PhD in Veterinary Sciences for Animal Health and Food Safety

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Curriculum vitae

Phd thesis

Project title: Clinical and clinicopathological characterization of canine chronic enteropathy: the in-field evaluation of inflammatory, microbiomic and metabolomic profiles.  

Background

Chronic enteropathy in dogs is a group of complex, nonspecific gastrointestinal (GI) disorders characterized by clinical signs lasting 3 weeks or longer and for which other causes (e.g., parasites, bacterial infections, cancer, and non-GI diseases) have been excluded. CE are classified according to the treatment response as food-responsive enteropathy, microbiota-related modulation-responsive enteropathy, and immunosuppressive-responsive enteropathy. All these form of CE, if severe enough to lead to loss of proteins into the intestinal lumen, described a syndrome named inflammatory protein-losing enteropathy (iPLE). IPLE affects mostly dogs and it is characterized by guarded prognosis, high rate of relapse and poor treatment response. The pathogenesis of CE and iPLE is still unclear but it seems to be multifactorial, arising from a complex interplay of genetic background, environment, intestinal innate and adaptive immune system, intestinal microbiome disbalances. Lymphatic disease and dysbiosis seems to play a key role in iPLE pathogenesis. A deeper comprehension of pathogenetic mechanisms could help the clinician to earlier detect the severer forms of the disease, identify alternative therapeutic approaches and improve the outcome minimizing time and invasiveness. To date, however, there is not still clear consensus about diagnostic utility and prognostic value of gastrointestinal biomarkers.

Specific aims of the project and methods

This project aimed to explore new markers of disease severity and therapeutic response, and secondly to investigate lipid dysmetabolism in a population of dogs affected by iPLE. 

Specifically, the research project consisted in three different developments:  

1. Retrospective investigation of blood ratios as potential markers of disease activity in 53 dogs with iPLE and 68 healthy dogs. Neutrophil to lymphocyte ratio (NLR), albumin to globulin ratio (AGR), C-reactive protein to albumin ratio (CRP/ALB) were calculated and correlated with disease severity score and other clinico-pathological variables, before and after one month of treatment. Ratios were also compared between responders and non responders dogs, classified based on CCECAI improvement. 
2. Investigation of serum lipid profile and fat-soluble vitamins in a population of dogs affected by iPLE. This was a prospective and control study; 51 iPLE dogs and 40 healthy dogs were enrolled. Serum concentrations of C-reactive protein, paraoxonase-1, tryglicerides, cholesterol, and lipoproteins obtained by electrophoretic separation were studied. Serum level of vitamin A, D and E were analyzed through high-performance liquid chromatography. 
3. Investigation of intestinal lipid malabsorption trhough the study of fecal microbiome and metabolome perturbations in dogs with iPLE. Sequencing techniques for the determination of Dysbiosis Index (DI) and gas-chromatography/mass-spectrometry assay were used for a targeted analyses and the determination of fecal long-chain fatty acids, sterols and bile acids. This was a prospective observational study conducted on 38 iPLE dogs and 47 healthy dogs. 

Results: 

1. NLR was significantly increased in iPLE dogs, as expected. It was also higher than a previous cut-off identified for dogs affected by chronic enteropathy. NLR was not associated to clinical disease activity and didn't improved at T1. AGR and CRP/ALB were significantly associated with clinical disease activity and response to treatment, since they correlated significantly with CCECAI score (negatively and positively, respectively) and significantly improved at T1. 
2. Dogs with iPLE showed altered serum lipid profile and fat-soluble vitamins profile.          Serum concentrations of Chol, HDLs, and PON-1 were significantly lower in iPLE dogs, to the contrary TGs, LDLs and CRP were significantly higher. Lipid profile didn't correlate with clinical severity, but Chol, HDLs and PON-1 were lower in the subgroup of severe hypoalbuminemia. HDL correlated negatively with CRP and positively with PON-1, LDL correlated negatively with PON-1. These results suggest pro-oxidative status, involving lipid profile, in dogs with iPLE. Fat soluble vitamins were decreased in dogs with iPLE suggesting severe intestinal lipid dysmetabolism and lipid malabsorption.
3. Only a subset (55%) of dogs with iPLE showed a dysbiotic status based on the determination of Dysbiosis Index. Fecal targeted metabolome analysis was clearly shifted in dogs with iPLE respect to healthy dogs. A subset of 30% of iPLE dogs showed significant fecal bile acid dysmetabolism, with an increase of total primary bile acids and decrease of total secondary bile acids and concurrent low abundance of C. hiranonis. Fecal long chain fatty acids and sterols were significantly increased in iPLE dogs. The increase of these fecal metabolites seems to be the result of severe mucosal damage and shedding, lymph vessels rupture and impaired intestinal permeability. Long-chain fatty acids and sterols didn't correlate with bacterial taxa and DI at Multi-omics analysis. At T1, fecal metabolome partially improved. In conclusion, intestinal lipid dysmetabolism is reflected by fecal content increase of long chain fatty acids and sterols and it is not microbiome-dependent.  

References

1. Dandrieux JRS. Inflammatory bowel disease versus chronic enteropathy in dogs: are they one and the same?: IBD versus CE. J Small Anim Pract. 2016;57(11):589-599.
2. Allenspach K, Wieland B, Gröne A, Gaschen F. Chronic Enteropathies in Dogs: Evaluation of Risk Factors for Negative Outcome. J Vet Intern Med. 2007;21(4):700-708.
3. Gianella P, Lotti U, Bellino C, et al. Clinicopathologic and prognostic factors in short- and long-term surviving dogs with protein-losing enteropathy. Schweiz Arch Tierheilkd. 2017;159(3):163-169.
4. Craven MD, Washabau RJ. Comparative pathophysiology and management of protein‐losing enteropathy. J Vet Intern Med. 2019;33(2):383-402.
5. Allenspach K, Iennarella-Servantez C. Canine Protein Losing Enteropathies and Systemic Complications. Vet Clin North Am Small Anim Pract. 2021;51(1):111-122.
6. Marsilio S, Pilla R, Sarawichitr B , et al. Characterization of the fecal microbiome in cats with infammatory bowel disease or alimentary small cell lymphoma Scientific Reports nature researcher (2019) 9:19208
7. Suchodolski JS. Diagnosis and interpretation of intestinal dysbiosis in dogs and cats. Vet J. 2016;215:30-377. Becher A, Suchodolski JS, Steiner JM, Heilmann RM. Blood neutrophil-to-lymphocyte ratio (NLR) as a diagnostic marker in dogs with chronic enteropathy. J Vet Diagn Invest. 2021;33(3):516-527.
8. Nakashima K, Hiyoshi S, Ohno K, et al. Prognostic factors in dogs with protein-losing enteropathy. Vet J. 2015;205(1):28-32.
9. Pilla R, Suchodolski JS. The Role of the Canine Gut Microbiome and Metabolome in Health and Gastrointestinal Disease. Front Vet Sci. 2020;6:498.
10. Otoni CC, Heilmann RM, García-Sancho M, et al. Serologic and fecal markers to predict response to induction therapy in dogs with idiopathic inflammatory bowel disease. J Vet Intern Med. 2018;32(3):999-1008.

Research activities

Publications

- Full articles in ISI journals

• F.Cagnasso, A.Borrelli, E.Bottero, E.Benvenuti, R.Ferriani, V.Marchetti, P.Ruggiero, B.Bruno, C.Maurella and P. Gianella. Comparative Evaluation of Peripheral Blood Neutrophil to Lymphocyte Ratio, Serum Albumin to Globulin Ratio and Serum C-Reactive Protein to Albumin Ratio in Dogs with Inflammatory Protein-Losing Enteropathy and Healthy Dogs. Animals 2023, 13, 484. https://doi.org/10.3390/ani13030484
• “Strongyloides Stercoralis infection and long-term follow up in a privately -owned dog from north-west of Italy”. Authors: Cagnasso F., Rambozzi L., Miniscalco B., Molinar Min A.R., Borella F., Borrelli A., Gianella  P.  Veterinaria Italiana 2023, 59 (3), x‑xx doi: 10.12834/VetIt.2969.19642.2
• Elena Benvenuti; Riccardo Ferriani; Paola Gianella; Pietro Ruggiero; Federica Cagnasso; Antonio Borrelli; Giuseppe Benvenuto; Loris Bertoldi; Enrico Bottero. “The fecal bacterial microbiota is not useful for discriminating between lymphoplasmacytic enteritis and low-grade intestinal T-cell lymphoma in cats nor for predicting therapeutic response”. American Journal of Veterinary Medicine 2024, 85, 4. https://doi.org/10.2460/ajvr.23.11.0251
• Federica Cagnasso, Jan S. Suchodolski, Antonio Borrelli, Franca Borella, Enrico Bottero, Elena Benvenuti, Riccardo Ferriani, M. Katherine Tolbert, Chih-Chun Chen, Paula R. Giaretta and Paola Gianella. "Dysbiosis index and fecal concentrations of sterols, long-chain fatty acids and unconjugated bile acids in dogs with inflammatory protein-losing enteropathy." 
  Front. Microbiol. 15:1433175. https://doi.org/10.3389/fmicb.2024.1433175

• Paola Gianella, Federica Cagnasso, Alessia Giordano, Antonio Borrelli, Enrico Bottero, Barbara Bruno, Riccardo Ferriani, Franca Borella, Sara Meazzi, Donatella Scavone and Saverio Paltrinieri. "Comparative Evaluation of Lipid Profile, C-reactive Protein and Paraoxonase-1 Activity in Dogs with Inflammatory Protein-losing Enteropathy and Healthy Dogs". Animals 2024, under minor revision.

- Full articles in non-ISI journals

• “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. 

- Book chapters 

- Congress abstract (with active participation)

• 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”. 75° Convegno SisVet, 15-18 Luglio 2022, Lodi, Italy.
• POSTER PRESENTATION: “A retrospective evaluation of Neutrophil to Lymphocyte ratio, Monocyte to Lymphocyte ratio and Platelet to Lymphocyte ratio in cats with obstructive uripathy”.  75° Convegno SisVet, 15-18 Luglio 2022, Lodi, Italy.
• ORAL PRESENTATION: “Microbiomic and metabolomic perturbations in canine protein
• losing enteropathy: an observational prospective study” EuroGut Congress, 12-13 May 2023, Edinburgh, Scotland.
• ORAL PRESENTATION: “Relationship between serum lipid profile, clinical and clinicopathological findings in 59 dogs with inflammatory protein-losing enteropathy”. International Scivac Congress, 26-28 May 2023, Rimini, Italy
• ORAL PRESENTATION: “Investigation of fecal microbiome and metabolome perturbations in dogs with protein-losing enteropathy”. 33rd ECVIM-CA annual congress, 21-23 September 2023, Barcelona, Spain

- Congress abstract (as co-author)

• POSTER: “Fecal microbiota analysis in cats with inflammatory bowel disease or small cell lymphoma before and after therapy: a prospective comparison study”. 32nd ECVIM-CA Congress, 1-3 September 2022, Gothenburg, Sweden.

Publications