Detecting Klossiella equi, a potential cause of renal disease, in Ontario Horses
John R Barta | Professor, BSc (Distinction), PhD Toronto and Elizabeth Zeldenrust | Graduate Student, PhD

Klossiella equi is a single celled, obligate intracellular parasite (Apicomplexa, Adeleorina) that parasitizes the kidneys of equids, including horses. The prevalence of Klossiella infections is unknown because infected hosts usually have no clinical signs. Most infections are found incidentally during necropsy. The infective stages (sporocysts) are difficult to detect in urine making diagnosis of infections in living horses challenging. It has been shown that this parasite can cause damage to equine kidneys in cases of heavy infection, especially in immunocompromised hosts.

The proposed research would attempt to establish prevalence of the disease by developing a molecular diagnostic test specific for this parasite. The DNA-based test could be applied retrospectively to previous parasite-positive post mortem cases as well as for detection of infections in live horses. Without knowing the infection status of a particular horse, it is difficult to associate an infection with this biologically unusual parasite with particular clinical presentations or evaluate any contribution to renal disease. This research will create a sensitive DNA-based test to detect K. equi and then apply this assay on kidney tissues, blood or urine from clinically normal horses in Ontario. The newly developed test could provide the prevalence of this potential pathogen, K. equi, in Ontario horse populations.

Klossiella equi was first described in Hungary (Baumann 1946) so it is unknown if the same parasite, or a different species, is responsible for infections in Ontario horses. The next step of this research will to do multi-locus sequence-based genotyping of Klossiella-positive samples from Eastern Europe and compare the European sequences to the sequence of the Klossiella already sequenced from Ontario horses.

Ultrasound examination for assessment of closure of the nephrosplenic space in Horses
Nicola Cribb | Assistant Professor, VetMB, MA Cambridge, DVSc Guelph, Dipl. ACVS

Currently, preventative laparoscopic surgery for nephrosplenic entrapment colic is the recommendation for horses to reduce the risk of disease of this type. The technique is well established but 2 recent reports in the scientific literature have highlighted our lack of knowledge of aspects of this surgery. The papers have highlighted that firstly, we currently do not know how long the preventative effects from the surgery last, and, secondly, we do not know whether the outcome from the surgery can be evaluated by ultrasound examination. In our research herd, there are 12 horses that have recently undergone closure of the nephrosplenic space by the PI. It is our aim to follow these horses for up to 3 years following their surgery to determine whether the adhesions last this length of time. These findings will be correlated with a concurrent ultrasonographic examination to see if this imaging modality can be used to determine closure of the space.

Culture-Based Molecular Profiling of the Colonic Bacteria of Horses with Typhlocolitis
Luis G. Arroyo | Associate Professor , Lic. Med. Vet., DVSc, PhD, DACVIM

Typhlocolitis is an inflammatory process of the cecum and colon that can lead to acute and severe diarrhea horses. Affected horses can develop life threatening hypovolemia, electrolytes and acid-base disorders, toxaemia and sepsis. The most common enteropathogens associated with diarreha in adult horses include Salmonella spp., Clostridium spp (mainly C. perfringens and C. difficile), Neorickettsia risticii (endemic areas) and coronavirus; but a causal agent for a large proportion of these cases (>60%) cannot be established. In recent years, culture-independent methods revealed that microbial communities of the cecum and colonic contents of healthy horses are dominated by a fairly stable population of Firmicutes and either Bacteroidetes or Verricomicobiota. This method also showed that there is marked decreased in microbial richness and diversity occurs humans and animal with gastrointestinal disease.

The aim of this study is to characterize the anaerobic bacteria within the large colon by combining culture enriched-based methods and molecular profiling of intestinal contents of horses with or without colitis.

Improving Fecal Microbiota Transplantation in Horses
Luis G. Arroyo | Associate Professor , Lic. Med. Vet., DVSc, PhD, DACVIM

Fecal transplantation has saved thousands of lives in other species, but the method needs to be refined before it can be used to treat horses with intestinal flora imbalances (e.g. colic and colitis). The intestinal flora, also called microbiota, is comprised by thousands of microorganisms, especially bacteria. It is now known that the intestinal bacteria are very important to maintain health and that several diseases can occur because of imbalances on the composition of this bacteria (called dysbiosis).

Diseases affecting the gastrointestinal tract are the main cause of mortality in horses and bacterial imbalances have been demonstrated in horses with colitis, colic and under stressful situations (e.g. post-partum and shipping). Recent efforts have focused on manipulation of the intestinal bacteria to treat and prevent dysbiosis. Microbiota manipulation can be achieved by controlling diet, by using therapies such as antibiotics and probiotics, or by transferring the bacteria from a healthy individual to a patient. This procedure is called “fecal microbiota transplantation” (FMT). FMT has been used with great success to treat other species, but our preliminary data demonstrated that current recommendations are not efficient in horses.

Therefore, a refinement of the method is necessary before FMT can be recommended to be used in horses. In this study, we propose to test new doses to successfully use FMT to restore the normal intestinal bacteria of horses with bacterial imbalance. We will also bypass the proximal portions of the intestine by delivering the microbiota directly into the large colon to try to increase FMT efficacy. Experimental Design: We propose to induce microbiota imbalance in a group of horses by treating them with oral antibiotics (TMS). Then we will evaluate the efficiency of FMT by comparing current recommendations (FMT once a day) with a group receiving the procedure four times per day. Fecal bacteria before and after FMT will be evaluated using next generation DNA sequencing. Furthermore, we will deliver FMT directly into the distal gut to determine if the FMT efficacy can be increase.

Equine Mesenchymal Stromal Cells Ability to Help Fight Bacterial Infections
Thomas G. Koch | Associate Professor, DVM Copenhagen, PhD Guelph

Antimicrobial resistance may be one of the biggest medical challenges in the coming decades. This issue transcends species and a one-health approach, including both veterinary and human patients, is therefore desirable to test new potential treatment tactics. One recent approach is the use of mesenchymal stromal cells (MSC) to eliminate microorganisms. MSC have demonstrated antimicrobial effects not only through the modulation of the immune response, but also through the secretion of antimicrobial substances. These properties may allow MSC to make currently ineffective antibiotics effective again against resistant bacteria. Understanding what these MSC secreted substances are may allow us to produce or isolate these substances and use them as regular drugs in combination with currently utilized antibiotics to develop new treatment strategies.

We have selected bacteria commonly affecting horses to prove the principle idea of the project that MSC can make antibiotics more effective. MSCs from five horses will be paired with three isolates of anti-microbial resistant bacteria and tested for antimicrobial susceptibility. MSC included in the MSC bacterial co-culture will be harvested following co-culture, and RNA will be isolated for nextgeneration sequencing (NGS) to evaluate which MSC genes could be related to this antimicrobial effect. This work has the long-term potential to discover new therapies that can either potentiate current antimicrobials or maybe even act as novel antimicrobials on their own. This foundational work will support future funding applications to support clinical trials in client-owned horses of natural diseases.

Isolation and Culture of Equine Mesenchymal Stromal Cells in Serum-Free Conditions
Thomas G. Koch | Associate Professor, DVM Copenhagen, PhD Guelph

Fetal bovine serum is a central component of current culture media for the isolation and expansion of mesenchymal stromal cells in veterinary medicine. The use of fetal bovine serum supplementation has many drawbacks including increased risk of adverse reactions and batch-to-batch variation that can make it more difficult to predict the efficacy of a given cell-based therapy. For these reasons, so-called serum-free media has been developed to support the isolation and expansion of human mesenchymal stromal cells. Few studies have tested serum-free media on equine cells and the results have been discouraging showing reduced growth rate and other cell alterations compared to cells maintained in fetal bovine serum. These disappointing results could be due to unique metabolic requirements of equine mesenchymal stromal cells or improper coating of culture vessels and poor adaptation protocols when using media developed for human cells.

We hypothesize that equine umbilical cord blood-derived MSC can be isolated and culture expanded in commercially available serum-free media. Our objectives are: 1) To compare the isolation success of equine umbilical cord blood-derived (CB) MSC in FBS containing media and one serumfree media on different surfaces. 2) To compare the functional properties of established CB-MSC cultures with regards to differentiation potential towards the adipogenic, osteogenic and chondrogenic cell fates as well as ability to suppress lymphocyte proliferation in vitro. 3) To determine if serum-free grown cells are cryotolerant. We expect that we can establish MSC cultures using serum free conditions. The ability to isolate and expand MSC under standardized conditions is an important step towards more standardized therapeutic effect due to reduced product variability. Reduced risk of adverse reactions is another important goal of this work.