Progress on Red Mark Syndrome

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Progress on Red Mark Syndrome 2017-01-13T13:46:05+00:00

History

Red mark syndrome (RMS) is a relatively new disease effecting rainbow trout and possibly some other salmonid species. The disease was first reported in Scotland in the winter of 2003, and has since spread throughout Great Britain. The disease has also started to be a problem in continental Europe, with reports of RMS outbreaks coming from Switzerland, Austria, Germany, France and other countries.

In the UK RMS is also known as cold water strawberry disease (CWSD) in contrast to warm water strawberry disease (WWSD), or strawberry disease (SD UK) for short, which is similar to RMS, although they are believed to be two different diseases. The main difference between them is that WWSD can be treated with vitamin C supplementation in the feed, whereas this does not clear RMS.

A disease with similar clinical signs has been reported in the USA, referred to as Strawberry Disease (SD USA), and although the name is the same as WWSD in the UK, its symptoms resemble those of RMS. To sum up RMS = CWSD = SD USA and WWSD = SD UK. The etiological agents involved in these diseases are still unknown.

Symptoms

RMS results in multiple skin lesions found mostly on the flank of the fish, but these can also be seen all over the body of the animal. Severe reddening of the skin and elevation of the wound is typical. The lesions are graded on a scale of 1 to 3 (Figure 1). Grade 1 lesions are defined as the presence of small spots with increased mucus production; Grade 2 lesions appear red in the centre of the lesion, while Grade 3 lesions are fully developed lesion covering the breadth of the fish.

(a)

The disease occurs when the water temperature is below 15ºC. Fish with RMS do not appear sick and continue to feed and grow. A large number of fish can be affected with RMS, but mortality is usually very low. The main problem with the disease is that it results in a downgrading of stock, which in turn results in substantial economical losses for the trout farmer.

Histology

Histologically, the dermis at the site of the lesions and the layers below it including the muscle, appears inflamed (Figure 2). There is a large infiltration of inflammatory cells such as lymphocytes, heterophils and macrophages, so much so that the lesions appear raised opposed to normal skin. Loss of scales is common at the lesion site. The kidney shows increased production of blood cells, but no pathology is visible in the liver, while increased activity is seen in the white pulp of the spleen (i.e. the immunological region of the spleen). Inflammation of the heart muscle has also been reported.

Aetiology

Although RMS has now been studied for several years, the cause of the disease has not yet been established. Hugh Ferguson and co-workers at the Institute of Aquaculture, University of Stirling, were first to make an association between RMS and Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS) (Ferguson et al. 2006). They detected the bacterium in lesions on the skin and in the heart from formalin-fixed wax-embedded tissue archive material from RMS-affected fish. DNA was extracted from these samples and a region specific for the bacterium amplified using polymerase chain reaction (Crumlish et al., 2007). Heart lesions commonly seen in RTFS were also observed which was another clue for a possible involvement of F. psychrophilum. The bacterium was not however isolated using standard bacteriology. F. psychrophilum is fastidious and requires a low nutrient medium which makes it difficult to isolate.

David Verner-Jefferies and his team at CEFAS, UK made an epidemiological study of RMS (Verner-Jefferies et al., 2008). They noted that 10-60% of fish developed clinical signs of RMS 2-3 weeks after the first signs of the disease were observed on the farm. The disease develops so that the fish that first showed the signs are healing while others are just starting to become ill. No reoccurrence of disease outbreaks was observed in the same batch once recovered. They found treatment was possible with broad spectrum antibiotics such as Oxolinic acid, Oxytetracycline and Florfenicol. Disinfectants like Chloramine T and formalin can also help to clear up the disease before harvest, but these are less effective than antibiotics. David Verner-Jefferies et al. were able to transmit RMS by stocking non-infected fish in the same tank as diseased fish. The last two observations suggest that RMS is caused by a bacterial agent. They recovered a diverse range of bacteria from the lesions on agar, but were unable to relate any one particular species to the disease. They also prepared DNA libraries from both infected and non-infected fish and again were unable to associate any one particular bacterial species with the disease, and although F. psychrophilum was found in some of the RMS fish, no firm association was made between this bacterium and RMS.

A group from the Washington State University made DNA libraries from the SD lesions and healthy looking skin (Lloyd et al, 2008). They found one particular sequence of interest in most of the lesions which was not present in the healthy skin. Analysis of this DNA showed that the organism involved was closely related to a Rickettsia sp and was therefore referred to as a Rickettsia-like organism (RLO). They also used primers specific for F. psychrophilum, and found no presence of this bacterium in the lesions. Rickettsia spp., which are intracellular bacteria, are well known pathogens of fish. Piscirickettsia salmonis is one that has been particular devastating to the salmonid industry in Chile. The American finding is very interesting from a UK prospective since RMS might be caused by the same bacterium.

Current Research

More research is clearly needed to find the cause of RMS. The association of F. psychrophilum with RMS has been investigated by two research groups, but both have been unable to confirm a link between this pathogen and the disease. Our team in the Aquatic Vaccine Unit at the Institute of Aquaculture (Matthijs Metselaar, Kim Thompson and Sandra Adams) have been using immunohistochemistry to look for RLOs in infected–RMS tissues. This is a method in which pathogen specific antibodies are used to identify bacteria in infected fish tissue. We used two different polyclonal antibodies and a number of different monoclonal antibodies prepared against Piscirickettsia salmonis. We observed positive staining (Figure 3) in skin lesions, spleen, kidney and liver of the infected fish, but not in normal skin from the same fish. We also obtained slight staining in the mucus layer around the lesions and in normal skin with polyclonal antibodies against F. psychrophilum, but not in any of the organs. DNA of the RLO was found in the lesions of all RMS-infected fish examined and also in the spleen and liver of some of the fish examined. These results help to confirm the role of a RLO in the disease.

To establish that an RLO is the cause of RMS requires isolation of the bacterium and proof of Koch’s postulate i.e. the organism must be found in abundance in all fish suffering from RMS; it must be isolated and cultured; the isolated organism should cause disease in healthy rainbow trout when they are artificially infected with the organism and then re-covered from these fish.

Isolation of an RLO by culture is very challenging because it is an intracellular organism. The same protocols are used as those for isolating viruses from fish by culturing infected samples on fish cell lines; however no antibiotics can be used to control contaminating bacteria as this would kill the bacterial pathogen. There are recent reports of the culture of P. salmonis on special agar plates and these may assist in the isolation of an RLO from fish with RMS.

Treatment of RMS

As mentioned above, it is possible to treat RMS with antibiotics and disinfectants. These can help clear the fish of RMS prior to harvest, but have major drawbacks because of a required withdrawal period and the additional cost of the treatment itself. Left untreated RMS will resolve without major effects or mortalities. There are some reports of systematic fallowing and disinfection of the ponds helping to irradiate the disease from the farm.

Prevention

Prevention is better than treatment this requires knowledge of the causative agent. Current research in the Aquatic Vaccine Unit at Stirling is focusing on this.

Drs Matthijs Metselaar, Institute of Aquaculture, matthijs.metselaar@stir.ac.uk. This PhD project is funded by Intervet Schering Plough Animal Health.

References

Crumlish, M. et al. (2007) Journal of Fish Diseases, vol. 30, no. 1, pp. 37-41.

Ferguson, H. W. et al. (2006). Veterinary Record, vol. 158, no. 18, pp. 630-632.

Lloyd, S. J. et al. (2008). Diseases of Aquatic Organisms, vol. 82, no. 2, pp. 111-118.

Verner-Jeffreys, D. W. et al. (2008). Diseases of Aquatic Organisms, vol. 79, no. 3, pp. 207-218.

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