Aquaculture in Chile has been practiced since the 1920s; however, it was not until the 1990s that aquaculture became an important sector here. Important species in Chilean aquaculture include salmonids, algae, mollusks, and turbot. Salmonids are the dominant species in Chilean aquaculture for both harvest volume and export value, their production reaching greater than 800-thousand tons in 2015. However, this growth has been accompanied by an increase in disease presence, requiring greater drug use to control. This increase in drug use is an environmental and public health concern for the authorities, the salmon industry itself, and the destination markets. In this chapter, we review the literature on drug use, antibiotic resistance, regulatory framework, and alternatives, with focus on Chile.
- food safety
1. Introduction: brief history of antibiotics use in Chilean aquaculture
Antibiotics have been used to treat animals since the 1940s, which was soon followed by the appearance of resistant bacteria [1, 2]. In 1969, the House of Lords in the United Kingdom published the “Swann report,” highlighting the excessive use of antibiotics in animals and its potential risks to human and animal health. The Swan report suggested that antibiotic use should be restricted and regulated. The accumulated evidence from Europe and North America supports the notion that antibiotic use should be regulated and restricted to specific clinical situations .
In Chile, between 1973 and 1976, the first commercial fish farming of salmonids in the Region of the Lakes was consolidated and has grown ever since . In the subsequent decades, four species of salmonids of commercial importance have been cultivated in Chile: rainbow trout (
Sixteen antibiotics are used in animal treatments in Chile, compared to three in the United States (US) and four in Norway . In the case of aquaculture in Chile, antibiotics have been mainly used in sea water Atlantic salmon farming, which accounted for 80% of the total use of antibiotics used for 2015, followed by 11% for coho salmon, 9% for rainbow trout, and 0% for Chinook salmon .
The consumption of antibiotics in the salmon industry in Chile has increased by 56% from 2005 to 2015, with a production increase for those years of 23.48%. The highest consumption was recorded in 2014, with a total use of 563.2 tons of antibiotics with 955,179 tons of salmonids produced. In 2016, there was a 30.66% decrease in antibiotic use compared to 2015, using a total of 382.5 tons of antibiotics to produce 727,812 tons of fish ( Table 1 ) [8, 9, 10, 11].
|Year||Antibiotics use annual quantity (ton)||Annual production of salmonids (ton)|
The most used antibiotics in the salmon farming industry in Chile are florfenicol and oxytetracycline. Florfenicol use has increased steadily since 2013 and accounted for 87% and 82.50% of the total antibiotics used in 2015 and 2016, respectively [8, 9, 10, 11]. Florfenicol is mainly used in the seawater stage to control piscirickettsiosis (SRS) caused by the Gram-negative facultative intracellular pathogen
In 2005, SRS was the most diagnosed pathology, accounting for 77.04% of the antimicrobials used in the year 2005 by the industry . This trend has been maintained; 89.3% of all antibiotics used for the year 2016 have been for the control of SRS at the seawater stage followed by 6.8% for the control of renibacterosis .
2. Antibiotic resistance
2.1. Mechanisms of
In the last decade, there have been significant advances in the knowledge of
It is known that within biofilms, microorganisms are more resistant to the action of chemotherapeutics and have better survival rates under adverse conditions . Recent findings have identified genes that have a role in the formation of
Some authors have suggested that
Recent research carried out on coding and noncoding transcript during an
2.2. Piscirickettsia salmonis resistance to antibiotics
As mentioned above, the use of oxytetracycline and florfenicol are mostly used to control
3. Analysis of the regulatory framework
In Chile, in its Article 86, the Fisheries and Aquaculture law prohibits the preventive application of antimicrobials in a preventive way in aquaculture, as well as any use harmful to human health. Subsequently, there have also been the supreme decree N°319/2001, Regulation on Protection Measures for the Control and Eradication of High-Risk Diseases for Hydrobiological Species; the exempt resolution N°8228/2015 , the Manual of Good Practices in the Use of Antimicrobials and Antiparasitics in Chilean Salmon Farming ; the exempt resolution No 5.125/ 2016, Manual on Food Safety and Certification ; and the Quality Assurance Program (PAC) for Fisheries and Factories Vessels . This regulatory framework is jurisdiction of SENAPESCA (National Fisheries and Aquaculture Service) institution that is part of and depends on the Ministry of Economy, Development and Tourism.
According to the current legislation, only pharmaceuticals for exclusive veterinary use registered or authorized for application in hydrobiological species can be used . The pharmaceutical products authorized by Servicio Agrícola Ganadero (SAG) are shown in Table 2 . The studies of effectiveness, adequate dosage, animal safety, and human food safety (toxicology) of these authorized pharmaceutical products are not available to the public or were not found.
|Generic name||Trade name and registry no.||Presentation/route of administration||Registered by||Withdrawal period (degree days)||Dose mg/kg lw * /day||Tolerance level in muscle tissue (μg kg−1)|
|Oxolinic acid 80%||Reg. N°441||Powder/oral||FAV S.A.||450||20 per 10 days||100|
|Litoflox Reg N°648||Powder/oral||Centrovet LTDA.||450||10–30 per 10 days|
|Bandrol Reg N°481||Powder/oral||Veterquímica S.A.||450||10 per 10 days|
|Amoxicillin 50%||Amox-Feed Reg N°121||Powder/oral||Veterquímica S.A.||300||70.4 per 10 days||50|
|Erythromycin 50%||Vetromic Reg. N°1402-B||Powder/oral||Centrovet LTDA.||500||75–100 per 21 days||200|
|Erythromycin 80%||Eritofeed Reg. N°616-B||Powder/oral||Veterquímica S.A.||500||92.5 per 21 days|
|Vetromic Reg N°1803-B||Powder/oral||Centrovet LTDA||500||75–100 per 14–21 days|
|Flumequine 10%||Flumepren Reg. N°79||Powder/oral||Centrovet LTDA||300||(−)||*** 500|
|Flumequine 50%||Reg. N°484||Powder/oral||FAV S.A.||300||12–25 per 10–12 days|
|Reg. N°646||Powder/oral||Centrovet LTDA||300||12–30 per 10 days|
|Flumequine 80%||Reg. N°442||Powder/oral||FAV S.A.||300||20 per 10 days|
|Flox-Feed Reg. N°478||Powder/oral||Veterquímica S.A.||300||10 per 10 days|
|Flumepren Reg. N°645||Powder/oral||Centrovet LTDA.||600||12–30 per 10–15 days|
|Florfenicol 50%||Florfenox Reg. N°1537||Powder/oral||Bayer S.A.||300||10 per 10 days||1000|
|Veterin Reg. N°1556||Powder/oral||Centrovet LTDA.||300||10 per 10 days|
|Duflosan Reg. N°1769||Powder/oral||Veterquímica S.A.||300||10 per 10 days|
|Florfenicol 50%||Duflosan L Reg. N°2264||Solution/oral||Veterquímica S.A.||100||10 per 10 days||1000|
|Aquafen Reg. N°1193||Powder/oral||Intervet Chile LTDA.||200||10 per 10 days|
|Reg. N°1598||Powder/oral||FAV S.A.||300||10 per 10 days|
|Oxytetracycline hydrochloride 20%||Terrivet F200 Reg. N°2252||Suspension for injection||Veterquímica S.A.||1060||** 20||200 (−) Tetracyclines|
|Oxytetracycline 50%||Terrivet Reg. N°149||Powder/oral||Veterquímica S.A.||600||75 per 15 days|
|Oxytetracycline 80%||Terrivet Reg. N°485||Powder/oral||Veterquímica S.A.||600||75 per 15 days|
|Reg. N°1595||Powder/oral||FAV S.A.||600||55–82 per 10 days|
|Zanil Reg. N°1380||Powder/oral||Centrovet LTDA.||600||75 per 10 days|
|Oxytetracycline 40%||Reg. N°309||Powder/oral||Laboratorio Veterinario Quimagro S.A.||600||13.57–20.75 per 10 days|
The Chilean authority supervises the use of pharmaceutical products in hydrobiological species, and in accordance with the provisions of the general and specific health programs, therapeutic treatments applied to populations of hydrobiological species should be prescribed by a veterinarian and the application of antimicrobials for prophylactic purposes is prohibited. Before the application of the antimicrobials, fish samples should be obtained for subsequent confirmation of the diagnosis by laboratory analysis . Farming facilities should keep records of antimicrobial treatments performed and antimicrobial treatments should be reported monthly through the Aquaculture Inspection System (SIFA) .
Extralabel antimicrobials can be prescribed by a veterinarian when the health of an animal is at risk, there is danger of death, or there is suffering of the animal; or when one of the following is fulfilled: dosage, timing, duration of treatment or route of administration for a registered product does not obtain the expected response; the product is temporarily unavailable on the market; or there is no registered product to treat a diagnosed condition .
The Manual on Food Safety and Certification (resolution No 5.125/ 2016) describes the norms and procedures that allow to guarantee the sanitary quality of the fishery and aquaculture products destined for international markets along the whole productive chain. Regarding the procedures for the control of residues of pharmaceutical products, each farm facility must demonstrate (and issue a declaration of guarantee) that the concentrations of residues of pharmaceutical products in fish do not exceed the limits established by the Chilean authority .
The analysis must be carried out in authorized laboratories, according to the Methods of Analysis of Residues of Pharmaceutical Products and Contaminants for Export Fishery Products. If the maximum allowable limits are not met, the withdrawal period should be extended and a new sampling should be carried out .
The government of Chile also maintains a Program of Surveillance and Control of Piscirickettsiosis, in which monitoring system and the application of control measures are established for this disease . Upon request, the Chilean authority issues fish farming centers a certificate stating that the fish are free of antimicrobial and/or antiparasitic treatments . The Quality Assurance Program (PAC) is a voluntary certification program, based on the concept of hazard analysis and critical control point (HACCP), which applies only to fishing plants and factory vessels. This program, however, is mandatory for all companies that are authorized to export to the European Union and the United States. The Chilean authority must approve the quality assurance plan for the industry and supervise its subsequent operation . The administrative procedures, work guides, and specific requirements of this program (PAC) are not publicly available.
The main Chilean salmon markets are the US and Japan. In the case of the US, the Food and Drug Administration (FDA) is in charge of regulating the use of antibiotics in fish, primarily through its regulation 21 Code of Federal Regulations (CFR) 123 ‟Procedures for the Safe and Sanitary Processing and Importing of Fish,” which aims to ensure the safe and sanitary processing of fish and fishery products (seafood), including imported seafood . The regulation mandates the application of HACCP principles to the processing of seafood as a preventive system of hazard control that can be used by processors to ensure the safety of their products to consumers. For the control of drugs for use in food of animal origin, direct medication or for addition to feed must be approved, conditionally approved, or index listed by the FDA (Federal Food, Drug, and Cosmetic Act Section 512) .
Under certain conditions authorized by FDA, unapproved new animal drugs may be used in conformance with the terms of an Investigational New Animal Drug (INAD) application (21 CFR 511)  and FDA’s Center of Veterinary Medicine (CVM) guide 1240.3025. When a drug is approved by CVM, the condition of the approval is listed on its label or in the labeling (21 CFR 514.1) ; this condition specifies the species for which the drug is approved for use, indications for use, dosage regimen, and other limitations such as route of administration and withdrawal time. Labeled withdrawal times must be followed to ensure that no harmful drug residues are present in the edible tissue of the animal when harvested for human consumption; tolerances for some drug residues in the edible tissue have been established .
Relatively few drugs have been approved for aquaculture in the US ( Table 3 ). This has led to the inappropriate use of unapproved drugs, general-purpose chemicals, or approved drugs in a manner that deviates from the labeled instructions . Studies establishing the effectiveness, adequate dosage, animal safety, and human food safety (toxicology) of these approved drugs are available to the public .
|Antibiotic||Dose||Approved for:||Route of administration||Tolerance level in muscle tissue||Withdrawal period|
|Terramycin 200 (oxytetracycline dehydrate)||2.5–3.75 g/100 lb. of fish/day|
Maximum dose = 8.33 mg/kg/day/10 days
|Salmonids: For control of ulcer disease caused by ||Administer in mixed ration for 10 days; do not liberate fish or slaughter fish for food for 21 days following the last administration of medicated feed.||2 ppm (As the sum of tetracycline residues)||21 days for disease control in salmonids|
7 days for marking skeletal tissue in Pacific salmon
|OxyMarine Oxytetracycline HCL soluble powder-343, TETROXY Aquatic||200–700 mg oxytetracycline/L of water for 2–6 h||For marking of skeletal tissues in finfish fry and fingerlings as an aid identification||Immersion||2 ppm (as the sum of tetracycline residues)||21CFR 556.500|
|Sulfadimethoxine/ormetoprim combination Romet-30||For control of furunculosis in salmonids (trout and salmon) caused by ||Medicated feed||0.1 ppm for each drug (21 CFR 556.490)||42 days (21 CFR 558.575)|
|Florfenicol Aquaflor||10 mg/kg fish per day for 10 consecutive days||For the control of mortality in freshwater-reared salmonids due to cold-water disease associated with ||Medicated feed||2 ppm in salmon muscle/skin||15 days|
In the case of Japan, fishery products are regulated by the Food Sanitation Act and the Food Safety Basic Act. The authorities involved with in the Food Sanitation Act are as follows: the Office of Import Food Safety; Inspection and Safety Division; Pharmaceutical and Food Safety Bureau; Ministry of Health, Welfare and Labor; and the Department of Food Safety. The purpose of the Food Sanitation Act is to prevent the occurrence of health hazard arising from human food so as to contribute to the protection of people health by conducting regulations and measures deemed necessary, from the view point of public health and for securing food safety .
The purpose of the Food Safety Basic Act is to promote comprehensive measures to secure food safety by laying down the basic principles of safety for food, defining the responsibility of the government, local authorities, and food-related businesses, clarifying the role of consumers, and establishing basic policies for developing measures. The authority concerned is the Consumer Affair Agency .
Antibiotic residue concentrations for edible products from food-producing animals are determined based on jurisdictional-specific regulations that result in the determination of a tolerance or maximum residue level (MRL) for specific drugs in a specific tissue for specific animal species and based on toxicological assessments. This index estimates the amount of substance in food that can be ingested over a lifetime by humans without significant risk to health . There are notable differences among MRLs or tolerances set by the different agencies regarding the two antibiotics most used in Chilean salmon farming ( Table 4 ).
Many methods have been developed for analysis of antibiotics in fish. HPLC and mass spectrometry (HPLC-MS/MS) is the most sensitive method for the detection of these antibiotics and is currently regarded as the tool of choice for analysis of antibiotic residues in animal-derived food [47, 49], having a limit of detection (LOD) in fish of 10.3 ng/l  and a limit of quantification of 20 ng/l for tetracyclines .
4. Alternative antibiotic treatment in salmon farming
Fish are considered as the earliest class of vertebrates to have both innate and adaptive immunity, though the latter defense mechanism is not as elaborate as in higher vertebrates. Unlike in mammals, the alternative complement pathway in teleosts is relatively high and can mediate the lysis of target erythrocytes from several species. These features, along with their potential to function at varying temperatures, suggest that the complement system is a powerful defense mechanism in fish [52, 53, 54], and they are in constant interaction with their surroundings and therefore could easily encounter potential pathogens. In the wild, fish can protect themselves using innate defense mechanisms (either constitutive or responsive) .
The various alternatives to the use of antibiotics can be classified according to the action toward the pathogen or host. Pathogen-directed strategies include inhibitors of growth and virulence genes, antibacterial compounds, and the phage therapy. Host-directed strategies include the improvement of health, stress prevention, stimulation of the defense system, and selective breeding for disease resistance .
One of the first lines of defense against bacterial infection is the withholding of nutrients, termed nutritional immunity. The most significant form of nutritional immunity is the sequestration of iron . Recent studies have detected a relationship between iron transporter glycoproteins and
Several bacteriophages have been isolated against the following pathogenic bacteria,
Promising results have been obtained in laboratory studies. However, high concentrations of bacteriophages in seawater can induce bacterial genetic variation. This occurs through mutation and bacteriophage-mediated horizontal transmission of genetic material between different bacteria mediated by bacteriophages [5, 69]. The use of phages can also influence bacterial community dynamics and ecosystem biogeochemistry. These influences differ depending on whether phages establish lytic, chronic, or lysogenic infections. The impacts of lysogeny are well studied at the cellular level, but ecosystem-level consequences remain underexplored .
Probiotics have been credited for producing improved nutrition, health benefits, reduced disease incidence, improving growth, health status, immunity, feed conversion, microbial balance, and water quality, as well as food production in an environmental-friendly way [71, 72, 73]. Probiotics in aquaculture can be live or dead preparations, including cellular/extracellular components of the microorganism(s), administered either as a feed supplement or to the rearing water. Probiotics can be used to control a range of bacterial pathogens in various fish species . For example, rainbow trout (
The Food and Agriculture Organization of the United Nations (FAO) has now highlighted the use of probiotics in aquaculture as a means of improving the quality of the aquatic environment . However, concerns have been voiced about the possible acquisition of antibiotic resistance and virulence genes via horizontal gene transfer, which might lead to safety problems if using live probiotics in an open aquatic environment. Probiotics can also affect host tissue and result in severe cell damage. To avoid this, probiotic strains must be recognized as safe for the cellular integrity of the host .
In the aquaculture industry, vaccination strategies include traditional inactivated and attenuated vaccines, as well as next-generation vaccines comprising recombinant, subunit, vectored, genetically engineered, DNA and peptide vaccines, reverse vaccinology, plant-based edible vaccines, and nanovaccines . Current vaccination protocols for
Veterinarians in charge of the salmon industry in Chile have used large quantities of antibiotics relative to its production volumes. In the years of highest production, an average of 600 g ton−1 produced was used. The antibiotics used by this industry are florfenicol and oxytetracycline for the control of
There are 12 different types of generic and 25 branded antimicrobials authorized for use in salmonids in Chile, with no specifications related to pathogens or diseases. This is in contrast to the US situation, where the FDA has approved just four antibiotics for specific uses and against certain pathogens.
The studies of the effectiveness, adequate dosage, animal safety, and human food safety (toxicology) of the authorized pharmaceutical products, as established by the Chilean authority, are not available to the public or were not found. This was also the case for the administrative procedures, work guides, and specific requirements of the Quality Assurance Program (PAC), whereas the effectiveness and toxicology studies of the FDA-approved antibiotics are freely available online. This absence of Chilean regulation and antibiotic data is concerning. To avoid chemical hazards and ensure food safety, we propose that a mandatory legal framework based on international regulations is needed in Chilean aquaculture. Antimicrobial treatment is required for an efficient production of animal products; however, antibiotics should never be used as a substitute for proper nutrition and hygiene management.
The alternatives to the use of antibiotics in Chilean salmon farming, such as the use of nutritional immunity, phytopharmaceuticals, probiotics, antimicrobial peptides, and selective breeding for disease resistance, require advanced research with