FAQ 310.2 Lecture Notes on Intro. to Fish MCB & Pathology 2021 PDF

Lecture Notes on FAQ 310.2 Introduction to Fish Microbiology and Pathology By Dr. Victor Eyo Department of Fisheries and Aquaculture Faculty of Marine Environmental Management Nigeria Maritime University Delta State...

Lecture Notes on FAQ 310.2 Introduction to Fish Microbiology and Pathology By Dr. Victor Eyo Department of Fisheries and Aquaculture Faculty of Marine Environmental Management Nigeria Maritime University Delta State 2021 INTRODUCTION Disease can be defined as any harmful deviation from the normal structural or functional state of an organism, generally associated with various signs and symptoms. Fish disease is the state of harmful deviation from the normal structural and functional state of fish caused by pathogens. Pathogens are those organisms that are capable of causing diseases such as fungi, bacteria, protozoa, virus etc. Fish disease can lead to morbidity and consequently mortality. Fish diseases is a simple association between a pathogen and host fish in their environment. For fish disease to break out, the fish may be stressed from several factors such as environmental factor (poor water quality), handling, overcrowding, poor nutrition etc. Epidemiology of parasite populations in water body Types of Fish Diseases There are two broad categories of disease that affect fish, infectious and non-infectious diseases. Infectious diseases: are caused by pathogens that are present in the environment or carried by other fish. They are contagious and can be controlled by various treatments. Infectious diseases are broadly grouped into the following categories: i. Bacterial diseases ii. Parasitic diseases iii. Viral diseases iv. Fungal diseases. Non-infectious diseases: are non-contagious diseases caused that are not caused by pathogens and cannot be cured by drugs or medications. Non-infectious diseases are broadly grouped into the following categories: i. Nutritional deficiencies: This occurs when there a deficiency in nutrients required by the fish for growth and other body activities. Nutritional diseases can be very difficult to diagnose. The broken back disease which is the deformation of the spinal column is found in catfish and is caused by lack of vitamin C. No blood disease is also found in catfish and is caused by folic acid deficiency. Affected fish suffers from anemia and may die. Nutritional disease condition seems to disappear when the diet is corrected. ii. Environmental diseases: Environmental diseases are related to water quality parameters such as low or high dissolved oxygen, high or low pH, high ammonia, high nitrite, natural or man-made toxins in the environment. Environmental diseases can be controlled by proper management of water quality of the environment. iii. Genetic anomalies: This results from the alteration of genes of fish. such diseases include as lack of a tail, presence of an extra tail, presence of only one eye, absence of barbell in catfish etc. The Significance of Fish Disease to Aquaculture In the wild, the problems of fish disease is not really noticeable because sick fishes are quickly removed from the population by predators. Furthermore, fish are not crowded in the wild like in culture systems. Disease is a simple association between a pathogen and a host fish in the aquatic environment. For disease to break out, the fish are usually stressed therefore, an effective disease management practice should be targeted at reducing stress. Disease rarely results from simple contact between the fish and a potential pathogen. Environmental problems including poor water quality and other stressors such as handling often contribute to the outbreak of disease. Under optimum conditions, healthy fish are able to fight off most forms of infectious diseases. But when fish are stressed, they become weak and unable to maintain their natural defenses against infectious diseases such as bacteria, viruses, fungi or protozoan parasites. Stressors include overcrowding, handling stress, poor water quality, inadequate nutrition and weather etc. These forms of stress may kill fish outright, in which case they can be considered non-infectious diseases. Fish disease is a substantial source of monetary loss to farmers due to increase in production costs when there is outbreaks of fish disease. When fish is lost because of disease, money invested in the dead fish is lost. Also, money used in disease treatment increases the production cost. Decrease in growth of the fish also contributes to production cost. Three main practices can be used in minimizing the possibility of disease outbreaks. They include: i. Maintenance of good water quality ii. Good nutrition iii. Elimination of contact with wild fish whenever possible. Signs of sick fish The most obvious sign of sick fish is the presence of dead, dying or moribund fish. However, a good observer can tell when a fish is sick before they begin to die. The signs are classified into two groups including the behavioral and physical signs. The following are behavioral signs that could be used to know if a fish is sick.  i. Sick fish often stop feeding or respond poorly to feeding and may appear lethargic. Healthy fish usually eat aggressively when fed at their feeding time. ii. Hanging in shallow water, gasping at the surface for oxygen. Pond fish should not be visible except at feeding time. iii. Rubbing against objects or walls of the ponds. This indicate something may be wrong such as itching caused by ectoparasites attachment to the skin. iv. Flashing in the pond The following are physical signs that could be used to know when a fish is sick. i. Presence of sores such as wounds, ulcers or hemorrhages in different body parts ii. Ragged or frayed fins iii. Abnormal body confirmation such as a distended abdomen or "dropsy" and exophthalmia or "Popeye" iv. Loss of mucus v. Loss of scales What to do if Fish are Sick The first thing to do when there is suspicion of sick fish is check the water quality of the fish tank or pond. Generally, water quality parameters that should be checked include dissolved oxygen, ammonia, temperature, nitrite, and pH, total alkalinity, total hardness, conductivity etc. These water quality parameters can be measured with water quality test kit or specific meters such as dissolved oxygen meter, thermometers, nitrite and nitrate meter and pH meter. High levels of ammonia is very fatal in tanks or ponds especially when there is overcrowding. Ammonia is measured using colorimeters. Where the pond owner does not have water quality kits, water samples in the suspected pond should be taken to a good water quality laboratory for measurement of the water quality parameters. Ideally, a good farm should be able to keep daily records on all farm activities should which could be used for immediate reference when a fish disease outbreak occurs. Such records should include the dates of stocking fish stocked, size and age of fish at stocking, source of fish, type of feed used, feeding rate, growth rate, daily mortality and water quality. This information is will be very useful in correct diagnosis and treatment of fish disease. A complete case history that will guide the diagnostician in correct diagnosis should include the following: i. Good records ii. A description of behavioral and physical signs exhibited by sick fish iii. Results of water quality tests FISH PARASITES AND PUBLIC HEALTH Fish parasites have become a major concern especially in countries where raw or undercooked fish is consumed which may become a threat to public health due to zoonosis. Zoonosis is the transfer of diseases from animals such as fish to humans. There are several fish-borne diseases related to digenea, cestoda and nematode due to consumption of fish infested with parasites. The commonest diseases associated with fish consumption are: 1. Opisthorchiasis 2. Intestinal trematodiasis 3. Diphyllobothriasis 4. Anisakidosis 1. Opisthorchiasis: is caused by a parasite group known as a digenean trematode belonging to the family Opisthorchiidae. 2. Intestinal trematodiasis: is caused by parasite groups known as a digenean trematode belonging to the families Heterophyidae and Nanophyetidae. 3. Diphyllobothriasis: is caused by a parasite group known as a cestodes belonging to the genus Diphyllobothrium. 4. Anisakidosis: is caused by a parasite group known as a anisakid nematodes with species such as Anisakis simplex, A. pegreffii and Pseudoterranova decipiens.  Some of these parasites can be ingested by consuming freshwater fishes but for fishery products originating from the ocean or marine environment, anisakid nematodes are a major risk for fish-borne zoonoses.  Digenean trematodes are the most common parasites causing fish-borne diseases globally but are restricted to freshwater fish as intermediate hosts and play a minor role in zoonoses associated with consuming marine fishery products.  Cestodes are found in both freshwater and marine habitats. Most Diphyllobothrium species are found in freshwater fish while some are found in marine fish such as Diphyllobothrium cameroni, Diphyllobothrium cordatum and Diphyllobothrium hians. Diphyllobothriasis is often asymptomatic but can cause abdominal and digestive discomforts.  Nematodes such as Anisakids are found in marine fish and are known to constitute a major threat to food safety of fishery products especially in European countries where raw or undercooked fish is consumed. After consuming fish flesh or viscera infected with anisakids, humans can suffer from direct infections and the triggering of allergies. Human anisakidosis is associated with mild to severe gastrointestinal symptoms such as epigastric pain, nausea, vomiting and diarrhoea. Food Hazard Reduction Measures in fish and fishery products (Control of zoonosis) Food hazard reduction in fish and fishery products is a very important measure that should be taken to control zoonoses. Measures to reduce parasites in fish and fishery products should begin during harvesting or processing of fish which include special handling practices such as reducing the number of parasites and inactivation of viable parasites by heat and freezing treatment. Heating or cooking is much faster than freezing and the recommended temperature is above 60 ̊C for a minimum of 1 minute. For freezing treatment, the fishery product or fish should be frozen for at least –20 ̊C for 24 h or –35 ̊C for 15 h to kill parasites except trematodes. To prevent marketing of heavily infected fish, measures are taken to either reduce the infection level or sort out visibly infected fish. Good hygiene and screening of fish for parasites before marketing for consumption is very crucial. Several handling steps may be performed by processors and include:  Heading   Gutting  Filleting  Skinning  Trimming The inspection of eviscerated fish must be carried out by an experienced person with a sufficiently high number of samples and be focused on the abdominal cavity, liver and roe intended for human consumption. For fish fillets or slices, visual inspection must be performed during trimming and after filleting/slicing. The commonly used method for the detection of parasites in the fish flesh is known as candling method. Candling method involves placing fish on a light table or candling table in a darkened room to visualize parasites present in the muscle. However, the candling method has some weaknesses, e.g. a successful detection of nematode larvae varies depending on the thickness of the fillet, the presence of skin on the fillet, oil content, pigmentation and the level of experience of the operator. BIOSECURITY AND QUARANTINE PROCEDURE IN AQUACULTURE Biosecurity in aquaculture refers to all kind of practices that reduces the risk of introducing an infectious disease and spreading it to the fish or other aquatic animals at a facility and the risk that diseased fish or animals or infectious agents will leave a facility and spread to other facilities and to other susceptible species. Biosecurity helps in reducing stress to the animals, thus making them less susceptible to disease and infections. Good biosecurity reduces the chances of exposing fish to pathogens thereby reducing economic losses from mortalities. Pathogen can easily invade a fish facility spreading from one system to another causing diseases and economic losses. This depends on the following factors including: 1. The biological characters such as the species cultured, immune status, age, life stage, and species susceptibility 2. Environmental parameters such as water quality (pH, temperature etc.), water chemistry, and husbandry practices 3. Nature and characteristics of the pathogen, such as biology and life cycle, potential host, survival on inanimate objects such as equipment. 4. Ability of facility staff to understand biosecurity principles and comply with biosecurity protocols. Objectives of biosecurity in aquaculture The objectives of biosecurity include: 1. Fish management: obtaining healthy stocks and optimizing their health and immunity through good husbandry. Growing healthy fish is important in fish management. Therefore, the fish seed such as eggs, fry, juveniles, sub-adults and broodstock should be thoroughly screened to be sure that they are healthy before collection. A good historical record such as source, age, water quality of the source, disease history, treatment method, growth rate, genetic makeup etc. is very useful in providing information required for a successful fish management. If these information are lacking, there is need to test or screen the fish shortly after collection. Good husbandry is another important practice in biosecurity. Any activity that stress the fish or cause injury to the skin, fins, gills or intestine will compromise or weaken their immune systems and making them more susceptible to disease. Water quality management, fish nutrition and feeding management, and proper handling methods prevents the fish from disease. Also, good preventive medical practices such as quarantine, routine observation, vaccination, and the use of immunos- timulants, probiotics, and diagnostics for disease management will help control diseases. 2. Pathogen management: Pathogens are disease-causing organisms such as bacteria, parasites, viruses and fungi. For pathogens to cause disease, they must relate with the environment and host (fish). The immune system status is a great determinant of disease outbreak. For optimal pathogen management, it is advisable to work with a fish health expert in order to get professional guidance with disease diagnostics and management.  Pathogenicity: Understanding pathogenicity is crucial in control and management of pathogens. Pathogenicity is the ability of pathogens to infect and cause disease to the host. Pathogens have different pathogenicity or ability to infect and cause disease. For instance, Aeromonas and Vibrio bacteria are opportunistic pathogens and cause disease only when water quality parameters are poor or when fish are stressed. Some pathogens can cause disease readily in susceptible fish species while some pathogens fall in-between these two groups.  Diagnostics: Diagnosis is useful in identifying pathogens and administering the correct treatment. Many pathogens can be diagnosed using routine tests. Some pathogens are not easy to diagnose because they can be either be hidden or present in very small numbers in the host. Most viruses survive within a specific temperature range and when fish infected with such virus is removed from water to be tested, the result may be negative giving the diagnostician the impression that the fish is virus free.  Disease control: Diseases can be easily controlled by good sanitary practices, disinfection or drugs application. Cleaning involves removing all foreign material such as dirt, soil, organic material etc. from objects by scrubbing them thoroughly. Disinfection involves applying disinfectant on all surfaces and is only effective if the recommended concentration is used for the recommended duration. Some pathogens such as viruses are difficult to treat so it is better to adopt good preventive practices and proper vaccination. However, repeated and wrong application of drugs can result in to resistance such drugs which is common in bacteria. Types of Chemical Disinfectants used in aquaculture Disinfectant type Products / chemicals Dose or dilution Contact time  Chlorine Sodium hypochlorite (liquid) 200 to 500 mg/L** 10 to 60 minutes for general disinfection. available chlorine Calcium hypochlorite Higher concentrations and longer contact (powder, granules, pellets) 5,000 to 10,000 mg/L times may be required for specific available chlorine pathogens and situations. 10 to 30 minutes to eliminate some more resistant types of mycobacteria and spores. When cleaning tanks, disinfect for 24 hours, neutralize, rinse, and allow to dry. Iodine/ Iodophor Providone-iodine 30 to 50 mg/L of free 10 to 30 minutes for general disinfection iodine Contains 9 to 12% available Prolonged contact time likely for iodine calculated on a dry Dilution depends on mycobacteria and spores. weight basis. product. Formulations may contain 1 A few seconds only; foot baths or net dips. to 10% providone-iodine, 200 to 250 mg/L free which is equivalent to 0.1 to iodine 1% available iodine. Alcohol Ethyl alcohol (ethanol) and 60 to 90% (v/v; volume 10 to 30 minutes for general disinfection isopropyl alcohol of alcohol to volume of (isopropanol) water) Hydrogen 3% Hydrogen peroxide 3 to 30%** w%; weight 5 to 30 minutes for general disinfection. peroxide percentage Aldehydes Formaldehyde 1 to 8% 10 minutes to 16 hours pH Calcium hydroxide (slaked pH > 11 >4 hours lime) Calcium oxide pH < 4 (quicklime) Hydrochloric acid Phosphoric acid A stepwise procedure to routinely clean and disinfect equipment 1. Remove dirt Manually 2. Clean extensively with a detergent or soap and water 3. Rinse 4. Apply a disinfectant with appropriate contact time. 5. Rinse again 6. Dry completely (preferably in the sun). 3. People management Managing people around the facility is very important for a successful biosecurity program. For biosecurity to be successful, managers, staff and visitors and other personnel must understand and follow the necessary protocols and practices. Otherwise, the risk of disease outbreak will be increased. Employees and visitors who do not follow established protocols will increase the risk of disease. Restrictions should be placed on sensitive areas such as hatchery except authorization is granted. areas should be restricted to authorized personnel. Visitors may pose a serious risk to the farm. Staff and visitors should be properly educated on biosecurity program and protocols. To eliminate the risk of disease introduction, the following facilities should be put in place at strategic locations. They include: i. Disinfectant footbaths ii. Hand-washing stand or alcohol spray bottles iii. Net disinfection stand iv. Showers v. Vehicle disinfection stations Quarantine: Quarantine is one of the most important fish management and biosecurity measures in aquaculture. Quarantine is the procedure whereby an individual fish or population is isolated, acclimated, observed and, if necessary, treated for specific diseases before its release into the farm for rearing or market sale. The principles of quarantine is applied for new fish coming into a facility, fish moving from one area or system to another within the facility, and resident fish that become diseased. A good quarantine system physically separate incoming fish from the rest of the facility or farm including water. Apart from shielding the incoming fish from pathogens, quarantine also allows the incoming fish to acclimate to water and feed and to recover from stress arising from handling and transport. Major components of quarantine include: i. All-in-all-out stocking ii. Isolation or separation iii. Observation and feeding response iv. Sampling and treatment. i. All-in-all-out stocking: This involves bringing fish in and maintaining them as a group from only one original source population throughout the quarantine period. It helps in preventing fish from being exposed to other pathogens not currently in that population. ii. Isolation or separation: A group of fish in quarantine should be physically isolated from other quarantined populations and from the resident populations. Despite isolating the incoming fish, appropriate sanitation and disinfec- tion measures must be applied to reduce cross-contamination between quarantined and resident populations. iii. Observation and feeding response: Fish should be observed for normal and abnormal appearance and behaviors throughout the quarantine period for early detection of so diseases. Swimming behavior, response to feed, lesions and ulcer on the skin and other attributes should be carefully observed. Loss of appetite is a very common and early sign of disease. Good nutrition will boost disease resistance. iv. Sampling and treatment: at the beginning of quarantine, the quarantined fish should be sampled for possible disease and at the end of the quarantine period, sampling should also be done to ensure that the quarantined fish are disease free. Also, sampling should be done whenever there is a sign of disease. The skin, fin and gills can be examined for parasites using microscopy. Also, blood culture can reveal bacterial infections. After sampling and examination, the results can then be used to improve quarantine methods and the type of drugs that will be effective. IDENTIFICATION, ISOLATION PREVENTION AND TREATMENT OF FISH DISEASES DIAGNOSIS OF FISH DISEASES History History for fish patients is of vital importance in diagnosing the disease affecting the fish. Information gathered in the history helps to direct and focus the physical examination and can aid in the selection of additional pertinent diagnostics. Most times, through a good clinical history, a preliminary diagnosis can be reached. The general history should focus on determining what species are affected, what clinical signs have been appreciated by the client and the time course of the disease. The client should be questioned about what treatments have been attempted and the success or failure of these treatment endeavors. Specifically, clients should be asked about the application of any chemicals, antimicrobials, antiparasitics or supplements to the water or the feed. Similarly, the client should be questioned whether any environmental alterations have been attempted, such as adjusting the water temperature or salinity. As compared to land-dwelling animals, fish patients have the added challenge that their water environment plays a critical role in their health. Therefore, questions regarding the environment and water quality must be investigated as thoroughly as questions regarding the fish patient itself. A current water quality analysis should be performed as part of any clinical examination and details regarding historical water quality results and trends should be obtained. General history questions to ask during the client account of the clinical history are as follows: GENERAL HISTORY QUESTIONS Common name and genus/species of the patient Age of the patient, or how long has the owner had the patient? Origin: Captive bred, wild-caught/imported, unknown Source: Pet store, breeder, farm, wild-caught, etc. What other species are present in pond/raceway/cage/ tank? What clinical signs has the owner appreciated? Duration of problem(s)? What species are affected? Any mortalities? Any changes in behavior noted? Any changes in appetite noted? Is a quarantine system in place? Any prophylactic treatments routinely performed? When was the last introduction of fish, invertebrates, plants? Does tank/pond/facility have history of previous disease(s)?  Have any treatments for the disease been attempted? Name, dosage and frequency of any chemicals, antibiotics, anti-parasitics or supplements that have been used to treat the fish Have any adjustments to water temperature, pH, salinity or other water quality parameters been made? What is the cleaning routine for the pond/raceway/cage/tank? Are cleaning tools specific for each pond/raceway/cage/tank? What disinfection agents are used? What type of food is fed? How much and how frequently? Are fish fed consistently all year round or does feeding routine change seasonally? How old is the food? Does food have a known expiration date? How is the food stored? Sample Selection Good sample selection is a very important step in proper diagnosis of fish diseases. Strictly random sampling in a population with low-level mortalities may lead to evaluation of healthy fish. The clinician should select moribund fish showing typical disease signs, along with a healthy individual for comparison, especially in species that the clinician is unfamiliar with. If possible, multiple fish should be chosen at different stages of disease, including at least one or more with early-stage disease, as secondary complications that mask initiating causes are often seen in much later stages of disease. Evaluation of freshly dead fish, although not ideal, can also be helpful, especially if disease prevalence is low. Selection of freshly dead (i.e., collected immediately after death) fish for evaluation will help to avoid postmortem artifact and a subsequent misdiagnosis. Although dead fish may have lesions that make assessment of “freshness” more challenging, freshly dead fish should have relatively clear eyes, good coloration, red to pink gills, and should not have a bad odor. Dead fish are often cannibalized, undergo rapid autolysis, and/or become overrun by secondary postmortem organisms such as fungi, bacteria, and parasites. For this reason, the relevance of wet mount findings from lesions on dead fish must be interpreted with caution. Although fish demonstrating clinical signs of disease are ideal specimens for antemortem diagnostics and necropsy, freshly dead, frozen, and formalin-fixed specimens may have diagnostic value. Freshly dead fish examined immediately or kept in a plastic bag in the refrigerator or on ice for 6–12 hours can provide almost as much information as moribund fish, whereas frozen and formalin-fixed fish are much less informative. Common external parasites (e.g., protozoans and monogeneans) often die or leave a dead host, and bacterial cultures collected from fish that have been dead for as little as 30 to 60 minutes may be contaminated due to rapid autolysis of the organs. APPROACHES TO DISEASE DIAGNOSTICS Numerous factors will guide the clinician’s approach to disease diagnostics for each case. If the affected animal is considered too valuable to sacrifice for financial or personal reasons (e.g., brood stock, rare or endangered species, strong human–animal bond), antemortem, nonlethal diagnostics can be performed. If the outbreak involves a population health scenario (aquaculture/production or less valuable fish in a large display system), a full necropsy of representative fish is a much more informative option. Other factors include size limitations, client budget, logistics (i.e., time, location, and resources), available equipment and supplies, and the rapidity and severity of the disease progression. PHYSICAL EXAMINATION Observational examination  An observational examination of the patient in the water should be performed as part of every routine examination.  This observational examination allows the practitioner to study the behavior of the patient in a manner that is less compromised by the stress of capture/handling or the sedative effects of anesthetics.  Ideally the patient would be observed in its home tank, pond environment or aquaculture setting. This allows for observation of the patient’s interactions with other tank mates if present and will allow for a more thorough assessment of the patient’s behaviors as compared to simply observing the fish in a small transport container.  A site visit to the patient’s living environment will also allow for inspection of the patient’s living quarters and life support systems, which can be very useful in detecting environmental problems.  During the observational examination, the patient’s swimming patterns and position in the water column should be noted. Sick fish may congregate together away from the remainder of the group or may isolate themselves from other fish.  Weak or moribund fish may be found at the water outlet. Fish with respiratory compromise may be found congregating at the water’s surface or at areas of relatively higher oxygen content such as inflow pipes, aerators or waterfalls.  Fish may also exhibit other characteristic signs of disease such as “Flashing” or scraping along the bottom or sides of an enclosure is commonly seen when fish are affected by external parasites.  Fish with respiratory disease will often have an increased opercular rate with exaggerated flaring of the operculum. “Piping” behavior, where fish repeatedly gape at the water’s surface, can also be seen in fish with respiratory compromise.  In addition to abnormal behaviors and color changes, any obvious lesions or physical abnormalities should also be documented during the observational examination with the intent of performing further inspection of these abnormalities during the physical portion of the examination. Direct examination  Following the completion of the observational examination, a direct or “hands-on” physical examination is generally necessary for a closer assessment of any abnormalities noted during the observational examination.  When performing the direct examination of the patient, general anesthesia is usually recommended to minimize stress and potential harm to the patient.  General anesthesia is also indicated to help protect the practitioner from harm when dealing with species that have the potential to be injurious.  Fish from the family Scorpaenidae (i.e., lionfish, scorpionfish, stonefish) and certain catfish species have venomous barbs or spines along their fins that can cause considerable pain or even death upon envenomation.  Several species of fish including eels, triggerfish, surgeonfish, pufferfish and most species of elasmobranchs have teeth, barbs, modified scales or spines that can inflict injury to the handler.  Other species such as electric eels, electric catfish and torpedo rays can produce strong electrical currents and must be handled with rubber gloves and appropriate precautions.  Prior to examining the fish patient, all necessary equipment for the procedure should be gathered and readied.  Handlers should wear powder-free latex or nitrile examination gloves, both to help protect the fish’s skin and to help protect the handler from possible zoonotic pathogens.  If the fish needs to be placed on a surface for examination, the surface should be covered in a non-abrasive material (e.g., synthetic chamois, plastic drapes or bubble wrap) to protect the fish.  Absorptive materials such as paper or cloth towels are generally not recommended, as these materials may wick moisture from the mucous layer and have the potential to cause microabrasions on the skin of the fish patient. External Biopsies  During direct examination, external wet mounts, or external tissue squash preps of small pieces of skin, gills or fin, can be sampled for microscopic evaluation.  Skin scrapes and fin and gill biopsies are easy to obtain and highly informative techniques in clinical examination of fish.  Use of a gloved hand or dark material to cover the eyes of the fish will help reduce stress, stimulation and movement. Discolored areas, excess mucus, hemorrhages, ulcers, erosions, masses or parasites should be sam- pled. Skin biopsy  For skin biopsy, a coverslip or the back of a scalpel blade is used to gently scrape any areas that appear abnormal (e.g., discoloration, thickened mucus, ulcers, erosions). The skin scraping is placed on a slide with a cover slip and examined using the microscope.  In patients without any “abnormal” external areas, other sites to target include portions of the body that are softer or have fewer scales or where water flow is reduced. Gills biopsy  Gill biopsies are typically collected using scissor blades or a pair of forceps to lift the operculum and clip a small section of the gill filaments.  The amount of gill tissue to collect and submit for diagnosis will depend on if the patient is terminal or if the sample is to be collected nonlethally.  In the latter, the diagnostician needs to be particularly careful not to cut the gill arch since this structure contains major arterial and venous vessels.  As with the skin, the diagnostician should target abnormal areas of the gill. If the fish has been euthanized, the diagnostician can cut a section of the gill arch and examine it microscopically or macroscopically if the sample is too thick for a compound microscope. Fin biopsy  Although fin biopsies are not as commonly sampled as gills or skin, in some cases examining a small section from each major fin could be of value.  In some fish, hard fin rays and darkly pigmented fins are difficult to evaluate. Like the skin, fin scraping can be collected and viewed using the microscope. Wet Mount Evaluation  Immediately prior to sample collection, the diagnostician should have prepared slides for biopsy samples by placing one or two small drops of water equidistant on a slide. This will allow for one or two different biopsy samples to be evaluated per slide.  A separate coverslip is then placed on each biopsy sample with an adequate amount of water to completely flood the area under the coverslip, and gentle pressure is used to flatten the tissue to allow penetration of light and easier visualization.  For external (i.e., skin, gill, and fin) samples, the use of dechlorinated freshwater (not distilled water) for freshwater fish and salt water for marine fish, respectively, is necessary.  During microscopic examination of squash preps/ wet mounts, the clinician should scan each preparation at x10 and x40 (i.e., low power). Most parasites such as Henneguya sp. and lesions of interest are visible at 40×–100× magnification. TREATMENT OF FISH DISEASES Treating fish is a very technical practice and there are three basic techniques involved. They include: 1. Adding drugs to the water 2. Adding drugs to the feed: 3. Administration of drugs to individual fish 1. Adding drugs to the water: There are three ways of treating fish by adding drugs to water and are explained as follows: i. Dip method: In this method, sick fishes or infected eggs are placed in hand net or trays and dipped into a concentrated solution in a treatment tank for few seconds. ii. Bath method: The most common technique used by many fish keepers. This involves bathing sick fish in a solution of therapeutic agents in ponds or tanks. It is used in treating fish suffering from external diseases such as parasitic, fungal and bacterial infections. There are three types of bath treatments and the difference between these three types of bath treatments is the concentration of the chemical used for treatment and the duration of fish exposure to the chemical. a. Dip bath: This is where fish is dipped into a concentrated chemical bath for a short duration of less than a minute. If the exposure period is prolonged, fatality may occur. This method is suitable for treating large numbers of fish in a short time. b. Short bath: This is where fish is bathed into a moderate concentration of chemical bath for 30 minutes to several hours. c. Long bath: This is where fish is bathed into a low concentration of chemical bath for many hours to days. This is the only method of administering a bath to pond fish. Long bath is the cheapest way of administering a chemical bath. iii. Flush method: Flush is a used in flow through system. A high concentration of the chemical is added and water flow is continuous and allowed to flush through the system. This method is mostly used in hatcheries. 2. Adding drugs to the feed: This involves the use of medicated feeds to treat sick fishes. The drugs can be mixed in gelatin or soybean oil heated at 40 – 50ºC and used in coating the feed before administering to the fish. 3. Injection of drugs to individual fish: Injection is used in treating individual fish that are valuable. Injection helps in delivering a precise dosage but may introduce stress to the fish by capturing the fish and always bringing the fish to the fish clinic for each injection. There are three way of injecting fish and the include intramuscular (IM), intraperitoneal (IP) and dorsal sinus (DS) injection. Intramuscular injection is used for fish greater than 13 cm in length and is best injected in the dorsal musculature which is just lateral to the dorsal fin. For intraperitoneal injection, it is advisable to starve the fish for 24 hours before injection to eliminate the risk of peritonitis caused by puncture of the stomach. Intraperitoneal injection should be made near the ventral midline. Intraperitoneal injection is commonly used for administering vaccines. Dorsal Sinus injection is mostly used when treating bacterial kidney disease in some fishes such as salmon. It is given at the dorsal sinus which runs near the dorsal fin. SUBSTANCES USED IN TREATMENT OF FISH DISEASES 1. Common salt (NaCl): Salt which is referred to as the aspirin of aquaculture is among the commonest drugs used in aquaculture. The methods of application, concentration and duration of application depends on the disease, species, weight and type of aquaculture unit. 2. Formalin: Formalin is used as a bath treatment to control ectoparasites of fish found in the skin, fin and gills. High formalin concentrations are effective in the control of fungi on fish eggs. For long bath, 15 to 25 mg/L (2 drops /gallon) may applied. For short bath, dose 250 mg/L for 30-60 minutes is used. 3. Malachite green: is effective for many external protozoan such as trichodina and fungal infections. Concentration of 0.1 ppm can be applied in pond or aquaria for three or four days. For short bath treatment, 2 ppm for one hour is effective. 4. Potassium permanganate: is used in controlling many bacterial, parasitic and fungal agents. Concentration of 2mg/L as a long- term bath for a minimum of 4 hours is effective. 5. Antibiotics: Antibiotics are used for bacterial infections. For antibiotics to be effective, sensitivity test must be carried out to be sure of the correct antibiotics to be used. The dose used depends on the type of disease, types of antibiotic, age and weight of the fish. Common antibiotics used in aquaculture include: Oxytetracycline 10-100mg/L per day Erythromycin 4-8 mg/L per day Chloramphenicol 10 mg/L per day Penicillin 8000-12000U.I/L per day Precautions Taken when Fish is treated Through Water 1. Do not feed the fish for 24 hours before treatment. 2. Using plastic buckets for mixing the drugs. 3. Calculate dosage accurately 4. Apply treatment during the time of the day when the temperature is lowest. 5. Carry out a trial for treatment on few fish as control before making the treatment on large scale. 6. Watch the fish continuously during treatment. 7. Only repeat the treatment if absolutely necessary but not within 30 hours of the first treatment. PARASITOLOGICAL ASSESSMENT INDICES These are indices used to quantify the parasites in an infected host. The common indices used include: 1. Abundance 2. Prevalence 3. Intensity 1. Abundance: This is the number of parasites per number of fish examined. Abundance = Number of parasites/Number of fish examined Prevalence: This is the number of infected fish per total number of fish examined. 2. Prevalence (%) = (Number of infected fish/Total number of fish examined)*100 3. Intensity: This is the number of collected parasites per number of infested fish Intensity = Number of collected parasites/Number of infested fish Class Work 1a. 100 specimens of the African Pike (Hepsetus odoe) were collected from Okerenkoko Estuary and examined for ectoparasites. Results obtained showed that 20 specimens were infested by 30 parasites, calculate the following: i. Abundance ii. Prevalence iii. Intensity Ib. Out of the 100 examined specimens, 60 were males and 40 were females and 20 parasites were found in the 15 males and 10 parasites in the 5 females. Calculate the Abundance, Prevalence and Intensity in relation to sex. 1c. If out of the 30 recovered parasites, 12 trichodina were found in the fin, 8 monogenea were found in the fins and 10 protozoan cysts were found in the gills, Calculate the Abundance, Prevalence and Intensity in relation to organ specificity. 1d. Differentiate between mixed and single infestation in fishes.