A Suitable HPLC-MS/MS Methodology for the Detection of Oxytetracycline, Enrofloxacin, and Sulfachloropyridazine Residues in Lettuce Plants.

Oxytetracycline (OTC), enrofloxacin (EFX), and sulfachloropyridazine (SCP) are critically important antimicrobials (AMs) in both human and veterinary medicine, where they are widely used in farm animals. Lettuce has become a matrix of choice for studying the presence of residues of these AMs in plants, as the concentrations of residues detected in lettuce can range from ng to mg. While several analytical methodologies have been developed for the purpose of detecting AMs in lettuce, these currently do not detect both the parent compound and its active metabolites or epimers, such as in the case of ciprofloxacin (CFX) and 4-epi-oxitetracycline (4-epi-OTC), which also pose a risk to public health and the environment due to their AM activity. In light of this situation, this work proposes an analytical method that was developed specifically to allow for the detection of OTC, 4-epi-OTC, EFX, CFX, and SCP in a lettuce matrix. This method uses acetonitrile, methanol, 0.5% formic acid, and McIlvaine-EDTA buffer as extraction solvents, and dispersive solid-phase extraction (dSPE) for the clean-up. The analytes were detected using a liquid chromatography technique coupled to mass spectrometry (HPLC-MS/MS). Parameters such as the specificity, linearity, recovery, precision, limit of detection, and limit (LOD) of quantification (LOQ) were calculated according to the recommendations established in the European Union decision 2021/808/EC and VICH GL2: Validation of analytical procedures. The LOQ for the analytes OTC, 4-epi-OTC, CFX, and SCP was 1 µg·kg-1, whereas for EFX, it was 5 µg·kg-1 dry weight. All calibration curves showed a coefficient of determination (R2) of >0.99. The recovery levels ranged from 93.0 to 110.5% and the precision met the acceptance criteria, with a coefficient of variation of ≤14.02%. Therefore, this methodology allows for the precise and reliable detection and quantification of these analytes. The analysis of commercial samples confirmed the suitability of this method.

Assessing the Effect of Oxytetracycline on the Selection of Resistant Escherichia coli in Treated and Untreated Broiler Chickens.

Oxytetracycline (OTC) is administered in the poultry industry for the treatment of digestive and respiratory diseases. The use of OTC may contribute to the selection of resistant bacteria in the gastrointestinal tract of birds or in the environment. To determine the effect of OTC on the selection of resistant Escherichia coli strains post-treatment, bacteria were isolated from droppings and litter sampled from untreated and treated birds. Bacterial susceptibility to tetracyclines was determined by the Kirby-Bauer test. A total of 187 resistant isolates were analyzed for the presence of tet(A), (B), (C), (D), (E), and (M) genes by PCR. Fifty-four strains were analyzed by PFGE for subtyping. The proportion of tetracycline-resistant E. coli strains isolated was 42.88%. The susceptibility of the strains was treatment-dependent. A high clonal diversity was observed, with the tet(A) gene being the most prevalent, followed by tet(C). Even at therapeutic doses, there is selection pressure on resistant E. coli strains. The most prevalent resistance genes were tet(A) and tet(C), which could suggest that one of the main mechanisms of resistance of E. coli to tetracyclines is through active efflux pumps.

Determination of five antimicrobial families in droppings of therapeutically treated broiler chicken by high-performance liquid chromatography-tandem mass spectrometry.

Antimicrobials are currently used in poultry for disease treatment. However, their excretion in bird feces may contaminate the environment. Considering this, the objective of this work was to quantify antimicrobials residues concentrations in therapeutically treated broiler chicken droppings throughout the post-treatment period. For this aim a multiresidue method using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was validated. Forty-eight male broiler chickens were distributed and treated with commercial formulations of 5 different antimicrobials. Results showed that oxytetracycline and 4-epi-oxytetracycline, presented the highest concentrations during all sampling period, detecting concentrations of 1471.41 µg kg-1 at the last sampling point (day 22 post-treatment). Florfenicol, tylosin, enrofloxacin, and ciprofloxacin were eliminated and detected in treated chicken droppings until d 18 post-treatment. Sulfachloropyridazine decrease gradually during post-treatment period until day 30. Results demonstrate that studied antimicrobials in treated chicken droppings were eliminated for prolonged periods, therefore becoming a significant route of residues dissemination into the environment.

Detection of Antimicrobial Residues in Poultry Litter: Monitoring a Risk through a Selective and Sensitive HPLC-MS/MS Method.

Tetracyclines, sulphonamides, and quinolones are families of antimicrobials (AMs) widely used in the poultry industry and can excrete up to 90% of AMs administrated, which accumulate in poultry litter. Worryingly, poultry litter is widely used as an agriculture fertilizer, contributing to the spread AMs residues in the environment. The aim of this research was to develop a method that could simultaneously identify and quantify three AMs families in poultry litter by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Samples of AMs free poultry litter were used to validate the method according to 657/2002/EC and VICH GL49. Results indicate that limit of detection (LOD) ranged from 8.95 to 20.86 µg kg-1, while limits of quantitation (LOQ) values were between 26.85 and 62.58 µg kg-1 of tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, enrofloxacin, ciprofloxacin, flumequine, sulfachloropyridazine, and sulfadiazine. Recoveries obtained ranged from 93 to 108%. The analysis of field samples obtained from seven commercial poultry flocks confirmed the adequacy of the method since it detected means concentrations ranging from 20 to 10,364 µg kg-1. This provides us an accurate and reliable tool to monitor AMs residues in poultry litter and control its use as agricultural fertilizer.

Evaluation of Antibiotic Dissemination into the Environment and Untreated Animals, by Analysis of Oxytetracycline in Poultry Droppings and Litter.

Oxytetracycline (OTC) is widely used in broiler chickens. During and after treatment a fraction of OTC is excreted in its original form and as its epimer, 4-epi-OTC in droppings. To address the transfer of OTC into the environment, we evaluated the dissemination of OTC and 4-epi-OTC from treated birds to the environment and sentinels, through the simultaneous analysis of broiler droppings and litter. Male broiler chickens were bred in controlled conditions. One group was treated by orogastric tube with 80 mg kg-1 of OTC and two groups received no treatment (sentinels). OTC+4-epi-OTC were analyzed and detected by a HPLC-MS/MS post the end of treatment. The highest concentrations of OTC+4-epi-OTC were detected in the droppings of treated birds 14-days following the end of treatment (2244.66 µg kg-1), and one day following the end of treatment in the litter (22,741.68 µg kg-1). Traces of OTC+4-epi-OTC were detected in the sentinels' droppings and litter (<12.2 µg kg-1). OTC+4-epi-OTC can be transferred from treated birds to the environment and to other untreated birds. The presence and persistence of OTC+4-epi-OTC in litter could contribute to the selection of resistant bacteria in the environment, increasing the potential hazard to public and animal health.

Cryptosporidium spp. and Giardia spp. in Free-Ranging Introduced Monk Parakeets from Santiago, Chile.

Monk Parakeets (Myiopsitta monachus) are medium-sized parrots that due to international pet trade currently exist as invasive species in 19 countries globally. Such is the case of Chile, where Monk Parakeets have thrived in the city of Santiago. Cryptosporidium spp. and Giardia spp. are worldwide distributed gastrointestinal parasites whose potential hosts include birds and humans. The present study sought to determine the presence of these pathogens in Monk Parakeets from Santiago. During the austral summers of 2017 and 2018, 207 Monk Parakeet nestlings were captured, and fecal samples were studied via microscopical analyses. Environmental data related to the trees in which the nestlings were captured were analyzed to establish the existence of infection clusters. Associations between spatial clusters, environmental variables, and the presence or absence of these pathogens were explored. In total, 33 samples were positive to the presence of one or both protozoa. Of the 33, Cryptosporidium spp. oocysts were detected in 10 nestlings (30%) while Giardia spp. cysts were detected in 25 (76%). Two nestlings presented poly-parasitism (6%). Statistical analyses established pruned trees as a potential protective factor against infection with these parasites. The present study corresponds to the second report of Cryptosporidium spp. in Monk Parakeets in Chile and the first worldwide report of Giardia spp. in these birds, emphasizing Monk Parakeet's potential role as a reservoir and pathogen disseminator, especially in urban environments.

First record of Ornithonyssus bursa (Berlese, 1888) (Mesostigmata: Macronyssidae) parasitizing invasive monk parakeets in Santiago, Chile.

Myiopsitta monachus is an invasive psittacine with wide distribution due to the pet trade. Its large communal nests and synanthropic nature contribute to its successful colonization of cities, from where it seems to be expanding in range and numbers. This is relevant with regard to pathogens that invasive species may harbor, especially when host populations thrive. We aimed to identify an abundant mite found in invasive monk parakeet chicks that had been collected in Santiago during 2017 and 2018. Through morphological and molecular identification of the 18S ribosomal RNA gene, we confirmed the presence of Ornithonyssus bursa. This was the first report of this mite in Chile. This mite is common in native and invasive monk parakeet populations and may affect other birds, including domestic fowl. Further, this mite bites people and can be a potential vector of pathogens such as bacteria or viruses. We conclude that this parasite was likely introduced with the parakeet and discuss possible ecological, health and economic consequences of this new potential pest.

First record of Ornithonyssus bursa (Berlese, 1888) (Mesostigmata: Macronyssidae) parasitizing invasive monk parakeets in Santiago, Chile / Primeiro registro de Ornithonyssus bursa (Berlese, 1888) (Mesostigmata: Macronyssidae) parasitando caturritas invasoras em Santiago, Chile

Abstract Myiopsitta monachus is an invasive psittacine with wide distribution due to the pet trade. Its large communal nests and synanthropic nature contribute to its successful colonization of cities, from where it seems to be expanding in range and numbers. This is relevant with regard to pathogens that invasive species may harbor, especially when host populations thrive. We aimed to identify an abundant mite found in invasive monk parakeet chicks that had been collected in Santiago during 2017 and 2018. Through morphological and molecular identification of the 18S ribosomal RNA gene, we confirmed the presence of Ornithonyssus bursa. This was the first report of this mite in Chile. This mite is common in native and invasive monk parakeet populations and may affect other birds, including domestic fowl. Further, this mite bites people and can be a potential vector of pathogens such as bacteria or viruses. We conclude that this parasite was likely introduced with the parakeet and discuss possible ecological, health and economic consequences of this new potential pest.

Resumo Myiopsitta monachus é um psitacídeo invasor amplamente distribuído devido ao tráfico de animais selvagens. Os grandes ninhos comunitários construídos e sua condição de espécies sinantrópicas contribuem para a colonização bem-sucedida das cidades, onde parece estar expandindo sua distribuição e número de indivíduos. Isso é relevante, quando se trata de patógenos que os invasores podem abrigar, especialmente quando as populações hospedeiras prosperam. O objetivo deste trabalho foi identificar um ácaro abundante, encontrado em filhotes de periquitos-monge introduzidos em Santiago, Chile, coletados durante 2017 e 2018. Por meio da identificação morfológica e molecular do gene do RNA ribossômico 18S, foi confirmada a presença de Ornithonyssus bursa, sendo o primeiro registro para o Chile. Esse ácaro é comum em populações nativas e introduzidas de periquitos-monge e pode afetar outras aves, incluindo aves domésticas. Além disso, esse ácaro pode picar pessoas e pode ser um potencial vetor de patógenos, como bactérias ou vírus. Conclui-se que esse parasita provavelmente foi introduzido com o periquito e foram discutidas as possíveis consequências ecológicas de saúde e econômicas dessa nova praga em potencial.

Interactions between Invasive Monk Parakeets (Myiopsitta monachus) and Other Bird Species during Nesting Seasons in Santiago, Chile.

The monk parakeet (Myiopsitta monachus) is considered to be one of the most invasive bird species because its unique ability among parrots to build their own communal nests. Currently, they are considered an invasive species in 19 countries and a pest-even in their native distribution-because of economic losses derived from their impacts. During the reproductive seasons of 2017 and 2018, we registered interactions between invasive monk parakeets and resident bird species in Santiago, Chile. We observed agonistic and affiliative interactions, and further, we described monk parakeets' nest occupancy by nine bird species, two invasive and seven native. For this reason, we consider that the monk parakeet is an allogenic ecosystem engineer with the potential to shape distribution and richness of sympatric species in urban environments. Our results contribute to an assessment of the implications of the monk parakeet's ecological invasion to other synanthropic species, and raise concern of other potential impacts, such as pathogen transmission derived from these interactions.

Assessment of Three Antimicrobial Residue Concentrations in Broiler Chicken Droppings as a Potential Risk Factor for Public Health and Environment.

Tetracyclines, sulfonamides and amphenicols are broad spectrum antimicrobial drugs that are widely used in poultry farming. However, a high proportion of these drugs can be excreted at high concentrations in droppings, even after the end of a therapy course. This work intended to assess and compare concentrations of florfenicol (FF), florfenicol amine (FFa), chlortetracycline (CTC), 4-epi-chlortetracycline (4-epi-CTC), and sulfachloropyridazine (SCP) in broiler chicken droppings. To this end, 70 chickens were housed under controlled environmental conditions, and assigned to experimental groups that were treated with therapeutic doses of either 10% FF, 20% CTC, or 10% SCP. Consequently, we implemented and designed an in-house validation for three analytical methodologies, which allowed us to quantify the concentrations of these three antimicrobial drugs using liquid chromatography coupled to mass spectrometry (LC-MS/MS). Our results showed that FF and FFa concentrations were detected in chicken droppings up to day 10 after ceasing treatment, while CTC and 4-epi-CTC were detected up to day 25. As for SCP residues, these were detected up to day 21. Noticeably, CTC showed the longest excretion period, as well as the highest concentrations detected after the end of its administration using therapeutic doses.

Determination of Chlortetracycline Residues, Antimicrobial Activity and Presence of Resistance Genes in Droppings of Experimentally Treated Broiler Chickens.

Tetracyclines are important antimicrobial drugs for poultry farming that are actively excreted via feces and urine. Droppings are one of the main components in broiler bedding, which is commonly used as an organic fertilizer. Therefore, bedding becomes an unintended carrier of antimicrobial residues into the environment and may pose a highly significant threat to public health. For this depletion study, 60 broiler chickens were treated with 20% chlortetracycline (CTC) under therapeutic conditions. Concentrations of CTC and 4-epi-CTC were then determined in their droppings. Additionally, this work also aimed to detect the antimicrobial activity of these droppings and the phenotypic susceptibility to tetracycline in E. coli isolates, as well as the presence of tet(A), tet(B), and tet(G) resistance genes. CTC and 4-epi-CTC concentrations that were found ranged from 179.5 to 665.8 µg/kg. Based on these data, the depletion time for chicken droppings was calculated and set at 69 days. All samples presented antimicrobial activity, and a resistance to tetracyclines was found in bacterial strains that were isolated from these samples. Resistance genes tet(A) and tet(B) were also found in these samples.