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Background: 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. Objective: We aimed our research to determine a possible complex formation between biological bone substrates, such as proteins, and oxytetracycline (OTC), a widely and legally used antibiotic in zootechny, which might determine a toxic effect on K562 cells. Method: 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 antibiotic free-treated chickens at 24, 48 and 72h of incubation. Results: cell culture medium with ground bone from chickens reared in the presence of OTC resulted significantly cytotoxic at every incubation time regardless of the bone concentration while 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. Conclusion: 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.
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DOI: 10.2174/187494450181101????
SHORT COMMUNICATION
Oxytetracycline-Protein Complex: The Dark Side of Pet Food
Alessandro Di Cerbo1,2,*, Antonio Scarano2, Federica Pezzuto3, Gianandrea Guidetti4, Sergio
Canello5, Diego Pinetti6, Filippo Genovese6 and Lorenzo Corsi1
1Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
2Department of Medical, Oral and Biotechnological Sciences, Dental School, University G. d`Annunzio of Chieti-
Pescara, Chieti, Italy
3Department of Clinical Science, University of Modena and Reggio Emilia, Modena, Italy
4SANYpet S.p.a., Research and Development Department, Bagnoli di Sopra (Padua), Italy
5Forza10 USA Corp., Research and Development Department, Orlando (FL), USA
6CIGS, University of Modena and Reggio Emilia, Modena, Italy
Received: January 09, 2017 Revised: March 27, 2018 Accepted: April 08, 2018
Abstract:
Background:
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.
Objective:
We aimed our research to determine a possible complex formation between biological bone substrates, such as proteins, and
Oxytetracycline (OTC), a widely and legally used antibiotic in zootechny, which might determine a toxic effect on K562 cells.
Method:
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 antibiotic free-treated chickens at 24, 48 and 72h of incubation.
Results:
Cell culture medium with ground bone from chickens reared in the presence of OTC resulted significantly cytotoxic at every
incubation time regardless of the bone concentration while 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.
Conclusion:
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.
Keywords: Antibiotic abuse, Oxytetracycline, Protein complex, Cytotoxic effect, Pro-inflammatory effect, K562 cells.
1. INTRODUCTION
Some studies reveal that the widespread antibiotic use in agriculture and aquaculture might contribute to the
* Address correspondence this author at the Department of Life Sciences, University of Modena and Reggio Emilia, Alessandro Di Cerbo, Modena,
Italy; Tel: 00390592055367; E-mail: Alessandro811@hotmail.it
4 The Open Public Health Journal, 2018, Volume 11 Di Cerbo et al.
development of resistance to antibiotics commonly used in human medicine [1]. This issue becomes important
particularly in the zootechnical due to its accumulation in animal feed and food with potential chronic consequences
deriving from its ingestion to the species fed with these foods [2].
Nowadays, antimicrobials use represents a serious concern particularly in two correlated fields i.e. medical and
agricultural [2 - 5]. In poultry, for instance, antibiotics are used to promote growth and to treat, control, and prevent
overcrowding diseases [2, 6, 7]. A routinely exposure to antibiotics induce a selection for resistant bacteria that can
persist on meat and in animal waste with a vertical transmission through maternal generations of breeding stocks [7].
Such bacteria can get in contact with humans in food-animal production facilities, in meat processing plants but also
consuming contaminated meat [6, 8 - 10]. Despite there is not a common consensus on the use potential antibiotic
resistance elicited by antibiotic used in animal food many countries have made substantial efforts to reduce the overall
use of antibiotics in food-producing animals, in the attempt to decrease the antibiotic resistance in animals, the
environment, and in human beings.
Moreover, Mueller et al. hypothesized that food allergens e.g. beef, fish and chicken could drag antibiotics and
hormones thus representing the cause for the onset of dermatological symptoms in cats [11]. Among pharmacologically
active substances, tetracycline (in particular oxytetracycline, OTC) and their metabolites present in meats and meat-
based foods for humans and pets were considered and studied [9, 10, 12].
We firstly hypothesized and observed the role of OTC as an underlying cause of some chronic inflammatory
pathology in vivo and in vitro as described by Di Cerbo et al. [8, 9, 13 - 17]. Due to its low cost and high efficacy [18],
OTC is widely employed in the intensive farming of poultry [2], livestock [19] and aquaculture [20]. However, OTC
has a high affinity for calcium, mainly present within bones, and a very low and long clearance in treated animals [21].
Further, pet food production, which mainly relies on poultry by-products [22], also avails itself of an important
percentage of bone meal (20-30%) [8] with a consequent dragging of OTC residues that are frequently found within
commercially available diets [10].
Despite the setting of maximum residue limits in foods by Food and Drug Administration [23] and World Health
Organization [24] OTC residues may still persist since bone is not considered as an edible tissue, thus making pet food
potentially dangerous [25].
In this paper, based on our previous results [6, 21], we aimed our research to determine possible interactions
between biological substrates, such as proteins, and OTC, which might lead to an increase or decreased toxic effect on
K562 cells mediated by the antibiotic. For this purpose we evaluated the potential toxicity of bone extract derived from
OTC-treated chickens according to standard withdrawal times and from antibiotic free-treated chickens.
2. MATERIALS AND METHODS
2.1. Cell Culture
K562 myelogenous leukemia cell line, were purchased from American Type Culture Collection (ATCC) (LGC
Standards srl, Milan, Italy). K562 cells were grown in RPMI supplemented with 10% Fetal Bovine Serum (FBS) 100
g/mL streptomycin, 100 U/mL penicillin, 2 mM glutamine (Euroclone Spa, Milan, Italy). The cells were cultured in a
humidified incubator at 37 °C with 95% and 5% CO2.
2.2. Conditioned Culture Medium Preparation
Conditioned Culture Medium (CCM) was obtained by incubating 10 mL of a RPMI 1640 cell culture medium with
124, 90, 6, 2.48 mg and 124 μg of sterilized ground bone from chickens reared in the presence (OTC-CCM) or absence
(BIO-CCM) of treatments with OTC and constantly shaken for 24-48-72 h at 37° C. After incubation, both CCMs were
filtered through 0.45 and 0.20 μm syringe filters (Sartorius Stedim Biotech, Goettingen, Germany) to remove any
residual ground bone particles and microbial contamination.
2.3. Determination of Cell Viability
Cell viability was assessed after 24-48-72 h of continuous exposure with the aforementioned concentrations. A Cell
Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) was used to measure the cytotoxicity on
K562 cells. Briefly, the K562 cells were plated on 96-well plates (Euroclone, Milan, Italy) at concentration of 5000 or
10000 cells/cm2. After exposure to desired concentrations of the different compounds, 10µl of CCK-solution was added
Protein Complex: The Dark Side The Open Public Health Journal, 2018, Volume 11 5
to each well and incubated for a period of 2 h at 37°C. Finally, absorption was measured at 450 nm using a multiplate
reader Multiscan FC (Thermo Scientific, USA). Dimethyl Sulfoxide (DMSO) 3% was used as toxic reference drug. Cell
viability was expressed as a percentage of that of the untreated cells (Control). For each concentration of tested
compounds, mean values of the mean absorbance rates from four wells were calculated.
2.4. HPLC-ESI/MS Analysis
CCM samples were analyzed using a HPLC-ESI/MS analyses were performed on a 1200 Series HPLC coupled to
either a 6520A Quadrupole/Time-of-Flight high mass spectrometer (QqToF) or a 6410B Triple Quadrupole mass
spectrometer (QqQ), (both from Agilent Technologies, California, USA) via an electrospray ion source (ESI). On the
HPLC-ESI/QqToF system, the chromatographic separation was carried out with 4 mL injection volume on an Agilent
Zorbax SB-C18 30 x 2.1 mm ID 3.5 mm ps column. Elution was performed at T=25 °C, with a flow rate of 0.3 mL/min.
were used as a mobile phase. A linear gradient elution starting at 0.5 min going from 2% (B) up to 40% (B) in 11
minutes then up to 95% (B) in 6 minutes was performed using water with 0.5% formic acid (A) and Acetonitrile (B) as
mobile phase components. The mass spectrometer was operated in positive-ion mode (ESI+) using “Auto MS/MS”
acquisition with MS and MS/MS scan ranges being 50<m/z<1000 Th and selection of the top 3 most abundant mono-
charged ions from each MS scan for their subsequent MS/MS spectra acquisition (active exclusion was enabled after 1
spectrum and released after 0.15 min). Targeted MS/MS analyses on the HPLC-ESI/QqQ included chromatographic
separation on an Agilent Zorbax SB-C18 30 x 2.1 mm ID 3.5 mm ps column at 0.3 ml/min with the following gradient
(mobile phases: A: water + 0.1 FA%, B: acetonitrile + 0.1 FA%): 0’ 98:2 A:B, kept for 0.5’ then B% was raised to 40%
in 11’, then B% was raised to 95% in 3’, hold for 6’ then columns was reconditioned at the starting conditions, for a
total runtime of 32 minutes.
ESI source was operated in positive mode at 3.5 kV, the Gas Heater was set to 300 °C, the gas flow was 8 l/min and
the nebulizer pressure was set to 25 psi.
Three transitions for OTC were chosen for Selected Reaction Monitoring, as shown in the Table (1).
Table 1. Selected Reaction Monitoring for the three transitions of OTC.
Precursor (m/z) Product (m/z) Fragmentor (V) CE (V) Dwell Time (ms)
461.5 426.4 106 17 400
461.5 286.1 106 25 100
461.5 201.2 106 29 100
A matrix-matched calibration curve was prepared, ranging from 1 ppb to 1 ppm OTC concentration on a diluted and
filtered CCM; samples were filtered, diluted at the same dilution factor of the calibration curve and analyzed. For
standard-addition method, the sample was spiked with OTC ranging from 1 ppb to 1 ppm.
2.5. Statistical Analysis
All data are presented as the mean ± SD of at least three different experiments done in quadruplicate. One-way
ANOVA analysis of variance with Dunnett’s post-test, were performed to compare differences between the groups, as
indicated in the figures (Graph-Pad 6 Software Inc., San Diego, CA, USA). P values < 0.05 were considered significant.
3. RESULT
Here we found that OTC-CCM resulted significantly cytotoxic at every incubation time regardless of the bone
concentration while BIO-CCM resulted significantly cytotoxic only after 72h of incubation Fig. (1).
Qualitative analysis was performed using the HPLC-ESI/QqToF assay in order to test for the presence of OTC (at a
concentration 1mg/mL) and its main derivatives N-Demethyloxytetracycline, N-Didemethyloxytetracycline, and
Apooxytetracycline. To this purpose, their presence was tested using their molecular ion signals eventually revealed on
their respective extracted ion chromatogram (EIC) in the ESI(+) MS experiments. Due to the QqToF high resolution
and mass accuracy (< 5 ppm) each EIC was obtained using a narrow extraction window (10 ppm) centered on the
molecular ion theorical m/z value. In order to account for the eventual suppression of OTC signals at the 1 mg/mL
concentration level, due to matrix effect in extracted samples or for its degradation/combination in the extracted sample
environment, extracted samples were as well spiked with OTC at 1 μg/mL and analyzed right away (t0) and after 24 and
48 hours (t 24, t 48). Neither OTC nor its derivatives were revealed in non-spiked OTC-CCM and BIO-CCM extracted
6 The Open Public Health Journal, 2018, Volume 11 Di Cerbo et al.
samples Figs. (2, 3) while OTC molecular ion ([M+H]+ at m/z= 461.155) was clearly revealed in t 0, t 24 and t 48
spiked samples with roughly the same signal intensity (within the instrument variability). This suggested the OTC
concentration being substantially below the tested 1 μg/mL level, while reasonably excluding strong
degradation/combination reactions of OTC in the extracted matrix environment at least within the tested 48 hours
period.
Fig. (1). Graphical representation of the effects of conditioned medium with bone with (OTC-CCM) and without (BIO-CCM)
oxyteracycline on K562 cell line at different incubation times (24, 48 and 72h) and at different bone concentrations (124, 90, 6, 2.48
mg and 124 μg). *p < 0.05, ***p < 0.001 vs Control (Ctrl).
Targeted MS/MS analyses were performed on the HPLC-ESI/QqQ to quantitate OTC in bone samples. Preliminary
tests showed a heavy matrix effect, thus we optimized the dilution factor and injection volumes for best results, ending
up with a 20-fold dilution and 20 μl of diluted sample injected.
Protein Complex: The Dark Side The Open Public Health Journal, 2018, Volume 11 7
Fig. (2). From top, ESI(+) MS EIC of m/z 461.155 ion shown in the retention window of OTC in OSSO BIO non-spiked, t0, t24 and
t48 samples.
Fig. (3). From the top, ESI(+) MS EIC of m/z 461.155 ion shown in the retention window of bone with OTC non-spiked at 0, 24 and
48h.
8 The Open Public Health Journal, 2018, Volume 11 Di Cerbo et al.
Matrix-matched calibration curve showed a good fit (r2 ≥ 0.999); unfortunately, the sample signal was below the
quantification limit. In order to increase the sensitivity because of the low concentration of OTC in analyzed samples,
standard-addition method was applied. In this case, by extrapolating the intercept of the curve on the y-axis we were
able to determine OTC concentration. By correcting the value for the dilution factor, the final determined concentration
was 200 ng/ml. As a proof of concept, we ran the same standard addition analysis on the organic bone extract; the curve
in this case showed an intercept with the y-axis significantly closer to the origin, indicating that the concentration of
OTC (if any) was significantly lower in this extract compared to the bone extract Fig. (4).
Fig. (4). Triple Quadrupole mass spectrometer (QqQ-ESI) molecular iron determination of OTC-protein complex.
4. DISCUSSION
Previously published data showed a significant cytotoxic effect of OTC powder at 24, 48 and 72h at a concentration
of 150 μg/mL (according to the amount of OTC expected to be provided to chicken during a standard treatment and
disclosed on the antibiotic package) and at 48 and 72h at 75 and 35 μg/mL [8]. Moreover, the cytotoxic effect was
clearly visible at OTC concentrations far below those previously investigated [10].
We recently claimed the unavoidable occurrence of oxytetracycline as a contaminant of meat, bone meal and
poultry by-products, which are widely present within commercially available pet diets and human foods (mainly
würstels and sausages) [8, 10]. In addition, we reported a discrepancy among OTC kibble concentration, OTC serum
concentration of dogs with dermatitis and otitis, diarrhea and general anxiety and in vitro OTC, 20% liquid or powder,
last cytotoxic concentration [10]. In fact, OTC kibble concentration was 19 μg/mL, serum oxytetracycline concentration
was 0.22 μg/mL while in vitro last cytotoxic concentration was 35 μg/mL for the OTC powder and 87.5 μg/mL for the
20% liquid OTC.
Despite our in vitro data are in agreement with those previously published [6, 10], it is worth noting that HPLC-
ESI/QqToF clearly evidenced the lack of OTC and its main derivatives within conditioned medium until 1 μg/mL.
However the results obtained with Triple Quadrupole mass spectrometer (QqQ) showed the presence of an OTC-protein
complex
This latter result helped us to rule out the toxic role of OTC itself and allowed us to speculate the presence of a
protein complex inside bone with OTC able to exert a cytotoxic and pro-inflammatory effect once released in the
medium after 24-48h of incubation. Moreover, it is reasonable to hypothesize that most of symptoms observed in the
dogs of our previous paper might be due to an overall inflammatory condition elicited by the daily intake of kibbles
made of chicken-derived bone with OTC. Although more researches are needed in order to figure out the chemical
composition of the complex and the molecular mechanism involved in the cell toxicity, our results revealed a new
possible explanation linked to the side effects of chicken-derived bone with OTC.
Protein Complex: The Dark Side The Open Public Health Journal, 2018, Volume 11 9
CONCLUSION
Our research represents a further insight into the overall landscape of antimicrobials abuse pointing out the possible
consequences on pets and humans well being with particular regard to the actual antibiotic resistance spread.
Unfortunately, WHO and FDA have set high OTC minimal residual limits within organs of farmed animals and do not
consider bone as a possible veihicle of contamination since considered as not edible [8, 26]. In this view our findings
rise new questions concerning the interactions between antibiotics, i.e. OTC, and organic substrates. Indeed our results
pointed out the formation of a complex which induce cell toxicity even more then antibiotic alone. The further question
to be answerd is wether this complex is able to induce antibiotic resitence too.
Since WHO ruled out an agenda on the study and the prevention of antimcorobial resistence (AMR) in the different
fields where the antibiotic were used, further studies with more detailed evaluations are needed to support our
preliminary observations, and to confirm a possible new scenario in the AMR.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Not applicable
HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
ACKNOWLEDGEMENTS
Decleared none.
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© 2018 Di Cerbo et al.
This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a
copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.
... For this reason, the admitted dosage of antibiotic administration and allowed maximal residual limits are regulated by stringent guidelines [9,10]. However, tetracyclines, in particular oxytetracycline (OTC), which can accumulate in the bone tissue of farmed animals, are not included in the target samples imposed by the European Community [9] though they can be present in mechanically separated meat (e.g., sausages, Vienna sausages) [11] as well as in dry pet food [12][13][14]. ...
... Thus, long-lasting consumption of such food might be related to some reported cases of adverse food reactions observed in humans [16,17] and pets [12,13,18]. Moreover, several in vitro reports evidenced the toxicity [14,[19][20][21], proinflammatory [19,22], and genotoxic [23] activity of bone powder derived from chicken treated with OTC according to withdrawal times but also of OTC in its liquid form as per generally used in zootechny. ...
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... The emergence of bacterial resistance in the last few decades has increased significantly and has enhanced the economic burden of infectious diseases [2,3]. This resistance emerged because of certain factors of which the improper and excessive use of antibiotics is a leading cause [4][5][6][7][8][9][10][11][12][13][14]. Additionally, certain bacteria are capable of developing or acquiring resistance to various antimicrobial drugs; this is regarded as the multidrug resistance (MDR) [15]. ...
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The emergence of bacterial resistance has increased the economic burden of infectious diseases dramatically during the previous few decades. Multidrug resistance (MDR) is difficult to cure in both gram-negative and positive bacteria and is often incurable with traditional and broad-range antibiotics. Therefore, developing techniques to increase the antibacterial activity of therapeutic drugs is essential. Metal-organic frameworks (MOFs) are extremely versatile hybrid materials made of metal ions coupled via organic bridging ligands. They have been widely used as an excellent vehicle for drug delivery due to their low toxicity, biodegradability, and structural stability upon loading and functionalization. The present study focused on the synthesis of mannose (MNS) coated MOFs with enhanced surface contact with S. aureus cells. The MNS coating on the surface of MOFs enhances their adherence to bacteria by binding to lectins present on the bacterial cell, resulting in improved VCM cellular penetration and activity against resistant bacteria. Various techniques, including, atomic force microscopy, DLS, TGA, FT-IR, and DSC, were employed to analyze MNS-coated MOFs. They were also evaluated for their efficacy against resistant S. aureus. The results indicated that when VCM was loaded into MNS-coated MOFs, their bactericidal activity rose dramatically, resulting in greater suppression of resistant S. aureus. AFM investigation of S. aureus strains demonstrated total morphological distortion after treatment with MNS-coated drug-loaded MOFs. The results of this work suggest that MNS-coated MOFs may be effective for reversing bacterial resistance to VCM and open up new pathways for improving antibiotic therapy for diseases associated with MDR.
... In light of recent in vivo acquisitions, it might be reasonable to hypothesize a possible link between the occurrence of a high rate of resistance to tetracycline in humans and pets and the occurrence of tetracyclines in their sera [37][38][39][40]. Moreover, several reports demonstrated that even respecting withdrawal times in intensive farming, a huge amount of oxytetracycline could be recovered in the bone of the animals, in particular chickens [41], and then transferred to the pet food chain, which employs a chicken bone percentage ranging from 20% to 30% for kibble production [38,[42][43][44]. Thus, the chronic intake of such food might account for the presence of tetracyclines' resistance in pets but also in humans, where the presence of chicken bone residues has been observed in Vienna sausages (data not shown). ...
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... Real infertility is difficult to diagnose in veterinary clinical practice and therefore most bitches are classified as generally subfertile or hypofertile subjects [32]. The present clinical evaluation was based on standardized protocols used in canine reproduction and its aim was to evaluate the effect of a specific nutraceutical diet on the reproductive performance of 36 bitches presenting evident reproductive disturbances and possibly correlate them with serum concentration of a contaminant, oxytetracycline (OTC), frequently detected in pet food [33][34][35][36]. We previously observed the usefulness of a nutraceutical diet enriched with Lepidium meyenii, Tribulus terrestris, L-carnitine, zinc, beta-carotene, vitamin E, folic acid, and an omega-6:omega-3 ratio of 4:1 in improving motility percentage, semen volume and concentration, and total number of sperm per ejaculation in 28 male dogs suffering from infertility associated with hypospermia [37]. ...
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Based on the raising of food-related disturbances in animals and the recent findings concerning the toxicity of oxytetracycline presence within pet food we compared the effectiveness of two commercially available diets on 36 bitches suffering from reproductive disturbances. We run a randomized clinical evaluation over a 90-day period. Bitches were randomly assigned to receive a control (CD) or a nutraceutical diet (ND) and were assessed for the presence of OTC within their sera before and at the end of the study. 16 out of 18 bitches went on heat within an average of 35.5 ± 3.1 days, and 15 of them became pregnant. In the CD group, 12 out of 18 bitches went on heat within an average of 54.1 ± 3.2 days, and 10 of them became pregnant. A significant decrease in mean OTC serum concentration was observed in bitches belonging to CD and ND group (**P < 0.01). The overall amelioration, in particular in the ND group, provides new insights for future studies involving the modification of diet in order to restore the normal physiology of the bitches suffering from reproductive disturbances.
... Recently, cell viability and HPLC-ESI/QqToF assays confirmed the cytotoxic effect of bone derived by intensive farming chickens treated with OTC at 24, 48 and 72h of incubation but ruled out the possible release of OTC and its derivatives from the bone until a concentration of 1 μg/mL [116]. ...
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Tetracycline antibiotics (TCs) have been widely employed to treat bacterial infections and other pathologic conditions in humans and pets. Although most of TCs have been almost all ruled out from the human clinical practice they are still used as growth promoters and to treat promiscuity and overcrowding pathologies in the intensive animal farming. As a consequence, TCs are commonly found in all ecological compartments with potential direct or indirect toxicological effects on animals and, generally, on all living organisms. Moreover, clinical and in vitro observations raised the hypothesis that the widespread of some adverse food reactions and, to a less extent, antibiotic resistance phenomena could be ascribed to the presence of TCs residues in edible and non-edible tissues of intensive animal farming intended for animal and human consumption. Such residues may pose serious health threat, depending on the type of food and the amount of residue present. The aim of this review is to provide new insights about the clinical uses of TCs in humans and animals and their potential toxic effects as residues in the environment or as food components..
... Although diets favoring seafood, poultry, whole grains, fruits, and vegetables have been related to a better fertility in women and better semen quality in men [8], we recently highlighted the role of contaminants, e.g., oxytetracycline, dragged by food, e.g., chicken bone and meat meal, able to exert proinflammatory and cytotoxic effects in vitro [18][19][20][21][22] and in vivo [6,[23][24][25][26][27]. ...
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Male dog infertility may represent a serious concern in he canine breeding market. The aim of this clinical evaluation was to test the efficacy of a commercially available nutraceutical diet, enriched with Lepidium meyenii, Tribulus terrestris, L-carnitine, zinc, omega-3 (N-3) fatty acids, beta-carotene, vitamin E and folic acid, in 28 male dogs suffering from infertility associated to hypospermia. All dogs were received the diet over a period of 100 days. At the end of the evaluation period no adverse effects, including head and tail anomalies percentage onset, were reported. Interestingly, motility percentage, semen volume and concentration and total number of sperms per ejaculation significantly increased. Further investigations on a wider cohort of dogs might be useful to better correlate the presence of oxyetracycline in pet's diet and the onset of infertility and clearly assess the action mechanism of an oxyetracycline-free nutraceutical diet.
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Background - Antibiotic use in human medicine, veterinary medicine, and agriculture has been linked to the rise of antibiotic resistance globally. We did a systematic review and meta-analysis to summarise the effect that interventions to reduce antibiotic use in food-producing animals have on the presence of antibiotic-resistant bacteria in animals and in humans. Methods - On July 14, 2016, we searched electronic databases (Agricola, AGRIS, BIOSIS Previews, CAB Abstracts, MEDLINE, Embase, Global Index Medicus, ProQuest Dissertations, Science Citation Index) and the grey literature. The search was updated on Jan 27, 2017. Inclusion criteria were original studies that reported on interventions to reduce antibiotic use in food-producing animals and compared presence of antibiotic-resistant bacteria between intervention and comparator groups in animals or in human beings. We extracted data from included studies and did meta-analyses using random effects models. The main outcome assessed was the risk difference in the proportion of antibiotic-resistant bacteria. Findings - A total of 181 studies met inclusion criteria. Of these, 179 (99%) described antibiotic resistance outcomes in animals, and 81 (45%) of these studies were included in the meta-analysis. 21 studies described antibiotic resistance outcomes in humans, and 13 (62%) of these studies were included in the meta-analysis. The pooled absolute risk reduction of the prevalence of antibiotic resistance in animals with interventions that restricted antibiotic use commonly ranged between 10 and 15% (total range 0–39), depending on the antibiotic class, sample type, and bacteria under assessment. Similarly, in the human studies, the pooled prevalence of antibiotic resistance reported was 24% lower in the intervention groups compared with control groups, with a stronger association seen for humans with direct contact with food-producing animals. Interpretation - Interventions that restrict antibiotic use in food-producing animals are associated with a reduction in the presence of antibiotic-resistant bacteria in these animals. A smaller body of evidence suggests a similar association in the studied human populations, particularly those with direct exposure to food-producing animals. The implications for the general human population are less clear, given the low number of studies. The overall findings have directly informed the development of WHO guidelines on the use of antibiotics in food-producing animals.