Aquaculture pharmaceuticals and biologicals: current perspectives and future possibilities

Advanced Drug Delivery Reviews 50 (2001) 229–243www.elsevier.com/ locate /drugdeliv

Aquaculture pharmaceuticals and biologicals: currentperspectives and future possibilities

*Zezhi J. Shao

Schering-Plough Research Institute, 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA

Abstract

With the continued expansion of cultured fin and shellfish species, aquaculture has become a key component of the animalhealth industry. The ever-increasing demand for quality pharmaceutical and biological products, to combat bacterial and viralinfections, calls for the development of modern formulations and novel drug delivery systems. The purpose of this paper is toprovide a general overview of important fish pathogens, manifested fish diseases, and control methods. Currently availablepharmaceutical and biological products are tabulated. Emphasis is placed on the extension of formulation and processtechnologies, commonly encountered in human pharmaceuticals, to the aquaculture arena. New research efforts in the area ofnovel vaccine delivery systems, i.e. enteric-coated beads and biodegradable microspheres, are also summarized. 2001Elsevier Science B.V. All rights reserved.

Keywords: Aquaculture; Formulation and process technologies; Medicated premix; Medicated feed; Fish vaccine

Contents

1. Introduction ............................................................................................................................................................................ 2302. Brief overview of cultured aquatic species ................................................................................................................................ 230

2.1. Salmon............................................................................................................................................................................ 2302.2. Rainbow trout (Salmo gairdneri)....................................................................................................................................... 2312.3. Flat-fish........................................................................................................................................................................... 2312.4. Carp................................................................................................................................................................................ 2312.5. Shellfish .......................................................................................................................................................................... 2312.6. Aquarium (ornamental) fish .............................................................................................................................................. 231

3. Brief overview of fish diseases ................................................................................................................................................. 2313.1. Non-infectious diseases .................................................................................................................................................... 2313.2. Infectious diseases ........................................................................................................................................................... 2313.3. Stress-mediated infections ................................................................................................................................................ 232

3.3.1. Bacterial septicemias .............................................................................................................................................. 2323.3.2. Skin and gill infections ........................................................................................................................................... 2333.3.3. Chronic diseases .................................................................................................................................................... 234

4. Pharmaceuticals in aquaculture................................................................................................................................................. 234

*Pfizer Global Research & Development, 170 Tabor Road, Morris Plains, NJ 07950, USA. Tel.: 1 1-973-385-7187; fax: 1 1-973-385-2397.

E-mail address: [email protected] (Z.J. Shao).

0169-409X/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 01 )00159-4

230 Z.J. Shao / Advanced Drug Delivery Reviews 50 (2001) 229 –243

4.1. Anesthetic agents ............................................................................................................................................................. 2344.1.1. Immersion (inhalational) anesthetic agents ............................................................................................................... 2344.1.2. Injectable anesthetic agents ..................................................................................................................................... 234

4.2. Chemotherapeutic agents .................................................................................................................................................. 2344.2.1. Medical disinfectants.............................................................................................................................................. 2354.2.2. Antiparasitic agents ................................................................................................................................................ 2354.2.3. Antibiotics and chemotherapeutants......................................................................................................................... 235

4.3. Development of premixes as a dosage form in aquaculture .................................................................................................. 2364.3.1. Current formulation strategies for aquaculture premixes ............................................................................................ 2364.3.2. Current processing technologies for aquaculture premixes ......................................................................................... 2384.3.3. Regulatory submission: additional quality requirements ............................................................................................ 239

4.3.3.1. Type of incorporation ................................................................................................................................ 2394.3.3.2. Homogeneity of drug in finished feed ......................................................................................................... 2404.3.3.3. Compatibility with typical feedstuff ............................................................................................................ 2404.3.3.4. Stability of medicated feed ......................................................................................................................... 2404.3.3.5. Shipping study .......................................................................................................................................... 240

5. Aquaculture vaccines............................................................................................................................................................... 2405.1. Anti-bacterial vaccines ..................................................................................................................................................... 2405.2. Anti-viral vaccines ........................................................................................................................................................... 2415.3. Future possibilities for aquaculture vaccine delivery systems............................................................................................... 241

Acknowledgements ...................................................................................................................................................................... 242References .................................................................................................................................................................................. 242

1. Introduction anguillarum and Vibro salmonicida in addition to thefurunculosis component.

Despite the fact that over 100 aquatic species are The aim of this article is to provide an overview ofcultured in the United States, the development and important fish pathogens, diseases and current phar-commercialization of new medicines for the treat- maceutical and biological products in the marketment of aquatic diseases are rather scarce. To date, place. Development work geared toward registrationthere are only five FDA approved products available and marketing, yet unique to aquaculture products,for use in finfish and shellfish hosts, of which 80% will be outlined. Future development strategies forare catfish, crawfish, trout, and salmon [1]. aquaculture pharmaceutical delivery systems will be

This situation is the result of a variety of factors addressed.ranging from lack of sponsor interest, concern overenvironmental impact of new chemical entities, andthe regulatory agency’s classification of aquatic 2. Brief overview of cultured aquatic speciesspecies as minor species. The small and diversifiedaquaculture industry presents financial challenges for Depending on the climatic zone, the species ofany pharmaceutical company to target. The tremend- cultured aquatic animals vary from shellfish toous investment in developing a pharmaceutical prod- finfish. The major farmed species in the US anduct for exclusive use in fish is not always rewarding, Europe, nevertheless, are salmon, rainbow trout,except for a few indications. The recent development carp, mussels, oysters and crayfish. Farming systemsin vaccines for aquatic use further diminishes the include lakes and highly intensive tank units.need for certain antimicrobial agents.

The vaccine field has made significant advance- 2.1. Salmonments in the aquaculture industry. Most notably, theintroduction in Europe in 1992 of oil-adjuvanted Atlantic salmon (Salmo salar) represents the mostvaccines containing antigens against furunculosis. important species cultured in Europe, with Norway’sThese vaccines are either monovalent against furun- production exceeding 10,000 tons [2]. Other coun-culosis or polyvalent containing antigens of Vibrio tries include the UK, Finland and Ireland. The

Z.J. Shao / Advanced Drug Delivery Reviews 50 (2001) 229 –243 231

unique nature of salmon farming lies in the fact that 2.6. Aquarium (ornamental) fishyoung fish from the egg stage to the time of smoltingare kept in fresh water hatchery systems and later There are a variety of fish species being re-transferred to sea-water cage sites as soon as they produced for ornamental purposes, i.e. Koi, goldfish,smolt. arrowanas, cichlids, etc. The economic reward for

the farming of these species can be substantial. Thequantities of aquaculture medications used for these2.2. Rainbow trout (Salmo gairdneri)hosts, however, are much less compared to fishfarmed for consumption purposes.Rainbow trout can be cultured in either fresh water

or seawater systems. For example, Denmark utilizesearth ponds for low stocking intensity farming, whileNorway produces trout exclusively in sea cages, each 3. Brief overview of fish diseasesholding up to 300 tons of fish. Trout farming can befor the purpose of restocking or producing table-size The very nature of intensive farming practicefish for consumption. imposes high risks on the well-being of the fish. The

factors contributing to the morbidity and mortality offarmed fish include stress, water quality, as well as2.3. Flat-fishdisease-causing organisms, i.e. bacteria, fungi, para-sites, and viruses.Flat-fish species currently being cultured include

This section describes the pathogens responsibleturbot and Dover sole, which needs to be reared atfor the most commonly observed fish diseases andhigh sea water temperatures to attain a high growththe symptoms of those diseases.rate. The major challenge in expanding flat-fish

farming is in managing young turbot larvae feedingon artificial diets since they have to be weaned on 3.1. Non-infectious diseasesrotifer and artemia species. Finding a species whichis in short supply for a good price drives flat-fish Non-infectious diseases can be attributed to ge-farming. Interests and efforts in farming flat-fish are netic reasons, inadequate nutrition, or poor waterprimarily in Europe. quality. Genetic disorders, as exhibited by infertile

eggs or abnormal fry, have become more common asa result of the establishment of faster growing strains2.4. Carpof fish. Nutritional problems caused by poor formu-lations are now rare, but improper storage of feedThe species most commonly grown are variousleading to rancidity can still cause malnutrition. Poorstrains of the common carp, Cyprinus carpio. Thewater quality is known to cause various catfishmain consumer populations are Asian and Eastdiseases, such as brown blood disease. Excess nitro-European communities. A weight of approximatelygenous metabolite waste in water leads to blue sac700 g can be achieved from the fry stage in about 2diseases of salmonid fry. Another example of wateryears with little labor involvement.quality-related symptoms is the so-called gas-bubbledisease, caused by supersaturation of the water2.5. Shellfishsupply with dissolved gas.

Shellfish currently being farmed are mussels(Mytilus edulis) and oysters (Crassostrea gigas). The 3.2. Infectious diseasesmost notable site for shellfish production is France.The farming technique involves seeding mussel spat Infectious diseases are the result of bacteria, fungi,on ropes suspended from a platform in a sea loch parasites and viruses. Various species of bacteria canwith good tidal flow. Harvesting is carried out when cause diseases in fish, which necessitates drugthe mussels reach a good size. therapy. Parasitic infestation of the fish is currently


another leading problem. Primary pathogens are 3.3. Stress-mediated infectionsbacteria and viruses.

The major bacterial pathogens that affect various The majority of infectious diseases of fish are thefinfish hosts are listed in Table 1, together with the results of opportunistic invasion of stressed fish byresulting diseases. Aeromonas salmonicida is a pathogens (bacteria and parasites) which normallyprimary bacterial pathogen in salmonids causing co-exist with the host.furunculosis, a severe septicemic disease. The majorsymptoms of the disease include reduced appetite, 3.3.1. Bacterial septicemiasand hemorrhages at the base of the fins, around the Vibio anguillarum inhabits the gut of healthy fish.gills and in the abdominal cavity. The challenge of Frequent handling of the fish (grading, transport)eradicating this disease is that a few causative leads to an outbreak of vibriosis, with symptoms ofbacteria persist after antibiotic treatment. These hemorrhages in the muscle and viscera and some-persistent bacteria can re-infect other fish upon times skin ulcers. Other well-known acute bacterialstressful handling or change in the environment, septicemias of farmed fish are caused by Aeromonasleading to recurrence of the disease. hydrophilia, Aeromonas salmonicida, Edwardsiella

The second important class of pathogens affecting tarda, Pseudomonas fluorescens, Vibio anguillarium,the salmonid industry is the virus. No means of Yersinia ruckeri, etc. Chemotherapy by means ofchemotherapy is available for treating these diseases. antibiotics and sulfonamides is used to combat acuteThe five common viral diseases are listed in Table 2. bacterial septicemias.

Table 1Major pathogens and diseases that affect finfish hosts in the USA [3]

Bacterial pathogen Disease

Salmonids (salmon, trout, and char)Aeromonas salmonicida FurunculosisRenibacterium salmoninarum Bacterial kidney diseaseVibrio anguillarum VibriosisVibrio salmonicida Hitra disease (coldwater vibriosis)Cytophaga psychrophilia Coldwater diseaseCytophaga columnaris Columnaris diseaseFlavobacterium branchiophilum Bacterial gill disease

CatfishEdwardsiella ictaluri Enteric septicemia of channel catfishEdwardsiella tarda Fish gangreneAeromonas hydrophila Motile aeromonad septicemiaAeromonas sobria Motile aeromonad septicemiaCytophaga columnaris Columnaris disease

Striped bass and hybridsPasteurella piscidium PasteurellosisVibrio spp. VibriosisCytophaga columnaris Columnaris diseaseMycobacterium marinum Fish tuberculosisStreptococus iniae Streptococcosis

Tilapia and hybridsAeromonas hydrophila Motile aeromonad septicemiaCytophaga columnaris Columnaris diseaseStreptococus iniae Streptococcosis


Table 2Characteristics of the important fish viral diseases [4]

Disease Susceptible Age of Usual temperature Geographicalfish susceptibility susceptibility range distribution

Channel catfish Channel Fry and . 258C Southernvirus (CCV) catfish fingerlings USA

Infectious hematopoietic Salmonids Mostly , 188C Japan,necrosis (IHN) , 1 year old N. America

Infectious pancreatic Salmonids Fry and up Any Worldwidenecrosis (IPN) to 20 weeks

Spring viremia Common All but mostly , 228C Europeof carp (SVC) carp yearlings

Viral hemorrhagic Salmonids (grayling, All , 148C Europesepticemia (VHS) trout, whitefish) and pike (mainland)

3.3.2. Skin and gill infections The most common skin and gill problems areInfections of the skin and gills are external caused by parasitic infestation, especially under

diseases caused by a variety of organisms, i.e. intensive farming conditions. The parasites attachbacteria, parasites and fungi. Examples of bacterial themselves to the skin damaging the epidermis,skin and gill diseases include columnaris, caused by thereby feeding on the underlying tissue and blood.the Cytophaga group of bacteria. Bacterial gill Heavy infestation can lead to skin lesions, reduceddisease leads to respiratory distress of salmonids, growth, and high mortalities. The most commonresulting in low-level but continuous mortalities if external parasites are shown in Fig. 1 [4].left untreated. Two types of louse infestation of freshwater and

Fig. 1. Fish parasites.


seawater fish deserve additional description. The fat, fish disease state, as well as environmentalArgulus fish louse occurs widely in freshwater fish factors such as pH and temperature. An inversefarms and can be visible to the naked eye. The louse relationship has been reported between weight ofscuttle crab-like over the fishes body to suck blood rainbow and brook trout and induction and recoveryby means of a proboscis. The most common salmon times to tricaine methanesulphonate [6].louse is Lepeophtheirus, which can be found in wildand farmed salmon. However, under high-intensity 4.1.1. Immersion (inhalational) anesthetic agentsfarming conditions, the number of sea lice inhabiting The only registered anesthetic agent in Norththe host can increase tremendously, causing severe America is tricaine methanesulfonate (TMS), andamage to the entire body of the fish. Fish louse analogue of benzocaine with superior water solu-injuries also lead to secondary infections of the open bility. The label withdrawal time for TMS in Canadawounds. is 5 days. A longer withdrawal time of 21 days is

maintained in the USA [7].3.3.3. Chronic diseases Other anesthetic agents are approved for use in

The most commercially damaging chronic fish Europe with varying withdrawal times. Benzocaine,disease is the bacterial kidney disease of the salmon, for instance, has a narrower safety margin compareddue to Renibacterium salminarum. The difficulty in to TMS, and Norway requires a 21 day withdrawaltreating this disease is that damage to the kidney and time. Metomidate, an imidazole-based nonbarbiturateviscera is widespread before the fish exhibits any hypnotic agent, is marketed for use in not-for-foodclinical symptoms. fish. A newly developed agent, AQUI-S, has been

Internal parasites causing chronic fish diseases reported to be ‘stressless’ to fish and environmentallyinclude nematode roundworms and cestode friendly [8].tapeworms.

4.1.2. Injectable anesthetic agentsInjectable anesthetic agents are used only when

4. Pharmaceuticals in aquaculture needed for lengthy surgical procedures, after the fishhas been sedated with an immersion anesthetic agent.

The use of pharmaceutical agents in treating fish Sodium pentobarbital, a CNS depressant, is the bestdiseases and modulating fish growth is rather un- choice due to its lipophilic characteristics resulting insophisticated. The primary means of administration longer duration of action. Ketamine hydrochloride,include water treatment, incorporation in feed (medi- which is widely used in laboratory animals, has alsocated feed), and by injection [5]. It needs to be been used in aquaculture [9].pointed out that the conditions of the water, theearth-pond, and equipment all play an important role 4.2. Chemotherapeutic agentsin the well-being of eggs and the growing fish.Similarly, adequate farming practices are also a Few new chemotherapeutic agents have beencritical component. For instance, newly introduced approved for use in aquaculture since the turn of thefish stock should be quarantined prior to release to century. This situation is caused by several factors,the pond/ tank. i.e. concern about the environmental impact of a new

chemical entity especially on non-target organisms/4.1. Anesthetic agents species, cost in developing a new product, dual

registration requirements for the product and medi-Anesthetic agents are used primarily in fish farms/ cated feed, etc. As a result, off-label use of chemi-

laboratories to provide analgesia and immobilization cals not approved for aquaculture and extra-label useof the fish prior to transportation /grading and for of approved chemicals (at higher dose, for newminor procedures. Many factors influence the effica- diseases) are widespread on farms in North Americacy of an anesthetic agent, i.e. number of fish in the and Europe. A review of the currently availablebath at a time (can be up to 2000), fish size and body chemotherapeutants is given below.


4.2.1. Medical disinfectants ide (Salartect , Brenntag; Paramove , Solvay-Inter-Medical disinfectants are used to disinfect fish ox) and synthetic pyrethroids such as cypermethrin

eggs and to clean ponds, equipment, etc. They (Excis , Vericore) and deltamethrin (Alphamax ,include iodophores, salts, organic chlorocompounds, Alpharma).aldehydes, hydrogen peroxide, quaternary ammo- Avermectins have not been approved for use innium compounds and antiseptic dyes. They are aquaculture, except in the United Kingdom whereprimarily intended to target fungi and ectoparasites. veterinarians can prescribe ivermectin under theChloramine-T and formalin are the most commonly cascade procedure when authorized products fail toused disinfectants in North America. The FDA has show effective control over sealice infestation.

approval the use of formalin (Parasite -S, Parasid - Nevertheless, off-label use of ivermectin premix inF, Formalin -F) in salmonids and penaeid shrimp. the treatment of sealice infestation of Atlantic sal-

Formalin is useful in the treatment of ectoparasitic mon is well known. Although ivermectin has demon-infections, especially by protozoa and skin and gill strated safety and efficacy in livestock and compan-flukes. Formalin forms paraformaldehyde, which is ion animals, its effect on the CNS of salmon couldtoxic to fish and must be removed prior to use. lead to death. Additionally, ivermectin has a pro-Formalin drums should not be stored below 408F. longed residence time in fish tissue and the drug

cannot be metabolized on excretion [11]. Extended4.2.2. Antiparasitic agents withdrawal times are expected to ensure depletion of

As shown in Fig. 1, ecto- and endoparasites are tissue ivermectin residues. Overall, its use in control-known to infest fish, particularly the salmonids. ling sealice infestation is limited to young salmonThese parasites include protozoa, trematodes, ces- shortly after transfer to seawater. Heavy sealicetodes, nematodes, and crustacea. An ideal an- infestation calls for newer and safer avermectintiparasitic agent possesses a wide therapeutic index, products.is effective against various stages of the parasite’s Other compounds with temporary approval inlife cycle, is not amenable to resistance development, certain European countries for sealice control include

and is environmentally friendly. diflubenzuron (Lepsidon , Ewos) and teflubenzuronAnthelmintic agents are drugs used to control (Calicide , Nutreco) [12], which are classified as

roundworms (nematodes), tapeworms (cestodes) and chitin synthesis inhibitors. These compounds areflukes (trematodes). Praziquantels (a given by the in-feed method and have the problem ofpyrazinoisoquinoline) is registered in Norway for use binding to marine sediments, creating environmentalagainst intestinal tapeworms. It is administered in concerns.feed with a withdrawal time of 14 days. Albendazoleand fenbendazole are currently being investigated for 4.2.3. Antibiotics and chemotherapeutantsuse against flukes and larval tapeworms in salmon. The use of antibiotics constitutes the bulk of

Dichlovos (Aquaguard , Novartis) and trichlor- prescriptions, especially in the 1980s prior to thephon are organophosphates used to treat sealice introduction of mineral oil-based vaccines. An esti-infestation by the immersion bath method in the mated 48 tons of antibacterial agents with an esti-salmon industry, except in the USA and Canada. mated 1.26 treatments /fish were used in Norway inThese compounds are cholinesterase inhibitors and 1987 [13].affect both the host and parasite alike. The therapeu- Several concerns cloud the widespread use oftic indices are low. Additionally, their use affects antibacterial agents in bulk quantities:other organisms in the aquatic environment. A newer

organophosphate, azamethiphos (Salmosan , Novar- 1. The degradation of residues in marine sediments.tis), has recently gained attention due to its higher It has been shown that oxytetracycline is so stablepotency and greater therapeutic index in Atlantic that a half-life cannot be determined [14]. Flor-salmon. It was reported to be effective against fenicol, on the other hand, has a half-life of onlychalimus, pre-adult and adult stage of the sealice 4.5 days.[10]. Other chemical agents include hydrogen perox- 2. Residues in marketed fish meat. To determine a


withdrawal time, studies needs to be performed at premix containing one or more new animal drugsdifferent water temperatures. intended for use in the manufacture of another

3. Emergence of drug-resistant bacterial strains medicated article or a medicated feed is defined as awhich may transfect humans. This is still a Type A medicated article, requiring full regulatorydebatable area where some researchers claim a approval.potential risk to public health while others have The technology in premix manufacture has notshown it to be a non-issue. changed drastically over the last decades. Neverthe-

less, the availability of new functional excipients andTetracycline has become the most popular drug the popularity of high-shear mixers /granulators have

therapy in aquaculture due to its demonstrated made it possible to manufacture homogeneous pre-efficacy, low cost and low toxicity. The most com- mixes of potent actives at low concentrations.mon use is via the oral route through topical The desired attributes of an aquaculture premixtreatment of water. Occasionally, injection of brood product are summarized in Table 4.fish is carried out for prophylaxis of furunculosis. Formulation and process technologies need to beDue to binding with sea-water borne divalent cations properly devised to achieve the above pre-requisites

21 21(Mg and Ca ), the bioavailability of tetracyclines of an aquaculture premix under development. Ain salmonids is low ( , 10%) [15]. successful development program relies on the selec-

Potentiated sulfonamides composed of a sul- tion of compatible and functional excipients and thephonamide (e.g., sulphadimethoxine) with a 2,4- design and validation of a robust manufacturingdiaminopyrimidine (e.g., ormethoprim) have been process.used for virtually all types of bacterial diseases in

fish [16]. Romet -30 (sulphadimethoxine:ormetop- 4.3.1. Current formulation strategies forrim), manufactured by Hoffmann-La Roche, is ap- aquaculture premixesproved by the US FDA for use in salmonids and Over the last decades, the commercial availabilitycatfish against furunculosis and enteric septicemia. It of many compendial grade excipients has graduallyis a type A medicated premix for incorporation into changed the landscape of premix formation. It was,feed rations. and still is, a common practice for premixes to be

Quinolones, oxolinic acid and flumequine are also formulated with naturally occurring carriers, such aswidely used in Europe for all types of bacterial ground corncobs, ground rice hulls, toasted soy flour,infections including classical vibriosis, cold water wheat middlings, vermiculite, various clays andvibriosis, furunculosis, and yersiniosis. Recently, limestone. These carriers are inexpensive to sourcehowever, the emergence of quinolone-resistant and provide excellent absorptive capacity for liquidstrains of bacteria has caused tremendous concern in drugs and drugs dissolved in solvents. For instance,areas of heavy use of the drugs [17]. ground corncobs, one of the most preferred carriers

Amphenicols have been used in Europe for many in the premix industry, is capable of absorbingyears. Recent new developments include the intro- approximately 40% water (by weight) while stillduction of florfenicol with demonstrated in vivo being a free-flowing powder. This property is im-efficacy against furunculosis [18]. parted by the heavy natural pore buildup in the

Drugs approved by the FDA for use in aquaculture corncobs. One advantage of having the drug solutionare listed in Table 3, together with species for absorbed into the corncobs is that the stability of theapproved use, indications and dosage regimen. drug can be improved, since the drug is protected

from the environment (air oxidation). The disadvan-4.3. Development of premixes as a dosage form in tages of using corncobs include quality variationaquaculture from lot to lot and the heavy microbial bioburden. It

is quite common to observe mold growth in corncob-Although liquids and semisolids can be adopted based formulations if the product is exposed to

for aquaculture use, the preferred dosage form for 408C/75% RH (an ICH condition). Analytically,the medication of fish feed is still the premix. A extraction of the absorbed drug from the corncobs


Table 3Drugs approved by the FDA for use in aquaculture

Drug Species Indication Dosage regimen Comments

Oxytetracycline Pacific salmon Mark skeletal tissue 250 mg/kg/day for 4 days Salmon , 30 g; 7 day(Terramycin ) withdrawal time

Salmonids Ulcer disease, furunculosis, 2.5 to 3.75 g/100 lb /day 21 day withdrawal time

bacterial hemorrhagic septicemia, for 10 days

and pseudomonas disease

Catfish Bacterial hemorrhagic septicemia 2.5 to 3.75 g/100 lb /day 21 day withdrawal time

and pseudomonas disease for 10 days

Lobster Gaffkemia 1 g/ lb medicated feed for 5 days 30 day withdrawal time

Sulfadimethoxine, Salmonids Furunculosis 50 mg/kg/day for 5 days 42 day withdrawal timeOrmetoprim (Romet -30) Catfish Enteric septicemia 50 mg/kg/day for 5 days 3 day withdrawal time

Tricanemethane-sulfonate Fish and Sedation /anesthesia 15–330 mg/ l (fish); Concentration depends(MS-222, TMS, Finquel ) other aquatic 1:1000 to 1:20,000 on desired degree of

poikilotherms (other poikilotherms) anesthesia, species, size,

water temperature, etc.

Formalin Salmonids, catfish, Protozoa and Tanks and raceways: up to Drug must not be subject (Paracid -F, Formalin -F, largemouth bass, monogenetic 170 ml / l up to 1 h at above to temperatures below 408F.

Parasite -S) bluegill trematodes 508F; up to 250 ml / l indefinitely Do not apply to ponds when

at below 508F. Earth ponds: water is warmer than 808F

15 to 25 ml / l indefinitely

Salmodi and Fungi of the family Tanks and raceways: 50 to 100 m / l

esocid eggs. Saprolegniaceae up to 4 h daily. Earth ponds:

Penaeid shrimp Protozoan parasites 15 to 25 ml / l single treatment

Sulfamerazine Rainbow, brook, Furunculosis 10 g/100 lb /day for up to 14 days 21 days withdrawal time;

and brown trout not currently available

Table 4Attributes of a high-quality aquaculture premix

Quality Attributes Comments

Physical Elegant product which is free-flowing Easy to meter;and dust-free low exposure to workersGood physical stability. No segregationduring shipping/vibration.Non-hygroscopic, non-electrostatic

Chemical Good stability of active in premix Compatibility of drug withand medicated feed excipients in the premix

and ingredients in the feedHomogeneity of active in premixand medicated feed

Efficacy Good bioavailability, efficacy

Other Environmentally friendly Low impact to otherorganisms in aquaticenvironment

Economic to manufacture


can be difficult, resulting in incomplete recovery. Type II [19]. A sample (typically 50 g) is loaded intoThe use of corncobs in a GMP-grade pharmaceutical a rotating drum and an air flow (4 l /min for 5 min)plant is generally viewed as unacceptable. takes the dust to a 0.45 mm glass fiber filter. The

Compendial grade carriers /diluents which are weight of fines deposited on the filter can besuitable for use in aquaculture premixes include measured gravimetrically and the active level in thecorn /maize starch, maltodextrins, dicalcium phos- fines can then be determined using analytical meth-phate, lactose and microcrystalline cellulose ods (e.g., HPLC). Staubel and Beutel [20] provided a(Avicel), amongst others. These excipients have good comparison of the dusting levels of somelower capacities for absorbing liquid drugs. How- commercially available premix products.ever, due to the wide range of grades currently To suppress the dusting level of dry blend formu-available, they can be used to function as carriers for lations, mineral oil and vegetable oil can be used,fine drug particles or as diluents for potent com- typically in the range of 1–3%. The use of oil oftenpounds. Maltodextrins, derived from partially hydro- leads to agglomeration problems, especially at alyzed starch, have the added advantage of being higher level of incorporation. Propylene glycol andgood flow aids. Fig. 2 illustrates the flowability index polyethylene glycol (PEG; various molecularof various starch–maltodextrin placebo blends. The weights) have been shown to reduce the dustiness offlowability index was measured using a Flodex dry blends with less-pronounced lumpiness /ag-Model 211-R1 (Hanson Research, Northridge, CA, glomeration problems. PEGs are known to self-oxi-USA). A flowability index of # 22 mm is desirable dize with time and therefore should not be used withand can be achieved at 40% maltodextrin level. drugs prone to oxidation. Fig. 3 demonstrates the

The dusting potential of aquaculture premixes is a effectiveness of propylene glycol in controlling theanother key parameter to follow and a prime target to dustiness of a starch /maltodextrin placebo blend. Itoptimize. The dust generated during the manufacture should be pointed out that, at higher levels ofand packaging operations of a premix and sub- incorporation ( . 4%), the flowability of the blendsequent use by farm workers causes safety concerns. will be greatly compromised.Additionally, it has been demonstrated that the activecan be more concentrated in the airborne fines than 4.3.2. Current processing technologies forin the product. aquaculture premixes

The standardized method of measuring dust levels The typical processing equipment used in theof feed premixes is by the Heubach Duster Meter, premix industry is ribbon blenders coupled with

comminuting mills. Ribbon blenders offer the advan-

Fig. 2. Flowability of corn starch–maltodextrin M-100 physical Fig. 3. Effect of propylene glycol concentration on premixmixtures. dusting potential.


tage of low purchase and maintenance costs, being 4.3.3. Regulatory submission: additional qualitycapable of processing large-volume batches, and requirementshaving certain advanced features (heating, vacuum The European Pharmacopoeia 1997 has estab-drying, etc.). Ribbon blenders, however, are low- lished a separate monograph on premixes for medi-shear mixers by design and do not provide high- cated feedingstuffs for veterinary use. Premixes are,intensity mixing actions. A high-shear mixer / therefore, to be considered a separate pharmaceuticalgranulator is recommended for dispersing actives of form, different from powders or granules for use invery low concentrations in the premix. drinking water or for oral use [22]. The registration

The effect of the Collette Gral in dispersing a of a premix product in the European Union andpremix of 0.2% active concentration is shown in Fig. North America requires the filling of complete4 [21]. The dual mixing and chopping actions of the CMC/quality sections, as would any other regularequipment result in an homogeneous active distribu- pharmaceutical product. Nevertheless, there are cer-tion in the premix blend. The top-entry choppers tain additional requirements for the registration of aprovide the added advantage of size reduction, premix.thereby eliminating the need for a separate milling Due to the intended application of premixes,step. additional information needs to be generated regard-

High-shear processors such as the Collette Gral ing the mixing properties of the premix with the feedseries and the Niro Fielder PMA series are widely ration to ensure an homogeneous distribution of theused in human pharmaceuticals for dry-blending and drug in the feed. The results from mixing studies canwet-granulation purposes. The extension of wet- be used to justify a particular type of compoundergranulation technology to the premix industry offers and associated mixing parameters. It is the regulatoryseveral advantages, including improved drug dis- agency’s position to use full production-scale batchestribution, reduced segregation during shipping, low for feed-medication studies, although smaller pilot-dusting and improved flowability. A milling step is scale batches are acceptable [23]. Stability programsneeded for sizing the dried granules with the flex- for medicated feed need to be established andibility of using different mesh screens to produce followed, in order to propose a suitable shelf-life.premixes of different particle sizes. Other considerations include:

Roller compaction represents another technologywhich can be extended to the aquaculture premixindustry. For moisture-sensitive drugs, roller com- 4.3.3.1. Type of incorporationpaction is a suitable method to produce dry wafers, Aquaculture premixes can be incorporated bywhich can then be sized to the form of a premix. either surface coating of fish feed pellets or co-

extrusion with fish meal ingredients to form pellets.In surface coating, the premix is typically suspendedin fish oil and the suspension is poured onto the feedunder mixing in a suitable mixer (ribbon or cement-type mixer). The premix can also be dusted onto thefeed pellets followed by the oil coat, which isreferred to as the double-coating procedure.

In the co-extrusion method, the premix is blendedwith feed ingredients in a mixer. The blend is thenconditioned and passed through an extruder underhigh heat and humidity conditions. The extrudedpellets are then dried and coated with oil, if desired.This method pre-exposes the active to the hightemperature (85–1108C) and humidity conditionsinherent with the extrusion process, potentially af-Fig. 4. Distribution of active in the Collette Gral mixer /

granulator. fecting drug stability.


4.3.3.2. Homogeneity of drug in finished feed 5. Aquaculture vaccinesRegardless of the type of incorporation method,

homogeneity of the drug in the finished feed needs to Although fish do not have bone marrow or lymphbe demonstrated. Sufficient samples should be taken nodes, the thymus, kidney, and spleen constitutefrom the mixer and analyzed using a validated important organs of the immune system. It has beenmethod. The sample size should represent the daily shown that specific antibodies and cell-mediatedintake of the target fish but should be less than 50 g. responses can be generated [24] upon antigenic

challenge. The immune system of the fish is alsoknown to be influenced by environmental factors

4.3.3.3. Compatibility with typical feedstuff such as water temperature and seasonal changes, i.e.Compatibility of the drug with fish meal and time of year [25,26].

additives (minerals, vitamins, preservatives, etc.)needs to be examined. 5.1. Anti-bacterial vaccines

Efforts in the early 1970s lead to the development4.3.3.4. Stability of medicated feed of vaccines against several bacterial strains, e.g.

The stability of the active in medicated feed Vibrio anguillarum, A. salmonicida, V. salmonicida,should be substantiated with data from three batches, and V. ordalii. Selected commercially available fishpreferably of commercial batch size. The fish feed vaccines, their types and application methods areused should be representative of the type intended listed in Table 5.for the species and age group (fry, fingerling, on- The first of the vaccines licensed by the USgrowing, brood, etc.). Medicated feed should be Department of Agriculture were those against Y.packaged in containers similar to the commercial ruckeri and V. anguillarum [28,29]. The popularitystyle and stored under various temperature /humidi- of these initial vaccines was due to the immersionty / light conditions. Based on collected data, a shelf- method as the intended means of administration.life for the medicated feed can then be proposed. Injectable vaccines containing antigens against A.

salmonicida, A. salmonicida plus Y. ruckeri, and A.salmonicida plus V. anguillarum were later made

4.3.3.5. Shipping study available. To enhance the performance against A.Medicated feed needs to be transported to simulate salmonicida, an adjuvant was incorporated in the

the effect of shipping /vibration on segregation of formulation. Aluminum salts and glucan produce aactive from the pellets. Samples are taken from top, moderate effect in improving the potency, whilemiddle, and bottom locations prior to and after oil-based adjuvants (mineral oil) provide a highershipping and analyzed for potency. level of protective immunity.

Table 5Commercially available anti-bacterial vaccines in aquaculture [27]

Disease Causative Protective Vaccine Vaccinationorganism antigen type method

Vibriosis Vibrio Lipopolysaccharides Bacterins Injectionanguillarum Immersion

Cold-water vibriosis Vibrio Lipopolysaccharides Bacterins Injectionsalmonicida Immersion

Yersiniosis Yersinia ruckeri Lipopolysaccharides Bacterins Immersion

Furunculosis Aeromonas A-layer protein Bacterins, Injectionsalmonicida some withsubsp. salmonicida adjuvants


The acceptance of injectable vaccines in aquacul- which the bacteria are inactivated with formalin andture was in part due to the heavy economic loss in emulsified with mineral oil or other oils. The mostthe 1980s as a result of an outbreak of furunculosis effective delivery route is by intraperitoneal injec-in salmon. Currently, injectable polyvalent vaccines tion. Although injectable oil-adjuvanted vaccinesare in demand because salmonid fish are prone to have been accepted by the industry for use in fishinfections by multiple bacterial strains. Almost all . 15 g, there are still major concerns over theirAtlantic salmon smolts are vaccinated with ‘triple side-effects. Intraperitoneal injection of these vac-vaccines’ against vibriosis, cold-water vibriosis and cines leads to the formation of unsightly granulomat-furunculosis 2–3 months prior to sea-transfer. ous lesions adhering to the viscera [35]. In addition,

It is well recognized that vaccination by injection mineral oil adjuvants are known to result in ayields the strongest immunity, followed by immer- reduction in weight gain of fish [36].sion and spraying methods with intermediate levels Other routes of vaccine delivery have been ex-of protection, followed by oral vaccination with the plored, e.g. topical by immersion [37], by bath [38],lowest immunity. or by hyperosmotic infiltration [39], oral [40–42],

At present, there are still no commercially avail- and anal [40]. The feasibility of the oral immuniza-able vaccines against two other important bacterial tion method was in fact demonstrated as early asfish diseases, bacterial kidney disease and salmonid 1942 by Duff [42]. The advantages of oral vaccinerickettsial septicemia. delivery in fish have been exemplified by Horne [43]

to include improved performance, easy application,5.2. Anti-viral vaccines increased flexibility and improved safety. The suc-

cess of the anal intubation method [41] furtherImportant fish viral diseases include infectious supports the hypothesis that enteral administration of

pancreatic necrosis (IPN), viral hemorrhagic sep- vaccines is feasible provided that the antigens beticemia and infectious hematopoietic necrosis (IHN). protected through the anterior gut.The development of vaccines for these diseases was Different approaches have been employed inrather unsuccessful until recently. The difficulty in recent years to formulate oral vaccine deliverydeveloping anti-viral vaccines lies in the fact that systems with certain advanced features, e.g. bio-these diseases occur primarily at the fry age of fish, degradable polymer as matrix [44] or modifiedmaking it difficult to use injection as the means of release [45,46].vaccine administration. Poly(DL-lactide-co-glycolide) microspheres con-

Early prototype vaccines containing inactivated taining human gamma globulin (HGG) were pre-viruses developed for immersion all resulted in pared using the water-in-oil-in-water (w/o /w) emul-insufficient protection [30–32]. The use of attenuated sion /solvent evaporation method and delivered oral-or avirulent forms of the viruses is regarded as ly to rainbow trout. It was found that microencapsu-unacceptable due to the residual virulence in targeted lated HGG was retained in the stomach for a longerspecies and virulence in non-target species. duration of time compared to soluble HGG. As a

A major advancement in the area of anti-viral result, proteolysis of surface-adsorbed antigen wasvaccine research was the result of recombinant DNA detected. Nevertheless, a significant amount of intacttechnology. An injectable anti-IPN vaccine based on antigen was found in the posterior intestine and inviral protein VP2 was formulated for use in Norway. the bloodstream. This provides evidence that poly-It is added to a polyvalent vaccine as a component in (DL-lactide-co-glycolide) protects the antigen, at leasta commercial oil /glucan-based formula [33,34]. partially. Unlike the mammalian intestine, which

absorbs the microspheres through the microfold cells5.3. Future possibilities for aquaculture vaccine of Peyer’s patches, teleosts do not appear to possessdelivery systems such patches. The mechanism of increased antigen

absorption is thus largely unexplained.The current art of aquaculture vaccine preparation The use of an enteric coating in protecting orally

still relies on the approach of whole broth culture, in delivered vibrio vaccine was explored by Lillehaug


[45] and Wong et al. [46]. In 1989, Lillehaug Acknowledgementsformulated two oral vaccines against vibriosis, oneof which comprised a slow-release matrix and the The author would like to thank Dr. Richardother consisted of lyophilized whole-cell granules Endris, Project Manager of Schering-Plough Animalcoated with 10% Eugragit L 100-55 for enteric Health, for valuable discussions and suggestions.protection. The slow-release matrix was formulatedwith saturated long-chain fatty acids, oleic acid,lecithin, glycerol, and the like. The enteric-coated Referencesformulation was produced by wet granulation of thevaccine with excipients, extrusion of the wet mass,

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[15] J. Pye-MacSwain, D. Rainnie, E. Cawthorn, G. Johnson,proaches, i.e. protection from proteolysis, improving Plasma pharmacokinetics and bioavailability of oxytet-permeability of the antigen and controlled /sustained racycline in Atlantic salmon held in seawater, Aquaculturerelease patterns for better immuno-response. Assoc. Can. Bull. 92 (3) (1992) 70–72.


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Aquaculture pharmaceuticals and biologicals: current perspectives and future possibilities