PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Giuseppe Quaranta

Curriculum vitae

Curriculum Vitae

Phd thesis

TITLE: Biological agents and antimicrobial resistance in mammals and birds hospitalized in a wildlife rescue centre

• Scientific background/state of the art

Wildlife rescue centres (WRC) represent a valuable source of data and information about wildlife diseases (1, 2). The monitoring of rescued animals allows to define the causes of disease, the potentially carried pathogens, as well as their microbiota and antimicrobial resistant (AMR) bacteria of which they could serve as a reservoir (3).

Emerging infectious diseases represent a growing threat for global health (4, 5); many of them are zoonosis and originate from wildlife (6). Intestinal microbiota is the sum of all microorganisms which colonize the gut and plays a critical role in host physiology and health (7, 8). Antibiotic resistance is an emerging risk, too (9). It develops due to the direct pressure exerted by antimicrobials overuse (10) but resistant genes can be also shared among bacteria through horizontal gene transfer (11).

Wild animals are considered important sentinels because of their distribution in the environment (12). Among wild animals, hedgehogs are small mammals which increasingly come into close contact with humans and domestic animals (13), becoming potential carriers of emerging pathogens and AMR bacteria (14). Wild raptors, instead, are predators at the top of the food chain; therefore, they can collect microorganisms from preys and are regarded as useful indicators of the spread of microorganisms in their habitat (15).

For these reasons, wildlife surveillance is an essential part of the One Health approach because it allows the monitoring of pathogens and AMR bacteria that can affect both animals and human beings (16).

 

• Aims

The aim of this PhD project is to evaluate the epidemiological role of WRC in the surveillance and characterization of the pathogens and the diseases affecting wildlife.

In order to fulfil this goal four objectives have been identified:

1. To detect the main causes of admission and, eventually, death of hedgehogs
2. To inspect emerging biological agents (such as viruses, bacteria and protozoa including the vector-borne ones) carried by hedgehogs and to associate their presence with potential anatomo-histopathological findings.
3. To describe the composition of faecal microbiota of hedgehogs and birds of prey and to analyse its changes following a hospitalization period.
4. To study antibiotic-resistance patterns of commensal bacteria (Escherichia coli and Staphylococcus) isolated from hedgehogs and raptors at the day of admission, after a hospitalization period.

Birds of prey will be sampled to fulfil objective C and D in order to analyse the differences of microbial composition and antimicrobial resistance among mammals and bird species occupying different ecological niches and how they will vary according to hospitalization.

 

• Materials and methods

In order to achieve these goals, adult hedgehogs and selected species of raptors rescued by Centro Animali Non Convenzionali (C.A.N.C.) of the Veterinary Teaching Hospital of Turin University will be sampled. Birds of prey focus of the study will be two diurnal (Falcus tinnunculus and Buteo buteo) and two nocturnal (Athene noctua and Strix aluco) species, selected because they are the most commonly admitted species in the centre.

To fulfil objective A, to assess an adequate number of cases for statistical evaluation, a minimum number of 30 hedgehogs (calculated with IBM® SPSS Statistics) will undergo a clinical evaluation to determine their cause of admission and a blood sampling will be performed to detect their metabolic profile. Animals dead during hospitalization will be submitted to a complete necropsy and histological/microbiological evaluations will be performed. These findings will be correlated with in vivo results.

Arthropods and selected organs will be collected during necroscopy to evaluate by PCR the potential presence of emerging and zoonotic biological agents (objective B) and their possible correlation with anatomo-histopathological findings will be examined.

At admittance and on the tenth day of stay an anal/cloacal swab will be collected and analysed through NGS technology (16S metagenomic approach) to inspect intestinal microbiota and its changes over hospitalization (objective C).

Bacterial isolation and identification of E. coli and Staphylococcus spp through MALDI-TOF analysis will be performed from anal/cloacal and nasopharyngeal swabs, taken on the day of arrival (and possibly on the tenth). On the isolated strains, Kirby-Bauer test, identification of integrons and of the main resistance genes through PCR will be carried out to inspect their mechanisms of resistance and, if possible, how it changes during hospitalization (objective D). Similar investigations will be done on selected birds of prey to fulfil the goals C and D. A minimum number of 15 samples for diurnal and nocturnal species will be collected.

 

• Expected results

In last decades a growing interest on wildlife diseases has developed (17) but knowledge available on this topic is still limited, as well as on pathogens and commensal bacteria carried by wild animals (18). At the end of this PhD project more information on the causes of admission and death of selected wild species at WRC are expected to be available and correlations among clinical and anatomo-histopathological findings will be disclosed. We expect to isolate the target microorganisms from hedgehogs and to identify histological modifications of their organs attributable to the presence of the microorganisms. Microbiota of the sampled species will be assessed and it is expected to be influenced and modified during hospitalization. Human activities are one of the main causes of antimicrobial resistance spread in the environment (19) and, since the significant anthropization levels in Piedmont region (e.g., the presence of densely populated towns, of farms and landfills even in rural areas), a high prevalence of AMR bacteria is expected to be isolated from wild animals (even if not directly treated with antibiotics).

 

• References

1. Fuentes-Castillo D, Farfán-López M, Esposito F, Moura Q, Fernandes MR, Lopes R, et al. Wild owls colonized by international clones of extended-spectrum β-lactamase (CTX-M)-producing Escherichia coli and Salmonella Infantis in the Southern Cone of America. Science of the Total Environment. 2019; 674: 554-562.

2. Gourlay P, Decors A, Moinet M, Lambert O, Lawson B, Beaudeau F, et al. The potential capacity of French wildlife rescue centres for wild bird disease surveillance. European Journal of Wildlife Research. 2014; 60: 865–873.

3. Camacho M, Hernández JM, Lima-Barbero JF, Höfle U. Use of wildlife rehabilitation centres inpathogen surveillance: A case study in white storks (Ciconia ciconia). Preventive Veterinary Medicine. 2016; 130: 106–111.

4. Heymann DL, Chen L, Takemi K, Fidler DP, Tappero JW, Thomas MJ, et al. Global health security: the wider lessons from the west African Ebola virus disease epidemic. The Lancet. 2015; 385: 1884–1901.

5. Morens DM, Fauci AS. Emerging infectious diseases in 2012: 20 years after the institute of medicine report. mBio. 2012; 3: e00494–12.

6. Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL, Daszak P. Global trends in emerging infectious diseases. Nature. 2008; 451: 990–993.

7. Cao J, Hu Y, Liu F, Wang Y, Bi Y, Lv N, et al. Metagenomic analysis reveals the microbiome and resistome in migratory birds. Microbiome. 2020; 8(1): 26.

8. Benson AK, Kelly SA, Legge R, Ma F, Low SJ, Kim J, et al. Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. Proceedings of the National Academy of Sciences. 2010; 107(44): 18933-8.

9. Santos T, Silva N, Igrejas G, Rodrigues P, Micael J, Rodrigues T, et al. Dissemination of antibiotic resistant Enterococcus spp. and Escherichia coli from wild birds of Azores Archipelago. Anaerobe. 2013; 24: 25-31.

10. Holmes AH, Moore LSP, Sundsfjord A, Steinbakk M, Regmi S, Karkey A, et al. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet. 2016; 387: 176–187.

11. Middleton H., Ambrose A. Enumeration and antibiotic resistance patterns of fecal indicator organisms isolated from migratory Canada geese (Branta canadensis). Journal of Wildlife Diseases. 2005; 41:334–341

12. Veldman K, van Tulden P, Kant A, Testerink J, Mevius D. Characteristics of cefotaxime-resistant Escherichia coli from wild birds in The Netherlands. Applied and Environmental Microbiology. 2013; 79: 7556–61.

13. Delogu M, Cotti C, Lelli D, Sozzi E, Trogu T, Lavazza A, et al. Eco-Virological Preliminary Study of Potentially Emerging Pathogens in Hedgehogs (Erinaceus europaeus) Recovered at a Wildlife Treatment and Rehabilitation Center in Northern Italy. Animals. 2020; 10(3): 407.

14. Garcias B, Aguirre L, Seminati C, Reyes N, Allepuz A, Obón E, et al. Extended-Spectrum β-Lactam Resistant Klebsiella pneumoniae and Escherichia coli in Wild European Hedgehogs (Erinaceus europeus) Living in Populated Areas. Animals. 2021;11(10): 2837.

15. Jurado-Tarifa E, Torralbo A, Borge C, Cerdà-Cuéllar M, Ayats T, Carbonero A, García-Bocanegra I. Genetic diversity and antimicrobial resistance of Campylobacter and Salmonella strains isolated from decoys and raptors. Comparative Immunology, Microbiology & Infectious Diseases. 2016; 48: 14-21.

16. Vidal A, Baldomà L, Molina-López RA, Martin M, Darwich L. Microbiological diagnosis and antimicrobial sensitivity profiles in diseased free-living raptors. Avian Pathology. 2017; 46(4): 442-450.

17. Michel AL, Van Heerden H, Crossley BM, Al Dahouk S, Prasse D, Rutten V. Pathogen detection and disease diagnosis in wildlife: challenges and opportunities. The Scientific and Technical Review. 2021; 105-118.

18. World Organisation for Animal Health (OIE). Principles and methods for the validation of diagnostic tests for infectious diseases applicable to wildlife. In Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 8th Ed. 2018. OIE, France.

19. Alcalá L, Alonso CA, Simón C, González-Esteban C, Orós J, Rezusta A et al. Wild birds, frequent carriers of Extended-Spectrum β-Lactamase (ESBL) Producing Escherichia coli of CTX-M and SHV-12 Types. Microbial Ecology. 2016; 72: 861–869.