Download A Fishkeeper`s Guide to Mycobacteriosis

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.
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