came back that weren't microplasma boss, plus we also developed the biour one.
And then at that time, the bios, Eliza was available, so we, we utilised that particular elizer. The initial strategy that we adopted was identify clinical cases in Coha. Managed cow grouping, avoiding the fresh cows in sick pens, having a lot of focus on milking procedures routines, so minimal stripping, wearing gloves, sanitising hands between cows.
If you've got a hospital string that's long, more than the number of units, and you have to go through twice, making sure you, you cluster dunk or just just decontaminate clusters between cows, and we would, initially avoid intra-memory treatment, pending the, you know, the cultures of all the animals and removal of infected animals. And once we got on top of it and things settled down again, and we thought we were in the clear, then we would, we'd go back to using intra memories. We'd also look at preventing transmission from cows to calves, so pasteurise the milk, if possible, pasteurised Clostrum, you have to have two different units for that.
So, it usually comes down to the cost and, and the size of the operation and whether that's viable to have. You know, costum pasteurizer and milk pasteurizer, or, or whether they would just not pull Calostrom and feed powdered milk instead. The other thing is to look for and manage environmental stresses.
So, in this regard, you know, nutrition, so making sure your transition re is going OK, comfort and cow time. So, if, if you've got cows standing around on concrete like you, or they're, they're working hard way through lots of mud, there's probably going to be more risk of like that lameness for, for more manifestation of the disease. In the beginning, we did a couple of whole herd cultures, but we Didn't find we were getting a lot, so we tended to go away from that and focus more on the clinical cases, and I'll talk a little bit more about that in a sec.
So what happened to herds, here's an example of a herd. So the initial outbreak, there was, 87 cases, 35 of those were in heifers average, Jay milk for heifers was 44, for cows was 96. Then they go along and and basically cull all the affected animals and continue monitoring all clinical mastitis, and we had the occasional one over time, not too many.
And then we had this little mini cluster again. Which is interestingly. 3 years down the track, which it's a little bit longer than 3 years.
So this was interesting because this was predominantly in heifers, so 36 of 44 cases were heifers, and average days in Mel for heifers was 23 versus the cows was 106. So what was interesting about this is when this initial outbreak occurred, the calves were being fed colostrum and waste milk without a pasteurizer. So these animals are cold, pasteurise was installed.
And we were looking to think, well, OK, 2 years' time when they carve, you know, we might get a flare up with these heifers that were fed that product. Becoming clinical and it didn't happen. But then down the track, we have heifers coming up with disease.
This is a year-round calving system, so you have young stock in the system all the time. So I suspect what's happening there is actually it's the aerosol transmission that just is, you know, going across the different groups. But most of the time, really not much was happening, and actually subsequent to this, there's been no more, no more outbreaks per se, occasional sporadic case but nothing significant on this particular farm.
This really raised to me the question of the, the question of host, pathogen and environment interactions. It doesn't appear to be as simple as just a host and a pathogen. There's something that seems to shift that balance between the two.
So, the additional questions well how can you prevent herds from, from becoming infected, i.e. By security.
And then the industry sort of was getting concerned because more herds were becoming infected and some of those herds were experiencing significant losses. So the question, so it really then was about disseminating existing knowledge because there is quite a lot of existing knowledge about mycoplasma. Regarding the, the disease and disease management, and, and so we ended up doing a bunch of talks and webinars and things to, to sort of get more information out there so more people, knew about it and could help different, you know, farms that were affected.
And then we set about to do some research, trying to answer a few questions. So, the studies that we've ended up doing, we looked at genetic characteristics of the organs, we've also looked at bulk tank serology for detection of infected herds. Looked at anatomical sites for detection of infected animals, looked at detection of subclinical infections, utilising herd test samples, looking at the consequences of exposure to mycoplasma, in young stock and then investigating the transmission role of bulls in infected herds.
Some of this has been published, so this is a bit about the genetics, and so we had isolates from 2006 through to 2015. And they also came across from multiple different states in Australia. And then we also selected islets from that we considered, you know, clinical, which was from joints, lungs, milk, lymph nodes, and then colonising which were from islets from the nose, from the penis or the proposed semen and vagina.
And then looked at the relatedness of the different organisms, and this is looking at single nude polymorphous and all the difference in those. This was looking at islets from New South Wales, Victoria, Queensland, Tasmania and South Australia, and they're actually disseminated the these, there was only 50 single nucle or snip differences across all the islets and the, Relatedness was distributed across the different states. And similarly, if we looked over time, they were distributed, the the the relationships were were tight.
And so the conclusion of that work was he had limited genetic differences across all Australian eyes it was 50 snips. The difference between the reference strain, which is BG 45 and the strand isolates was 318, and so the conclusion is that we really only have one strain or genotype circulating in Australia, and, and I didn't, So the graph, but also there was no difference between clinical and sub-clinical or the carrier sort of status. So it didn't look like disease reflected a change or a different strain of the organism.
It wasn't that the ones that were causing disease were one strain and the ones that were colonising were another, they were basically all the same. Just for comparison in Denmark in between 1981 and 2001, there was a 50 snips difference in the strain that they had, and that was only 3 steps different to PG 45. So it was basically the same organism as that one.
But then in 2011, they had a new genotype come in, which is 5000 steps different. So, and, and what was interesting is their 1981 to 2001 was quite virulent, and the one apparently in 2011 is less, less vile. So if our genetics are not different per se, then the question is, What is the driver for disease?
And I guess I'm coming back to this concept of the, the outcome is a trial between environment host and pathogen, and the environment can shift the, the benefit towards a host, where the host can deal with it and it can be subclinical. Alternatively, under adverse environmental conditions, the way it can go. Towards the pathogen or favouring the pathogen either by I guess survival of the organisms so that the challenges dose is greater or or alternatively by compromise to the host so that the host is less capable of dealing with it.
And that seems to be a little bit of a feature in that scenario, I showed you where the heifers that many outbreaks seemingly, they, they were having some issues with, ketosis and DAs and, and, so that may have been something that, you know, provided the, the background in which the pattern that was sort of not causing much of an issue, re-emerged. Looking at biosecurity for identification of infected herds, there's been obviously lots and lots of work done in New Zealand, and I think, well, they're definitely ahead of where we are, or where we, where this work was at. But I'll just give you some background.
So here we have a herd that, was milking a couple of 1000 cows. They've had, the, these bars represents a number of islets at that specific time. So they're having islets intermittently, and then they have a big, big cluster here.
So there's a many sort of outbreak scenario there. We started looking at serology of bulk tank milk in August of 2013, and then followed it through at different times for a while. And what was interesting in this scenario is that There's quite a lot of time where there's not much shedding occurring, and if you do cultures of the bulk tank milk, it comes back negative.
And then, obviously, the serology stays up longer than, the tent remains positive. So I guess that was the take home message was that, in herds that we, you know, we're working with, we would find that the, the presence of the organism in the bulk tank was infrequent. But yet, we still would have considered them an infected herd, and you would have this scenario of things popping up, you know, a year or so later.
So it hadn't left her guessing. So we did a little study looking at 192 bulk tank samples, which were derived from 19 infected herds and 6 non-infected herds and the significant variables at the end of the day was Time since start of carving was highly significant, so you're more likely to pick it up if it was closer to carving, and then time since outbreak, which is, makes sense sort of this right here, time since initial mycoplasma outbreak. They basically end up in the final model being the, the important variables to to predict whether you were going to pick it up.
And this sort of shows you some of the issues, and that is that, although serology was better, it, it it was accurate from 39% of the samples were Elyzer positive, as opposed to 24% PCR positive, 1% culture positives was in the infected herds. So you can see that most of the time the PCR and the culture were not going to identify the herd correctly. Even though the Eliza was better, it still was missing some.
We're missing a lot. And it did, it was influenced by when you sampled relative to the time since, started carving. We worked with herds that were seasonal split calving and also year-round, and this is just started from year round.
And this was the threshold for the test when they would be considered positive. And when we looked at you know, the distribution of Results for her, it did correctly classify the 6. Non-infected herds, and we, you know, non-infection's a good question, what is non-infected, in this case it was herds that we'd worked with for, For a long time where we'd never seen a clinical case, never seen any signs that were consistent with mycoplasma, and during our, you know, we don't, we didn't do lots of mastitis cultures on those farms, but we had done them as required, and we hadn't pulled up any mycoplasma from those farms, versus farms that we got involved with, which did have mycoplasma.
So, the negative herds were negative, but you can also see that positive herds, while frequently were positive, they were also negative as well. So the conclusion was The assay is designed to detect organism in bulk a detect an organism that's in the cultural PCR have a low sensitivity. I didn't point it out, but when we were looking and it, it was only sort of came in late.
If you looked at serology of the hospital milk, it generally was higher than the, bulk tank. So it may be more sensitive. And certainly, if you're doing cultures, of milk from a herd, You're better off to be sampling the hospital herd because mycoplasma cows are more likely to be in the hospital than in the bulk tank.
The BIOX, Eliza had improved sensitivity, and could have application as a screening test, however, sensitive is not high, and we required to completely evaluate specificity, i.e., we only looked at 6 negative hertz, so this was more of a sort of have a look to see if what possibilities were.
It it did imply that serology probably was going to be a better way for screening. Herds and using culture or PCR, but it wasn't a definitive, the definitive work, you could say. .
And the, the issue, I guess, for industry, even if you do, you know, several PCRs on a herd that you're considering buying stock from, you, you couldn't be sure that those animals, that herd was truly going to be negative. You would have to do repeated testing over quite a period of time, and you'd probably want to combine serology with, Culture and PCR, you know, the more testing you do, the more confident, you would be, but the practicality was not good. Just to give you a bit of an update, on what's out there now is, there's a couple, well now they're a lot, well, this is Western Blo, which is not a, a practical sort of test for most labs to be doing in, in large numbers, but there is the ID screenizer that came out subsequent to the work we did and, It has a sensory 93.5 as opposed to the BIOX one, which was 49.1 in this comparative study, so this is what I believe that New Zealand's using and it makes sense because it's a, it's basically a has a higher sensitivity than the BIOX test.
And its specificity is good as well, which is important. So, the other question is, how do I detect infected animals? So if you think about it from the farmer's perspective, they're going, well, I've got this problem, I want it gone.
How do I, you know, how do I go in and sample the animals and get rid of them? That was obviously an important question. You know, could, could we, you know, suggest that we can go and eradicate it from a farm?
And we had a little bit of a, an opportunistic scenario that occurred, and, This was, we had 16 cows on a farm that had clinical mastitis that had been diagnosed with the PCR about 7 to 13 days before we resampled them, so, we went in and took nasal eye, And vaginal swabs and the culture and PCR and also collective blood for serology, just to see, well, you know, if we have animals that aren't milking, is there another site that we could sample that might be useful for picking up some clinical infections, hoping that potentially it would provide us an option for non-lactating animals. The result was Basically suggested no, so this was the cycle threshold of the initial milks, and most of them were shedding a lot, the exception would be like the 36 and 32s, not so much on 36 there, but these are low cycle thresholds, which means there was lots of organism in the milk, . But when we looked at noses, none of the cows came up positive, none of the eyes came up positive for bovis, and then 3 of the 16 came up positive from the vagina, so none of the sites we checked here in cows that have been recently clinical.
Were picked up on alternate sites, which, you know, wasn't what we were hoping to find, but, it just basically suggested it's for animals that aren't lactating and where you can't get milk, those sites may not be always that rewarding. There may have been some limitation, well there was some limitation. I swabs were swabs, probably, 115 centimetre swabs, and the organism hangs out in the tonsils, so ideally it would be better, probably, you know, in retrospect to have had a longer swab.
Eliza, on the other hand, for the 16 animals, 15 of the 16 did come back positive. So the sensitivity for this cohort was reasonable. You, you would expect if an animal had a very recent infection, it may not be, you know, seropos, but what this doesn't evaluate is the specificity and the issue we have, done a lot of alliances and.
You will go into herds where they're no disease apparent, and you will have positive animals. So, on an individual cow level, I, I would be very, I wouldn't recommend going in and doing lisense on everyone and, and, and saying that, well, not with this test anyway, of saying that if they were EISer positive, that they are definitely infected, because that would be, you know, it wouldn't necessarily correct. So conclusions, low detection of M.
Bovis in nose, eyes, and vagina of 16 clinically infected cows with mastitis. Sampling these sites in non milking such individual subclinical cars is probably gonna be unrewarding. Serology had a better sensitivity individual animal level, however, especially for this particular lizer is, well, there's been that published, well, that was on bulk tanks, so again, it's not on individuals, so we, I can't really say.
The other question is how do you detect subclinical infection and We were interested in really the scenarios where you've had an outbreak, you've gone in, you've, Cleaned up the hospital herd, which is really important in mycoplasma herd. If you don't clean up the hospital, then it'll just keep spreading and you'll just in keep infecting more cows as they come in for, you know, massages from other causes or lameness or matritis, you'll end up just, accumulating if you mix them with the fresh cows, that will really, You know, exacerbate the problem. Let's say you've cleaned up the hospital herd, and then the question is, well, what's, what else is going on out there?
You know, we've got others in the herd, we want to get rid of this and get it settled down. So, we, as I mentioned, we did do some herd whole herd cultures, and it's, it was quite a lot of work, it's quite a lot of expense, and we didn't see a lot of animals coming in, so we thought, well, it's an alternative way. And, and what we were interested in was, Being able to utilise her test samples because, obviously that would take a sample that was already collected.
It's obviously a risk because of her test sample is not a sterile sample on the, It's not collected aseptically from each cow and, There could be carryover of milk from one cow. To, to, to the next cow through the sampling equipment, so, we tried to design it accordingly. To, to mitigate that risk.
So, we had 4 mycoplasma bovis infected herds. And then when we started talking about, you know, how to sample them, how, how to do the pools, and, you know, whether we look at so, you know, some people were looking at somatic cell count and saying, well, just sample the high somatic cell count one so we were sort of saying, well, is that, is that reliable? Because we're a little concerned that it may not be.
Anyway, so we'll go through what we did, so. You know, a milk sample from every cow, we did individual culture, and then we pulled samples, for PCR. So the, these were pulled by milking bale, on the rotary or milking cluster in the herring bones.
So effectively, if there was carryover, we wanted to, not contaminate our pools across. Bales or clusters, because then it would be really difficult to interpret. So we kept track of milking water, so we knew the order that cows came in and, and then we pulled by bail.
So the idea was if a if a bail came up positive, we could then go back to the individuals and we could, we would know the orders. So, let's say the first, You know, they say the 10th count was positive, and then the next 2 were positive, it would, it would be then easier to interpret, you know, how much carryover was an issue, and, and make it easier in the future to interpret what was going on. We also had somatic cell count data for these cows.
And, it also been a bulk tank milk sample on for culture on PCR. So, Number of cows, 497 in the 1st, 475, 816, 444, for example, number of initial MO PCR positive pools, so only one in these three and none in herd number 2. Number micro bovis, PCR positive cows, so it's only 1 cow in this 3rd and 1 cow in this herd.
This was in the main milking group, OK. Then we had our hospital groups and there was a mycoplasma bovis not suspected, so, the number of cows in the pool was 55 and 19, and it was not detected. And then number of cows is suspected, so they separated some animals that they suspected may have mycoplasia, there's, 2 here, none of them can positive.
13 here, 5 of those were positive. This illustrates a point, and that is that once herds have experience with mycoplasma, they actually tend to get pretty good at picking. In mycoplasma infected cows.
So sometimes our experience was that we would find that they would, we would go ahead, do cultures or PCRs for people, and then we'd say, Oh, we've got the result, it's positive. They'd say, Oh yeah, she's, she's over here in the cult paddock. Or they'd already separated them out.
So, and that's one of the things that can be a problem for some herds too, because if you've got a larger herd, where there's quite a lot of employed labour, and there's turnover of labour, if, if you have, loss of corporate memory, that can actually be a contributed to recurrence of mycoplasma. Because people who have experience with it, who know what to do with it, who recognise it, pull it out, manage it, if they turn over, then the next crew is not familiar with it, you go back to where you started. So the follow up on these herds, herd one monthly bulk tank sample, MIOS PCR negative for 12 months.
So, they obviously got rid of the positive animal. Herd 2, no, new clinical mysvis case 7 months later, her 3, was positive 9 months later, and her 4, No future evidence of any MO's cases for 3 years, so I wouldn't say it was negative, like the herds, you know, cleared it's possible, but it, That that's, I guess the problem is, once it, once mycoplasma infected, because it can colonise without signs, and it can go fairly long periods of time without showing up, it's hard to make that call to say I heard is or is not infected anymore, even if there's no disease. So conclusion for this one was detection of subclinical infection using PCRL culture post outbreak can be unrewarding.
So what we mean by that is that you, you know, you can spend quite a lot of money for not all that much yield. So we've sort of, we do reckon, you know, people approach us about Investigating multiplasma and herds, we really focus on cleaning up the hospital, focus on your clinical infections, focus on your milking routines, focus on grouping of cows, focus on calf milk. And that does seem to work reasonably well.
Chasing subclinicals, you know, expensive, and, if you've got good people in the parlour who are tuned into what microplas looks like, they'll usually, the cows that you need will be in the hospital. We didn't have enough data, enough cases to really look at somatic cell count as a predictor. Now the question was, what's the role of bulls?
So we followed some bulls through some in infected herds, and you didn't really know what the prevalence was going to be either. So this has been published also. So we had 5 seasonal calving mycoplasma bovis infected herds.
There was 150 bulls that were managed in a central facility in the offseason and during the breeding period. 88 weeks of AI followed by a bull breeding period of 12 to 17 days. So we did a pre and post-joining assessment, the pre-party involved a breeding soundness evaluation, semen and swab culture and PCR in serology.
So The semen or propecial swab pre-breeding, didn't find any mycoplasma virs. I guess the important point here is there is other mycoplasma, particularly mycoplasmavigenic alium, which is quite common in bulls, and if you're just doing culture and you don't have a way to speciate, well then you would, Think that you had lots of, well, you would have lots of like plasma but it's not boverse so it is important to identify what species. You also get a coral plasma, so that's kind of found, confound culture andless you speciate because on a plate they can look the same as.
Is mycoplasma. Post-breeding, there was 4 balls out of 118 at that point. That came up with mycoplasma.
So it is possible for bulls to become, colonised. It was interesting, these bulls also sero converted and and it was quite significant. So number of bulls sample pre-breeding is here.
Erro prevalence average is 8.5. Cerro prevalence post-breeding on like this farm here, it went up to 69%.
This is a number of all with sero negative pre-breeding, so that's sero conversion, . So we, we got up on, you know, the sort of conversion instance, 40, 40, 65, 44. So what was interesting about this is that none of these bulls looked sick or got sick per se, but a lot of bulls sort of converted, so I guess I take home point from that is that there's a lot of dissemination of mycoplasma in an infected herd.
That isn't occurring via milk as well, so presumably that's occurring by aerosol, no, I guess that's what I would assume, . You know, the fact it wasn't common venere either there was obviously a small amount but not enough to explain this sort of conversion, so I think the clinical signs of micropas are the tip of the iceberg. So conclusion, bulls can become colonised.
We haven't got data as for how long they will be colonised, and, for how long they'll shed in semen. Subclinical infections were common. Transmission occurring through pathways of the milking, and then microplasmbo genitalium, the most frequent species isolated from the prebulls.
And then, this is our last question was what about heifer replacements and con which have been exposed to microplasma contaminated milk, cos, As, you know, if you're going to keep them with replacements, what's the risk for your herd, and then if you sell them, what's the risk for other people? So there's been several studies looking at shedding, between birth and weaning, but there hasn't really been that longitudinal one through to when they carve. And so we were interested in that longer term one of, OK, what's important is, is, you know, are they going to break down at carving?
Are we gonna have a, you know, a problem there? So this was a longitudinal study, heifers exposed to microplasmophobic contaminated milk, 9 herds, 1 control. 50 heifers per herd, we had autumn born, spring born, central heiferrearing facilities, so sample time points were post-weaning, pre-breeding, pre-carving, calving, and post calving.
And the samples were I know, vagina, lostrum milk, and then obviously blood for serology. So this is the autumn 250 cars, autumn 250 springs and following them through, that's post-weaning. So the mycoplasma bovis is the red ones, so here.
And then you notice we, as we get older, There's less red. And then we get to post-carving, and there was one. That was isolated from the nose, and one heifer post-carving, but we did not see, any disease in this cluster, you know, after carving, and there wasn't shedding in, lostrum post-calving.
So, I guess this was more like that scenario I showed you early on with the herd that where they'd fed them, the, the contaminated product, and those heifers didn't break with disease. These didn't break with disease. Not to say that heifers can't break with disease, because we've also seen that happen too, but, the message, I guess, and we've, we generally, you know, people have in Australia, where it's, we're not eradicating the disease, if people have exposed heifers, we would usually, that some will die, some they'll treat successfully, some will treat the poor doers and then you get rid of those.
The other ones that look fine and are growing, we generally recommend that they hang on to them. The ELISA results, they were sero positive or high proportion were seroposit, post-weaning, that declined pre-weaning. Further decline pre-carving, then you get a slight rise post carving, so there, there might have been a little bit more there than we picked up on cultures, that's probably likely because the sensitivity of our methods would be somewhat limited.
So minimal detection of persistence of MOvis colonisation, isolation decreases with increasing age. Antibodies just declined with increasing age until carbon, and there was a slight rise, we might have had some limitations with our sampling site or detection methods, it's possible that we missed some, . Yeah, the heifers exposed to microbes prior to when he did not break with disease calving, but we also have evidence that, Exposed heifers represent a potential source of infection to non-infected farms, i.e., even though there wasn't a lot, there was still one for sure, that, was still colonised, even though it didn't look sick, didn't show signs of disease.
And this is really the kicker, this is really the issue from a biosecurity perspective. If you move animals, even if they look well, from an infected herd, then you could potentially disseminate the organism to other farms. So the biosecurity risks, definitely livestock, colostrum milk.
So, you know, moving colostrum or milk between farms would be a risk. And then obviously within a farm, they can be a risk in an infected herd. Biological products could be a risk that we didn't prove it, but we did have a question as to service providers potentially being a conduit for infection between two herds, .
I, I, I haven't got any data on manure spreading or effluent disposal across properties, but there is literatures say it can survive for a period of time and . In manure piles, so that would probably be a risky thing to do between farms. I've mentioned this service for biological products, but I guess I was getting it was, we, we know that it can be, can get into semen.
It obviously it's not a common event to be a problem in frozen semen. There is antibiotics in frozen semen. Presumably bulls are tested for mycoplasma before they freeze it as well, but Yeah, I guess it would not be impossible per se.
Service providers, I've mentioned already, I think that is it. So thank you for the invitation to to present at the meeting, disappointing that we've all had to stay home, and, and all the best. Thank you.