**2. Antibiotic residues in pet food and adverse food reactions**

In the last 35 years, a surprising increase of skin and gastrointestinal (GI) diseases in both *cats* and *dogs* has been observed [1]. For instance, *in vivo* studies have widely demonstrated that the most commonly responsible ingredients for the onset of cutaneous and gastrointestinal adverse food reactions are beef, dairy products, wheat, and to some degree, lamb, soy, and fish [2–5]. However, only in a few cases were such adverse food reactions clearly ascribed to the presence of food additives such as dyes, preservatives, or even antibiotics [6–8].

One of the main symptoms of skin‐related diseases is severe itching, which may lead to self‐ inflicted injuries caused by obsessive scratching, while frequent gastrointestinal symptoms are vomiting and diarrhea, which continue to persist after therapy. These phenomena suggest paying particular attention to the history of several examined cases in order to determine their primary and real cause.

We recently identified a specific compound, oxytetracycline (OTC), as the possible underly‐ ing cause of most inflammatory pathologies both *in vitro* [9, 10] and *in vivo* [11, 12]. OTC belongs to the class of tetracyclines, which are the most widely and legally used antibiotics in intensive farming, for example, poultry [10], livestock [13], and aquaculture [14], due to their low cost and efficacy [15]. Unfortunately, OTC also has a high affinity for calcium‐rich tissues such as bone and teeth [16] and can remain fixed for extended periods in treated animals even respecting withdrawal time [10]. Moreover, pet food production relies on meat (mainly poultry) by‐products, which are mechanically separated [17, 18]. This kind of sepa‐ ration, and the common use of important percentages of bone meal mixed with meat meal, generates a bone‐based meal‐bearing OTC residues that is present in commercially available diets (canned, semi‐moist, and especially dry) in a percentage of 20–30% and can accumu‐ late within pet's body.

Odore et al. and Di Cerbo et al. recently demonstrated the significant *in vitro* toxicity of milled bone from chickens treated with OTC, either alone or diluted 1:4, toward peripheral blood mononuclear cell (PBMC) culture (\*\*p < 0.01 and \*\*\*p < 0.001, respectively) [9, 10]. Conversely, bone derived by chickens untreated with OTC did not show any cytotoxic effect (**Figure 1**).

Furthermore, Di Cerbo et al. have recently shown the *in vitro* ability of the OTC to induce a significant interferon (IFN)‐γ release from human T lymphocytes and non‐T cells [9].

Besides the ability to induce mortality of both the T lymphocytes and non‐T cells after a 48‐h co‐incubation, OTC was also able to induce the release of pro‐inflammatory cytokines in the first 10–12 h of challenging. More in detail, T lymphocytes increased their IFN‐γ production once exposed to OTC or to the culture media conditioned with the bone derived by OTC‐ treated chickens in order to resemble the same conditions of intensive farming [15].

Both the innate immunity (non‐T cells, mainly represented by natural killer (NK) lymphocytes) and the acquired immunity (T lymphocytes, CD8+ , and CD4+ ) [19, 20] resulted to be influenced by the OTC toxicity (**Figure 2**).

**2. Antibiotic residues in pet food and adverse food reactions**

326 Superfood and Functional Food - An Overview of Their Processing and Utilization

In the last 35 years, a surprising increase of skin and gastrointestinal (GI) diseases in both *cats* and *dogs* has been observed [1]. For instance, *in vivo* studies have widely demonstrated that the most commonly responsible ingredients for the onset of cutaneous and gastrointestinal adverse food reactions are beef, dairy products, wheat, and to some degree, lamb, soy, and fish [2–5]. However, only in a few cases were such adverse food reactions clearly ascribed to

One of the main symptoms of skin‐related diseases is severe itching, which may lead to self‐ inflicted injuries caused by obsessive scratching, while frequent gastrointestinal symptoms are vomiting and diarrhea, which continue to persist after therapy. These phenomena suggest paying particular attention to the history of several examined cases in order to determine their

We recently identified a specific compound, oxytetracycline (OTC), as the possible underly‐ ing cause of most inflammatory pathologies both *in vitro* [9, 10] and *in vivo* [11, 12]. OTC belongs to the class of tetracyclines, which are the most widely and legally used antibiotics in intensive farming, for example, poultry [10], livestock [13], and aquaculture [14], due to their low cost and efficacy [15]. Unfortunately, OTC also has a high affinity for calcium‐rich tissues such as bone and teeth [16] and can remain fixed for extended periods in treated animals even respecting withdrawal time [10]. Moreover, pet food production relies on meat (mainly poultry) by‐products, which are mechanically separated [17, 18]. This kind of sepa‐ ration, and the common use of important percentages of bone meal mixed with meat meal, generates a bone‐based meal‐bearing OTC residues that is present in commercially available diets (canned, semi‐moist, and especially dry) in a percentage of 20–30% and can accumu‐

Odore et al. and Di Cerbo et al. recently demonstrated the significant *in vitro* toxicity of milled bone from chickens treated with OTC, either alone or diluted 1:4, toward peripheral blood mononuclear cell (PBMC) culture (\*\*p < 0.01 and \*\*\*p < 0.001, respectively) [9, 10]. Conversely, bone derived by chickens untreated with OTC did not show any cytotoxic effect (**Figure 1**).

Furthermore, Di Cerbo et al. have recently shown the *in vitro* ability of the OTC to induce a

Besides the ability to induce mortality of both the T lymphocytes and non‐T cells after a 48‐h co‐incubation, OTC was also able to induce the release of pro‐inflammatory cytokines in the first 10–12 h of challenging. More in detail, T lymphocytes increased their IFN‐γ production once exposed to OTC or to the culture media conditioned with the bone derived by OTC‐

Both the innate immunity (non‐T cells, mainly represented by natural killer (NK) lymphocytes)

, and CD4+

) [19, 20] resulted to be influenced

significant interferon (IFN)‐γ release from human T lymphocytes and non‐T cells [9].

treated chickens in order to resemble the same conditions of intensive farming [15].

and the acquired immunity (T lymphocytes, CD8+

by the OTC toxicity (**Figure 2**).

the presence of food additives such as dyes, preservatives, or even antibiotics [6–8].

primary and real cause.

late within pet's body.

**Figure 1. Percentage of PBMC undergoing apoptosis**. On the *x*‐axis, different cell incubations and conditioned cell culture medium dilutions, after 12h (B) and 24h (A) of incubation, are shown. OTC‐CCM indicates the conditioned cell culture medium challenged with a ground bone of chickens treated with OTC; the C‐CCM indicates the growth medi‐ um challenged with a ground bone of chickens treated without OTC, while OTC alone indicates a growth medium with the addition of 1 μg/ml of OTC. The "ctr" indicates the incubation in the growth medium with Annexin V stain‐ ing, which has been used as a control of the apoptosis that occurs in the cells when in a culture without any other incubation is maintained; \*\**p* < 0.01, and \*\*\**p* < 0.001 (with the permission of John Wiley and Sons) [18].

**Figure 2. Percentage of IFN‐γ production in CD4+ and CD8+ T lymphocytes and in non‐T cells.** The bar column graphs represent the mean values of the percentage of IFN‐γ‐producing cells. On the *x*‐axis, different cell incubations and conditioned cell culture medium dilutions are shown. OTC‐CCM indicates the conditioned cell culture medium challenged with a ground bone of chickens treated with OTC; the C‐CCM indicates the growth medium challenged with a ground bone of chickens treated without OTC, while OTC alone indicates a growth medium with the addition of 1 μg/ml of OTC. The condition indicates as "ctr" refers to basal IFN‐γ production. All the cell cultures (ctr, OTC alone, OTC‐CCM, and C‐CCM) were maintained in a growth medium added with PMA and ionomycin to induce cyto‐ kine production. Panels A, B, and C show IFN‐γ production in CD4+ T lymphocytes, CD8+ T lymphocytes, and in non‐ T cells, respectively; \* *p* < 0.05, \*\**p* < 0.01, and \*\*\*\**p* < 0.001 (with the permission of John Wiley and Sons) [18].

In this context, it is known that IFN‐γ represents the main cytokine involved in the immune response [21], as well as a crucial element in the onset of impaired tissue homeostasis condi‐ tions, typically related to autoimmunity or chronic inflammation [22–30].

These observations clearly reinforce the potential toxicity associated with chronic consumption of poultry bones and derivatives by pets and pave the way for a new concept of food sensiti‐ zation due to contaminant presence as main enhancers of inflammatory processes, which typically characterize skin and gastrointestinal diseases. Although the Food and Drug Administration [31] and World Health Organization [32] have recently established maximum residue limits in foods, antibiotic residues in foods may still be present [33] thus explaining the persistence of dermatological manifestations in many pets. Moreover, international laws do not impose an antibiotic concentration evaluation in bones and fat, which are considered inedible, thus making pet food dangerous for pet's health [34].

A wide number of scientific reports suggest the possible toxicity and harmfulness of OTC toward human and pet health as a consequence of the consumption of meat derived from intensive farming [15, 35–40].

All of these data may explain why chicken proteins, widely considered hypoallergenic and highly effective from a dietary point of view, play an important role in the etiology of several inflammatory pathologies. It is worth noting that the similarities between these phenomena and food allergies, atopy, and Flea allergy dermatitis have been observed. In spite of the limited evidence that canine food allergy is suggested to resemble a type I hypersensitivity reaction to allergens ingested by food, it cannot be excluded that non‐IgE‐mediated food allergies may also occur. Although literature reports have been evidenced that the prevalence of food allergies in *dogs* and *cats* is still unknown, the impressive number of cases is not justified merely on the basis of increased allergy spreading in civilized societies. Furthermore, it has been observed that 25% of the *cats* with both chronic GI and skin problems do not clinically express food allergies, while the remaining 75% had only gastrointestinal problems. On the other hand, there are no data regarding food allergies related to gastrointestinal problems in *dogs*. In addition, food allergies can be often confused with pyoderma, pruritic exudative dermatitis or "hot spots."

Based on our recent studies, we investigated the sera of 24 *dogs* with food‐adverse reactions, that is, itching, diarrhea, otitis, dermatitis, conjunctivitis, overnight fasting, vomiting, flatu‐ lence, interdigital pyoderma, and anal sacs repletion for the presence of any haptens which might be responsible for such conditions by means of an enzyme‐linked immunosorbent assay (ELISA) (FS0059, IDLABS™ Inc. Biotechnology, PO Box 1145, Station CSC, London ON N6A 5K2, Canada) according to the manufacturer's instructions [12].

Results indicated the presence of OTC and doxycycline in all animal sera. Although only eight out of 24 *dogs* (33%) showed antibiotic concentrations above the ELISA detection limit (7.5 ng/ ml or ppb), all the remaining *dogs* presented serum levels of both antibiotics. OTC serum levels ranged from 2.61 to 56.04 ng/ml (6.30 ± 2.12; mean ± standard error of the mean), whereas doxycycline serum levels ranged from 1.28 to 22.84 ng/ml (5.20 ± 0.89; mean ± standard error of the mean). Our preliminary clinical investigation further confirmed the haptenic toxic‐ sensitizing mechanism due to prolonged subliminal oral intake of OTC‐enriched bone‐meal‐ based feeds derived from animals grown under a chronic tetracycline administration regime.
