Detection of Quinolones in Commercial Eggs Obtained from Farms in the Espai´llat Province in the Dominican Republic

Introduction.

Between 2000 and 2010, the production of table eggs in the Dominican Republic (DR) nearly tripled, increasing from 58,000 to 150,000 tonnes (58,000,000 to 150,000,000 kg) ⁽²¹⁾. The increase is partially due to a higher average consumption of eggs in the DR (10.3 kg per person) than the world average (8.3 kg per person). The DR also exports table eggs, with 135 million eggs per month going to Haiti alone.

Antibiotics, such as macrolides, are commonly used at subtherapeutic levels in the poultry industry due to a growth-promoting effect. Delivered in the feed, these antibiotics stabilize the gut population and reduce susceptibility to disease ⁽²⁾. Quinolones, however, are used therapeutically because they are very effective against chronic respiratory diseases in poultry ⁽²⁰⁾. The European Union and the U.S. Food and Drug Administration (FDA) allow fluorquinolone use in broiler chickens but has set maximum residual limits on the concentrations of residues that can be present in edible tissues ^{(4, 22)}. However, in the European Union and United States, these drugs are not approved for use in laying hens due to the possibility of transferring and accumulating the drugs in the eggs.

Enrofloxacin, a commonly used quinolone, is deethylated to ciprofloxacin in vivo. This pharmacologically active metabolite is used in human medicine because it is active against several zoonotic pathogens, including Salmonella, Campylobacter, and Shigella; therefore, administration of ciprofloxacin can begin prior to precise diagnosis ⁽¹⁴⁾. However, poultry are frequently colonized with Campylobacter and Salmonella, and administration of quinolones to poultry has led to selection for resistant strains that jeopardize human treatment with ciprofloxacin⁽²⁴⁾.

Previously, we reported the presence of quinolone residues in chicken meat obtained from retail establishments in the DR⁽¹⁹⁾. In this study, we found that 6.6% of 135 retail poultry meat samples contained quinolone residues at concentrations that were higher than the residue maximum limits established by food industry authorities, including the FDA and European Authority of Food Safety. This implied that drug withdrawal protocols in poultry meat production were not being followed, and these residues could impose a health hazard to consumers. Given our previous findings and the large consumption and production of eggs in the DR, the aim of this study was to determine if quinolone residues were present in table eggs, produced in laying hen farms in the Espai´llat Province. Additionally, any levels of residues detected in egg samples were quantified.

Table 1. Percentage of egg production in each municipality and number of farms (separated by rearing system) from which commercial eggs were obtained in the Espai´llat Province in the DR.

Detection of Quinolones in Commercial Eggs Obtained from Farms in the Espai´llat Province in the Dominican Republic - Image 1

Materials and methods.

Experimental design and sampling. A transversal study of egg-laying hen farms in Espai´llat Province, located in the North Central portion of the DR, was conducted. The province consists of four municipalities: Moca, Jamao, Cayetano Germose´n, and Gaspar Herna´ndez. In this study, three of the four municipalities were sampled because there were no producers available to sample in Gaspar Herna´ndez. At the time of this study, there were a total of 98 egg producers in Espai´llat Province, and according to data from the governor’s office, Espai´llat Province was the leading egg producer within the DR ⁽¹⁷⁾. Data regarding each producer that was sampled included the size of the producer in terms of egg production (small scale, ,30,000 eggs per day; medium scale, 30,000 to 60,000 eggs per day; or large scale, .60,000 eggs per day), the type of production system (caged or floor laying system), and location within the province.

Collection and processing of samples. In total, 48 farms were sampled, and three eggs from each farm were collected (n ~ 144). The sampling was repeated twice more with at least 1 month between sampling times, for a total of 342 eggs collected and analyzed (Table 1). Half of the samples were collected from hens raised on the floor and the remaining from hens raised in cages. All the samples taken were identified, marked, and transported to the Microtech Laboratory located in Moca (Espai´llat Province, DR).

Eggs and yolks were deposited and gently beaten in a clean beaker until homogenized. From the homogenized egg, 1 ml of the mixture was combined with 2 ml of distilled water. Negative controls of sterile distilled water and positive controls consisting of commercially available quinolones (enrofloxacin 10% or norfloxacin 20%) were included in the study. For positive control preparation, 25 ml of each commercial quinolone was diluted in 2 ml of sterile distilled water. After dilution, 25 ml of the diluent was mixed with 1 ml of egg verified to be antibiotic free.

Quinolone assays. All quinolone detection was conducted as we have previously described ⁽¹⁹⁾. Briefly, we used the Equinox test (Zeu-Immunotec, Zaragoza, Spain), a colorimetric assay based on the inhibition of an Escherichia coli strain that is sensitive to quinolones. Following the manufacturer’s instructions, the lyophilized E. coli was reconstituted with the Q medium (Zeu- Immunotec). In each well of a microtiter plate, 50 ml of the egg sample, prepared as described in the previous section, or controls were mixed with 200 ml of the E. coli culture suspension. The microwell plate was covered with foil and incubated at 37 ¡ 1uC until negative control wells had turned to brown-orange (approximately 18 to 20 h), as directed by the manufacturer. For qualitative analysis, wells turning blue were considered positive, while negative wells were brown. The plates were analyzed spectrophotometrically at 400 and 750 nm, and quantitative results (optical density) were obtained as the difference between the values of the two readings, as previously described ⁽¹⁸⁾.

Data analysis. Prevalence data for quinolone residues were sorted by municipality, rearing system, farm size, and season and were analyzed by using the Fisher’s exact test with SAS for Windows (version 6.12, SAS Institute Inc., Cary, NC). A probability of P , 0.05 was a prerequisite for statistical significance.

Results and discussion.

In this study, we found that 51% of the eggs collected contained quinolone residues. Thus, it can be inferred that some of the hens were administered antibiotics and these eggs were collected within days of administration because residues in eggs can be detected for days after antibiotics are withdrawn. Specifically, if enrofloxacin is delivered by intramuscular injection to laying hens, residues can be detected in eggs 48 h after administration and persist in both yolk and albumen for 9 days after withdrawal^{(6, 10, 12, 13)}. When enrofloxacin is delivered in the water, higher concentrations of the residue persist in the egg than when the drug is delivered intramuscularly ⁽¹⁰⁾. Furthermore, the secondary metabolite ciprofloxacin persists for 7 to 10 days, dependent on route of administration.

Eggs obtained from Jamao had the highest percentage of samples positive for quinolone residues (67%), while 56 and 25% of eggs from Moca and Cayetano Gemose´n were positive, respectively (Table 2). However, only the differences in prevalence between samples from Moca and Cayetano were statistically significant (P ~ 0.0073; Table 2). Furthermore, the concentration of residues was higher (200 mg/kg) in eggs collected from Jamao than in eggs from the other two municipalities. A study conducted in South Korea reported only 3 of 120 eggs collected from six cities were positive for enrofloxacin ⁽³⁾, which is a lower prevelance than in our study. The differences in prevalence rates may be due to legislation and enforcement, as South Korea has stringent regulations for quinolone use in laying hens⁽⁸⁾.

The samples obtained from small-scale farms had a higher prevalence of quinolone residue (69%) compared with the prevalence in medium-scale (50%) and large-scale (40%) farms, but only the difference between small and large farms was statistically significant (P ~ 0.0037; Table 2). This difference may be attributed to farm practices because small-scale farmers have relatively limited access to professional services and may use empirical methods to diagnose and treat diseases that, in many cases, may not be infectious. Also, small-scale farmers have less economic resources, leading to more deficient infrastructures, and decreased biosecurity, resulting in more infections ⁽¹⁾.

In the eggs analyzed from egg-laying hens raised on the floor, quinolone residues were detected in 61% of eggs, while 40% of eggs from hens raised in cages contained residues, and this difference was statistically significant (P ~ 0.0193; Table 2). Egg-laying hens raised on the floor have more contact with pathogens, which exposes them more frequently to enteric and respiratory diseases and parasites, than do hens raised in cages ⁽⁹⁾. Thus, egg producers increase the use of antibiotics to prevent and control diseases^{(7, 15)}. Furthermore, the litter-pecking behavior commonly observed in hens raised on the floor can contribute to the consumption of antibiotic residues that can be transferred to the eggs. Antibiotic usage in floor-rearing systems can result in residue accumulation in the litter ⁽⁵⁾.

Table 2. Percentage of eggs testing positive for quinolone residues obtained from 48 farms in three municipalities in the Espai´llat Province of the DR, sampled three times between July and August^a.

Detection of Quinolones in Commercial Eggs Obtained from Farms in the Espai´llat Province in the Dominican Republic - Image 2

^a Percent positive samples calculated for factors including the rearing system, district location, and production size.
^b Numbers within the column under the same factor with different letters have statistically significant differences (P , 0.05).
c Producer size was defined as small scale, ,30,000 eggs per day; medium scale, 30,000 to 60,000 eggs per day; or large scale, .60,000 eggs per day.

The lowest percentage of eggs positive for quinolone residues was collected in August (19%). Similarly, the concentration of the residues in the eggs collected in August was the lowest (50 mg/kg). Eggs sampled in July and September were 71 and 63% positive, respectively, and the average concentration of residues for all these samples was 100 mg/kg (Table 2). The differences in percent positives between August and the other 2 months were statistically significant (P , 0.0001). July through September is considered summer, with an average temperature of 32uC, and the temperature does not fluctuate throughout the season. Heat stress can predispose the birds to infectious diseases requiring treatment ⁽²³⁾. However, this would not explain the significant differences in the number of positive eggs during these months, because the temperature was consistently hot throughout the study. Thus, other explanations might include a lower prevalence of disease in August, requiring antibiotic treatment, or if quinolones were administered, sampling may have occurred at a time when residues were depleted or undetectable in the eggs.

Quinolone residues are highly heat stable and may persist after treatment, so the concern for residues is significant. Concentrations of ciprofloxacin and norfloxacin in milk were reduced by only 12% when heated to 120uC for 20 min ⁽¹⁶⁾. A study reported that enrofloxacin concentrations in chicken meat did not change due to boiling or microwaving, and residues were displaced from the meat to the water ⁽¹¹⁾. However, this same study determined that roasting or grilling increased the concentration of enrofloxacin due to moisture removal from the meat during cooking. In summary, the stability of quinolones, the high consumption of eggs in the DR, and the high prevalence of contaminated eggs indicate that quinolone usage in laying hens presents a risk to consumers.

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