PhD in Veterinary Sciences for Animal Health and Food Safety

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Curriculum vitae

Phd thesis

Title: The in-field evaluation of new blood inflammatory, fecal microbiome and metabolomic biomarkers for the characterization of severe forms of Chronic Enteropathies in dogs and cats.

State of the art

The term canine and feline chronic enteropathies (CE) refer to a heterogeneous group of idiopathic diseases characterized by persistent gastrointestinal clinical signs. They are classified according to the treatment response as food-responsive enteropathy (FRE), antibiotic-responsive enteropathy (ARE) and immunosuppressive-responsive enteropathy (IRE).^1,2 In case of inflammatory loss of proteins across the gut lumen, the term protein losing enteropathy (PLE)³ is preferred. Inflammatory PLE affects mostly dogs and it is characterized by guarded prognosis, high rate of relapse and poor treatment response.^4,5 The pathogenesis of CE and inflammatory PLE is multifactorial but still unclear,⁴although intestinal microbiome and its interaction with the patient seems to play an important role.⁶ In addition, although some blood prognostic biomarkers have been studied (vitamin D, cobalamin, blood urea concentration, etc..), the results are not convincing yet^7,8 and lipid profiles are unknown. A deeper comprehension of pathogenetic mechanisms could help the clinician to better characterize clinical phenotypes of CE and inflammatory PLE, further explore the intestinal dysfunction and better identify the appropriate therapeutic approach while minimizing time and invasiveness. To date, there is no evidence of specific dysbiosis patterns that can help to differentiate CE phenotypes^6,9, and no biomarkers are available to assess temporal changes in disease clinical activity and predict clinical course of CE and inflammatory PLE¹⁰.

Aims

To evaluate the diagnostic utility of unexplored tests/biomarkers and fecal microbiota/metabolome profiles, in order to better characterize different CE phenotypes/inflammatory PLE, improve therapeutic approaches and disease outcome.

Specifically, the research project will consist of four different developments:

• Fecal microbiota analysis of cats diagnosed with IRE and low-grade alimentary lymphoma before and after therapy
• Evaluation of selected CBC inflammatory indices in dogs with inflammatory PLE and exploring their correlation with disease severity (canine chronic enteropathy clinical activity index, CCECAI)² before and after therapy
• Evaluation of the lipid profiles of dogs diagnosed with inflammatory PLE and exploring their correlation with disease severity before and after therapy
• Evaluation of fecal microbiota and selected fecal metabolites (bile acids, fatty acids, sterols) of dogs diagnosed with inflammatory PLE and exploring their correlation with disease severity and follow-up

Materials and methods  

All different project developments will be organized as multicentric prospective comparison study. Statistical power analysis will be performed for every project development, to assess an adequate number of cases for statistical comparison; a minimum number of 30 cats and 50 dogs it is expected.  will be collected from dogs and cats referred to the VTH of Turin University and referral clinics of the professional association Endovet before and after 1-month of therapy. Inclusion criteria will be defined basing on previously published diagnostic pathways^5,11 Control groups will be built including healthy dogs and cats with overlapping signalment respect to study groups. Ethical approval will be requested.

• Fecal microbiota analysis from diseased (IRE and low-grade alimentary lymphoma) and healthy cats will be performed via16S rDNA gene (Biomolecular Research Genomics, Padua)
• Selected inflammatory indices (see aims, point 2) from healthy dogs and dogs with inflammatory PLE will be calculated and correlated with CCECAI
• Serum concentrations of cholesterol/triglycerides will be measured. Plasma lipoprotein agarose gel electrophoresis (dedicated kit) will be performed (Department of Veterinary Sciences, Milan University). Samples from healthy dogs and dogs with inflammatory PLE will be analyzed
• Sequencing techniques (PCR based assays) will be used to analyze fecal microbiota. Fecal bile acids, fatty acids and sterols will be measured using a gas chromatography-mass spectrometry assay (GC/MS) from healthy dogs and dogs with inflammatory PLE (GI Lab Texas A6M University, USA)

Expected results

The hypotheses related to the different developments of the PhD project are, respectively:

• Fecal microbiota analysis should help to discriminate IRE from low-grade alimentary lymphoma (a disease similar to IRE but with a different prognosis) and help in the prognostic prediction
• Inflammatory indices should be altered in dogs with PLE and should correlate with CCECAI. These results will help in the prognostic prediction and will guide different therapeutic protocols
• The analysis of lipid profile will help to better characterize the pathogenesis of lipid loss in inflammatory PLE. Eventual correlations with disease severity will help to guide a more aggressive therapy
• Fecal microbiota and metabolome profile analysis may provide a more accurate and complementary alternative to traditional diagnostic tools (histopathology). Furthermore, correlations with disease severity will possibly help in the prognostic prediction and to guide different therapeutic protocols.

Research activities

- Partecipation at 75° Convegno SisVet. Oral Presentation “ Neutrophil to Lymphocyte ratio, Monocyte to Lymphocyte ratio, Platelet to Lymphocyte ratio, Albumin/Globulin ratio and C-Reactive protein/Albumin ratio in dogs with inflammatory protein losing enteropathy”

- Poster Presentation: 75° Convegno SisVet “ A retrospective evaluation of Neutrophil to Lymphocyte ratio, Monocyte to Lymphocyte ratio and Platelet to Lymphocyte ratio in cats with obstructive uripathy”. Data 15-18 Luglio 2022

Pubblicazioni: 

• “Critical review on the usage of antimicrobials and on the restoration of the intestinal environment in canine acute and chronic diarrhea” Authors: 1 Gianella P., 2 Pietra M., 1 Cagnasso F., 2 Galiazzo G., 3 Marchetti V., 3 Pierini A., 4 Boari A., 4 Crisi P. E., 5 Furlanello T., 6 Marchegiani A., 6 Cerquetella M.                                            Veterinaria Year 35, n. 6, December 2021, pg 313-323.

Publications