Ichthyophthiriasis
Ichthyophthiriasis is caused by the sub-epidermal ciliate protozoan Ichthyophthirius multifiliis. It is regarded as one of the most pathogenic diseases of freshwater aquarium fishes. Ichthyophthirius multifiliis invades the skin and gills of freshwater fish and is commonly referred to as Ich or whitespot. Most species of freshwater fish are susceptible to infection by this virulent parasite, although some may be more so than others. Infections have been reported in both cold and warm water species.
Visible spots on the fish (the clinical sign for which the disease is named) are individual parasites known as trophonts. However, a single white spot does not necessarily represent a single trophont, since aggregations of trophonts can occur in one large white spot as a result of multiple entries at a single site. The mature parasite is very large, ranging in size from 40 to 800 µm in diameter, and becomes easily visible owing to the opacity of the cytoplasm in the fish’s skin and the formation of a somatic cyst around the parasite. Immature forms of Ichthyophthirius multifiliis are smaller and more translucent in appearance.
In aquarium systems, ichthyophthiriasis outbreaks are more common due to the confinement of fish under “unnatural” condition and the exponential increase in parasite numbers. Fish may maintain low, subclinical infection, while encysted tomonts may persist in the aquarium. Transition from nonclinical enzootic to epizootic clinical infection is often stress -related, prompted by adverse aquarium conditions such as overcrowding, improper feeding and poor water quality.
Chronic ichthyophthiriasis infection will cause serious damage to the skin, fin and gills. Corneal infection will impair vision. The infective stage invades the integumentary epithelium and becomes established in the basal layer of the epithelium just above the basal membrane. Cellular damage in low to moderate infections remains restricted to the infected site. In addition to the damage caused to epithelial cells by the feeding and expanding parasites, in heavy infections mass exodus of parasites from the epithelial layer, having completed their scheduled growth, causes its erosion and detachment from the basal membrane. In some infections, parasites cause widespread damage of the inner layer of the epithelium. Prolonged infection also induces epithelial proliferation and haemorrhagic inflammation, causing the integument to become severely disintegrated. Haematological and clinical data from heavily infected fish reveal evident physiological dysfunction resulting apparently from both direct pathological damage induced by the parasite and as a byproduct of the stress response. If the disease is left untreated, outbreaks can result in 100% mortality. Even very young fry (larvae) can be infected causing substantial mortalities. Severe damage of the skin epithelium may also lead to secondary bacterial or fungal infections.
Life Cycle The life cycle of Ichthyophthirius multifiliis is a direct one that requires no intermediate host and has both a fishassociated stage and a free-swimming stage. Ichthyophthirius multifiliis has a well documented life cycle and involves the invasion of the skin and gills by freeswimming, 40 µm long, pear-shaped theronts that burrow several cell layers deep into epithelial tissue of the skin and gills to feed on mucus and tissue. Infection is followed by a growth phase in which the theront matures into a trophont (parasitic feeding stage) that grows inside the host epithelium. Trophonts feed on epithelial cells and grow into 500–800 µm diameter cells, causing extensive damage to skin and gills, which in severe infections results in mortality. By maturity, which is reached in 2 days at 25-28°C (3-4 days at 21-24°C), the parasite evacuates the host tissue and settles within 2-6 hours on a substrate (gravel, plants, aquarium glass, etc.) to form a cyst-encapsulated tomont (reproductive stage). Parasites evicted from the tissue before the scheduled time for their spontaneous departure, fail to develop into tomonts and eventually die. Within the cyst, tomonts undergo division to form tomites to be released as theronts (infective, free-swimming stage), which after release will seek a suitable host. The division of tomonts into theronts, in temperatures of 25-28°C, is completed within 15-20 hours.
After being released, the free-swimming theronts can infect a new host or re-infect the same host, thus compromising its health status. With subsequent rounds of infection the number of parasites continues to increase, and each wave of re-infection becomes more deadly than the last. By the second or third re-infection the fish population is usually overwhelmed and fish begin to die. Although the disease is usually fatal, fish that survive mild infections develop immunity to subsequent infections.
Invasion of theronts (20-50 µm long) into the host integument is facilitated by the excretion of a sticky substance. In the absence of a suitable host, theronts will lose their infective potential within 24 hours at 24-28°C. Higher temperatures hasten trophont maturation and tomont division, but at lower temperatures, slower development allows the growth of larger trophonts (0.8-1.0 mm in 5-10°C vs. 0.5-0.7 mm in 20-24°C), yielding tomonts with higher numbers of theronts. In lower temperatures the survival of the theronts is prolonged, thus, allowing more time to locate a host. Low temperatures do not interrupt propagation; a full cycle is completed at 20°C in 3-5 days, at 15°C in 7-14 days and at 10°C in 21-35 days. Data on the effect of other environmental parameters is less conclusive, although it has been suggested that dissolved oxygen levels below 1 mg/L affect parasite reproduction.
Some researchers have reported that Ichthyophthirius multifiliis can multiply directly by dividing underneath the fishes’ top skin layer, bypassing the usual three-stage life cycle. When this occurs, one can see multiple cells of similar size lined up or in clumps underneath the thin layer of host cells. The disease is not treatable when it becomes established to this degree and reproduces in this manner, because it does not need to leave the host where it would ordinarily be vulnerable to treatment.
Diagnosis Identification of the parasite is necessary to conclude that the fish has an ichthyophthiriasis infection. Look closely at the clear parts of the fins for cloudiness and/or white pinhead size spots. Rainbowfishes infected with this parasite typically develop small white spots on the body, fins and gills. If the infection is restricted to the gills, no white spots will be seen. The gills will appear swollen and be covered with thick mucus. In some cases, the small white spots coalesce together forming larger white spots. Hemorrhagic patches appear on the bases of the fins, body and mouth. Fin rot sometimes appears on some affected fish. The presence of white spots along with associated haemorrhages and behavioural changes is considered the ultimate indication for the presence of an infection with ichthyophthiriasis.
Early warning signs include loss of appetite and listlessness. The fish will scratch or flash on the sides of the aquarium or other objects. As the disease progresses the fish may congregate near the filter outlet or appear to gasp for air. The fish may produce a thick mucous over the skin in an attempt to protect itself. Occasionally the mucus will come off removing many of the Ichthyophthirius multifiliis cells and leave the skin dry. When this occurs, the outer defence against infectious invaders is removed and essential salts and body fluids are lost. Within six days, if left untreated, the fish will stopped feeding, appear lethargic and swim near the water surface. Death is likely after about 10 days. Skin scrapping under a microscope will revealed the presence of different developmental stages of Ichthyophthirius multifiliis. The most predominant stage will be the mature trophont with the C-shaped macronucleus.
Treatment Treatments have typically focused on killing the infective theront or the detached trophont to stop the reproductive cycle and prevent spread of the disease. There are currently no chemicals that will kill Ichthyophthirius multifiliis cells while they reside in the fishes skin or gills; they can only be kill when they are in the water, and therefore all current therapies require a cyclical retreatment program, for which purpose malachite green together with formalin has for many years been the most effective composition.
The first step in the successful treatment of ichthyophthiriasis disease in aquarium fishes is early recognition and proper diagnosis. The organism can only survive if live fish are present for completion of its life cycle. If only one parasite is seen, the entire system should be treated immediately. Ichthyophthirius multifiliis is an obligate parasite and capable of causing massive mortality within a short time, and in severe cases, control may be impossible. Because both trophont and tomont stages are resistant to practically all externally applied antiparasitic chemicals, a single treatment is not sufficient to treat the parasite. Infection can be effectively controlled only by destruction or elimination of the free dividing tomonts or the tomites (theronts) they release. Repeating the selected treatment every other day at water temperatures from 20-25°C will disrupt the life cycle and control the outbreak.
Daily vacuum-cleaning of the aquarium is also beneficial, as the encysted forms are physically removed from the environment. The use of one of the gravel-cleaning devices is recommended. These devices are very effective at removing uneaten feed and faeces, resulting in improved water quality and other environmental conditions.
There are two main drugs which have been found to be highly effective for treating Ichthyophthirius multifiliis in freshwater systems: malachite green and formalin. Formalin is a generic term, which describes a solution of 37 -50% formaldehyde gas dissolved in water. These chemicals can be used separately, but are particularly effective when used together, because they exert a synergistic effect; that is, together they give a greater effect than the sum of their separate individual capabilities. The combination of both chemicals is less toxic than either drug used separately.
Malachite green and formalin are toxic poisons. Malachite green acts as a respiratory poison, damaging the cell's ability to produce energy to drive vital metabolic processes. Formalin is a powerful disinfectant used to kill microorganisms or as a preservative for biological specimens. It works by reacting with cell proteins and nucleic acids - altering both structure and function. Formalin is more toxic in soft, acidic water and also removes oxygen from water. Each 5 mg of formalin removes 1 mg of dissolved oxygen from the water.
The malachite green/formalin (37%) combination is used at a concentration of 0.15 ppm and 25 ppm respectively for 24 hours. They exert a mild anti-bacterial effect but in most circumstances will not destroy biological filtration bacteria, although they may have a slight effect for a short period.
As you might expect both of these chemicals are affected by variations in water chemistry. Both can be deactivated by high dissolved and particulate organics such as fish waste, detritus and algae. In water that is high in dissolved organic wastes the chemicals will oxidise the organic wastes rather than attacking the parasites. This has the effect of lowering the effectiveness of the chemical dosage. It is a good idea to do a 50-75% waterchange and a thorough cleaning of the aquarium to reduce the level of organic wastes before starting the treatment. This includes the removal of particulate matter (faeces, uneaten food, detritus, etc.); the removal of algae from tank walls and the removal of particulate matter from the filter (change filter wool or wash sponge etc.).
Treat the fish every other day for a total of three to five treatments. Change 50-75% of the water in between the chemical treatments. In simple terms, treat on day 1, waterchange on day 2, treat again on day 3, waterchange on day 4 etc., etc. If the fish is heavily parasitised, you may not see any remission of the disease until after the third treatment. Treatment effectiveness should be evaluated after the third treatment to decide whether to continue with the treatment schedule. If you use the correct method and dose of malachite/formalin and the fish do not show some signs of improvement within 3 treatments, you may have misdiagnosed the problem.
Chemotherapy is ineffective when the parasite penetrates into the fish’s skin or gills; however, treatment of free-living theronts and recently detached tomonts is known to be effective. The chemicals must be at full strength when the theronts are free-swimming in the water. This is why it is important to observe and be aware of the cycle of appearance and disappearance of the spots on the fishes. The treatments (including the water changes) should still be done when the spots have disappeared or decreased in number.
It is often suggested that the temperature of the aquarium water be raised. This is because the free-swimming, infective stage theronts are heat sensitive and raising the temperature several degrees above the normal aquarium temperature tends to kill them. In addition, increased temperatures enhance the fishes’ immune responses. The treatment consists in increasing the temperature of water with infected fish to 30-32°C and maintaining it there for ten days. After treatment, the water is allowed to gradually cool to the original temperature.
However, given that temperature stress might have been a contributing factor in the start of the disease it is not logical to change the temperature again. Also, increasing the temperature, as many aquarists do, might actually increases the stress on the diseased fishes. As the water temperature increases the dissolved oxygen content is reduced proportionately. For instance, increasing the temperature from 20ºC to 30ºC decreases the dissolved oxygen level by more than 17%. Additionally, the metabolic rate of the fishes also increases as the temperature goes up. This causes the fishes to consume more oxygen at a time when the increased temperature causes a decrease in the oxygen concentration.
Other Treatments Several alternative medications for treatment of ichthyophthiriasis have been developed, but there has been some debate as to how effective they are compared to the widely used malachite green and formalin combination. For many years, malachite green has been used (often in combination with formalin) to treat fish infected by Ichthyophthirius multifiliis. In recent years, however, there have been strong moves against malachite green application, especially with respect to its use in food fishes. This is because the chemical is believed to have potential teratogenic, mutagenic or carcinogenic attributes. However, a recent study (Rintamäki-Kinnunen et al. 2005) has shown that most of the new treatments are not as effective as the malachite green and formalin combination.
The study reported that with the exception of one treatment, all were less reliable than the malachite green and formalin products they are supposed to replace. The study tested the performance of formalin, potassium permanganate (KMnO4), chloramine-T, hydrogen peroxide and two new chemicals called Per Aqua® and Desirox®. The latter two products, which are mixtures of acetic acid, peracetic acid and hydrogen peroxide, were also tested on their own, and in conjunction with formalin. The results showed that all of the chemicals were able to successfully lower the parasite burden so that the mortality rate dropped within a month of the fish picking up an infection. However, with the exception of one product, none were as good as the malachite green - formalin mixture. Large differences in parasite burden and mortality occurred among the replicates in all except the Desirox-formalin tests, which means that they were not as reliable as the malachite green – formalin combination. It was also evident that the chemicals and their concentrations must be planned carefully to suit the conditions of each situation.
Peracetic acid containing products have been studied for control of Ichthyophthirius multifiliis and other pathogens for several years. In one study Straus & Meinelt (2009) using a 4.5% peracetic acid solution on theronts isolated from Notemigonus crysoleucas and exposed to 0.225 mg/L peracetic acid resulted in 70% mortality in 2 hours, whereas theronts isolated from Xiphophorus hellerii and exposed to 0.225 mg/L peracetic acid had 95% mortality by 4 hours. With a 40% peracetic acid solution, the highest mortality of the theronts isolated from Notemigonus crysoleucas exposed to 0.40 mg/L was 80% by 1 hour, whereas theronts derived from Xiphophorus hellerii exposed to 0.40 mg/L resulted in 95% mortality by 3 hours. Differences in peracetic acid toxicity can be attributed to different strains of Ichthyophthirius multifiliis.
Peracetic acid is relatively toxic to fish, which leaves a rather small treatment margin. Meinelt et al., (2007) exposed Sander lucioperca to increasing peracetic acid concentrations (0, 0.5, 0.9, 1.3 and 1.9 mg/L) for 24 hrs, and observed increasing mortality starting at 0.9 mg/L and 100 % mortality was observed at 1.9 mg/L.
Sodium chloride (NaCl) A continuous well-aerated salt bath of 2-5g/L until disease controlled (may be for up to 20 days) has been reported as effective in controlling ichthyophthiriasis. One study found that fish maintained at 4g/L salt for 23 days showed a gradual reduction of white spots and survival was 100%. There appear to be significant differences among species and possibly families in the tolerance of larval and fry stages to salt.
The efficacy of salt in controlling and preventing ichthyophthiriasis in the silver perch (Bidyanus bidyanus) was evaluated in aquaria. Concentrations of 2 or 3g/L salt controlled infestations of Ichthyophthirius multifiliis, and fish were free of both theronts and trophonts by day 8 at temperatures of 17.3-21.3°C and by day 6 at 19.2-23.5°C. Fish treated with 1g/L salt remained infested and all fish in a control treatment (0 g/L salt) died.
Copper sulphate (CuSO4) Copper sulphate at concentrations of 0.1-0.2 mg/L have been reported to control ichthyophthiriasis, but higher concentrations of 0.25-1.0 mg/L were toxic to some fish species, and 0.05 mg/L was ineffective. However, copper sulphate is for specialist use only as it is highly toxic and requires removal from the aquarium after treatment. It is inadvisable to use this compound where other treatments are available.
Sometimes three to four daily transfers of fish to clean tanks will effectively reduce infection, while enabling the fish to develop tolerance to reinfections. Maintain temperature at around 24-28°C. Management techniques such as alleviate stressing conditions by improving water conditions, reducing stocking densities, etc., can also be helpful.
Remarks Increasing reports are continually being published indicating that different fish species and populations have significance difference in their resistance to Ichthyophthirius multifiliis. These differences in susceptibility were attributed primarily to environmental factors and/or genetic make up of the host (Gleeson et al. 2000). Wild-caught rainbowfish originating from three isolated populations were infected with a quantified dosage of Ichthyophthirius multifiliis in a controlled environment.
Melanotaenia eachamensis from Dirran Creek were much more susceptible to ichthyophthiriasis than were Melanotaenia splendida from the Lake Tinaroo or Bluewater Creek populations. When the highly susceptible Dirran Creek rainbowfish were crossed with rainbowfish from a fourth population, Melanotaenia eachamensis (Lake Eacham), they produced hybrids with significantly higher resistance than pure-bred Dirran Creek, but not higher than pure-bred Lake Eacham fish. Hence, intraspecific hybridisation increased resistance to Ichthyophthirius multifiliis infection in Melanotaenia eachamensis.
Hosts from all three populations were much less susceptible to infection on their second exposure to the parasite. However, the Bluewater Creek population was better able to acquire immunity to Ichthyophthirius multifiliis than either the Dirran Creek or Lake Tinaroo populations. It was tentatively suggested that there may be a link between the heterozygosity of populations of rainbowfish and their initial ability to resist infection by Ichthyophthirius multifiliis.
Although infection with Ichthyophthirius multifiliis is often lethal, a number of studies have indicated that sublethal infections in the host are able to induce an acquired resistance against re-infection. Under laboratory conditions fish have been routinely immunised by exposure to controlled numbers of parasites. Serum and mucus antibodies from immune fish have immobilised free-swimming theronts in vitro, suggesting several potential antibody-mediated mechanisms of protection.
Spontaneous recovery from infection and resistance to reinfection of recovered fish indicates that fish are capable of developing defence mechanisms against Ichthyophthirius multifiliis. Spontaneous recovery has been observed in both natural infections in natural habitats and in captivity.
The potential for spontaneous recovery varied with fish species. Infection in scaled fish regressed faster than in smooth skinned fish. After recovery, fish were resistant to reinfection or retained a merely subclinical chronic infection. The infective stages were also shown to be unable to penetrate the skin of resistant fish or reacts in such a way which forces the parasite to leave the body without completing its life cycle in response to the immune reaction. The likelihood of maternal immunity could be raised as another assumption for the mild infection in some fish species. It is well documented that maternal antibodies passed from mothers to their offspring directly via eggs or indirectly in mouth-brooding fish via mucus of the buccal cavity.
A number of reports assumed the presence of more than one strain of Ichthyophthirius multifiliis which differ in their pathogenicity to different fish species. This assumption was based primarily on the wide distribution of the parasite, subtle variation in cell morphology and serotypic variations among isolates based on immobilisation antigens. In 2006 a team of parasitologists from the Department of Infectious Diseases at the University of Georgia’s College of Veterinary Medicine studied two forms of Ichthyophthirius multifiliis and found that one of them killed all of the fish infected while the other only killed half.
In a recent study (Sun et al. 2009) the presence of symbiotic bacteria was identified living with Ichthyophthirius multifiliis. However, the physiological relationship between Ichthyophthirius multifiliis and its resident bacteria is unclear. It is not known if the endosymbionts contribute to the growth of Ichthyophthirius multifiliis, if they contribute to the severity or pathogenicity of infection, or if they provide their host with any selective advantage. It has not been determined if they influence the immune response of fish infected with Ichthyophthirius multifiliis. It is possible that they may simply be parasites of this parasitic ciliate.
Researchers found the presence of three classes of bacteria associated with the protozoan: Alphaproteobacteria (Rickettsiales), Sphingobacteria and Flavobacterium columnare. The presence of the first two as endosymbionts living within the cytoplasm of the protozoan was confirmed by microscopy, while the Flavobacterium is thought to adhere to the protozoan through association with cilia.
The methods used for isolation of Ichthyophthirius multifiliis theronts and tomonts were designed to minimise contamination of Ichthyophthirius multifiliis with other environmental bacteria from the water used to collect tomonts and hatched theronts. Ichthyophthirius multifiliis tomonts were individually collected and washed such that a minimal volume of water was transferred between washes. Theronts were collected by centrifugation and washed extensively in sterile water.
Despite this, Flavobacterium columnare was found on 50% of the tomont or theront preparations. Thus, the association of Flavobacterium columnare with Ichthyophthirius multifiliis appears to be extracellular through interactions with cilia and enmeshment in mucus secreted by theronts and tomonts.
Because of the close physical association of Flavobacterium columnare with the surface of Ichthyophthirius multifiliis, it is possible that the parasite serves as a carrier of bacteria to fish. Flavobacterium columnare was isolated from two different strains of Ichthyophthirius multifiliis: one that was in laboratory culture for 15 years, and recently isolated from a wild fish. This suggests that Flavobacterium columnare are commonly found with Ichthyophthirius multifiliis populations and are not lost even after long-term passage in the laboratory.