Multidisciplinary approach to Cheetahs affected by gastrointestinal disease: study of the immune profile and of the fecal proteome

“One Health” concept links human health, animal health and the environment with a great potential to mutually influence the health of all species. Infectious diseases associated with wildlife can worse negative public perceptions of wild animals and public support for conservation requiring conscientious effort and urgency to avoid an undesirable outcome 2 . The exponential growth of the human population led to ecologic drastic changes related to human encroachment on wildlife habitats 3 . During the 20th century, growth of human population, invasion of wildlife habitats, changes in agricultural practices, domestication of wild animals and ecotourism have damaged the conservation of wildlife 4,5 . Establishing and maintaining protected areas are key tools for biodiversity conservation. However, this approach is insufficient for wide-ranging species. Protected areas, created for conservation purposes, do not always adequately conserve biodiversity, many terrestrial protected areas within human-dominated systems are isolated from one another increasing the risk of species extinctions 6 . The relationship between isolation and extinction is the basis of the “metapopulation theory” that many spatially distinct subpopulations reconnected by movement of individuals, leading to genetic exchange and the possibility of re- establishing extirpated subpopulations 7 . Numerous emerging infectious diseases have arisen from, or have been identified in, wildlife with implications for human health. For this reason, wildlife conservation can be considered an important resource of protection8 . Since 1960 the IUCN Red List has made multiple lists for a compendium of animals and plants conservation. To date, more than 134000 species have been assessed for The IUCN Red List, of these 26% are mammals 9 . In thisthesis, different aspectsrelated to an animal classified as vulnerable by the IUCN Red List, the cheetah, will be focused. The aim is to investigate the gastrointestinal physiopathology of cheetahs, with a morbidity rate of 95% in captive animals, correlating it with the immune response. Different studies compared diseases in captive and free-ranging cheetahs showing the prevalence of stress-induced pathologies 10. The reduction of the population has led several cheetahs to be housed in zoos, in conditions that do not allow carrying the physiological behaviors of the species 10. For this reason, some pathologies are defined as probably stress-induced. The present research project is based on a multidisciplinary approach to the physiopathology of “cheetahs” GI diseases. In the first part of the study, starting from a common viral disease of cats (feline infectious peritonitis - FIP) in which the immune system plays a key role in disease progression, methods of monocytes’ isolation from peripheral blood were investigated, and then macrophages were studied regarding phagocytosis and respiratory burst activities. The method was firstly performed from samples of cats with feline infectious peritonitis (FIP) and cats’ samples positive for Feline coronavirus but not FIP, and then performing the same evaluations in samples from healthy cheetahs and from subjects with GI disease associated to Helicobacter spp. In the second part of project, the study of the cytokines profile on the same samples was performed, comparing results between animal populations (healthy vs diseased cheetahs). In the third line of research, fecal proteomics to investigate possible differences in healthy and diseased cheetahs was performed

Study of Macrophage Activity in Cats with FIP and Naturally FCoV-Shedding Healthy Cats

Coronavirus frequently infects humans and animals, showing the ability to recombine and cross over to different species. Cats can be considered a model for studying coronavirus infection, in which feline coronavirus (FCoV) represents a major enteric pathogen related to gastroenteric disease. In this animal, the virus can acquire tropism for macrophage cells, leading to a deadly disease called feline infectious peritonitis (FIP). In this study, monocyte-derived macrophages were isolated by CD14-positive selection in venous whole blood from 26 cats with FIP and 32 FCoV-positive healthy cats. Phagocytosis and respiratory burst activities were investigated and compared between the groups. This is the first study comparing macrophage activity in cats affected by FIP and healthy cats positive for FCoV infection. Our results showed that in cats with FIP, the phagocytic and respiratory burst activities were significantly lower. Our results support the possible role of host immunity in Coronaviridae pathogenesis in cats, supporting future research on the immune defense against this systemic disease

Therapeutic approaches to coronavirus infection according to “One Health” concept

Coronaviridae constantly infect human and animals causing respiratory, gastroenteric or systemic diseases. Over time, these viruses have shown a marked ability to mutate, jumping over the human-animal barrier, thus becoming from enzootic to zoonotic. In the last years, numerous therapeutic protocols have been developed, mainly for severe acute respiratory syndromes in humans. The aim of this review is to summarize drugs or other approaches used in coronavirus infections focusing on different roles of these molecules or bacterial products on viral adhesion and replication or in modulating the host's immune system. Within the "One Health" concept, the study of viral pathogenic role and possible therapeutic approaches in both humans and animals is essential to protect public health

Canine Blood Group Prevalence and Geographical Distribution around the World: An Updated Systematic Review

“Blood group” or “blood type” refers to the blood group system comprising red blood cell antigens and a specific pattern. Many studies have characterized dog blood groups by the prevalence of the Dog Erythrocyte Antigen (DEA), Kai, and Dal antigens in different geographic areas and by using a variety of methods. Some pioneering studies on blood groups, upon which others were subsequently based, were conducted by Bowdler, Colling, and Hall in the 1970s and 1980s. Our results show that most relevant studies covered the European and American continents, and the methods used to identify DEA, Kai, and Dal groups evolved from 1999 to 2020, albeit without a single method based upon specificity and sensitivity. However, the existence of rapid commercial kits for the determination of the DEA 1 group makes this group the most widely used in clinical practice. Through a systematic review, our aim was to illustrate the countries in the world where different blood groups have been identified with reference to the different methods used and the prevalence of those groups among dog breeds

Updates on Laboratory Evaluation of Feline Cardiac Diseases

Laboratory tests can be altered in cardiovascular diseases and the investigation of specific tests or biomarkers may provide additional information about myocardial damage. Traditional laboratory tests, such as cell blood count, serum biochemistry, and coagulation, can be useful in investigating patients, but are not specific. However, markers like Troponin and Natriuretic Peptides may possibly furnish further data on myocardium damage and can be used in both studying and monitoring cats with cardiac disease. Moreover, the evaluation of the thyroid profile is very important as hyperthyroid cats concomitant cardiovascular diseases are very common and they can also be a direct consequence of endocrinopathy. The purpose of this manuscript is to provide the widest possible overview of what is present in the literature about the feline clinical pathology of heart diseases through a rational division of the main alterations of traditional tests and biomarkers

Evaluation of the Fecal Proteome in Healthy and Diseased Cheetahs (Acinonyx jubatus) Suffering from Gastrointestinal Disorders

Fecal proteomics allows for the identification of proteins and peptides present in stools and is useful in finding possible new biomarkers for diagnosing and/or monitoring gastrointestinal (GI) disorders. In the present study, we investigated the fecal proteome in healthy and diseased cheetahs (Acinonyx jubatus). Captive individuals of this species frequently show gastrointestinal disorders characterized by recurrent episodes of diarrhea, rare episodes of vomiting and weight loss, associated with Helicobacter spp. infection. Fecal proteomic evaluation has been performed by two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. In healthy cheetahs, the results showed the presence of the following proteins: collagen alpha-1 (II) chain, transthyretin, IgG Fc-binding protein, titin, dystonin, isopentenyl-diphosphate Delta-isomerase 1, sodium/potassium-transporting ATPase subunit alpha-1 and protein disulfide-isomerase A6. The presence of albumin isoforms was found only in diseased cheetahs. The present paper reports the study of the fecal proteome in the cheetah, evidences some differences between healthy and diseased patients and confirms, once again, the potential of fecal proteomics for the study of the GI environment, with promising developments regarding the identification of new diagnostic/monitoring markers

ROLE OF XENOSIALIZATION IN THE PATHOGENESIS OF COLITIS IN THE CANINE MODEL: POSSIBLE POSITIVE EFFECTS OF NEW PROBIOTICS BLENDS

Inflammatory bowel disease (IBD) is a heterogeneous state of chronic intestinal inflammation with no exact known cause. In dogs, IBD is expressed histologically with lymphoplasmacytic inflammation (LPL) of gastrointestinal tract. The study evaluates the possible correlation between microbiota, dietary absorption,enteric expression of N-glycolylneuraminic acid (Neu5Gc) (xenosialization) and intestinal inflammation (xenosialitis) in dogs with IBD. Neu5Gc is indeed synthesized from its N-acetyl precursor (Neu5Ac) by cytidine-5′-monophospho-N acetylneuraminic acid hydroxylase (CMAH), absent in humans, and polymorphic in dogs

Faecal proteome in clinically healthy dogs and cats: Findings in pooled faeces from 10 cats and 10 dogs

Background: In the scientific literature, there are only a few manuscripts available on small animal faecal proteomics. Methods: In the present pilot study, this evaluation was performed using pooled faecal samples from 10 clinically healthy dogs and, for the first time, in 10 clinically healthy cats by mean of two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. Results: Our results showed the presence of nine (albumin, alkaline phosphatase, chymotrypsin-C-like, cytosol aminopeptidase, elastase-3B/proteinase E, immunoglobu-lins and nuclear pore membrane glycoprotein 210) and 14 (albumin, caspase recruitment domain-containing protein, chymotrypsin-like, deleted in malignant brain tumours 1 protein-like, hypothetical protein LOC107375, immunoglobulin, kallikrein-1, superox-ide dismutase, transthyretin precursor, interstitial collagenase-like) different proteins in canine and feline faeces, respectively. Conclusion: These preliminary findings document the presence of a range of proteins in the faeces of apparently healthy dogs and cats and may serve as a basis for larger, prospective studies to establish reference proteomic data against which diseased popu-lations can be compared

Fecal Protein Profile in Eight Dogs Suffering from Acute Uncomplicated Diarrhea before and after Treatment

Acute diarrhea is a very frequent condition affecting dogs; nevertheless, little is known about what happens in the GI tract during such conditions. Proteomics allows the study of proteins present in a specific biologic substrate, and fecal proteomic investigations have been recently implemented to study GI diseases in dogs. In the present study, the fecal protein profiles of eight dogs suffering from acute uncomplicated diarrhea at the time of inclusion was investigated for the first time, and then the same patients were followed, replicating two further evaluations at two subsequent time points (after 2 and 14 days from the first presentation), with the aim of gaining possible new insights regarding the pathologic changes in the gastrointestinal environment during such conditions. Two-dimensional gel electrophoresis (2-DE) was performed, followed by mass spectrometry. Nine spots, corresponding to four (groups of) proteins (i.e., albumin, alkaline phosphatase, chymotrypsin-C-like, and some immunoglobulins), showed significant differences at two or more of the three time points investigated, almost all behaving similarly and decreasing at T1 (2 days after the onset of the condition) and significantly increasing at T2 (14 days after the onset), mainly evidencing a reaction of the organism. Further studies including a greater number of patients and possibly different techniques are needed to confirm the present finding

Fecal Proteome Profile in Dogs Suffering from Different Hepatobiliary Disorders and Comparison with Controls

In the present study, the fecal proteomes of clinically healthy dogs (HD = n. 10), of dogs showing clinical, ultrasonographic, and/or laboratory evidence of different hepatobiliary dysfunction (DHD = n. 10), and of dogs suffering from chronic hepatitis (CHD = n. 10) were investigated with an Ultimate 3000 nanoUPLC system, coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer. Fifty-two different proteins of canine origin were identified qualitatively in the three study groups, and quantitative differences were found in 55 proteins when comparing groups. Quantitatively, a total of 41 and 36 proteins were found differentially abundant in the DHD and CHD groups compared to the control HD, and 38 proteins resulted dysregulated in the CHD group as compared to the DHD group. Among the various proteins, differently abundant fecal fibronectin and haptoglobin were more present in the feces of healthy and DHD dogs than in chronic ones, leading us to hypothesize its possible diagnostic/monitoring role in canine chronic hepatitis. On the other hand, the trefoil factor 2 was increased in DHD dogs. Our results show that the analysis of the fecal proteome is a very promising field of study, and in the case of dogs suffering from different hepatobiliary disorders, it was able to highlight both qualitative and quantitative differences among the three groups included