PhD in Veterinary Sciences for Animal Health and Food Safety

TITLE: Role of inhaled mineral particles in equine lung inflammation

● Scientific background/state of the art

Equine asthma (EA) is an umbrella term that encompasses chronic lung inflammatory disorders of adult horses. The clinical presentation of EA varies in severity from subtle to life-threatening. It is incurable in its severe form. Worldwide, EA is highly prevalent, causing economic losses in the racehorse industry and rising welfare issues for the geriatric equine population.

The etiology of EA is multifactorial and only partly explained. Available evidence supports air quality as a relevant determinant of disease presentation. Inhaled organic particles, such as endotoxins, fungal spores, or molds derived from horse feed and bedding, cause airway inflammation in affected horses, associated with clinical signs. Data suggest that other still undefined factors are implicated, however.

Among inhalable dusts, mineral particles have received a little attention in EA studies. Evidence exists in support of a high pro-inflammatory potential of some of them, such as silica, one of the most prevalent components of horse riding, racing, and training surfaces. Silica toxicity within the lung is due to its strong oxidative properties and high bio- persistence, as it eludes alveolar macrophages’ phagocytic ability while inducing their pro-inflammatory phenotype.

This project hypothesizes that inhaled mineral – especially silica – dusts originated from equine surfaces are implicated in EA pathophysiology as a currently unrecognized determinant of lung inflammation.

● Aims

Systematic research has been previously done to better understand prevalence and risk factors associated with equine lung diseases, with particular attention to the role of air particulate matter on respiratory health. Searches were performed in January 2023 in the electronic Database PubMed. The search string used was: “Standardbred horse asthma”, “Thoroughbred horse asthma”, “Standardbred horse IAD”, “Thoroughbred horse IAD”, “horse air quality” and “horse air dust”.

Based on the knowledge gaps on the levels of exposure of horses to mineral particle dusts, on the dynamics of deposition and bio-persistence of such mineral dusts in the equine lungs, and on the toxicity of many mineral species composing equine surfaces, the first aim of the proposed project is to validate a protocol for quantification of the equine lung mineral load as well as its clinical and pathological effects.

Given the potential toxicity of the mineral dusts derived from equine surfaces, collecting experimental evidence on their pathogenicity by controlled exposure of living horses was considered unethical. Instead, as a second aim of the proposed project, we plan to prospectively and regularly monitor the level of exposure to inhaled mineral particles and the respiratory health of horses stabled in a selected group of riding and/or racing structures, investigating risk factors for both mineral dust increased exposure and lung inflammation.

● Materials and methods
AIM 1 - Validating a protocol for quantifying equine lung mineral load and its clinical and pathological effects.

Task 1.1_ Equine lung mineral load is assessed at 5 specific sites for each lung using postmortem samples dried and chemically digested. Residual inorganic particles are filtered, and their quanti/qualitative assessment performed by scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM-EDS) or Raman spectroscopy. Lung lesions and refringent particles are classified by 2 experienced pathologists, using light and polarized microscopy.

Task 1.2_In a subset of cases, Task 1 is performed in conjunction with postmortem transtracheal wash (TTW) and bronchoalveolar lavage (BAL). The joint analysis of their cyto/histological profile mineral load is performed.

AIM 2 - Monitoring inhaled mineral particles exposure and their impact on equine respiratory health, investigating risk factors for increased mineral dusts exposure and lung inflammation.

Task 2.1_Surface mineral analysis and regular air sampling is performed in 6 equine riding centers throughout a year for mineral load and composition. Meteorological data, management practices, and equine respiratory health are monitored by data collection and/or validated questionnaires.

Task 2.2_On a subset of randomly chosen horses in each stable, respiratory secretion sampling is performed to validate horse respiratory health questionnaire data.

Task 2.3_Risk factor analyses.

● Expected results

Aim 1 will provide the first data on normal ranges of equine lung mineral particle load and their deposition dynamics. This, in turn, will define a simplified sampling method to this aim (preferred mineral deposition site across the lungs, reliability of light/polarized histology to predict the quality/quantity of lung mineral load). Aim 1 will also inform on the reliability of in vivo respiratory sampling (TTW, BAL) in the assessment of equine lung mineral load and its effects on respiratory health.

Aim 2 will assess real situations studying at the same time the mineral composition of riding surfaces, air samples obtained from the breathing zone of horses and riders, and respiratory secretions from the horses working in the same environment. We expect to gain new and significant data from this sampling scheme, which will help defining the role of recent and past mineral exposure in equine lung inflammation and identifying any relevant risk factor in disease development.

During my internship-equivalent period at the University of Turin, I participated into a project that aims to investigate the structure-function relationship of the airway smooth muscle in healthy and asthmatic horses. This project has led to national and international collaborations, and preliminary results obtained have been presented at scientific meetings and symposium.

 

Presenter when the name is underlined

1. Sica B. (2022) “Rottura del tendine prepubico: fattori di rischio, trattamento e prognosi” [ENG: “Rupture of the prepubic tendon: risk factors, treatment and Prognos”], During a seminar on “Peripartum diseases of mares” hold in the context of the Internship educational project, Department of Veterinary Sciences, University of Turin (18 February 2022). – Oral presentation
   
   
2. Sica B, Bullone M. (2022) “Prevalence and risk factors associated with aberrant geometric airway smooth muscle arrangement in equine severe asthma”, In: Game of Research 2022, Department of Veterinary Sciences, University of Turin, Italy (23 December 2022). – Oral presentation
   
   
3. Sica B, Parrilli AP, Memoli G, Romolo A, Bullone M. (2023) “Preliminary analysis for the optimization of 3D airway smooth muscle visualization in micro- and submicron-CT images”. In: Proceedings of the 76^th Congress of the Italian Society of Veterinary Science (SISVET), 21-23 June 2023, Bari, Italy. – Accepted as oral presentation
   
   
4. Romolo A, Sica B, Pavan C, Tomatis M, Ardit M, Di Benedetto F, Capella S, Belluso E, Turci F, Bullone M. (2023) “In vitro preliminary investigation of the toxic potential of equine riding surfaces: let the mineral hunt begin!”. In: Proceedings of the 76^th Congress of the Italian Society of Veterinary Science (SISVET), 21-23 June 2023, Bari, Italy. – Accepted as oral presentation
   
   
5. Sica B, Pavan C, Tomatis M, Romolo A, Memoli G, Turci F, Bullone M. (2023) “Exposure to respirable silica in equestrian environments and its potential contribution to lower airway inflammation in horses”. In: 41^th VCRS (Veterinary Comparative Respiratory Society) SYMPOSIUM, 23-25 October 2023, Providence, RI (US). – Accepted as poster