Hello, everyone. My name is Valerias Corsa. I'm currently an instructor at the centre for Companion Animal Studies at Colorado State University.

I obtained my veterinary degree in Argentina in Univers Nacional de la Plata, and then I finished a PhD programme in the department of Clinical Sciences at Colorado State University. And my research interest is sonotic diseases of companion animals. So today I will talk a little bit about GR biosis in companion animals.

So in this talk, I prepared an updated review of Galiadonalysis infections in cats and dogs with a special emphasis in the diagnosis, so not the potential and treatment. And I hope this can be useful for you. And Gal analysis, as everyone knows, is one of the most common parasites infecting humans and animals worldwide.

In humans, it is estimated that it can cause approximately 184 million cases of human illness per year. And the overall prevalence in dogs is approximately 15% and in cats, 12%. Currently, there are 8 Giardia spec species described.

Some of them are host specific such as, for instance, Garda Ardai and and Cittai that are specific for birds. Gauris and microdi are specific from for rodents, and the species that infects mammals is Gardodealis. Anter analysis is considered a species complex that comprises 78, I'm sorry, 8 assemblies or subgroups, if you want to say it in another word.

And these assemblshes can be host specific such as assemblies C and D that infects specifically dogs, assemblish F is specific for ellips and establishes A and B are shared among different animal species and also with humans. So these two assemblies are considered sonotic ages. There are several cycles of transmission among individual species, as you can see on, on the other side.

And then sometimes these cycles of transmission Overlap between the species, so you can have zoonotic transmissions if the direction of the infection is from animals to humans or anthropozoonotic transmissions if the infections are from humans, environment and animals. TI has two life stages. 11 is the cyst, and cysts are the environmental resistance stage.

They are responsible for the transmission. And usually transmission occurs in, in animals by direct ingestion of of cysts from faeces or by indirect ingestion of contaminated food or water or infected by species or trans transport host. The other form of giardia is are the trophosoides.

Trophosoides have the motile form and are found in intestinal tract. In the dogs are, they are usually found from the duodenum to the ileum and in cats are more in euro and illi. The phosoides are not responsible for transmission among animals in general because they are susceptible to inactivation by environmental conditions.

So the cysts are responsible for transmission and cysts can be, can remain infective in the environment from days to weeks if the environment is moist enough. And usually the young animals that are, that are infected, they shared a large amount of cysts in the environment and they are a major source of contamination. Here, I wanted to include the maximalist excretion by different species, just to give you an idea of the amount of contamination that infected animals can, can live in the environment.

In dogs and cats, the rebate period range between 5 to 16 dogs, I'm sorry, 5 to 16 days in cats and 5 to 12 days in dogs. Usually the the shedding peak of occurs doing days 2 to 7 is usually during the first week of infection. And clinical disease is, is supposed to happen by a combination of factors such as disruption of the normal flora, induction of inflammatory bowel disease, induction of intestinal vielial cell apoptosis, induction of motility disorders, production of, of toxins.

GRA can cause infections that can range from asymptomatic with completely no clinical signs, absence of clinical signs, to symptomatic infections that could be, could be seen as acute, intermittent or chronic diarrhoea. The mechanism why GRD are cause this disease in some animals and not in others is not completely elucidated, but it's estimated that it can be caused by a combination of factors such as the infective strain, the host immune response, the host immune status, co-infection with other GI pathogens, and the composition of the microbiome. A recent study done in our laboratory show no differences between clinical signs and assemblies in dogs from the US.

And it is known that co-infections with maybe with other protozoans like with ya foetus or with a Pyridium can exacerbate clinical seno of infections. And usually regarding age, there is a higher prevalence of GRA in young animals and children. Clinical signs, as I said, when diarrhoea is present, usually in young animals, diarrhoea is acute.

While in older animals, you can find them from being acute, intermittent, or chronic. Diarrhoea is usually mucoid, pale, soft, and steata may be present. Blood is not usually present because this organism is not enteroinvasive.

Usually animals are afebrile, they do not vomit, and they show mild to moderate discomfort. And as I said in the previous slide, co-infections or any immunosuppressive condition can exacerbate clinical sites. So, CAPC, I don't know if you're familiar with CAP C.

CAPC is the Companion on Animal Parasitic council and it's a non-profit organisation composed of veterinarians, university researchers, and private practise clinicians, and people who are interested in parasitic diseases. Their, their goal is to generate awareness of parasitic infections in companion animals and they generate periodical updates and guidelines for the treatment of small animal parasites. Usually what CAPC recommends is like animals in the first year of age should be, not animals, cats and dogs should be tested between 2 to 4 times a year by faecal flotation for the presence of parasites.

And after the first year of age, between 1 or 2 times a year. The faecal screening for diarrhoea is, they recommend to do faecal flotation with a deformation using zinc sulphate or shredded sugar for detection of all cysts, eggs, and cysts. Wet mounts for detection of tropozoys, rectal rectal cytology, and cryptoporidium screening by the use of fluorescent antibody ads.

GRDA is one of the most common misidentified, underdiagnosed, and overdiagnosed parasites. It's very easy to confuse GRDA with pseudoparasites, with yeast, and also if we are not used to, to see them in, in microscopic examinations, it's, they can easily, they can cyst can be easily missed. So in this case, they would be underdiagnosed.

And GRA can be diagnosed by direct smear, faecal flotation. Antigen detection in faeces by immunoassays, and immunofluorescent assay detection for cyst detection and PCR if we want to determine the assemblage infecting the data. Troposoides can be detected by faecal smear.

Sophosoides are only found in fresh samples. So if the sample, if the sample have been refrigerated or they sold, you're not going to be able to see the trophosoides. So usually you can, what you can do is grab a little bit of diarrhoea, put them in the, in the slide with a drop of saline, and then observe under the microscope.

And at the 100X magnification, you will, you will be able to see the falling leaf motion. And at 400 magnification, structural characteristics of the geotroposoides can be seen, such as the pair body shape, the flagella, the two nuclei. It's important to remember that if we are looking in for tropozoids in cat and in cats and rarely in puppies, we have to differentiate a troposoides of GRia from troposoides from tri-trachoonas foetus or petatrichomonas hominis.

And we can do that for the type of motion and also we have to consider that usually titacommona foetus or pentarichomonas comin also cause large bowel diarrhoea and it's usually found in cats from cataries. CRDI can be identified by faecal flotation and faecal flotation can be passive floatation. Passive floatation is when you use just an, an amount of pieces and mix them with a float with the solution media and then mix, let them sit and and then collect them.

The a drop from the supernatant or put the covert slip on top of the tube and observe under the microscope. If this is the only procedure that you are going to use, it is recommended that at least you test 3 samples over a period of 1 week before ruling out the presence of GRA. And this is because passive flotation is less sensitive than a faecal flotation with centrifuriation, and also because GRD has intermittent patterns of shedding and dogs and cats may shed low number of cysts.

The recommended method to to screen for Gaddisy is the faecal flotation with centrifugation. And the solutions that can be used are shredded sugar or zinc sulphates, although the zinc sulphate is the recommended solution for floating GRD assists. Because she the sugar can distort the cyst.

Although both solutions can be used, they just, the, the cyst looked different in each solution. Commercialised that kits are available for detection of GRD antigen faeces. However, it is important to consider that the antigeninin can continue even weeks after clinical responses on those.

So we sample that has been positive. And then the, that animal was treated and then there's no more diarrhoea in that animal, then they can remain positive for a while. So it's not clear for how long after the diarrhoea resolved, the anti-shell would be present in faeces.

So CAPC recommends for that specific reason to use the commercial Llysas only in animals with diarrhoea and do not use the LISA in clinically healthy animals or if you want to evaluate response to treatment once the diarrhoea resolved. CRDy can also be detected by direct immunofluorescent antibody acids. There is a commercial kit to detect well, there are several probably commercial kits to detect GRD assist and cryptoporidionosis in human faeces that can be used for detection of cryptoporidium and Gia in the pieces of cats and dogs.

It's a test that is more sensitive than faecal flotation, so it has been considered a gold, a gold standard or a referent test in a studies in which different diagnostic tests were compared. Because it requires a a fluorescent scope, to observe the cyst. It is only performed at diagnostic laboratories, and it's a test that is recommended in cases of chronic small bowel diarrhoea because it can also detect ryptopyridium osis.

And these coinfections are very, are very common in, in. In the next two slides, I just wanted to show you the results of a few studies that compare diagnostic tests for the diagnosis of Giardia. And I only highlighted the test with the highest sensitivity and specificity.

So as you can see are the immunofluorescent antibody assay and the The antigen test. This is the other slide. And if we want to know which assemblage is infecting that particular cat and dog, we have to perform PCR because GADA analysis assemblies are morphologically identical and we would, wouldn't be able to identify them by by microscopical examination.

So the most commonly genes used for detection of tiao analys assemblies are the ADS ribosomal larinna, the glutamate dehydrogenase, triose phosphate isomerase, and beta galin genes. It is recommended to do a multi-locus genotypic, geno genotyping testing if we want to do a sam determination because sometimes the results of one gene do not agree with the results of another one. You can see here in the In this table that some of the genes are assemblished type that sample as assemblage T and then another gene type that assemble type that sample assemblage A1.

So by one gene, that sample has the those specific assemblage and by the other gene, it shows the Sonotic assemblage. And commercial laboratories offer different types of PCR assays. So it depends on the objective.

Of, of, of, what, what do you want to know. If you want to screen the sample for GAA and other parasites, if you want to know if that dog is infected just with GADA and other parasites, you can do a multiple, multiplex screening PCR that will detect GAA and other pathogens. If you are interested only in knowing the genotype infected.

That sample, there are other assays for the for that purpose. But in the case of the genotype in PCRs, these PCRs are usually less sensitive than the multiplex screening VCRs. So false negative results should be expected due to inhibitors in the faeces and also due to the the, the lowest sensitivity of these techniques.

Since we are talking about established determination, I just want to talk a little bit about the synotic potential of GRD. So, as I mentioned before, And GRD analysis is a semblage complex that has several assemblagees that are hosts specific and some semblagees that are considered potentially sonotic because they are shared between animal, animals and humans. I also want to repeat that Each, each, each animal species have an individual cycles of transmission among the specific species, but sometimes these cycles of transmissions overlap and the infection can be both ways.

It can be zoonotic or anthropozoonotic depending on the direction of transmission. A meta-analysis studies show that the overall prevalence of GRD in dogs is 15%, and the majority of the isolate type from dogs around the world type assemblish is C or D that are the dogs specific assembs. In cats, the overall prevalence was 12% and approximately 60% of the isolate type from cats, we assemblish F, that is the cats specific genotype, and the rest of the establishes the type assemblish A, that is the zoonotic genome.

In dogs, it was established that those may have two cycles of transmissions. When animals tend to be grouped together, like in kennels or just a large group of animals of the same species, the host adaptive strains will predominate and in environments contaminated with seeds from humans and other animals, so not the sonotic sablishment. Humans are infected with assemblies A and B, and among those assemblies A's and B's, they are subassemblies that some of them are human specific, some others are animal specific, and sometimes they may overlap.

And DNA from dog and cats specific establishes have been sporadically detected in humans. It's not very good. And there were several studies in which they evaluated samples, they tested samples from animals and humans living in the same house, in the same household.

However, the, these studies were inconclusive and they were, they were different, different results were found. There were studies in which dogs and humans in the same household share the same sams like beef, for instance. And there were other studies in which humans harbour their own genotypes, and dogs and cats have their own dog and cat, specific genotypes.

So, both, both findings have been reported. And it's important to, to, to say that these studies were based on the detection of Giaia DNA in each species and not in demonstration of infection. Here I wanted to show you this pie chart that showed the sonotic assemblages A in yellow and B in light blue.

So, this is from a very recent study and they show that the percentage of, or the proportion of each assemblage infected each animal species and humans. And you can tell that from this pie charts that the ones that have a higher proportion of yellow yellow and a light blue are equine, rodents. Rabbits and non-human primates.

So as a conclusion, there is really a lack of evidence of direct transmission of GR analysis between, between a human and animals. The fact that the same genotype has been found in human and animals in the same household only shows cross-species transmission. In order to prove zoonotic transmissions, identical subtypes of geo analysis in both humans and animals living in the same household and in that and showing that the infection occurs first in animals and then in humans needs to be demonstrated.

Another finding of this recent review is that, assemblage A has been detected in less than 10% of, companion animals, livestock, and, and other animals. So the sonotic transmission of assemblage A is less common than it was previously believed. However, they, insisted in that future research should be focused on subtyping of, of assemblage B.

And the sonotic impact of of the analysis should be focused on other types on other species. It should be focused more in horses, rodents, rabbits, and non-human primates, because as I showed you on the previous slide, there were the animals that have a higher A, a higher proportion of Sonotica sandwiches. I also wanted to talk a few minutes about dog parks.

In 2019, there were more than 800 parks in, in the US and the benefits of attending the parks are, are very clear. For both humans and animals, they can both exercise and socialise. But infected dogs can expose other dogs and humans to zoonotic pathogens in their faeces.

And there were some studies that detected an association between dogs attending dog parks and the presence of enteropathogens. So, in this table, I summarise the latest studies done in dog parks. 5 are from the 5 studies are from the US and another one is from this one.

And from what you can see in these tables, Dogs attended dog parks harbour parasites, they harbour Giardia and cryptoporidium, and dogs attending dog park had are more likely to tested positive for Giardia and cryptoyridium. And so it is true that dogs attending dog parks harbour parasites. However, the finding of this studies shows that those that visit the parks, yes, they were more likely to be GRDA positive than other dogs that do not attend the parks.

But there was no association between being GRD positive and showing clinical disease. That means to show diarrhoea. And in two studies, the one from California and the one from Colorado, a group, the, the genotyping on the GRD isolates, and those, the dogs who were infected with host adaptive genotypes.

They harbour ablishes C and D that are the host genotypes. And it was also observed that off off leash activity showed a positive association with transmission of CRD infection. It was more the off-leash activity than attending the park alone.

And this could be explained that if dogs are running alone, they can, they can drink raw water and water can be contaminated with Giardia and so they were going to be more likely to be positive for GRD. So since the benefits of attending dog parks outnum outnumber the disadvantages for both dogs and and their owners because they can socialise and they can be active. It is important to, to tell owners to minimise environmental contamination and to, to, to be very responsible with the disposal of.

If you want to find more current information about not only GRA, this, I only focus this slide on GAA but on sonotic pathogens, and especially sonotic parasites, in, in companion animals. The CDC website has a, a section that is called GRA parasites and, parasites and pets or something, something like that, and it keeps giving, it keeps updating information about the importance of all the pathogens and their and their owners. And then I also included CAP C and ECAP that both are the European and the American Council for a Companion Animal Parasites.

So they always provide updated guidelines on s sonosis and non-soonotic. And then going back to the last part of the presentation, that is treatment, the main goal of treatment is to eliminate diarrhoea. And there is TRA has several, they, they, T can have specific antimicrobial susceptibility patterns, but susceptibility testing is not available in commercial labs.

So the treatment should be evaluated in each individual basis in that particular cat though. It is unknown if different, GD analysis that, assemblies responds different to treatment. And in the US there are no drugs approved for the treatment of GR diasis in cats and dogs, and evanel albendazole are approved in some European countries.

The first choice for GArea treatment in cats and dogs are the benzimidazole drugs. And this is because they do not induce intestinal biosis, and they are recommended to be used also when there are current infections with cestos or nematodes. I included the CAC doses and the recommendations are between 5050 milligrammes per kilogramme once a day for 3 to 5 days when used as monotherapy.

And 50 mg per 50 milligrammes per kilogramme once a day. For 5 days, if they are administered in met with metronidazole, because sometimes you have to do a combination of therapy. Ferventel is approved in some countries to to use as a treatment for giardia and it can be the combination of ebanel, pirantel and rasciuantel used at the administered at the level dose of three consecutive days, days is generally very effective.

Albendazole is also efficient against chardia, but we do not recommend the use because it can cause bone marrow suppression in cars. The second choice of treatment are the nitromedazole class, and we said it's the 2nd choice because they do cause an effect in the intestinal micro microbiome, so those will have this biosis. And the recommended dose is between 10 to 25 milligrammes per kilogramme twice daily for 5 to 8 days when used as monotherapy.

In the cases that it requires to be used in a combination with fambendazole, it's 25 milligrammes per kilogramme twice a day for 5 days. And in the case of cats, we did a study many years ago in our lab, and we, we showed that the benzoate form was better tolerated by cats than the US before. And you have to be careful with chronic therapy or with acute high doses of metronidazole because they can cause GI and neurologic toxicity.

And they, it is the recommended dose a drop, if it's if there's current infections with Clostridium perfringe. Pronidazole has also been found effective against Giardia. But since it's the only recognised treatment for taemona foetus, we only recommend it to be used in coinfections with GR analysis and trityomonayus.

There are other drugs that can be used for the treatment of GRD, are others that have been used in the past, but I'm only using the I'm only showing you the ones that are recommended by CAP C. And these two are new, relatively new drugs are not commonly used drugs in cats and dogs. Anoin it has a broad spectrum against protozoan and Gram-positive bacteria and it has been Using shelter dogs with diarrhoea that were either infected with Giardia or infected with Giardia and cryptoridium.

And it's supposed to cause, I mean, it shows no adverse effects and the dogs become negative and have normal stools after 5 to 10 days after treatment. Another drug that had been used in humans for quite a while is nitooxanide. Nittooxanide is the only drug approved for the treatment of human crypto cryptooridosis in the US.

And it has been used in several studies for the treatment of GRiodoalis infections and Gadonalis cryptoporidium coinfected cats and dogs at different doses and it worked in, in, in several studies and it shows a, a reduction in cyst shedding. But it has to be considered that it, it had to be before using it has to be remembered that it's the GI irritant, it cause vomiting. In dogs and actually vomits were very frequent and it's not effective in the absence of an appropriate immune response.

So again, as a consensus of parasitologists and clinicians in general, the main goal of the treatment is to eliminate diarrhoea and treatment should be evaluated in individual basis. And it is recommended not to treat animals that test positive for Giardia. And, but do not have symptoms or, and do not show diarrhoea.

So it's not recommended to treat animals that are positive with aria, but they have foreign tools. Sometimes treatment is recommended because owners want to treat or because animals are in a high-risk environment, such as kennels, scatters, or breeders, or when there is high incidence of diarrhoea in puppies or in households that have small children or there are immunocompromised humans. So, in this case, this, you know, you, you can't read.

Another thing that I wanted to, to talk about is the follow-up testing. And follow-up testing should be done between 24 to 4 48 hours after the completion of the therapy, if clinical sites have not been resolved if dogs are still having diarrhoea. And if dogs are having diarrhoea, yes, analyzer test can be used for retest, for testing.

If dogs do not have diarrhoea, the follow-up testing should be done by faecal flotation. And this is because we don't know for how long the addition is shared after finishing the trip. So, Eliza, ELISA test should not be used .

To, should not be used in animals that do not show diarrhoea. I also included in the, in the treatment part of the, of the talk, a little, a little bit about probiotics because it has been extensively used for prevention or for treatment of gastrointestinal disease. And I included the findings of a recent review.

And they, in this, in this review, what they looked basically was they look for studies of acute or chronic GI disease. And they look if the administration of probiotics, decrease clinical signs and improve faecal consistency. So if probiotics help in the resolution of diarrhoea.

As the conclusion of this systemic review, they found a limited effect of probiotics on reducing all these Signs. In some studies, the administration of probiotics reduced diarrhoea and improved clinical signs while in others, it didn't. And the conclusion is like there is a need for a larger, for larger multicentric studies.

And in this table, I'll try to, I try to summarise a little bit the studies that the systemic review show in which the administration of probiotics actually caused significant improvement of clinical science or thesis. So these are the list and these are the ones that they found that the work and the reference, the review references in the bottom of the slide if you want to check it. Again, some studies in the in, in the rotor modent enterococcus rium are less in diarrhoea and this shedding in rodents.

But when it was used in, in dogs that were subclinical with GR shelter dogs with diarrhoea. And the results were variable. It's also important to remember that these animals indicate that it's not common, but if they are, they are showing vomiting, they are vomiting or they have small bowel diarrhoea, highly digestive diets, bland diets are recommended.

If large bowel diarrhoea is, is happening, high fibre diets are recommended. As part of the treatment, it is important to wash the dogs also to shampoo the dogs with chloroidine shampoo. And this is important to remove the cysts from the coats.

In this way, you prevent the infection of the, of the dogs by licking themselves or also by, also by reducing the, the environmental contamination. CRDia fortunately, is, is, is a parasite that is easily killed and regular disinfectants such as household bleach or Guatemariaorion compounds, following the manufacturing, delusions, work well eliminating GRA. It's also important to have rigorous hygiene like a daily removal of faeces, personal hygiene of animal cares, thorough, wash of the hands, cleaning the environment, food and water containers, and litter boxes should be washed, with boiling water and detergent.

Just basic, hygiene rules should be applied in general. And another way also of preventing not only Giardia but any other gastrointestinal pathogens is to prevent and if possible, to prevent cats and dogs from hunting, to feed them, commercial diets or cooked meals because raw meat diets have been found to have to harbour parasites. Some, and another bacteria that are GI, that can cause GI disease.

And it's also important to enforce a leash laws and require owners to remove faeces, the, the faeces deposits from their pets just to, decrease environmental contamination. So as a conclusion, I saw that if I have to conclude all this talk, I just want to restate that apparently there's limited sonotic risk from from companion animals from the last reviews, and based on what I show you in the slides, and we have to remember that infections could be, and the direction of the infections could be or anthroposonnotic. And in terms of treatments, treatments, They are recommended only with two animals with diarrhoea or clinical signs and to evaluate clinical response of treatment with centrifugal flotation and to have only To use only the faecal elyzer in animals with diarrhoea.

So that's all I have for today and I just want to thank you for the opportunity of giving this talk and I'm happy to receive any questions anytime. Thank you so much for your attention.