Gianandrea Guidetti

Epiphora is defined as tear overflow due to impaired tear drainage by way of the nasolacrimal duct. We evaluated a combined therapeutic approach for epiphora based on drug and a commercially available nutraceutical diet administration over a 30 days period in dogs poorly responsive or unresponsive to the only immune-suppressive
treatment. Forty-five client-owned household dogs of different breeds (19 females and 31 males) and aged 6.5 years were enrolled. Schirmer tear test-1, conjunctival inflammation, corneal keratinization and blepahritis were evaluated before and at the end of the evaluation.
Schirmer’s test value significantly decreased from 22.96 ± 0.37 mm, at T0 to 18.86 ± 0.24 mm, at T30 (***p < 0.001), conjunctival inflammation significantly decreased from 1.30 ± 0.11, at T0 to 0.14 ± 0.04 at T30 (***p < 0.001), corneal keratinization significantly decreased from a T0 value of 0.17 ± 0.05 to a T30 value of 0.07 ±
0.03 (*p < 0.05) and blepharitis significantly decreased from 0.64 ± 0.1, at T0 to 0.03 ± 0.02 at T30 (***p < 0.001). This clinical evaluation represents the first evidence of the usefulness of a specific nutraceutical diet as a reliable tool to improve pharmacological treatment of epiphora.

In the present study, we investigated the efect of a pharmaceutical formulation (OTC – Ossitetraciclina liquida 20%) used as antibiotic, on the mechanical properties of K562 cells by using the Micropipette Aspiration Technique (MAT). This formulation has been shown to increase in a time dependent way the infammation and toxicity in terms of apoptosis in in vitro experiments on K562 and other types of cells.

Severe adverse reactions of the organism to environmental elements have been dizzily rising in humans and pets over the last 50 years. Such reactions can be expulsive (vomit, diarrhea, dandruff, and abundant secretion or excretion) or driven by an inflammatory process (which has been considered as healing process) in charge to destroy every toxic introduced into the body. Thus, it is clear that if a contaminated food is assumed daily, the inflammatory process becomes inevitably chronic.
Most common inflammatory processes of dogs and cats originate from this condition, which we observed to be frequently caused by welldefined contaminants: toxic residues of oxytetracycline (OTC). In fact, once everything containing in this compound is eliminated, all inflammatory processes tend to rapidly and spontaneously regress. Here, we reviewed and discussed the problem related to the amount of pharmacological and chemical substances, which are used to increase the production
of fruits, vegetables, intensive farming-derived meat and fish, milk, eggs, and grain. Such substances can persist within the products in variable amount and, gradually or rapidly (often in a few hours), poison the organism causing reactions such as allergies, anaphylactic shocks (not so frequent), autoimmune diseases (fortunately not so frequent but continuously increasing), and inflammatory processes, the most common reaction. In this context, nutrition, as a daily and frequent habit, should be taken seriously into account; given that wild animals do not seem to have the same pathologic reactions, there are no doubts that many foods deriving from intensive farming have become a poison rather than a remedy.

Worldwide antibiotic abuse represents a huge burden, which can have a deep impact on pet and human health through nutrition and medicalization representing another way of antibiotic resistance transmission.
We aimed our research to determine a possible complex formation between biological bone substrates, such as proteins, and Oxytetracycline (OTC), an approved antibiotic for use in zootechny, which might determine a toxic effect on K562 cells.
Cell viability and HPLC-ESI/QqToF assays were used to assess potential toxicity of bone extract derived from OTC-treated chickens according to standard withdrawal times and from untreated chickens at 24, 48 and 72h of incubation.
Cell culture medium with ground bone from chickens reared in the presence of of OTC (OTC-CCM) resulted significantly cytotoxic at every incubation time regardless of the bone concentration while cell culture medium with ground bone from chickens reared without OTC (BIO-CCM) resulted significantly cytotoxic only after 72h of incubation. HPLC-ESI/QqToF assay ruled out the possible presence of OTC main derivatives possibly released by bone within culture medium until 1 μg/mL.
The presence of a protein complex with OTC is able to exert a cytotoxic effect once released in the medium after 24-48h of incubation.

In the last decades, adverse food reactions have increased considerably in dogs and cats. In this study we report on the possible onset of food intolerances symptoms, including otitis, diarrhoea, generalised anxiety, and dermatitis in a cohort of 8 dogs consuming commercial diets. All dogs received an organic chicken-based diet for 15 days. We performed analysis of blood biochemical parameters, kibble composition, and oxytetracycline (OTC) serum concentration before and after 15 days of organic chicken-based diet supplementation. We hypothesised that a chronic intake of contaminated food enhanced by the presence of nanoparticle aggregates might be at the base of the onset of pharmacologic or idiopathic food intolerances.

Food allergies and food intolerances are clinically difficult to discriminate. Most often, along with cutaneous adverse food reactions or CAFR, they are classified as adverse food reactions, whose causes are numerous, including toxic compounds.
Eighteen indoor-housed domestic cats with evident clinical symptoms related to CAFR (drooling, back and neck intense itching, neck eczema, chronic conjunctivitis and stomatitis) involving skin lesions were studied. Cytological evaluations of ear, skin and gingival swabs revealed an increased turnover of keratinocytes while the oxytetracycline ELISA determination showed an unexpected high amount of oxytetracycline in all cats at the first visit. All cats were then randomly assigned to receive a standard (SD group) or a nutraceutical diet (ND group) for 60 days.
In the ND group a significant reduction of the mean serum concentration of oxytetracycline, pruritus intensity and skin lesion severity (**p<0.01, ***p<0.001, and ***p<0.001, respectively) was observed after 60 days, and associated with a significant improvement in the clinical picture. Although a direct correlation between oxytetracycline presence within cat sera and CAFR-related symptoms has never been described, this study highlights the benefit of a specific nutraceutical diet supplementation in improving clinical symptoms and skin lesions in cats with CARF.

Urolithiasis accounts for 15% to 23% of cases of feline lower urinary tract disease (FLUTD), with struvite uroliths occurring more frequently, followed by calcium oxalate, ammonium urate, cystine, and xanthine calculi. In this clinical evaluation, we tested the efficacy of a commercially available nutraceutical diet in 33 cats affected by struvite uroliths. Results clearly indicated a significant urine color, turbidity, pH, RBC, WBC, weight and proteins decrease (***p < 0.001, *p < 0.05), and a significant decrease of struvite uroliths in all treated cats. The nutraceutical diet enriched by botanicals, such as, Hieracium pilosella, Urtica dioica, Lespedeza spp, Vaccinium macrocarpon, Taraxacum officinale formulated with DL-methionine, and a controlled addition of minerals and amino acids resulted particularly effective for struvite uroliths management. This work can pave the way for a new, safe, and long lasting natural approach to treat struvite uroliths.

Functional foods provide health benefits if they are consumed on a regular basis as part of a varied diet. In this review, we discuss the availability and role of functional foods in pet nutrition with a focus on dogs and cats. Indeed, functional foods modify gastrointestinal physiology, promote changes in biochemical parameters,
improve brain functions and may reduce or minimize the risk of developing specific pathologies. This evidence derives largely from clinical studies while only limited evidence is available from studies in dogs and cats. Therefore, functional food consumption should be further investigated in pet nutrition to understand how
dietary interventions can be used for disease prevention and treatment.

Background. Oxytetracycline (OTC), which is largely employed in zootechnical and veterinary practices to ensure wellness of farmed animals, is partially absorbed within the gastrointestinal tract depositing in several tissues. Therefore, the potential OTC toxicity is relevant when considering the putative risk derived by the entry and accumulation of such drug in human and pet food chain supply. Despite scientific literature highlights several OTC-dependent toxic effects on human and animal health, the molecular mechanisms of such toxicity are still poorly understood.
Methods. Here, we evaluated DNA damages and epigenetic alterations by quantitative reverse transcription polymerase chain reaction, quantitative polymerase chain reac-tion, chromatin immuno-precipitation and Western blot analysis.
Results. We observed that human peripheral blood mononuclear cells (PBMCs) expressedDNAdamage features (activation ofATMand p53, phosphorylation ofH2AX and modifications of histone H3 methylation of lysine K4 in the chromatin) after the in vitro exposure to OTC. These changes are linked to a robust inflammatory response indicated by an increased expression of Interferon (IFN)- and type 1 superoxide dismutase (SOD1).
Discussion. Our data reveal an unexpected biological in vitro activity of OTC able to modify DNA and chromatin in cultured human PBMC. In this regard, OTC presence in foods of animal origin could represent a potential risk for both the human and animal health.

Functional foods provide health benefits if they are consumed on regular basis. Some nutraceutical pet diets have been demonstrated to exert health benefits in vitro and in vivo while also exhibiting palatability to the animals. The aim ofthis chapteris to provide an overall update of commercially available pet diets with proven efficacy against pathologies with an inflammatory background. Research on pet food is still scarce and biased. The ultimate success of functional pet foods will depend on delivering bioactive components in a predictable and assured mannerto effectively reduce the risk of disease and/or support the body. Our investigations outlined the improved health status of sick dogs by means of a commercially available nutraceutical pet diet approach. Therefore, additional investigations into the consumption of functional foods in domestic animal nutrition should be done in order to study dietary interventions for disease prevention and treatment.

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, the traditional ways to monitor behavioral changes in dogs are complicated and not efficient. In the current clinical evaluation, a new, simple monitoring system was used to assess the effectiveness of a specific diet in positively modulating the intense and restless activity of 24 dogs of different ages and breeds. This protocol describes how to easily and rapidly evaluate improvement in a set of symptoms related to generalized anxiety by using a specific sensor, a mobile phone app, a wireless router, and a computer. The results showed that dogs treated with specific diets showed significant improvement in the times spent active and at rest after 10 days (p < 0.01 and p < 0.05, respectively). These dogs also showed an overall significant improvement in clinical and behavioral symptoms. A specific sensor, along with its related hardware, was demonstrated to successfully monitor behavioral changes relating to movement in dogs.

The well-being of dogs can be affected by changes in human lifestyle, eating habits and increased stressors that lead to behavioural disorders including fear, hyperactivity and anxiety, followed by negative affective moods and poor welfare. This randomised, controlled clinical evaluation involved 69 dogs, 38 males and 31 females, of different breeds, with behavioural disorders related to anxiety and chronic stress. They were fed a control diet or a nutraceutical diet (ND group) for 45 days. Neuroendocrine (serotonin, dopamine, β-endorphins, noradrenaline and cortisol) and stress (derivatives of reactive oxygen metabolites (dROMs) and biological antioxidant potential (BAP)) parameters related to behavioural disorders were evaluated at the beginning and end of the study period. Results showed a significant increase in serotonin, dopamine and β-endorphins plasma concentrations (*P<0.05, *P<0.05 and **P<0.01, respectively) and a significant decrease in noradrenaline and cortisol plasma concentrations in the ND group (*P<0.05). dROMs significantly decreased in the ND group (*P<0.05) while BAP was not affected. This study demonstrated for the first time that a specific diet significantly and positively affected neuroendocrine parameters and dROMs. These results open significant perspectives concerning the use of diet and nutraceuticals in the treatment of behavioural disorders.

Fifty dogs of different breeds affected by immune-mediated cKCS were equally distributed and randomly assigned to receive either a standard diet (control, n = 25) or the nutraceutical diet (treatment group, n = 25) both combined with standard immunosuppressive therapy over a 60 days period. An overall significant improvement of all clinical parameters (tear production, conjunctival inflammation, corneal keratinization, corneal pigment density and mucus discharge) and the lack of food-related adverse reactions were observed in the treatment group (p < 0.0001).
Conclusions: Our results showed that the association of traditional immune-suppressive therapy with the antioxidant/anti-inflammatory properties of the nutraceutical diet resulted in a significant amelioration of clinical signs and symptoms in cKCS. The beneficial effects, likely due to the presence of supplemented nutraceuticals in the diet, appeared to specifically reduce the immune-mediated ocular symptoms in those cKCS-affected dogs that were poorly responsive or unresponsive to classical immunosuppressive drugs. These data suggest that metabolic changes could affect the immune response orchestration in a model of immune-mediated ocular disease, as represented by cKCS.

Several extrinsic factors, like drugs and chemicals, can foster autoimmunity. Tetracyclines, in particular oxytetracycline (OTC), appear to correlate with the emergence of immune-mediated diseases. Accumulation of OTC, the elective drug for gastrointestinal and respiratory infectious disease treatment in broiler chickens, was reported in chicken edible tissues and could represent a potential risk for pets and humans that could assume this antibiotic as residue in meat or in meat-derived byproducts. We investigated the in vitro anti-inflammatory properties of a pool of thirteen botanicals as a part of a nutraceutical diet, with proven immunomodulatory activity. In addition, we evaluated the effect of such botanicals in contrasting the in vitro proinflammatory toxicity of OTC. Our results showed a significant reduction in interferon- (INF-) γ production by human and canine lymphocytes in presence of botanicals (p<0.05). Increased INF-γ production, dependent on 24-hour OTC-incubation of T lymphocytes, was significantly reduced by the coincubation with Haematococcus pluvialis, with Glycine max, and with the mix of all botanicals (p<0.05). In conclusion, the use of these botanicals was shown to be able to contrast OTC-toxicity and could represent a new approach for the development of functional foods useful to enhance the standard pharmacological treatment in infections as well as in preventing or reducing the emergence of inflammatory diseases.

Tetracyclines, which represent one of the most commonly used antibiotics for poultry, are known to be deposited in bones, where they can remain, despite the observation of appropriate withdrawal times. The aim of the study was to determine the concentration of oxytretracycline (OTC) residues in the bone and muscle of chickens, following the oral administration of a commercially available liquid formulation, and to test their cytotoxic effects on an in vitro cell culture model. Seventy-two 1-day-old broiler chickens were randomly allotted into 2 groups (control and treated animals). OTC (40 mg/kg BW) was administered via drinking water during the 1 to 5 and 20 to 25 days of life periods. At the end of the trial, the birds were slaughtered and the OTC residues in the target tissues were measured by means of liquid chromatography (LC) – tandem mass spectrometry (MS/MS). Cytotoxicity was assessed by evaluating the pro-apoptotic effect of the bone residues on the K562 erythroleukemic line and on the peripheral blood mononuclear cells (PBMC). In all the animals, the OTC residues in the muscle were far below the established MRL of 100 μg/kg. The OTC levels in the bones of the treated animals were instead found in the parts per million (ppm) range. Cell cytotoxicity was assessed by evaluating the pro-apoptotic effect of OTC bone residues on the haematopoietic cell system. This in vitro system has revealed a significant pro-apoptotic effect on both the K562 cell line and PBMC cultures. This result suggests potential human and animal health risks due to the entry of tetracycline residues contained in the bones of treated livestock into the food-chain. This could be of concern, particularly for canine and feline diets, as meat, bone meal, and poultry by-products represent some of the main ingredients of pet foods, especially in the case of dry pet food. Further studies are needed to define the underlying mechanisms of cytotoxicity and to evaluate the in vivo toxicological implications due to the observed in vitro effects

Antibiotics are widely used in zoo technical and veterinary practices as feed supplementation to ensure wellness of farmed animals and livestock. Several evidences have been suggesting both the toxic role for tetracyclines, particularly for oxytetracycline (OTC). This potential toxicity appears of great relevance for human nutrition and for domestic animals. This study aimed to extend the evaluation of such toxicity. The biologic impact of the drug was assessed by evaluating the proinflammatory effect of OTC and their bone residues on cytokine secretion by in vitro human peripheral blood lymphocytes. Our results showed that both OTC and OTC-bone residues significantly induced the T lymphocyte and non-T cell secretion of interferon (IFN)-γ , as cytokine involved in inflammatory responses in humans as well as in animals. These results may suggest a possible implication for new potential human and animal health risks depending on the entry of tetracyclines in the food-processing chain

Clinical appearance and evolution of Canine Leishmaniosis (CL) are the consequence of complex interactions between the parasite and the genetic and immunological backgrounds. We investigated the effect of an immune-modulating diet in CL. Dogs were treated with anti- Leishmania pharmacological therapy combined with standard diet (SD Group) or with the immune-modulating diet (IMMD Group). CD3+ CD4+ Foxp3+ Regulatory T cells (Treg) and CD3+ CD4+ IFN-γ + T helper 1 (Th1) were analyzed by flow cytometry.

Biological aging is characterized by a progressive accumulation of oxidative damage and decreased endogenous antioxidant defense mechanisms. The production of oxidants by normal metabolism damages proteins, lipids, and nucleotides, which may contribute to cognitive impairment. In this study 36 dogs were randomly divided into four groups and fed croquettes of different compositions for 6 months. We monitored derivatives of reactive oxygen metabolites (dROMs) and biological antioxidant potential (BAP) levels in dogs’ plasma samples as well as brain-derived neurotrophic factor (BDNF) serum levels at the beginning and at the end of the dietary regime. Our results showed that a dietary regime, enriched with antioxidants, induced a significant decrease of plasma levels of dROMs ( < 0.005) and a significant increase in BDNF serum levels ( < 0.005) after six months. Thus, we hypothesized a possible role of the diet in modulating pro- and antioxidant species as well as BDNF levels in plasma and serum, respectively. In conclusion the proposed diet enriched with antioxidants might be considered a valid alternative and a valuable strategy to counteract aging-related cognitive decline in elderly dogs.

The aim of this evaluation study was to assess the possible role of a specific nutraceutical diet in relieving main clinical symptoms of chronic bilateral otitis externa (occlusion of ear canal, erythema, discharge quantity, and odor) in 30 adult dogs. Thirty dogs of different breeds (mean age ± SEM; 6.03 ± 0.15 years and mean weight ± SEM; 32.01 ± 1.17 Kg; 53.3 % males, 46.6 % females) with evident chronic clinical otitis symptoms were equally divided and randomly assigned to receive either the nutraceutical diet (ND group) or a standard diet (SD group) over a period of 90 days. In all cases a topical pharmacological treatment was given. The nutraceutical diet, also endowed with anti-inflammatory and antioxidant activities, significantly decreased the mean score intensity of all symptoms after 90 days of intervention (P< 0.0001) with the exception of Malassezia pachydermatis infection which was only slightly reduced. Our investigation is one of the few evidence-based results where a commercial nutraceutical diet has been proven effective, in combination with drugs, in relieving otitis externa-related symptoms. This study opens new insights into otitis externa clinical management providing evidence of efficacy of a combined therapy with drugs and a specific nutraceutical diet.

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized placebo-controlled crossover clinical evaluation, we assessed the effectiveness of a dedicated dietary supplement to improve chronic halitosis in 32 dogs of different breeds and ages. This protocol describes how to evalute the presence of oral volatile suphur compunds, e.g. methyl mercaptan, hydrogen sulfide and dimethyl sulfide, by means of a portable gas chromatograph device coupled with a syringe, which was used to collect the breath, and a dedicated software, which allows the operator to monitor each compound concentration during each measurement, in a relatively short time (8 min). A significant modification of halitosis parameters was observed after 30 days since the beginning of treatment (p <0.05), while a long-lasting effect was still observed even 20 days after the suspension of the treatment. Portable gas chromatograph, which is also widely used in clinical practice, can be therefore used to confirm and control halitosis in humans and animals. Even though human and animal species present some differences, this innovative and alternative therapy for halitosis management might be extended to human clinical practice as an adjuvant dietary approach.

Oxidative Stress (OS) is considered an underlying mechanism by which dysfunctional metabolism occurs in obese subjects but there are still very few studies in canine species. The purpose of this study was to evaluate simultaneously the effects of diet on body weight, oxidative and inflammatory status in a group of 12 adult dogs. The dogs were fed a maintenance diet, integrated with natural antioxidants, for a period of 6 months. At the beginning and the end of the trial, Body Condition Score (BCS) was evaluated and haematological (CBCs, Complete Blood Counts), oxidative parameters (d-ROMs, derivatives of Reactive Oxygen Metabolites; BAP, Biological Antioxidant Potential; Retinol; α-tocopherol) and inflammatory parameters (Fibrinogen; CRP, C Reactive Protein) were performed. Significant differences (p < 0.05) emerged about BCS, hematocrit (HCT), number of platelets (PLT), d-ROMs, BAP and retinol while no differences were for α-tocopherol, fibrinogen and CRP between the two periods of different diet. In this study, dogs showed an oxidative imbalance documented by the increase in d-ROMs and the reduction of BAP and retinol. Inflammatory parameters don’t change in relation to body weight like an alteration of the oxidative status could precede the onset of inflammation. The role of oxidative stress and of integration with antioxidants should be taken into special consideration in the dietary treatment in overweight dogs.

We evaluated and analyzed the role of functional foods in pet diet in order to extend the link of food consumption in human nutrition.
Methods: We analyzed some specific nutrients in pets on the basis of historical literature reports claiming some effectiveness in cancer prevention. Other on going focused areas are metabolic and immunological imbalance and chronic joints inflammatory conditions.

We investigated the serum level of possible new haptens in 15 heavy meat consumers for sport fitness affected by various kinds of food intolerance and who had ever been administered antibiotics in their life for clinical problems.