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A Fishkeeper's Guide to Mycobacteriosis Compiled by Adrian R. Tappin Created 4 June, 1999 Updated 1 September, 2008 B efore the end of the nineteenth century (1897), mycobacteriosis had been described by a group of French scientists. They reported that Mycobacterium piscium (a name that is now obsolete) was a pathogen of diseased carp, Cyprinus carpio. They reported an outbreak of mycobacteriosis in pond carp in the garden of a sanatorium for tuberculosis patients. Isolated cultures were not pathogenic to warm-blooded laboratory animals. The first report of mycobacteriosis in a marine fish was noted by Von Betegh in 1910. In 1926, when J. D. Aronson studied a number of saltwater fish, which had died in the Philadelphia Aquarium, USA. He found that the fish had a tuberculosis-type disease with large nodules or ulcers throughout many of their tissues. From the nodules, Aronson cultured a bacterium, which he named Mycobacterium marinum. Injections of bacteria from this culture killed Goldfish, Frogs, Pigeons, and Mice but had only minor effects on Guinea Pigs and Rabbits. M. marinum was subsequently shown to also be a human pathogen when it was isolated again much later in a swimming pool-associated outbreak of human granulomatous skin lesions, although in this report the mycobacterium was mistakenly given a new species name, M. balnei, a name that is no longer used. Since these earlier times, much research on mycobacterial diseases in fish has been carried out. Mycobacteriosis is now known to affect a wide variety of aquarium and wild fish species of both freshwater and saltwater worldwide. Mycobacteriosis is usually a sub-acute to chronic disease of fish where the etiologic agent is an acid-fast bacillus in the genus Mycobacterium. Two terms are used to describe the disease, either “piscine tuberculosis” or “mycobacteriosis”. The term “tuberculosis” was previously used to describe diseases of fish, which involved any acid-fast bacilli. Since the typical tubercular inflammatory response observed in mammals was absent in fish, Parisot & Wood (1960) suggested that “mycobacteriosis” was a more appropriate expression for the disease. Although M. marinum was considered the primary causative agent of mycobacteriosis, a great number of Mycobacterium species associated with tubercle granulomas in aquarium and wild fish populations have been found. However, it has to be considered that the risk of infection in fish maintained in captivity is significantly higher. Pathogenic mycobacteria are most commonly reported in aquaculture and aquarium fish. Mycobacteriosis has been reported to cause massive mortalities among fish grown in intensive aquaculture systems. Aquarium fish are also subjected to intensive culture conditions and are thus susceptible to mycobacterial infections. Most likely, the aquarium environment offers a more favourable condition for propagation of mycobacteria (temperature, water, oxygen, etc.). The potential for disease among aquarium fish depends on the contact rate among infectious fish and number of susceptible fish. Under aquarium conditions, the frequency of that contact is vastly increased. Beyond overcrowding and confinement, aquarium fish are also subjected to other stressors such as handling, fluctuating temperatures, poor water quality, and social stresses. High numbers of mycobacteria have been correlated with warmer temperatures, low dissolved oxygen and pH. Such factors exacerbate the susceptibility of fish to disease and thus further increase morbidity and mortality in the population. Mycobacteriosis is one of the most commonly diagnosed bacterial diseases of aquarium fish. In 2005, a team of biologists from the University of Ljubljana, Slovenia, analysed 35 infected aquarium fish using several different methods, including Ziehl-Neelsen staining; Polymerase Chain Reaction; Restriction Fragment Length Polymorphism; cultivation and Hain Life Science's GenoType Mycobacterium assay. The analyses managed to detect the presence of the mycobacteria in 29 of the 35 fish analysed. The findings showed that seven were infected by M. fortuitum; six were infected by M. gordonae; six by M. marinum; three by M. chelonae and one by M. peregrinum. Five of them were infected by unidentifiable species of mycobacteria and one was probably a combination of two species. Another study of aquarium fish was conducted in Italy from June 2002 to May 2005. Two surveys were carried out, one of aquarium fish sent to a laboratory for diagnosis, and the other of prevalence of infection by mycobacteria in aquarium fish imported into Italy. In the first survey, 387 fish were examined and Mycobacterium species were isolated from 181 (46.8%) fish. In the second survey 127 batches of aquarium fish from different countries were examined. Mycobacterium spp. were isolated from 38 (29.9%) batches. The following species were found: M. fortuitum, M. peregrinum, M. chelonae, M. abscessus, M. marinum, M. gordonae, M. nonchromogenicum and M. interjectum. There was a high prevalence of infection independent of the presence of macroscopic lesions. Mycobacterium fortuitum and M. chelonae were more prevalent than M. marinum in the samples examined. In 2006, a study on the presence of mycobacteria in healthy fish and aquariums found that the incidence of the pathogens is ‘quite high’. Samples taken from home aquaria found that 18 of 42 samples taken from 19 fish contained various forms of mycobacteria. The study also showed that mycobacteria were present in the water itself, with 75.4% of samples testing positive. Mycobacteria were also found in snails and crustaceans. Further studies of home aquaria in 2006 found mycobacteria in 201 of 325 (61.8%) samples. Ecology Morphologically, mycobacteria are pleomorphic, acid-fast, non-motile, non-sporulated, Gram-positive, non-branching rods, 0.2–0.6 µm in diameter and 1.0–10 µm long. They are slender in shape and can be beaded or barred. Over 100 species of mycobacteria are recognised. In spite of recent profusion of new mycobacterial species, recent reports document that 30% of mycobacterial isolates from water do not belong to any of the identified species. Therefore, there are a number of species yet to be scientifically described. Mycobacteria are not typically classified by staining as Gram-positive or Gram-negative, but instead are acid-fast bacteria. Mycobacteriosis is not the same disease as pulmonary tuberculosis (TB) in humans. Contagious mycobacteria that cause serious disease in humans are obligate pathogens and include the M. tuberculosis complex (the cause of pulmonary tuberculosis) and M. leprae (the cause of leprosy). Mycobacteria such as M. fortuitum, M. marinum and other species are collectively termed “atypical” or “nontuberculous mycobacteria” to distinguish them from the species that cause human tuberculosis. They are true inhabitants of the environment and can be found as saprophytes, commensals, and symbionts. Mycobacteria have the characteristics of both a specialist bacterium, adapted to persist within intracellular environments, such as free-living aquatic amoeba and vertebrate hosts, as well as of a generalist, equipped to survive under changing and extracellular environmental conditions. Atypical mycobacteria are common in all natural ecosystems, including water, soil, food, dust, and aerosols. However, some species are also pathogenic for humans or animals, causing pulmonary and cutaneous disease, lymphadenitis, and disseminated infections. Atypical mycobacterial infections are transmitted by ingestion, inhalation, and inoculation from environmental sources rather than from person to person. Atypical mycobacteria have been isolated from public water distribution systems and from samples from various other sources, including homes and hospitals. Sources include hot and cold-water taps, ice machines, heated nebulizers, showerhead sprays, etc. Such “environmental” mycobacteria can survive in water, which contains chlorine, or varies over a wide range of pH and temperature conditions. Optimum growth temperatures vary widely according to the species and range from 25°C to over 50°C. In some cases, temperatures of up to 70°C are required to inhibit the organism. They are also documented as being able to survive in temperatures below 0°C. It is speculated that the water thus serves as a natural habitat and may serve as a means of transmission. Even though environmental mycobacteria can survive outside of an animal host saprophytically, they can also be pathogenic for a wide variety of aquatic fauna and humans. Mycobacteria are very hardy, surviving in harsh environments, and are naturally resistant to many antibiotics. Mycobacteria are considered one the most resistant microorganisms. in ejected droplets, their preference to attach to surfaces, and to phagocytosis by macrophages and protozoa. High hydrophobicity leads to their concentration at air-water interfaces, where organic matter is concentrated by the same process of preferential adsorption to rising air bubbles. Adsorption to bubbles leads to concentration in droplets ejected from water to air (Aerosolisation). The high concentration of mycolic acid and the hydrophobic surface characteristics of mycobacteria are also primarily responsible for the high resistance of the group to chemical disinfection. The very high resistance of mycobacteria to ozone and chlorine based disinfectants allows the organism to persist and grow in aquatic systems. M. fortuitum and M. chelonae are more resistant to chlorine disinfection than most other species. Aquatic mycobacteria will colonise biofilms at solid-water and air-water interfaces, which seem to be an important replication site in oligotrophic habitats. Rapidly growing species are especially adept at forming biofilms. M. fortuitum can form dense biofilms within 48 hours. They produce an extracellular matrix that makes them more difficult to eradicate compared to their free-swimming counterparts. Different species vary in their susceptibility to biocides (e.g. M. marinum biofilm cells are more sensitive and M. fortuitum is more resistant to biocides than their single cell forms). Biofilms are capable of forming on all aquarium system components, incorporating the various microflora present in the water. Samples taken from substances such as plastic and rubber (sponge filters, etc.) typically have higher concentrations of mycobacteria than substances like glass. M. fortuitum and M. chelonae have been found to form biofilms on Silastic rubber sealant. Mycobacteria species are commonly found growing in activated carbon and biological filtration media. Pathogenic microorganisms released from the biofilm are capable of causing recurring exposure to disease in both fish and humans. Most pathogenic mycobacteria are slow growers, the most notable exception being the rapid-growing M. fortuitum complex. Rapidly growing species are M. abscessus, M. chelonae, M. fortuitum and M. smegmatis. Slow growing species are M. kansasii, M. simiae and M. marinum. Although they can be divided into fast or slow growing groups according to their replication in laboratory medium, the many species have a number of characteristics in common. It should be noted that rapidly growing mycobacteria still grow significantly more slowly than most bacteria. Environmental mycobacteria exhibit great variation in growth rates (2 to 48 hours doubling times). Many mycobacteria species adapt readily to growth on very simple substrates, using ammonia or amino acids as nitrogen sources and glycerol as a carbon source in the presence of mineral salts. A study in 1992 analysed 50 biofilm samples obtained from water-treatment plants, domestic water supplies and aquaria and found 90% of the samples contained mycobacteria. A similar study in (2003-2004) found mycobacteria were most frequently isolated from biofilm (43.3%) and drinking water (32.4%). The most frequently isolated species were M. avium (6.5%), M. fortuitum (3.2%) and M. flavescens (3. 2%). A high percentage of the isolates were not determined or determination was not successful (76.2%). In 2006, a study of home aquaria found mycobacteria in 201 of 325 (61.8%) samples. In this group of samples, mycobacteria were most frequently isolated from biofilm (77.1%), plants (68.0%) and sediment (65.9%). Mycobacteria were also isolated from 12.5% of fish food (pellets). Dry pelleted feed for fish is not suitable for mycobacteria and hence the percentage of mycobacteria is lower. Either external association with or outright invasion of plants is also possible. The prevailing species were M. fortuitum (13.9%), M. marinum (8.0%) and M. gordonae (4.5%). Mycobacteria are the most hydrophobic of bacteria. The presence of fatty acids, lipids and waxes in the cell wall of mycobacteria is responsible in part for the extreme hydrophobicity of the cells. The high hydrophobicity leads to adsorption to rising air bubbles in water and their enrichment A biofilm is a consortium of microbes that adhere to either abiotic or biotic surfaces. The rapid colonisation of biofilms by mycobacteria has been explained by the hydrophobicity of the mycobacterial cell. It has been proposed that mycobacteria could be the first colonisers, since they can form biofilms without the presence of other microbes and also under low-nutrient conditions. The development of biofilm is part of an overall survival strategy that permits bacteria to survive in often harsh, low-nutrient environments. By immobilising themselves, bacteria encounter a greater flux of nutrients, thus enhancing their survival prospects and regrowth potential. Not only are the biofilms sources for more cells, but biofilms also provide mycobacteria with physicochemical protection against environmental stresses such as osmotic changes, UVirradiation, dehydration and disinfecting agents. Suggesting that biofilm growth alone renders mycobacteria more resistant to antibiotics and disinfecting agents. Sharrer et al. (2005) presented a hypothesis that recirculating aquatic systems that treat with UV-irradiation provide selection pressure for bacteria that embed within particulate matter or that form bacterial aggregates, because this provides shading from some of the UV-irradiation. Even if this hypothesis is invalidated, achieving total inactivation of bacteria in recirculating aquarium water using only UV-irradiation appears to be difficult. Source of Infection Mycobacteriosis disease outbreak in aquarium fish is reported to be related to management factors. However, even the healthiest aquarium can harbour the bacteria. A variety of fish bacterial pathogens are always present in an aquarium, even if the system is maintained in optimal condition. Most of them are ubiquitous in aquatic environments and the nonexpression of their virulence could be ascribed to a good management of the system and to a good physiological status of the fish. Moreover, the presence of bacteria described as producers of inhibitory compounds, suggests that the indigenous microbiota can control pathogenic organisms in aquarium systems. Although there is no firm evidence to confirm that environmental stress can cause mycobacteriosis infection, it has been suggested that an unnatural environment, such as an aquarium, may actually promote the disease. Fish should be maintained under optimal conditions. Inappropriate aquarium management can result in abnormal stress and a reduction in the normal resistance of the host. Overcrowding, accumulation of waste and organic matter in the water and increasing water temperature (above 28°C) may all be predisposing factors. Attention to water quality and good nutrition will assist the fish in fighting these chronic infections. Poor nutritional health can greatly enhance the progression and severity, and reactivation of disease. Once present in an aquarium, infection rates can vary from 10 to 100%. The severity of the disease is influenced by a number of interrelated factors, including bacterial virulence, the kind and degree of stress exerted on the population of fish, the physiologic condition of the host, and the degree of resistance inherent within specific populations of fishes. Mycobacteriosis is thought to be acquired through the ingestion of mycobacteria present in the environment, and which usually have their origin in detritus derived from dermal lesions, faecal material, urine or exudates etc. shed by diseased animals that contain mycobacteria. The sources and modes of transmission in fish may be related to the infection of invertebrates, such as freshwater snails, daphnia and shrimp. Live feed for fish, comprising daphnia, mosquito larvae, tubifex, and oligochaetes collected from both the wild and those bred in captivity in the central region of Thailand, were found to be contaminated with M. marinum, M. fortuitum, M. chelonae and other Mycobacterium species. The entry of mycobacteria through skin and gill lesions caused by injury or parasitic infection should also be considered. After the organisms enter the body, they may cause skin lesions or spread to other organs through the circulatory or lymphatic system. It is suspected that vertical transmission (transmission from parent to offspring) may occur through egg or sperm products. A report from an Australian fish hatchery has provided the evidence that mycobacteriosis can be introduced by eggs and transmitted to the F1 generation. This observation does not confirm that ovarian transmission takes place, as the egg surface may be contaminated by peritoneal fluid containing mycobacteria. However, research in 1994 confirmed the transmission of mycobacteria in Siamese fighting fish (Betta splendens), via transovarian passage. Acid-fast bacteria were found in the ova of diseased female Siamese fighting fish, using the fluorochrome technique. Transovarian transmission has also been reported in Xiphophorus maculatus. The observation of mycobacteria in the piscine ova and tubercle granulomas in the ovary wall suggests that transovarian transmission is a definite possibility. The implementation of preventive measures in controlling chronic mycobacteriosis is particularly relevant, due to difficulties in treatment, and different fish species probably have different levels of sensitivity. Rainbowfishes seem to be particularly susceptible to mycobacteria infection and it is very common among captive populations. The rainbowfish family may differ from other fish in their immunologic response to mycobacterial organisms. Breeders should maintain separate young brood fish populations and avoid using older brood fish. Under pathology examination, mycobacteria have been found in apparently healthy rainbowfishes. Therefore, fish should be obtained from specific pathogen-free sources and quarantined when received. Knowledge of the origin and aquarium practices of your source can help you prevent potential problems. Mycobacteria are widespread in Asian fish farms and are commonly found in imported ornamental fish from that source. Some experts in the aquarium trade believe that mycobacteria infections are becoming more common in imported fish. Clearly, prevention and appropriate routine disinfection should be viewed as the primary means to control mycobacteria in aquarium facilities. The chronic nature of mycobacteriosis means that it is often too late for any remedial action to be taken once the first cases have been observed and diagnosed. The same protocol can be used in quarantine systems, at least on a periodic basis, to prevent potential concentration of mycobacteria. Although fish should be quarantined for at least 4–8 weeks before being placed in their main aquarium, most fish become clinically affected after a longer period of time. Direct lethal sampling of a quarantined population, with histopathology and culture, may be necessary to detect subclinical infections. Equipment such as nets, hoses and containers must be thoroughly surface-disinfected before use and immediately afterwards by immersion for 20 minutes in a fresh aqueous solution of a commercial iodophor compound with a pH of seven and a concentration of active iodine (as I2) of 150 ppm. At the conclusion of the disinfection process, any residual iodine can be neutralised by adding crystals of sodium thiosulphate. Control of mycobacteriosis in aquarium systems is extremely difficult once an infection has occurred. In most cases, when mycobacteria pathogens have gain entry into the aquarium setting, eradication or depopulation of infected animals followed by disinfection of the aquarium and restarting with clean stock may be the only reasonable choice available to the aquarist. Fish that have survived an epizootic disease and have recovered may be latent carriers, posing a significant risk to the entire population. It is generally believed that infected fishes are the main source and reservoir of mycobacteria in aquaria. Dead fish, which have died from mycobacteria infection, and live carrier fish, can spread these bacteria. Obviously, the practise of feeding sick rainbowfishes to your pet Saratoga or fluffy feline has its risks. Dead fish should be destroyed by burning. Under no circumstances should fish from an infected population be sold, moved or given away. Clinical Signs Early signs of mycobacteriosis may be subtle or unapparent, and fulminate clinical signs often do not develop until the disease has become widely systemic. Clinical signs of mycobacteriosis are not specific to the disease and often resemble other diseases. They can vary in occurrence and severity and infected fish may manifest few or no external signs of disease. Clinical signs can vary between fish species and the species of mycobacteria can also influence the clinical symptoms observed. Mycobacteriosis is generally a chronic, slowly progressive disease. The acute form of the disease occurs rarely. It is characterised by rapid morbidity and mortality with few clinical signs. The chronic form of the disease is most commonly seen and it may take months to years for the number of organisms to grow to readily detectable numbers. There is ample evidence that these organisms are capable of adapting to prolonged periods of dormancy in tissues, and that this dormancy is responsible for the latency of disease. Because of the slow progression of the disease, younger fish infected with mycobacteriosis show no external signs. As fish age or are stressed, the infection becomes more serious. It is difficult to specify the length of incubation (the time from infection to the appearance of the first signs of the disease). The incubation period varies greatly and depends on susceptibility, temperature, and severity of exposure. If clinical signs develop, emaciation, cachexia (wasting, loss of weight), exophthalmia (pop-eye), ascites (dropsy), skeletal deformities (curvature of the spine), haemorrhagic and dermal ulcerative lesions or loss of scales may be observed. Other signs of infection can be seen in the gills, which are paler than normal and show thickened areas on some filaments. Small lesions may be observed around the mouth and vent. Changes in cutaneous pigmentation include a fading of normal colour in aquarium fish or change in colouration. Affected fish generally exhibit lethargic behaviour, isolation, abnormal swimming behaviour, floating impassively on the surface of the water, with concurrent loss of appetite. Poor growth, panophthalmitis and retarded sexual maturation may also occur. Affected fish populations may show chronic low-level mortality, and increased susceptibility to parasitic infection. Acid-fast staining and mycobacterial culture should be used to evaluate any group of fish showing chronic, low-level mortality and spawning difficulties, regardless of whether they show external signs of the disease. Diagnosis Unfortunately, there is no non-lethal method available to identify infected individuals, especially those in early to mid stages of disease. Slow mycobacterial growth rates contribute to the late onset and chronic effects of mycobacteriosis. By the time clinical signs and low-level mortality are observed, the disease may already be entrenched in a population. Methods for detection of infected individuals have yet to be developed. The techniques for diagnosing mycobacteriosis in fish are continually evolving, but clinical signs and gross pathology may give an initial indication of infection with mycobacterial species. Most cases of mycobacteriosis are not identified or more often, are simply misdiagnosed. It is recommended that infected fish be submitted to a laboratory for identification. Diagnosis of mycobacteriosis depends on clinical and histological signs and identification of the bacterial pathogen. Aquatic mycobacteria can be detected in tissue sections using Ziehl-Neelsen staining, and characterisation is usually based on growth rate, pigmentation, optimal growth temperature and biochemistry. However, these tests are protracted, tedious and often unsuccessful. Definitive identification of the type strains, M. marinum, M. fortuitum and M. chelonae, is however, not possible using these conventional methods. Isolation of the pathogen can provide definitive diagnosis. Identification of the mycobacterial pathogen via polymerase chain reaction (PCR) is probably best for providing a reliable and timely diagnosis. Hobbyists Diagnose The most common bacterial infections in aquarium fish are caused by organisms such as Aeromonas, Pseudomonas, Mycobacterium and Flavobacterium. Aeromonas has been found to be the most common. All of them cause opportunistic skin infections often caused by injury or parasitic infection. Mortality increases significantly once bacteria enter the circulatory system. Aeromonas, Pseudomonas and Flavobacterium generally have short incubation period and rapid progression of infection. Clinical signs are generally reached within one week of the initial infection of the disease. On the other hand, mycobacteriosis is a chronic disease and it may take months to years for infected fish to show any clinical signs. Aeromonas infections can cause 100% mortality amongst fish in 21 days. The average mortality rate of Pseudomonas can be as high as 50% during the first 21 days with continued mortality for another 7-14 days. Within 36 hours of infection with Flavobacterium, fish will show areas of greyish discoloration. Once established, the infection can spread quickly and cause high mortality rates. In contrast, mycobacteriosis infected fish populations generally show low-level mortality. Therefore, if you have an infected rainbowfish with a lesion that has not changed that much for more than 21 days, then I would suggest that in all probability it will be a case of mycobacteriosis. Chronic mycobacteriosis infections manifest themselves primarily as swollen white patches or lumps on the body that turn into red or pale lesions. Fish with only skin infections may have several types of concealed lesions. Both the dermis and epidermis are eroded and the underlying musculature becomes severely necrotic. At this stage, the infection has usually become systemic and the infection on the surface of the skin may occur throughout the peritoneum and musculature. Internally the liver, kidney, and spleen may be impaired. These diseases do not hang around waiting for the average hobbyist to decide what infection the fish have. Fish diseases and identification is, at present, difficult. It requires specific laboratory sampling and testing which takes considerable time when bacterial diseases require quick application of treatment. Experienced aquarium hobbyists can and do make accurate, presumptive diagnosis's based on examination and assessment of the clinical signs, and then apply affirmative control measures. However, economics and other factors will determine the appropriateness of the selected treatment. The cost of treatment may exceed the value of the fish in the aquarium or pond. An aquarist with a large-scale breeding set-up stocked with valuable or rare and endangered fish, for example, would probably be wise to spend the money on proper diagnose. On the other hand, if the loss only involved common species, then spending a lot of money for a fish health professional and treatment may not be economically sensible. Post-Mortem In the acute form of the disease, mortality can occur without the presence of typical lesions, although this is uncommon. Mycobacteriosis is often characterised by necrosis and granulomatous inflammation in internal organs. Histologic examination exhibits a diffuse infiltration of macrophages and reticuloendothelial cells and the presence of mycobacterial organisms scattered throughout infected organs. Formation of multiple discrete granulomas is the most distinctive lesion of chronic mycobacteriosis infection. Soft granulomas are composed of centrally located caseous material in a sheath of epithelioid cells and macrophages, which are themselves surrounded by fibrous tissue. Hard granulomas are formed at an earlier stage of the disease and lack a defined epithelioid layer and caseous necrosis. In general, granulomas vary in size, often ranging from 80 to 500 µm, and usually contain necrotic cellular material and varying numbers of intracellular mycobacteria. The spleen, kidney and liver are the most commonly affected. Peritonitis and oedema may be apparent. In severe cases, visceral organs will be swollen and fused by whitish membranes around the mesenteries large caseous necrotic areas. Treatment Mycobacterial infections of aquarium fish should be considered non-treatable. Clinically infected fish or infected populations of fish should be humanely euthanised and immediately destroyed by burning. Unlike most other bacterial fish diseases, there is no cure for mycobacteriosis and it will progress despite your best efforts. The infection will continue, resulting in chronic health problems and eventually, mortality in the whole population. The application of antimicrobial compounds in an attempt to treat established cases of this infection is unadvisable, as the results are not always fully successful, and the application of such compounds could contribute to the appearance of antimicrobial-resistant strains of the bacterium. There are several reasons why systemic mycobacteriosis should not be treated. There is a lack of information on the bioavailability of most chemotherapeutic agents in fish, as are successful well-documented clinical trials. In fact, no chemotherapeutic agent is approved for the treatment of mycobacteriosis in aquarium or food fish. Efforts to eliminate infection in affected populations with prolonged use of antibiotics have not been successful as mycobacteria are mostly resistant to conventional antibiotics. Finally, mycobacteriosis has zoonotic potential. In most situations, the customary treatment for infected fish or populations is euthanasia, especially in breeding situations, and the disinfection of the aquarium before restocking. If several fish become infected in the same aquarium, it is usually assumed that the survivors are carriers and that they be treated accordingly. Following depopulation, the entire system, especially the filters and substrate, must be thoroughly disinfected with a mycobactericidal product. In addition, all equipment that has been in contact with the infected fish should be disinfected. Gloves should be worn when handling infected fish or cleaning contaminated tanks or other equipment. Hands should be washed thoroughly afterwards with 70% isopropyl alcohol and a bactericidal soap. Break down the original infected aquarium and any other tank use as a treatment or quarantine tank and disinfect them with a strong chlorine solution. Use Calcium hypochlorite 65% to disinfect any tanks, which are in the vicinity of others housing live fish. Granular chlorine does not volatilise as readily as liquid chlorine (Sodium hypochlorite). In a poorly ventilated fishroom, fumes from liquid chlorine can cause fish kills in adjacent tanks. Concentrations of 2001000 mg/L available chlorine for 60 minutes should be effective for disinfections of tanks, substrate, and submersed equipment (keep filters running during treatment). Always use chlorine with caution as repeated use and extended exposure of the silicon sealant to strong chlorine solutions will destroy or render the adhesive bond ineffective on glass aquariums with disastrous results. Chlorine will dissolve synthetic material like sponge filters, but most plastics are unaffected. Calcium hypochlorite is an oxidising agent and should not be exposed to intense heat, acids, or organic compounds because it is a fire hazard, particularly if wet. In some cases, explosion may occur. Always wear eye protection and rubber gloves when handling large quantities of chlorine. Chlorine can be neutralised by adding Sodium thiosulfate to the solution (7.5 grams of Sodium thiosulfate will neutralise the chlorine present in 5 litres of a solution of 200 mg/L). However, disinfection is not always successful due in large part to the resistance of many species of mycobacteria to common disinfectants. Mycobacteria are resistant to many commonly used bactericidal agents at standard dosage rates, including chlorine and quaternary ammonium compounds. Mycobacteria can be highly resistant to chlorine disinfection. As much as 10,000 mg/L available chlorine has been reported necessary to kill mycobacteria. Bacterial biofilm in an aquarium can harbour the organism even after aquariums and equipment are disinfected; indeed, biofilm bacteria appear to be more resistant to disinfection than free organisms. Veterinarians at the National Aquarium in Baltimore, USA recommend using chlorine to clean the tank and substrate, etc., and then spray 65-90% isopropyl alcohol onto the glass, and allow it to dry. 70% isopropyl alcohol can destroy Mycobacterium tuberculosis in 5 minutes. They recommend the alcohol as they found that chlorine does not kill all mycobacteria. They use chlorine to remove/oxidise organic material to assure the alcohol contacts all mycobacteria in/on the tank. Remove all residues of disinfectant from the aquarium before reuse. (Denise Petty DVM, pers. comm. 1998). Antibiotic Treatment Antibiotics are generally not a hundred percent effective for treating fish against mycobacteria infections. A number of therapeutic measures have been attempted with limited efficacy or feasibility. Continuous use of antibiotic drugs can also introduce drug resistant strains of the bacterium. Resistance to commonly used antibiotics is an emerging problem in the ornamental fish industry. During a 12-month screening program on fish samples collected from two German ornamental fish importers newly imported from four different Asian countries of origin (Hong-Kong, Thailand, Singapore and Sri-Lanka) were examined within two weeks of importation. Two fish species were examined from each country of origin. For each sample, 15 fish for each group were sacrificed, dissected and examined. After differentiation of the bacteria found, antibiogrammes were established on Müller-Hinton-agar. Following substances were involved in the testing procedure: Chloramphenicol, Trimethoprim-Sulphonamide, Oxytetracycline, Furazolidone, Chlortetracycline, Enrofloxacin, Flumequine, Oxolinic acid, Amoxicillin, Gentamicin, Neomycin, Colistin and Florfenicol. The inhibition test was carried out at three pH-ranges: pH 6 / 7.2 / 8. A total of 250 positive bacterial agents could be detected. Most of the findings were of facultative fish pathogenic nature. Only few specific bacteria could be identified. A total of 13 cases of mycobacteriosis were detected after Ziehl-Neelsen staining. In ten samples, Flavobacterium columnare was identified. Two cases of Aeromonas salmonicida subsp. achromogenes and of Vibrio anguillarium infection were recorded. In the case of the facultative fish pathogenic bacteria, mainly motile Aeromonads were found, most of them Aeromonas sobria. Furthermore, Pseudomonas sp. (33 cases) and Myxobacteria (30 cases) were identified. 35 other bacterial and 38 mycotic (fungal) agents were isolated. High disparities were observed between the rates of resistance of the different antibiotic substances tested. The lowest rate of resistance was found for Florfenicol (13.4%) while Oxytetracycline showed the highest rate (90.1%). In general, the resistance situation for substances used frequently in ornamental aquaculture (Tetracyclines, Furazolidone, potentiated Sulfonamides) is very unfavourable. In contrary, the rate of resistance for Florfenicol, Colistin and also for Enrofloxacin was low. Comparing the rates of resistance of the four countries of origin showed that the Thailand strains were less resistant than those from the other three countries. The differences between fish species within one country of origin were not very high. The average rates of resistance for the different countries were between 47.1% for Thailand and 63.3% for Sri-Lanka and are considered to be quite high. The causes for appearance of antibiotic resistance can be manifold. Breeding facilities of ornamental fish in Asia are rarely supervised by fish health services. On the other hand, these breeding farms and trading farms are under economic pressure to produce as much fish as possible at low cost prices. In addition, the transport by air must be carried out at low cost. In order to achieve this, the fish are transported at high stocking densities as to save “heavy” transportation water. All these factors lead to the situation that environmental conditions are not optimised. Consequently the condition of the fish is lowered which makes them more susceptible to diseases. To prevent disease outbreaks chemotherapeutants of all kind are used. These substances are applied prophylactically under uncontrolled conditions often using wrong dosages. The uncontrolled use of antibiotic substances leads to the situation that fish come in touch with different antibiotics and chemotherapeutants at an early age and bacterial resistances are built up. Furthermore, antibiotic substances (mainly tetracyclines) are used in shipping water. After transport by air, the fish undergo prophylactic treatments to prevent disease outbreaks in the importers holding facilities, although examination possibilities are much better in the importing western countries. Therefore, the very high rate of antimicrobial resistance in ornamental fish is probably mainly due to the uncontrolled application of antibiotic drugs. Appropriate application of antibiotic drugs should only be done as far as possible after carrying out antibiogrammes testing and supervision by a fish health professional. There are some reports that a combination of antimicrobials, including penicillin, streptomycin, ethambutol, cycloserine, cotrimoxazole, rifampicin, tetracyclines and kanamycin has been used and thought to be effective. Among these drugs, cotrimoxazole or rifampicin with ethambutol have been used most frequently. Treating infected Pearl Gourami (Trichogaster leeri) by applying penicillin to the skin of the fish, and treating black widows (Gymnocorymbus ternetzi) with terramycin by a continuous bath have both proven successful means of treatment. Mycobacteria infections in Siamese fighting fish were successfully controlled by the addition of kanamycin (100 mg/L) to aquarium water so as to maintain a permanent bath of the antibiotic over a 5-day period. This antibiotic has also been successfully used to control mycobacteriosis outbreaks in three-spot gouramies (Trichogaster trichopterus) using the same treatment protocol. On the other hand, it was found that feeding rifampin to striped bass (6 mg/100g of food for 60 days) was not effective as a treatment. It was later confirmed that mycobacteria were resistant to rifampin, whereas cycloserine reduced mortalities caused by the bacterium. Isoniazid and rifampin have, however, been recommended as treatments for valuable exotic marine fish. Recently, it was reported that M. marinum isolated from snakehead fish and Siamese fighting fish in Thailand were susceptible to amikacin, sarafloxacin and kanamycin. In addition, external lesions related to a mycobacteria infection were treated with a series of enrofloxacin injections and found clinical resolution of the disease signs. However, none of these studies were complete clinical trials, and almost nothing is known about the pharmacokinetics of anti-tuberculosis drugs in fish. Moreover, the question remains whether these treatments completely eliminate infection. A likely scenario is that treatments only eliminate overt clinical signs and these treated fish become asymptomatic carriers. In addition, even if treatment is attempted, it is further limited by the prospect of long-term antibiotic therapy and by the potential for developing resistant bacteria. Therefore, many veterinarians and fish health professionals recommend destruction of infected fish, which often means destroying whole ponds or tanks of fish. Fish health has been a relatively small discipline of veterinary attention in the past because of many factors, the most important of which is the perceived value of aquarium fish. As more people invest in expensive species, such as koi and various reef species, the demand to provide a higher level of care for these animals is increasing. This trend is also evident in the commercial food and bait fish industry, where aquaculture producers are expecting improved standards of care for populations of fish that are worth millions of dollars. With increasing numbers of aquarium and aquaculture operations, veterinarians will be expected to have the abilities and knowledge to diagnose and treat aquatic species and provide a standard of care commensurate with other commonly treated animal species. Ultraviolet Light Ultraviolet (UV) disinfection is a well-established technology and has been around for more than 50 years. In the late 1800, researchers first discovered the germicidal effects of sunlight, and systems based on fluorescent tube technology have been operating since the 1950’s. The UV lamps are similar to household fluorescent lamps, except that fluorescent lamps are coated with phosphorous, which converts the UV light to visible light. Advances in UV technology have resulted in more efficient lamps and more reliable equipment, and therefore, the use of UV technology has increased dramatically. There are presently several manufacturers of UV disinfection equipment with a large number of lamp configurations, types, and intensities. Research is continuing into new types of UV systems, such as pulsed output lamps. Mercury vapour lamps are the source of UV light for all systems, except for the pulsed UV system. Low-pressure mercury lamps are more efficient in converting electricity to germicidal UV light, but the total UV output is much weaker than from a medium-pressure lamp. The Low-Pressure, Low-Intensity (LPHI) mercury lamps have design features to maintain mercury pressure at an optimum level under high discharge currents. However, the purpose of using a UV steriliser is not always to exterminate all bacteria present in the water, as the energy required to achieve this would be excessive. In fact, UV equipment is used mostly in aquatic systems to maintain bacterial populations below dangerous levels. UV equipment consists of a cylindrical chamber containing one or more quartz tubes (permeable to UV), producing ultra-violet radiation. Water flowing through the chamber is exposed to UV-C radiation produced by special lamps. The best solution for aquarium systems seems to be low/mediumintensity equipment. In addition to the power of a UV lamp, it is also necessary to know the useful life span (usually 2500-10000 hours). During this period, the UV dosage of the lamp will progressively decrease until it reaches a value close to 50-60% of the original dosage, which is considered the end of its life span. Ultraviolet radiation is similar to visible light in all-physical aspects. It has a shorter wavelength just before the violet end of the visible colours. In scientific terms, UV radiation is electromagnetic radiation just like visible light, microwaves and x-rays. Electromagnetic radiation is transmitted in the form of waves that are described by their wavelength or frequency as well as their amplitude (strength or intensity). For radiation in the UV region of the spectrum, wavelengths are measured in nanometres (nm). UV light can be categorised as UV-A, UV-B, UV-C or vacuum-UV, with wavelengths ranging from about 40 to 400 nanometres. The UV-A range causes “sun tanning” in the human skin. The UV-B range causes “sun burning”. The UV-C range is absorbed by DNA and thus can cause cancer and mutations. This is also the range that is most effective in inactivating bacteria and viruses. The Vacuum-UV range is absorbed strongly by water and air and thus can only be transmitted in a vacuum. The most effective UV radiations are UV-C (200-290 nanometres) and UV-B (290-320 nanometres). UV-C light disinfects water by permanently deactivating bacteria, spores, moulds, viruses and other pathogens, thus destroying their ability to multiply and cause disease. The maximum effectiveness occurs at between 240 and 280 nm, with the most effective wavelength typically at 254 nm. Although, recent research has shown that UV radiation at wavelengths between 263 and 275 nm are the most effective for the deactivation of particular target organisms. The intensity of UV radiation is measured in the units of milliwatts per square centimetre (mW/cm2), which is energy per square centimetre received per second. In addition, it is measured in the units of milliJoules per square centimetre (mJ/cm2), which is energy received per unit area in a given time. Most scientists and engineers in the UV business now use the units “mJ/cm2” (milliJoule per square centimetre) or “J/m2” (Joule per square meter) for UV dose [1 mJ/cm2 = 10 J/m2]. The old term “mW-s/cm2” (milliwatt-second per square centimetre) is equivalent to “mJ/cm2”, since a “W-s” is the same as a “J” (Joule). [1000 microwatt = 1 milliwatt]. Overall dosages are calculated by multiplying the lamp output by the time the water is exposed to the light, with the final dosage commonly expressed as milliJoules per square centimetre (mJ/cm2), which is equivalent to milliwatt seconds per square centimetre. One milliJoule (mJ) corresponding to one milliWatt (mW) per second. If lamp output is 10 mW/cm2 and residence time of water inside the sterilisation chamber is three seconds, total UV-C dosage applied equals 30 mJ/cm2. The mechanism of kill involves the absorption of photons of UV energy by the DNA (RNA in some viruses), which fuses the DNA and prevents replication. DNA (Deoxyribonucleic acid) consists of a linear chain of nitrogen bases known as purines (adenine and guanine) and pyrimidines (thymine and cytosine). These compounds are linked along the chain by sugar-phosphate components. The DNA of most forms of life is double stranded and complimentary; the adenine in one strand is always opposite thymine in the other, and linked by a hydrogen bond, and guanine is always paired with cytosine by a hydrogen bond. The purine and pyrimidine combinations are called base pairs. When UV light of a germicidal wavelength is absorbed by the pyrimidine bases (usually thymine) the hydrogen bond is ruptured. The dimer that is formed links the two bases together, and this disruption in the DNA chain means that when the cell undergoes mitosis (cell division) the DNA is not able to replicate. Microorganisms may, however, become viable again in the presence of visible light (photoreactivation) if UV disinfection is inadequate. It has been proven that with UV irradiation there is always a non-negligible degree of reactivation. Under laboratory conditions, a UV dose of 2.7 mJ/cm2 results in a 5- LOG10 reduction in Vibrio salmonicida, Vibrio anguillarum and Yersinia ruckeri, and a 3-LOG10 reduction in infectious pancreatic necrosis virus at a UV dose of 122 mJ/cm2. However, actual fish culture conditions may require longer exposure or higher dose, because factors such as total suspended solids can affect UV transmittance and bacteria may be protected by an envelop of particulate matter. For example, in a recirculating aquaculture system it was observed that UV intensity greater than 1800 mJ/cm2 was required to achieve a not quite 2LOG10 reduction in heterotrophic bacteria. It was also found that UV-irradiation within a recirculating aquaculture system produced inconsistent inactivation or no inactivation of heterotrophic bacteria, Aeromonas (hydrophila and punctata), and Flavobacterium columnare. Sharrer et al. (2005) presented a hypothesis that recirculating aquatic systems that treat with UV-irradiation provide selection pressure for bacteria that embed within particulate matter or that form bacterial aggregates, because this provides shading from some of the UV-irradiation. Even if this hypothesis is invalidated, achieving total inactivation of bacteria in recirculating aquarium water using only UVirradiation appears to be difficult. Log reduction is used in reference to the physical-chemical treatment of water to remove, kill, or inactivate microorganisms such as bacteria. During the UV process, the rate of destruction is logarithmic, as is their rate of growth. Thus, bacteria subjected to UV-irradiation are killed at a rate that is proportional to the number of organisms present. The process is dependent on both the exposure and the time required to accomplish the desired rate of destruction. “Log” stands for logarithm, which is the exponent of 10. For example, log2 represents 102, or 10 x 10 or 100. Log reduction stands for a 10-fold or one decimal or 90% reduction in numbers of recoverable bacteria. Another way to look at it is: 1-Log reduction would reduce the number of bacteria by 90%. This means, for example, that 100 bacteria would be reduced to 10 or 10 reduced to 1. 5-LOG refers to 10 to the 5th power or reduction in the number of microorganisms by 100,000-fold. For example, if a water sample contained 100,000 microorganisms, a 5-Log reduction would reduce the number of microorganisms to one. It must be remembered that the efficiency of sterilisation using UV-irradiation is strongly conditioned by the way in which this radiation is transmitted in the water (transmittance). The transmittance can be drastically reduced by the presence of suspended solids. For this reason, prefiltration is a must on all UV applications to be effective. High colour, turbidity, dissolved and suspended solids, presence of metals and organic matter reduce the amount of UV radiation reaching microorganisms and necessitate higher doses of applied radiation for effective disinfection. Units require regular cleaning and maintenance to remain effective. The UV dosage is also influenced by other factors such as the variation of the water flow inside the radiation chamber or the temperature of the water to be treated. Ideally, a UV disinfection system should have a uniform flow with enough axial motion (radial mixing) to maximise exposure to UVirradiation. The path that an organism takes in the reactor determines the amount of UV-irradiation it will be exposed to before inactivation. A reactor must be designed to eliminate short-circuiting and/or dead zones, which can result in inefficient use of power and reduced contact time. Total dissolved solids should not exceed approximately 500 mg/L. There are many factors that make up this equation such as the particular make-up of the dissolved solids and how fast they absorb the available UV energy. Calcium and magnesium, in high amounts, have a tendency to build up on the quartz sleeve, again impeding the UV energy from penetrating the water. Suspended solids need to be reduced to a maximum of 5 microns in size. Larger solids have the potential of harbouring or encompassing the microorganisms and preventing the necessary UV exposure. Turbidity is the inability of light to travel through water and should be less than one nephelometric turbidity unit (NTU). Over one NTU can shield microorganisms from the UV energy, making the process ineffective. An additional factor affecting UV is temperature. UV levels fluctuate with temperature levels. If the temperature of the water exceeds a certain threshold value as specified by the manufacturer, UV lamps can break. Hence, the water temperature should always be monitored. If the temperature exceeds the limit, the UV reactor should be shut down. Calcium carbonate (hardness) is one of the rare compounds with decreasing solubility at higher temperature. Hence, at elevated temperatures calcium carbonate may be precipitated which may reduce the UV transmittance. Photoreactivation In certain cases, bacteria and other microorganisms are capable of repairing their DNA following damage by ultraviolet radiation. Known as ‘photoreactivation’, it is a natural defense mechanism that has evolved over millions of years. While some microorganisms need visible light in order to repair their DNA, others can repair their DNA without light (‘dark repair’). This self-repair ability poses obvious problems when UV disinfection technology is used to treat aquarium water. Photoreactivation has been known since 1949 when Albert Kelner, while doing research into the lethal effects of radiation, noted that the damage induced in bacteria by UV exposure was altered by exposure to visible light. Since this initial discovery, photoreactivation has also been observed in fungi, algae, and higher plants and animals, including humans. Although photoreactivation occurs in many species, the photoreactivating ability among species varies. Previous studies on bacteria suspended in liquid, or on agar plate surfaces, have been conducted showing that photoreactivation occurs with mycobacteria, and that the length of exposure to photoreactivating light is an important factor affecting the extent of photoreactivation. Photoreactivation in liquid suspensions was highly correlated with the dose of UV used, and that damaged cells were continuously repaired during exposure to common fluorescent light. David et al. (1971) indicated that to inactivate 90% of M. tuberculosis and M. marinum cells, 7 and 22 sec of irradiation at 11,000 to 197,000 ergs was required, respectively. However, both species were shown to be capable of photoreactivation. 56% of the M. tuberculosis cells and 40% of the M. marinum cells were photoreactivated when they were irradiated with visible light for 1 hour with a 116 watt; 120-volt filament bulb located at a distance of 40 cm. M. smegmatis required 30 sec of irradiation at 243,000 ergs of energy. In the same study, M. fortuitum required 22 sec @ 178,200 ergs [1 microwatt (µW) = 10 erg/second (erg/ s)]. McCarthy and Schaeffer (1974) reported a 1.7-log increase in M. avium counts following exposure to 9 mWsec/cm2 and a 3-hour photoreactivation. They also noted that the 14-day post-exposure incubation period for M. avium provided ample opportunity for significant dark repair of UV lesions. It has been reported that M. smegmatis possessed photoreactivating enzyme, which is able to repair UV damage not only in phage DNA but in its own genome as well. Postirradiation exposure of M. smegmatis to visible light increased the numbers of surviving colonies. M. smegmatis was the most resistant to inactivation and demonstrated the greatest degree of photoreactivation after exposure to visible light for one minute. M. marinum was the least resistant to inactivation and demonstrated a lesser degree of photoreactivation. UV-irradiation is lethal to mycobacteria, but each species has its own particular tolerance. Varying intensities of UVirradiation are required for removal of different microorganisms, with the recommended dose being of the order of 35,000 – 1,000,000 µWs/cm2. However, the suggested dose does not take in to account the photoreactivating ability of the different mycobacteria species. It has become increasingly clear that a great variety of factors determines the loss or survival of biological activity following UV-irradiation. It should be pointed out that ultraviolet irradiation only kills cells, including mycobacteria, if irradiation occurs in the dark. In addition, UV-light may only cause partial damage to bacterial cells and may enhance mutation (e.g. UV-resistant strains). Ozone Application The use of ozone has also gained popularity for the enhancement of water quality and bactericidal disinfection. While UV-disinfection targets on DNA-destruction, ozone reacts with the surfaces of microbial membranes. However, the efficacy of ozone for mycobacterial fish pathogens has not been well established. Preliminary studies have shown that M. fortuitum is resistant to ozonation. It was concluded that ozone residual is the controlling factor in the inactivation of M. fortuitum. It has also been reported that ozone can favour the growth of mycobacteria and other heterotrophic bacteria by increasing the assimilable organic compounds and also by increasing the amount of microbially available phosphorus. When using ozone, continuous monitoring is required to maintain the desired levels for disinfection and to protect fish from overdose. Typical dosage levels for ozone disinfection within aquarium systems are between 0.01 - 0.10 mg/L water flow with retention time for treatment between 0.5 and 20 minutes. In contrast to UV, ozone is generally added before the mechanical and biological filter elements as it decomposes dissolved and solid organic material and thus improves the performance of mechanical filtration and reduces the load on biological filters. It could also potentially improve the disinfection efficiency of subsequent UV-irradiation. Combining ozone dosages of only 0.1-0.2 min mg/L with a UV-irradiation dosage of >50 mJ/cm2 provides an advanced oxidation process that could consistently produce a posttreatment water nearly free from free-swimming bacteria and bacterial colonies. Ozone diffuses in all directions, even inside biofilms where it can reach hidden microorganisms. It has been demonstrated that microorganisms seek shelter from UV rays inside the biofilm. However, UV-irradiation is also effective at dissolved ozone destruction. In a recirculating system used for salmonid production, a UV-irradiation dose of 49 ± 1 mW s/cm2 removed 100% of the dissolved ozone when the inlet ozone concentration was <0.10 mg/L. Therefore, UV-irradiation could be used to prevent dissolved ozone residuals from reaching the fish in recirculating systems that use ozonation for disinfection, i.e., when a dissolved ozone residual is maintained at the outlet of ozone disinfection chambers. Summary Ultraviolet and ozone sterilising units can help reduce overall pathogen numbers in an aquarium system, but they will not prevent the spread of pathogens within the system. The variable nature of mycobacteria populations alter the efficacy and predictability of the disinfection process. The innate resistance of mycobacteria and the presence of organic matter, turbidity, excessive numbers of organisms, exposure times or dilution use concentrations, pH, temperature, and water hardness may all affect treatment. Personal Experience Case # 1: In early 1997, I transferred some 4-year-old Goyder River rainbowfishes from their present aquarium to a larger 600-litre aquarium. I had raised 30 individuals and decided to split them in half. The 600-litre aquarium contained a mixture of fully-grown rainbowfishes. Most of them were more than 4 year old with some specimens as old as 9 years. Over the ensuing weeks the Goyder's, one by one, stopped feeding and started to ‘hang’ just below the surface of the water. Apart from laboured breathing, and looking as though they just had a very large meal, no other symptoms were apparent - death followed soon after. When only 3 individuals were left from the original 15 that had been transferred, I decided that I needed some confirmation of their disorder. I took the remaining three fish to veterinarian, Dr. Stephen Pyecroft BVSc of Aquatic Diagnostic Services International Pty Ltd for examination. Stephen's diagnose showed that “Disseminated caseating pyogranulomatous inflammation possibly due to Mycobacterium infection” and “Hepatic Lipidosis” (fatty liver disease). He commented, “The severity and chronicity of the pyogranulomatous inflammation suggests this is the primary disease process. Special stains have shown the presence of acid-fast bacilli consistent with Mycobacterium spp. These organisms were found in the macrophages in the liver and kidney. The hepatic lipidosis is quite severe and could well be associated with hepatoencephalopathy although histological evidence of this was not detected in the brain sections examined. The lipidosis was found in all those examined.” “The fish I concentrated on for the histopathological examination definitely showed the greatest degree of pathology and because of the diagnose of mycobacteriosis we must suspect that the total clinical picture observed is due to this problem. There is no ignoring the fact that these fish on the whole were over weight and that the hepatic lipidosis present would have eventually caused their demise had the TB not caused their final problems.” “The picture is still not that clear and I personally believe that the nutritional imbalance leading to the lipidosis is the major management factor that will need to be addressed. However the fact that a mycobacterial infection is present must, in these fish, be accepted as the primary cause of disease.” What that means in layman terms is that the fish were overweight and infected with mycobacteria. My conclusion from all this was that the 600-litre aquarium was the culprit and knew somewhere down the track that I would have to destroy all the fish and sterilise the tank with chlorine. This belief was confirmed as I continued to have disease outbreaks in this aquarium with some fish displaying similar symptoms while others also developed external lesions. This aquarium was treated with a strong chlorine treatment and all fish and plants destroyed. It is interesting to note that the remaining 15 fish, two years later and 6-years old, in the original aquarium were still doing well, albeit on a somewhat reduced and modified diet, and showed no external signs of the disease whatever. Case # 2: About 8 months after the above episode I presented Stephen Pyecroft with six young (1-year-old) specimens of Melanotaenia oktediensis. All six specimens had what I refer to as “Blackhead Disease” in varying degrees. This disease exhibits itself as a black darkening of one side of the head only. Two of specimens also had small skin eruptions on one side of the body and one also showed the darkening skin colouration along one side of the posterior portion of its body. The most severely affected fish would swim with their head up and tail down and showed an increased respiratory rate. This disease (blackhead) seems to be common among rainbowfishes as I have seen it often and many other hobbyists have spoken to me about this problem. It also seems to be particularly prevalent among Goyder River rainbowfish. Stephen found that most of the fish had enlarged kidneys, which had a granulated pale colour and protruded beyond their normal position. Granulomas were also present in the spleens and around abdominal organs. Acid fast (ZiehlNielsen) stains were preformed on impression smears from most of the affected organs and the presence of acid fast bacteria was confirmed. The Diagnose: Disseminated granulomous inflammation - nephritis, hepatitis, and peritonitis. Stephen comments were “As we have discussed before, the dark areas on the skin are most likely due to a malfunctioning in either the pigment cells or the nerves that control the pigment cells in that area of the skin. The findings of a generalised infection with Mycobacterium species would be suggestive that the localisation of the dark pigmentation is due to the formation of local abscesses, which are then causing the expression of the major clinical sign. Most of these cases of “blackhead syndrome” in rainbowfish that I have investigated have had a primary infection with mycobacteria. There may be other primary causes of this distinct clinical sign but in these fish it was piscine TB.” Zoonosis Mycobacteriosis is different from most other fish diseases that you are likely to experience in your aquarium. This is because mycobacteria are capable of causing disease in humans. Human infections caused by pathogens transmitted from fish or the aquatic environment, are quite common. Reports on mycobacterial infection of the skin have been appearing with increasing frequency in medical literature. In the past, human outbreaks of mycobacterial infections were sporadic and most commonly associated with contaminated swimming pool water. For this reason, the skin infection was termed swimming pool granuloma. Chlorination practices used today have greatly minimised the frequency of outbreaks from these sources (However, the apparent loss of M. scrofulaceum from the environment and its replacement by M. avium is thought to have resulted from the widespread chlorination of drinking water). Since then, several authors have noted the association of the skin infection with aquariums and today it is generally referred to as “fishkeeper’s disease”. Despite this innocuous nickname, infections often warrant long-term medical treatment and sometimes, even surgery. Mycobacteria infection of aquarium hobbyists typically occurs when mycobacteria gains access through skin abrasions and generally produces superficial and self-limiting lesions involving the cooler parts of the body such as hands, forearms, elbows and knees, following direct contact with an infected fish, or contaminated aquatic environments. Clinical observations typically involve painful or painless reddish nodules at the site of infection, usually adjacent to a cut or scrape. However in several instances lesions developed in an ascending proximal fashion strongly suggesting sporotrichosis. Sporotrichoid skin lesions have a characteristic lymphangitic spread with nodules that ascend proximally along lymphatic vessels. Aquatic mycobacteria can also occasionally spread to the internal body systems of humans and have been isolated from pulmonary lesions, and from synovial fluid and muscle. Allergic dermatopathies have also been reported on the skin of aquarists handling water in which affected fish have been reared. The incubation period of atypical mycobacterial infection in humans can range from one week to two months, but may take up to nine months to develop disease. M. marinum infections on the skin appear as brown-red papules or as granulomatous nodules with central ulceration, whereas the cutaneous infection with M. fortuitum or M. chelonae may be presented as cellulitis, abscess, nodules, sporotrichoid-like lesion, sinuses and ulcers with serosanguineous or purulent discharge. M. chelonae and M. fortuitum are two wellrecognised human pathogens. They are principally responsible for post-infection abscesses, wound infections and corneal ulcers. With M. chelonae, inflammation relapses are very frequent and therefore surgical intervention is often necessary. Anyone who suspects they may have been exposed to mycobacteriosis from handling infected fish should contact their physician and inform them of the nature of the exposure. Diagnosis and treatment may be difficult, especially in view of emerging antibiotic resistance in fish pathogens. The significance of fish mycobacteriosis as zoonosis is evident from case reports published in scientific papers. Ninety-nine publications dealing with the infection of 652 cases of humans with M. marinum appeared between 1966 and 1996. Of those infections, 76% were associated with the aquarium environment. M. fortuitum was first detected in human skin lesions in 1938. One of the first infections of an aquarium keeper due to M. marinum was reported in 1958, and two more cases turned up in California in 1962. These were a 37-year-old woman and her 18-year-old son, who ran a pet shop. Both of them cut their fingers while cleaning a particular freshwater fish tank. Some weeks later, they developed a number of nodules near the injury - small, slightly tender swellings that periodically enlarged and discharged pus. M. marinum was isolated both from the nodules and from the fish tank. In 1970, a similar case was reported in Sweden, which identified daphnia as the possible source of infection. M. marinum was not only found in the patient's skin lesions, but also in snails and fish in the patient's aquarium, and in mud in a pond from which daphnia had been collected and fed to the fish. Also in 1970, three more cases of M. marinum infection were reported in California, all of aquarists who cut their hands just before or during work on a tropical aquarium. The first cases in the Southern Hemisphere were reported from Auckland, New Zealand, in 1971. One was a tropical fish keeper at the Auckland Zoo, another a pet store owner, and a third a part time assistant in a pet store. M. marinum was isolated from all three cases. Tanks at the zoo and the pet store, and the elephant pond at the zoo, from which the keeper collected daphnia, were checked for the presence of M. marinum. It was found in five tanks at the zoo, and in dead fish but not at the other sites. In Australia, several cases of “fishkeeper's disease” have been reported. In Queensland alone, 29 culture-proven cases of mycobacteriosis infections were recorded by the Tuberculosis Reference Laboratory between 1971-1990, two of which resulted in amputation. However, “fish keeper's disease” is not a focal infection of the skin. A case of mycobacteria infection contacted from mouth syphoning water from a fish tank has been reported. It concerned an individual who experienced a throat infection that would not get better, and was eventually diagnosed as fishtank granuloma (Practical Fish Keeping, Jan. 1998). So next time you do a water change and take a big suck on the end of the syphon hose - just think of this article. Mycobacterium species are particularly significant among infections transmissible from fish to humans. These species included M. avium complex, M. fortuitum, M. gordonae, M. marinum, M. scrofulaceum, M. terrae, M. ulcerans, and M. xenopi. Today the list continues to grow, with the possible addition of M. chelonae, M. immunogenum, M. abscessus, M. kansasii, M. szulgai, M. simiae and M. palstre. There has also been an increase in the number of potentially pathogenic mycobacterial species whose transmission route is associated with water. Literature Abalain-Colloc, M. L., D. Guillerm, M. Saläun, S. Gouriou, V. Vincent, and B. Picard (2003) Mycobacterium szulgai isolated from a patient, a tropical fish and aquarium water. European Journal of Clinical Microbiology and Infectious Diseases 22: 768-769. Adams, R.M., Remington, J. S., Steinberg, J. and Seibert, J. (1970) Tropical fish aquariums. A source of Mycobacterium marinum infections resembling sporotrichosis. Journal of the American Medical Association 211(3): 457-461. Aronson J.D. (1926). Spontaneous tuberculosis in salt fish. Journal of Infectious Diseases 39: 315-320. Ashburner, L.D. (1977) Mycobacteriosis in hatchery– confined chinook salmon (Oncorhynchus tschawytscha) in Australia. Journal of Fish Biology 10 (6): 523-528. Australian Drinking Water Guidelines (2004) National Health and Medical Research Council and the Natural Resource Management Ministerial Council. Bataillon, Dubard, and Terre (1897) Un nouveau type de tuberculose. Comptes Rendus Hebdomadaires des Séances et Mémoires de la Société de Biologie. Tome QuatriemeDixieme, Serie Quarante-Neuvieme de la Collection. Pages 446-449. Belič, M., Miljkovič, J. and Marko, P. B. (2006) Sporotrichoid presentation of Mycobacterium marinum infection of the upper extremity. A case report. Acta Dermatoven APA 15(3): 135-139. Beran, V., Matlova, L., Dvorska, L., Svastova, P. and Pavlik, I. (2006) Distribution of mycobacteria in clinically healthy ornamental fish and their aquarium environment. Journal of Fish Disease 29(7): 383-393. Beran, V., Matlova, L., Horvathova, A., Bartos, M., Moravkova, M. and Pavlik, I. (2006) Mycobacteria in the animal's environment in the Czech Republic. Veterinarski Arhiv, Journal of the Faculty of Veterinary Medicine, University of Zagreb: 76 (Supplement): S33-S39. Bernstad, S. (1974) Mycobacterium borstelense isolated from aquarium fishes with tuberculosis lesions. Scandinavian Journal of Infectious Diseases 6: 241-246. Bhatty, M. A., D. P. J. Turner, and S. T. Chamberlain (2000) Mycobacterium marinum hand infection: case reports and review of literature. British Journal of Plastic Surgery 53: 161–165. Center for Food Security and Public Health (2006) Mycobacteriosis. College of Veterinary Medicine Iowa State University. Chinabut, S., Y. Kanayati, and T. 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