Hi everyone, and thank you very much for joining us to listen to how to investigate and treat hepatopathies and equine patients. I know it's a bit of an odd time, but thankfully with things like the webinar that we're able to crack on and keep the CPD flowing. So this this evening we're going to take a bit of a, a round trip around how first of all to undertake the clinical investigation.

And that's gonna range from what the clinical signs are, most of us know them, but what they actually mean for the patient. The blood tests, how to interpret them, and again what that prognosis might be. Is ultrasound really relevant, and I'm sorry, that's all spelled a little bit wrong there.

And biopsies, how do we interpret them? So really what we need to know is, does the horse really have a hepatopathy? Can we confirm it, and if so, what is the prognosis and how are we going to treat it and all these steps will get us to that final decision making treat.

So, what are the clinical signs? You know, we're talking about horses with true hepatic dysfunction on this slide, and thanks to Andy and Victoria for the pictures. But the main signs of those horses with a marked dull demeanour, lethargy, inappetence, weight loss, photosensitization, particularly over the white areas of the skin, not, not the coloured areas.

Hepatic encephalopathy and jaundice. Some of the more uncommon clinical signs can be colic, edoema and coagulopathies as the liver produces the majority of the clotting factors within the horse. But in these cases, however, adequate hepatic function often persists despite the presence of mild to moderate degree of liver disease, leading to subclinical hepatopathies, for which the diagnosis can require further diagnostic procedures.

It has been suggested that approximately 60% of the liver requires sorry, 60% of the liver function is required to be removed before you start to see functional liver failure and precipitation of clinical signs. This implies that clinically normal subjects may well be suffering from liver disease, and also that the presence of clinical signs of hepatic dysfunction suggests a considerable degree of liver pathology. So what does this mean in the real world?

In many cases, what it means is that there'll be very mild or non-specific signs in those sources, or even none. And quite often, blood work will pick up liver disease as an incidental finding, rather than the, the initial thought of the vet involved. One small note of caution is that horses in quite intense race training will have elevated GGTs quite frequently.

Nobody really knows why that is the case, but what is important is that you don't dive into those cases and get a liver biopsy as soon as you possibly can, because most of the time it's not warranted. So are the clinical signs prognostic? .

Andy did a whole set of three different papers, and we're going to go through each of one of those at different stages of this lecture. In about 2003, they're the most sort of up to date on the whole big picture of liver disease. But in clinical scoring, he said that found that 18% of his 81 horses that he looked at, which had both bloods and biopsy as well as clinical presentation, had a score of 0.

We're going to go into exactly what that means in a moment, i.e., normal.

54% were mild and non-specific. 23% were mild to moderate and only 5% were severe, and all of these had marked abnormalities on their histopathology. How was that performed though?

So grade 0, they're completely normal, absolutely no indication of any problems at all systemically. Grade 1 was mild and non-specific, where the clinician really did not suspect that liver disease was involved in the case. Grade 2 was where they had a tentative diagnosis but did not feel committed to liver disease, and grade 3 was confident of the clinical diagnosis.

In that grade 2 and grade 3, other than the clinical signs we've already mentioned, they included pruritus, PUPD corinitis. And then for every single case, whether it was mild or severe, they recorded whether hepatic encephalopathy was involved, if it was positive or negative. And that could include a marked, a number of, sorry, different clinical signs, including marked depression, confusion, disorientation, ataxia, blindness, head pressing, all the rest.

But from my anecdotal experience, and we see quite a few of these that live in most often Little Shetlands, we see a lot of bilateral laryngeal paralysis, particularly in the smaller horses. Also, I see a lot of head pressing and the persistent walking. For me, the persistent walking seems to be a very pathonemonic clinical sign.

And by that I mean they walk nonstop in the stable. They'll often walk through you, and just, there is absolutely no way anyone can make them stop walking. What's important here as well though is that they found that 40% of those horses presenting with hepatic encephalopathy, so 6 out of 15 of them, did survive beyond the six month window that they used as their cut off.

So just because there are severe clinical signs does not mean we should be considering euthanasia in all of these cases. So do clinical signs act as a prognostic marker? I've already mentioned that hepatic encephalopathy, you know, a number of them survive.

But what they found was that those with grades 0 and 1 had a very good success rate beyond 6 months. As soon as you start getting into the mild to moderate, you start to see quite a significant drop, and then those in severe clinical signs, grade 3, only 14% of the cases did survive beyond the six month period. The Kaplan Meyer sort of just really demonstrates how rapidly they dropped off as well, with a number of them dying within the first few days of, of diagnosis.

What can we summarise from the clinical markers, clinical symptoms, sorry. Firstly, that clinically normal horses may well have significant hepatic disease. And so if you do find those blood works that are abnormal, it's worth further investigation to ensure that you're not missing the early window to treat them.

And when signs of hepatic insufficiency are present in that grade 3 window, then the, the prognosis is guided to poor. But there are a number that will survive, especially, especially with hepatic encephalopathy. And therefore, treatment is warranted if the owners are willing, as long as the horse is not in an ethically compromised situation.

But what about haematology and biochemistry? We all use our blood work as a very good way of diagnosing liver disease, and often pick things up as well. But can we truly rely on whether these blood works do tell us what the prognosis is, of course is, and we have historically relied on blood work to do so.

What Andy found was that there was a significantly increased risk of non-survival in association with AP GGT, bioassice, globulins, red cells, and white cells. And they were specifically at the values mentioned on the slide here. Anything less than that, and we didn't really see any association with non-survival.

Sometimes there was a leaning towards non-survival, say in the GGT in that slightly 2 to 3, 400 range, I think it was. But it was not statistically significant. Also increased AST which I haven't mentioned on the site in GLDH, were also a good diagnostic value, but only when used in combination with the above test.

So alone, they didn't really tell us much, but they really did indicate a bit more when linked together. However, no single combination or sequential test was able to dis discriminate fully between horses with and without biopsy confirmed liver disease. And therefore reliance on the use of non-invasive tests for the prediction of the presence or absence of significant liver disease may lead to frequent diagnostic errors.

Although certain positive results did reliably predict the presence of liver disease, negative tests in themselves were invariably poor predictors of the absence of liver. So in other words, caution should always be used when looking at our blood work and deciding what and how we should progress with the clinical case. Interestingly, one very sensitive marker was albumin, when they found that when it was less than 30 grammes per litre, and in combination with the other clinical, signs and values was consistent with quite severe liver disease.

It should be noted though that albumin alone or albumin with mild changes on the others should be interpreted with caution, because it is seen in a high number of other diseases in courses such as protein losing enteropathies, nephropathies, and lack of nutrition. But having a little look at some more detail on, on these blood works. What they found was that GGT and AP raised together without many others, was often linked with biliary hyperplasia, whereas AST and GLDH was often hepatocellular in origin.

Excuse me. They all, the other thing to note, and this is, not from the study, but the half-life of both GGT and GLDH are very, very different, and I think it's important in being able to interpret your results and also guide the owners how treatment is going. GDT's Half-Life is approximately 30 days, whereas GLDH is approximately 7 days.

So in those cases where you have an acute insult, you should have both elevated. But as you move forward and if treatment is successful, it may well be that your GLDH is returned to normal or very nearly normal, whereas your GGT is taking a long time to come down. And that doesn't, that means you're having success, even if it doesn't look fantastic.

Because if you think, if you have a 1400 GDT which isn't out of the question in some of the acute and severe cases, that may take a month to come down to 700 and then 350 the month afterwards. So don't be too dismissive of a, a bad result. But what we can see is that when we look at some of the individual tests, where sensitivity is the percentage of disease horses with an abnormal result, and specificity is the percentage of normal horses with a normal result.

You can see that GGT is very sensitive, so it's very easily picked up in horses with abnormal results, but it's not very specific. Whereas when we move down, albumin is very specific. As is total bile acids and total bilirubin.

And what I'm trying to bring to the forefront here is that no one test on its own is useful. You have to look at everything in combination so that you can be sure you're getting the right answer. So in summary on biochemistry and haematology.

Blood results are often diagnostic, and they are sometimes useful for prognostic guidance, but are frequently misleading, so care should be taken. The other thing to note is that the thought behind the red blood cells, elevation that I mentioned earlier is that often these severely affected horses, those ones with patic encephalopathy, are dehydrated concurrently, and the white blood cells is that there is often an ongoing inflammatory state within that liver and therefore system as well. So I think it's important also to just say, well, don't always assume that because it looks like liver disease, it is definitively liver disease.

So this is a case of hepatic encephalopathy. And what we can see is that the, sorry, this is a case that looks very much like hepatic encephalopathy, and what we can see is that the normal behaviours we mentioned earlier, dull demeanour, lethargy, blindness, ataxia, are all present in this horse. It's a yearling, and so it should be pretty bright, and it's a quarter horse, but they are, they are still meant to be bright in this.

You can see that it walks straight through the cone, blind and doesn't give a damn that it's absolutely tangled up in the cone. Intestinal hyperiminemia, as this case was, is fairly infrequent but still present out there. And it is often secondary to diarrhoea, they think it's secondary to Clostridium or delii.

The diagnosis can only be made on on ammonia, and it's quite a good point to mention that ammonia is very, very useful as a diagnostic tool in our liver diseases. But the problem is that it is very unstable. It only lasts 20 to 30 minutes, and the longer you hold on to it, the higher the value goes.

So if you want a definitive yes, then just hold on to it for 24 hours and submit it to us. Otherwise, get to a clinic within 20 to 30 minutes. Treatment in these cases is with hypertonic and lactulose, which will come onto it a little bit, as well as metronidazole for the suspected clostrid infection.

So, ultrasound examination of the liver. It's a process that is very frequently undertaken, and I think does play a significant role in our cases. But Andy found that the majority of ultrasonographic abnormalities detected in their study were non-specific and subjective.

Nevertheless, horses in which abnormalities were found were almost 4 times less likely to survive compared with those whose whose livers were judged ultrasonographically normal. However, the finding that 58% of their horses, which is 14 out of 24 with ultra sonographic abnormalities, survived beyond the 6 month limit, really does limit the clinical application of the findings in the individual case. But what are you looking for?

In those cases where you are doing an ultrasound, you want to look for generalised hyperecogenicity, hepatic atrophy, atrophy, patomegaly. So for the size of the liver, we are normally assessing it in regards to the costochondral junction. And so I would expect them live to be well short of the costochondral junction on the right hand side.

If you're getting close, then you're starting to get concerned. Dilate blood vessels, as you can imagine, is very, very subjective, focal anechoic areas and rounded liver margins. Remember when you are scanning the edge of the liver that it is very easy to subjectively make it rounded, so keep scanning and keep looking and make sure you are absolutely consistent about it.

But which histopathological features correlated with the changes on the ultrasound. They found that moderate fibrosis did, severe fibrosis didn't in their study, but they attributed that to the fact that they only had 2 cases with severe fibrosis and therefore deemed that it's more likely it is also increased in severe fibrosis does cause ultrasound abnormalities. Severe hemosiderosis as well as moderate to severe biliary hyperplasia all cause abnormalities within your ultrasound.

But when to perform a liver biopsy is obviously a really good question. A blood screen will only detect liver insult, but it won't tell you whether a horse definitively has or has not got liver disease. How to treat it or what the prognosis is.

The only way to do this is to use a liver biopsy. In short, you'd perform a liver biopsy if you had clear clinical signs or on the basis of blood work. When the clinical signs are present, you can be sure that there is, or fairly sure that there is liver disease.

But you can't tell how severe it is or what the prognosis is. It could range from a cirrhotic end stage liver all the way through to an acute and severe liver acute onset hepatopathy, which actually often respond very well to treatment. Equally, if the horse just looks a little under the weather and you run a few blood tests, you can usually form suspicion of liver disease.

But again, you wouldn't know what the prognosis or treatment might be. So, as I've mentioned, biopsy will confirm the presence of liver disease. It will guide your treatment selection, also offer a very good estimate of prognosis.

For me, it is a minimally invasive treatment, diagnostic, with relatively low risks when performed under ultrasound guidance and in the hands of somebody who knows what they're doing. But just remember that those if you find one case of liver disease on the yard, it probably is the tip of the iceberg. So it's best to start testing the rest of the herd mates and you can do that with either just a GGT or ideally a full hepatic screen in each of those cases.

But why not to perform a liver a liver biopsy? Hepatic lipidosis theoretically can cause the liver to rupture when they are so full of fat that they're bulging against the liver capsule. And passing the needle through it may cause a rupture.

Now, that's something that's that's linked with human rather than horses. I haven't yet seen a case of hepatic libiosis that was that severe, without knowing that I'm treating the, the triglycerides anyway. More though, we're looking at colic because there are two specific types of colic that are associated with transient liver insults, and transients the important part.

If we were to see a liver enzyme elevation on blood tests following about a colic, I definitely wouldn't perform a liver biopsy for at least 2 to 3 weeks, beyond that, that colic. And that's because when we look at two types of colic, firstly, proximal enteritis. Those with proximalentritis were 12 times more likely to have an elevated GGT compared to those with small intestinal obstruction.

We don't see much proximalentritis in the UK, but it is definitely an occurrence, and we do see a few of them in the hospital every year, so it is something to think about. More significantly though, colon displacements, particularly a right dorsal displacement, the large colon, was often associated with elevations in GGT. In these cases, it's thought that the elevation is caused by pressure on the common bile duct, which is then relieved when the, the displacement is resolved.

It's really important to consider that situation, as I have been referred plenty of horses that had elevated GTTs following a colic episode. But when not to perform a biopsy, very easy to consider that the, the former, . Oops.

Yes, so hydatid cysts are a . A relatively common finding in the UK, and leave them well alone. They're normally not affecting the liver function, and they will present as a round, perfectly spherical anechoic structure.

If the liver needs biopsy, then try and avoid them, because if you, do punch them, there is a risk of releasing metacestos. They can be treated with triclobendazole, but in the vast majority of cases they don't actually cause too much of a problem. The hyperchoic starry knight's liver that we do see very, very frequently in the aged horse is often incidental, and I will find it on many an abdominal ultrasound scan.

It does obviously reflect an underlying pathology, normally a chronic inflammatory state leading to mineralization in multiple areas. But it can be seen in in livers that are functionally very normal and therefore any biopsy should only be undertaken in line with any blood work that has been performed in the clinical parameters. Otherwise leave well alone.

I've tried to biopsy these a number of times in my earlier career, mostly with complete failure because the the spots, the anechoic spots are mineralized, so your biopsy needle just flies past it. This is a bit more of an extreme case, sorry, I'll stop it with the sound going. This, it was presented with a severe hemoabdomen, and on ultrasound scan these masses within the liver.

This was one that I decided to not undertake a liver biopsy, and the reason for that is to consider the main questions whenever you perform a procedure, whether it's blood work or a liver biopsy. What are your treatment options and will it change the plan of the the treatment in that horse? So in this case, the most likely diagnosis is hemangiosarcoma.

If I were to introduce a needle into that abdomen, I'm risking a peritonitis on top of the hemo abdomen. Also with something such as hemangiosarcoma, there is no treatment option, there is no surgery, there is no chemotherapy. So in doing so, I'm risking a quicker demise of that patient with absolutely nothing useful to be gained by it.

So always ask those questions before you do the treatment, sorry, do the procedure. When we look at practical considerations of the liver biopsy. A recent study, sort of, well, recent in 2014, showed that blind techniques are unlikely to, obtain satisfactory liver tissues.

They found that when they scanned over the 10th to the 14th rib spaces on the right hand side, as seen in the picture, running from the tubercoccate to the elbow. There were no horses in that group that had suitable thicknesses of liver in all four sections of the the rib spaces. And therefore, there's no justification to do this blind.

We have such ready access to to ultrasound, it seems beyond belief that we would still be doing it blind. You have two options when you do a transabdominal ultrasound and liver biopsy. One is to do it indirectly, in other words, you find a suitable location and insert the needle through the skin perpendicular to the skin and into the liver.

I'll show you why that isn't exactly ideal in a slide or two. The other is that you do it directly. So you're going to visualise the whole of the biopsy procedure, ensuring that the needle is guided into exactly the right place of the liver that you want.

I find that easiest with an ultrasound guide, they're available for the majority of transabdominal probes. And those guides, although expensive, can then be used for a number of other things such as facet injections, hip injections, and obviously biopsying anything else. So, yes, it's a bit of a, an outlay, but it is well worth it.

Also, fragmentation of samples is a real problem for the pathologist. Any fragmentation can affect the portal areas and therefore the interpretation of the results. Using an automated firing device stops that fragmentation and also makes it a lot, lot easier.

So this, you know, a true cut is probably what is most applicable in the sort of non-hospital based situation due to the cost involved. A Galini, which is an automated firing device, is the best. It has a lot more force, so it gets through a fibrotic liver a lot easier.

But you are talking about 400 pounds to buy the Galini itself. When using this, one of these two cuts, this is from Cook Medical, just remember that as you insert the needle, that firing of the needle goes forwards and the sheath comes over it. So in other words, the throw of the biopsy is about 2.5 to 3 centimetres beyond the tip of the needle as you insert it.

And that plays a significant role when we move on to the next slide. The other thing to note, as you saw at the end of that video, is removing the biopsy sample has to be done very carefully. Ideally with a scalpel blade or similar so that you lift it off gently and put it in formalin, needles do work, but there is an increased risk of fragmentation when you do that.

So, the principles of the biopsy, I'm going to go through how I do a biopsy and why we do different stages of it. Firstly, adequate sedation is absolutely essential. In the majority of cases, ditomidine and butterphenol is going to be more than enough, because it's a relatively non-painful procedure.

If though you've got a young horse or a fractious horse, I do like using a combination of ditomidine, ACP and morphine. You're going to identify the site. Normally on the right hand side, as there's a a lot more liver on the right, you can also use the left hand side cranio ventrally towards where the heart is.

But remember there are increased risks with that location. Once you've identified it, you're going to determine the depth of the biopsy, and this is where I was talking about perpendicular versus an acute angle. This is your very normal view of the right hand side with the liver and the right dorsal colon sitting ventral to the to the liver.

If we were to do a direct stick without ultrasound guidance, you're going to go perpendicular to the skin. You've got about 3 centimetres of skin and subcutaneous tissues, and then approximately 4 centimetres of liver. If you imagine that the throw of that needle that we were just talking about is 3 centimetres, you can only barely be in the liver before you have to, you would start hitting the right dorsal colon.

So ideally coming in just an acute angle with an ultrasound guided, either using a guide or just doing it freehand. Obviously you have to get through a bit more depth of approximately 5 centimetres, but then suddenly you've got 9 centimetres of depth to use of the liver. Remember that the tip of that arrow as we go down into the liver is really close to the large hepatic vessels, so you really don't want to be going down to that depth.

Also, the other thing to mention is if you are using a guide that will have about 3 or 4 centimetres of of equipment that's going to reduce the depth of that needle. So do be aware of that if you are doing something that's very deep in an abdomen. Once you have decided on your location, you're going to clip and scrub the area, prepare it as aseptically as possible.

I sort of often treat this as a clean procedure rather than a sterile procedure. And then infiltrate local anaesthetic. Subcutaneously and intramuscularly, absolutely, it's unlikely that you'll get to the peritoneum.

And you can sometimes if you're lucky, but do remember that a lot of horses will react when you start introducing the biopsy needle as you get to the peritoneum. I use then a stab incision, a 15 blade. This is because the, you don't want to blunt the biopsy needle or get it caught up in the skin and drag hair or anything like that through into the liver.

I've had plenty of pieces of hair on my liver biopsies and it's never caused a problem, but if you can avoid it, all the better. These stab incisions are obviously very small, and don't generally need suturing, but if there is a concern that the horse is going to be going out in the field and getting muddy or anything like that, then I would recommend either a non-absorbable or absorbable suture just to keep the site closed. Now in this picture you can see that I'm using a Galini, the blue, firing gun in my right hand and a biopsy guide.

They do make it so much quicker and so much easier. I can take biopsies in in seconds with that because there's no trying to align the ultrasound with the biopsy needle itself. Make sure you get yourself in a comfortable position so that you, then the biopsy and then the ultrasound scanner are all nicely in line.

There's nothing worse than twisting and it'll only make you rush and get a bad sample. Once you've got your samples, normally before that, sorry, I have administered non-steroidals because they've shown that in humans it is an uncomfortable procedure. And I will give some antibiotics as well.

I normally send them home with some non-steroidal so that at least if they do have a mild colic following this, they can have a little bit of bee. But I do say to the owners, if there are any concerns that the horse is collicking beyond just a little bit sad and maybe off its food, that they do get in contact with a vet immediately. What else might you biopsy though?

It's really important to consider your risks whenever you do a procedure. If you don't understand the risks, you can't try and negate them by good technique. You may biopsy some intercostal muscle, but that doesn't matter.

Diaphragm as well is pretty robust and really it, it doesn't matter too much if you get a little section of that. Intestine obviously is not something you want, and if you are aware that you have got an intestinal biopsy, then make sure that you send that horse home with an ongoing course of antibiotics for 5 days or so. But if it happens and you find out later, most of the time they don't, it doesn't cause any problems.

Kidney in very rare situations, we've seen those on the right hand side, you know, if it hasn't bled by the time it's gone home, it's going to be OK. Lung again is relatively common, so if it floats, repeat your biopsy because that's gonna be lung or intestine. And then from the left-hand side, spleen is a high risk part because they lie in such close proximity.

If you biopsy it, most of the time it's not a problem, although there is a slight increased risk of bleeding when compared to the liver. And in very rare written circumstances, people have managed to biopsy the heart from the left sided approach. I'll be very impressed if anyone can manage that.

But what are the risks beyond those? Infection and pain we've already mentioned, particularly with regard to if you do get an intestinal biopsy. But what about haemorrhage?

Oh sorry, then this, sorry, this was a study that we was in 2014, the one we've already mentioned. And this just really brings home why you shouldn't be, why you should be using ultrasound guidance. In those horses where they scan those four rib spaces, when liver was not visible in any of the intercostal spaces, there's 55% of the time it was lung, 36% of the time it was intestine, or 9% of the time it was both.

So it really does show that there really is not an indication for using a blind biopsy now there is ultrasound. You know, obviously these things can happen when you are doing an ultrasound guided biopsy, but the risk is absolutely much smaller. But what about haemorrhage then?

So, this was a study that was done in 2008, looking at whether or not haemorrhage was associated with a liver biopsy. We know that in liver disease, there is a reduced production of clotting factors and therefore a theoretical increased risk of haemorrhage. They looked at 33 cases that were biopsied, of which 58% had clotting abnormalities.

In 12% of their cases for horses, there was evidence of some bleeding, and I would say that's pretty high. I don't remember the last liver biopsy I performed that had evidence of of bleeding within its abdomen following the procedure. There was no clinically adverse effect in those cases, and there was no association between clotting, abnormalities and bleeding.

So I don't even test any horses for clotting dysfunction before I do a liver biopsy anymore because whatever the answer is, I would still do the procedure. That said, I do always scan following a liver biopsy, the area that I biopsy to make sure there's no hematoma forming. And also there's no, free fluid in the abdomen that wasn't there prior to it.

And if there is, then I normally keep them in the hospital for 24 hours to make sure that, we haven't caused any problems. But how to avoid complications. And again, I don't think I can say this enough, ultrasound just proves the point.

This is the same horse, one rib space different. On the left hand side, we've got the 11th, the 11th intercostal space, which looks like a perfect site to biopsy. Whereas we move forward into the 12th, sorry, backwards into the 12th intercostal space, and suddenly you've got a big blood vessel coming in, as well as the duoten ventrally.

So really choose your spot wisely. But how many biopsies should be taken? This is a real question that's been going on in human medicine for quite some time.

It's well recognised that a small sample or a small number of samples will underestimate the severity of the liver disease present. This is largely er because not enough portal areas will have been sampled, . And The fewer the portal samples, the less likely you are to get a definitive diagnosis.

In humans, they've found that at least 10 portal tracts are needed to be examined. Beyond that number though, the histological grade of and the fibrosis won't get any worse, and therefore it won't alter the severity of the disease. So how do we link that into into our work within horses?

There is at this moment in time, no golden number for the number of portal tracts that are required for a a biopsy. But Victoria and Andy, along with Alistair Foo over at Rossdale had taken a prospective study that was started a while ago, sadly sort of sitting in the eves waiting to be published. And what they did is they looked at multiple biopsies, multi-site biopsies from hepatopathy cases.

Preliminary results suggest that biopsies from different locations in the liver didn't change the outcome. But the scores of portal pathology varied within the same sample and between biopsies in the same horse. So, just as it is in the humans, it probably looks like it's the same in horses.

There will be a golden number of portal tracts that needed to examine a liver reliably to estimate portal pathology. We don't know what it is, but common sense would say at least start with the human number of 10 and go from there. To illustrate this, they looked at 2 biopsies and only 63% of those two biopsies had greater than 10 complete portal tracts.

Whereas when you took 3 biopsies, 93% had 10 complete portal tracts. As such, the recommendation is to take 33 biopsies in the majority of times unless you really feel like you have done a fantastic job with the 1st 2. Real judgement though should be taken if you get 3 absolutely terrible samples, obviously continue until you get a couple of really good ones.

You're taking your your sample and now you've got to decide what to do with the information. I think that's really important is knowing. When you start all these procedures is how to interpret them and what to do with the information.

Once you go to histopathology, it will confirm the presence of liver disease. Sometimes you get nice easy definitive diagnosis such as amyloidosis, neoplasias or marked xenophilic infiltrates associated with multisystemic xenophilic epitheliotropic disease. More likely you're going to be looking at septic cholangio hepatitis if you're, that's pretty rare though, as we'll come on to PA toxicity associated with ragwort.

Hemocirrhosis or something sort of very non-specific chronic active hepatitis or spotty necrosis. But again, Andy and and Alistair looked at the histopathological scoring and tried to associate it with non-survival. What they found was that severe fibrosis was very much associated with a poor prognosis, and so they graded it at a 4.

Moderate was also moderate, it was also moderately associated with poor survival. Irreversible cytopathology, only if moderate or severe. Inflammatory infiltrate when it was severe.

Hemo hemosideration accumulation. And this one really was specifically only in the severe cases. The rest really weren't even weighted enough to to include in their total scores.

And final biliary hy final, finally, excuse me, biliary hyperplasia, too many Y's in there. But what does this mean? How do we interpret those results?

Wonderful to have a score. And Andy's paper is readily available online, it's actually, it's really useful to have in the back of your car if you are doing these. The biopsies were scored, and rather than having a continuum which proved very difficult to understand as to what the prognosis was for each one, they grouped them a little bit.

So, the first two at 0 and 1, there was absolutely no difference really. One horse died in the horses that had a grade of 1, but that's just deemed not statistically significant, and therefore they're both 0 and 1 acts as a reference. If you had a score of between 2 and 6, those patients were 12 times more likely to die within 6 months than they were.

When we look at a score of 7 to 14, and it was 50 times more likely to die than the references. So this gives you a really good guide to be able to tell the owners what the chance of success is when you get these biopsies. But when we look at them a little more specifically, what are we dealing with?

. So one of them is septic cholangio hepatitis. I think it's something that we're all very much taught at university and theoretically plays a significant role in in these cases. But in reality, this is not what is going on.

What you're looking for in the report is a neutrophilic portal infiltration and specifically neutrophilic. It's very, very rarely diagnosed. So Tom Divers over in, in Cornell, reviewed his paperwork and found only 9 cases in 8 years.

And I would say in America, it seems to happen far more frequently than it does in the UK. Bettina Dunkle over at the RBC 2015 looked at it again, found 17% had mixed infiltrating portal areas. And just remember, a mixed infiltrate really does fit more with a chronic active hepatitis, rather than a truly septic cholangio hepatitis.

When Andy reviewed multi-site biopsies of 93 cases that we had seen, not one single one of those was diagnosed with septic cholangio hepatitis. So what does this mean? For me, it means that empiric anti microbial use in suspected liver cases that have not been biopsied is, is incorrect.

You know, we are striving towards having an antimicrobial stewardship as much as is humanly possible. And this is a case where there really is no indication to use antibiotics until proven otherwise. I think if you were to present me with a pyrexic course with marked elevations in liver enzymes, SAA high, and the liver looking unhappy on an ultrasound, then I would be pushed into the corner and say, yes, you probably should use something such as TMPS, but I haven't, I don't remember a case that that's ever been, the situation.

Ragwort toxicity, I think is an under-investigated and overestimated disease in the UK. Again, historically and in all the paperwork that's out there, ragwort toxicity is the one thing that kills horses. That's what all the studies and all those layperson magazines say.

In reality though, it's a very rare cause of liver disease in the UK. You're looking for megalyotosis, megalocytosis, and biliary hyperplasia in those. In one pathology group, 8% of liver samples had evidence of megalocytosis, but there was no indication as to whether or not that was associated with true liver disease or if that was an incidental finding.

In an interesting beaver study in 2014, horse vets, on average, said that they see two cases per year. But what's really important in that group of horses, that only 20% of the suspected cases seen by vets were confirmed histopathologically. So really the chances are that the vast majority of those cases were not ragwort toxicity and were just assumed to be so.

I think it's important to avoid a presumptive diagnosis because the prognosis for a toxicity is very different to that of the others. If re toxicity has occurred chronically over time, the liver is going to be severely fibrose and therefore the prognosis will be guarded. In some cases where you have acute onset of chronic disease, then there is a good chance of survival.

But again this is where biopsy takes place, it takes precedence. Obviously, if you see ragwort to ragwort in the field or ragwort in the feed, then do make sure that the owner removes it or removes the horse from that environment to ensure that it does not, is not the cause and does not contribute to any ongoing liver disease. Hemosideriin deposition, what you're looking for in the report is, deposition of hemosiderin in greater than 50% of the hepatocytes.

Only once it hits greater than 50% of hepatocytes was it associated with any change in the prognosis of that case. Excess dietary iron is deposited as hemosiderin into the hepatocytes. Now there is discussion as to whether or not therapeutic phlebotomy every 2 weeks creates an iron drain.

Anecdotally, people do say that it does, but I would really ask for caution, as there are a couple of studies that show that there is absolutely no change in the iron load within that horse when phlebotomy is carried out every 2 weeks. And in fact, it just causes a lot of hassle and a lot of mess. Collating agents are available, but they are incredibly expensive for, for a horse.

And when we really think about the iron stores in the horse, they are huge. To get an iron deficient horse is virtually impossible because of the large amount of iron both in the liver and in spleen amongst other areas. Whenever you do get a diagnosis of this or you are considering it as a diagnosis, it's really important to review the diet meticulously to minimise further iron intake.

And that includes all supplements. There are plenty of muscle and red cell supplements and contain a lot of iron. There are even some liver supplements that still contain iron, even though in the vast majority of cases that would be contraindicated is it's only going to overload the liver further.

Like many of these things, it's important to look at the wider picture as well, because quite often, if one horse is eating that diet, many horses will also be eating that diet. And therefore look at any other horses for evidence of emphatic insult. Chronic active hepatitis is one of our more common diagnoses, I'd say it's the majority of the ones I deal with when they come in for biopsy.

And what you're going to look for is a mononuclear infiltrating the portal areas. There is no definitive diagnosis as to why this is ongoing in the horse, but the aim of treatment in all cases is to reduce inflammation and therefore reduce the risk of fibrosis developing. What we don't know is whether or not fibrosis is reversible in horses.

What they do know is that in rats and now in humans is that beta blockers such as enalapril will reduce the fibrosis present in the liver. So in some of our really severe cases, I have recommended propranolol due to a cost effective basis rather than enalapril, to try and reduce any fibrosis. Obviously this is not evidence based within the equine population, but quite often our medication trial therapies are not.

The mainstay of treatment in these cases is going to be ongoing prednisolone. In some cases you may recommend a course of dexamethasone is a more potent glucocorticoid to start with. Azathioprine can be added in those cases that are refractory to prednisolone, as a further immunosuppressant.

Some other treatments that can be added, pentoxifiin acts as a real logic agent, so it's going to really alter the size of the red cells, so they can fit through the capillaries if there's fibrosis ongoing. But also acts as reducing neutrophilic burst and therefore ongoing inflammation. Vitamin E is an antioxidant and will bind any free radicals that are present within the lipid bilayer of the liver.

And so can reduce any further insults to the hepatocytes. If there is a suspicion of fluke, then triclobendazole can be used. Now, I would, this is another point of caution for me, is that the diagnosis of fluke, I think, is very much overdone.

But if you have real suspicion, then triclobendazole can be used. The study was undertaken at Liverpool, a good few years ago, and as of yet, I haven't seen any results, which does always make me slightly concerned as to what the results may well be, about flu and liver disease. But what, what about spotting necrosis?

This is another common one for me, this one and chronic active hepatitis are my two most common diagnoses. You're looking for focal aggregates of neutrals in hepatic parenchyma. And what's important is comparing this to your septic cholangio hepatitis.

This is just spotty aggregates with very little else going on rather than being around the portals. It appears that these are possibly associated with a toxic insult, something such as aflatoxin or fumicin. And it's common finding in biopsies from liver disease outbreaks.

So when this is many horses showing signs of liver disease or abnormalities on blood work rather than just a one-off horse. What we've managed to do at Liver at Lihook is we started to look at some hay, and he did this, and has released the results, and there's some promising results from that. But what, and I'll show you the slide on that in a second, but if you do see spotting necrosis, it's absolutely imperative that you look at all the herd mates again.

You know, these are, that case that you're dealing with is always the tip of the iceberg in spotting necrosis. So get a GGT as a minimum from every single other horse. When you're dealing with these cases, get the forage source changed as your first thing to do.

If they're on hay, even just another source of hay will do the job, otherwise, moving on to haulage is ideal. If they're severe enough, then steroids can be administered otherwise. Out and keep a close eye on those, those values.

But mycotoxins, previously aflatoxin was assumed to be the cause of the majority of spotting necrosis, liver disease, and it still does play a role, as far as we are aware. Andy recently investigated the presence of other microtoxins in forage. And so looking at about 60 or 70 different mycotoxins, samples were analysed from 29 case premises and 12 controlled case premises.

So where we knew there was no liver disease going on whatsoever. What he found was that microtoxins were present in 72% of case samples and 75% of control samples. So in other words, if you do get a diagnosis of mycotoxins, you know, if you get a client who has sent their sample off to one of these companies, and they're suddenly recommending a lot of the supplements that absorb mycotoxins, care should be taken.

If no horses are showing clinical signs, it's probably a waste of money. What they did find though was that mycotoxins found only in case samples, and 99 of those was fumicin B1. Aflatoxin B2 and G2 were found in both case control cases and control samples, but previous studies have found a correlation between naflatoxicosis and liver disease, so it still is very much something that we consider real.

So Anti found no clear association between naflatoxin contamination and forage with liver disease, but fuminitin B1 was definitely seen in case samples. So now as such, we are offering tests for humanitin B1 and aflatoxin and hay samples, and have found it very useful in diagnosing the cases. It is important though to note that when liver disease has occurred due to consumption of mycotoxins, it may well be that actually it happened two weeks ago, and just because you get a positive or a negative now doesn't mean that that sample is truly reflective of what was going on.

And more often than not, the hay is not going to look bad. Most samples look completely normal, it's not that sort of white mould that you see everywhere. It's, it's far more insidious.

But what about viral hepatopathies? This is something that's really come to the forefront in our, our understanding of hepatopathies in the last sort of 5 or so years thanks to a group over in Cornell now spreading throughout the world. They've looked at multiple things.

Initially, this all started following a tireless disease outbreak when a group of horses all got serum sickness, essentially. So when you get liver disease following injection of a serum associated with your species. So this was tetanus antitoxin, of equine origin.

They then did viral screens on the serum, the, the, sorry, the tetanus antitoxin, as well as all those forces that were positive for hepatopathies. Initially they found this TDAV, this Tyler's disease associated virus, and they were very pleased with themselves because they felt that this was the cause of the, the clinical symptoms. It was later found through COx postulates that did not occur, and TDAV seems to be a virus that is just around and endemic and not really causing much of a problem.

It does have a tropism towards liver cells but doesn't seem to cause any hepatopathies. So they started to widen their search with further viral screens and found that 3 other 3 total flab of viridase and 1 part of a virida seemed to be possibly associated with these viral hepatopathies. Though a lot of research, they found that the part of virus was also found following treatment with tetanus antitoxin.

So they tried fulfilling Cox's postulates again, and the Pavvavirida was given to horses, via tetanus antitoxin, and those horses were shown to have biochemical clinical signs associated with hepatitis. And what's interesting is that these Pavavirida horses seem to have a very broad range of clinical signs from very mild even not showing signs but quite marked biochemical changes all the way to a formin and fatal hepatopathy. So it does seem to be a very, very important virus that we need to be looking for.

What is, though, should be noted, sorry, is that there seem to be, it seems to be fairly endemic, with 13 out of 100 horses in a different situation testing positive for virus, the virus on PCR and 15 out of 100 were s positive. So it is out there causing non-clinical disease or very mild and non-specific clinical disease. They've also found that the non-primated patchy virus is seems to be causing temporary elevations in liver enzymes and definitely has a tropism towards hepatocytes.

It doesn't seem to cause the severity of clinical signs that we see, we see with the parvovirida, but it does seem to still be very important to monitor and see if it is going to cause any problems. As such, we, we're offering those to the Apache virus and parvovirus, and we do see a few parvoviridae positive horses, you know, throughout the year. And it does seem to be that, you know, most recently a couple of horses were found dead and positive for it, so it is an important virus to be looking for.

But what does that mean for treatment? And it's slightly frustrating because the answer is we don't have any viral antiviral treatments for these. So it's a case of monitoring and also probably steroids.

What they have found though is initially they assumed that it was going to be purely a transmission via serum or something along those lines, but there does seem to be a horizontal transmission in a minority of cases. Nobody really knows what that horizontal transmission is at the moment, they haven't found it shedding anywhere, but it definitely is, has occurred as some horses with absolutely no evidence of previous plasma administration or or equine origin drug administration did have evidence of parvovirus. So what are your treatments?

We've talked about them a little bit, but I'm just gonna bring them all together to summarise this talk a little bit over the last couple of slides. If you have suspect mycotoxicosis, so an outbreak of horses showing mild to severe clinical signs, then check all of your hi mates, first of all. Firstly, change your forage source.

Most people are gonna complain and you're gonna have a lot of angry people because it's a lot of money normally. But changing it will often lead to good resolution of clinical signs without any need for treatment. If those values of GGT aren't too high, you know, you're talking the low 100s, then what I'd recommend is change the forage source, recheck in a couple of weeks.

If it's high enough, then obviously start steroid treatment and then monitor as well. Viral hepatitis is, again, as I've already mentioned, as the horizontal spread is possible check all her mates. And then again monitor and or steroids depending on the severity of the values that you're looking at.

Chronic active hepatitis cases, you're going to be looking at a multiple of treatments, steroids being your mainstay, possibly adding azathioprine if you think that you're struggling to get complete resolution. Vitamin E and possibly beta blockers. I think the last one really is only for those cases when you're trying your hardest, and the owner is completely aware that you are, cascading very much, into an unknown.

But what about hepatic encephalopathy? We've sort of talked about the, the majority of cases. These are the rare ones which need immediate referral in my opinion, otherwise you're not going to be successful.

But in the, you know, in the short term, it might be that you need to deal with bilateral laryngeal paralysis. This is particularly true in my sort of hands for small horses, you know, the Shetlands, the miniature ponies, things like that. If you are concerned that there is bilateral laryngeal paralysis, then I would recommend a tracheostomy sooner rather than later.

It's a lovely easy procedure when the horse is standing and not trying to kill you or itself, but as soon as they start to suffocate, they are an absolute nightmare. If there is a suspicion of hyperaminemia, and to be honest, if you're even slightly suspicious of it, I would start latulose. It's very limited, I've never caused diarrhoea in a horse with lactulose, and the, the dose is meant to be 0.2 mL per kg every 12 hours, but often I'll give it every 6 hours in those severe cases.

What it's going to do is bind ammonia to ammonium within the GI tract and so it gets defecated out rather than absorbed into the blood. The resultant brain edoema, secondary to the hyperaminemia, hypertonic or manital. Recent studies have looked more that hypertonic is better at reducing brain edoema in general, not specifically associated with hepatic encephalopathy.

Manitol has a slightly longer acting effect. What I'd recommend is that if you trial it and you are successful, then you can keep bolusing it throughout the 24 hour period. If there is absolutely no improvement following hypertonic or Manitol, then don't keep trying because it's not gonna have an effect.

Obviously the liver inflammation itself and also the brain inflammation secondary to the edoema. Dexamethasone IV is what you want to be doing. That's the most potent glucocorticoid we can use in a horse.

Some of these horses will have gastric impactions as well. They, the surmised cause of this is that there's release of toxins that the liver should be dealing with causing neuronal damage. If that's the case, then a lot of these cases don't do very well, but if you are going to treat it in an indwelling nasal gastric tube with a trickle of fluids, it will hopefully resolve that gastric infection.

Many of them will be inappetent, so IV fluids are essential, but again, weigh this up against your hypertonic use, and parental nutrition, but really care should be taken with parental nutrition because liver failures are going to struggle with any protein in administration. So, and obviously you don't want to be causing a hyperlipemia as well. So they really do need a lot of intensive care.

Any ongoing toxic insult, vitamin E will help reduce that. And also reducing fibrosis, as I've already mentioned the beta blocker propranolol and colchinine is one that the evidence very much proves that it doesn't really do anything, but some people still do use it, so don't be surprised if it's out there. Some other therapies, milk thistle in humans has been shown to help with with humans with cirrhotic liver secondary to alcohol abuse.

So it may help. It doesn't seem to cause any harm in horses. There's a few papers out there that, you know, it's very limited response, but it's not causing harm, so why not in some cases.

Sammy, used a lot in humans and small animals, generally it is far too expensive to use in horses in my hands and has limited evidence as well. But to conclude, I hope that we've shown why and how to perform a liver biopsy, how we can use ultra guided, and that, you know, the liver biopsy is essential to give you a diagnosis and a prognosis. One really good point is don't dispense empiric antibiotic hepatopathies, because there really is very limited evidence of operative disease, in, in the vast majority of cases.

The histopathological prognostic scoring system gives you a good guide as to being able to tell the owners what might happen over the next 6 months. And really that I think for me the most important is screening your herd mates when you diagnose a new case of liver disease. The vast majority of these are outbreaks rather than individual cases.

And as such, looking wide is, is really important. Well thank you very much for listening, and I hope that was helpful.