Greek farmers could benefit from early disease detection
with rapid diagnostic kits

Dr. PANOS VARVARIGOS, reports on the potential benefits of rapid field testing for bacterial fish diseases.

 

    


VETCARE Ô
VETERINARY SERVICES TO AQUACULTURE AND
DISTRIBUTION OF FISH HEALTH PRODUCTS

Copyright (c) Dr. Panos Varvarigos.

 

    

Introduction

Outbreaks of bacterial septicaemias, in larval or juvenile tanks in hatcheries or acute sudden disease with sharply elevated mortalities of on-growing sea-bream (Sparus aurata), sea-bass (Dicentrarchus labrax) and trout (Oncorhynchus mykiss), frequently lack pathognomonic symptoms or lesions. Outbreaks occur in particular, subsequent to stock handling or due to natural stressors. In these cases urgent diagnosis is necessary and the appropriate treatment needs to be applied in order to prevent the escalation of the death toll.

Professional assistance is mobilised in order to identify the causative bacterium, usually by means of sampling fish tissues for bacteriological examination. However, the process of bacterial culture incubation, purification and further biochemical testing in order to identify the implicated bacterium is not only costly, but more importantly, time consuming.

Prime example is Photobacterium damsela subsp. piscicida (formerly known as Pasteurella piscicida), which is the causative agent of pasteurellosis in marine water fish. Pasteurellosis is particularly serious in its acute form during the larval and juvenile stages of sea-bream and during the early on-growing stage of sea-bass. The bacterium requires 24-36 hours of incubation at 25oC on TSA, BHI or blood agar prior to producing visible colonies. Even then, the colonies may not be pure since other opportunistic bacteria interfere (such as Aeromonas spp., Vibrio spp.) demanding further subcultures in order to isolate the bacterial strain in pure culture for identification.

In hatcheries there is an increasing need to certify fry batches as specific pathogen free. Sea bass fry has to be free from vibriosis and sea bream free from pasteurellosis prior to delivery to the on-growers. Evidently, rapid processing of a statistically significant number of randomly selected fish is required for such screening against latent carrier fish.

  

        

Available diagnostic kits

In recognition of the above problems, the Greek fish farmers increasingly take advantage of in vitro diagnostic kits, which have become available in recent years.

      

These comprise the AQUARAPID range of diagnostic kits manufactured in Norway by BIONOR A/S as well as the range of KWIK-DTECT kits manufactured in the U.S.A by DIAG-XOTICS INC.

The BIONOR kits comprise among others the: Aquarapid-Va kit, testing for Vibrio anguillarum, the Aquarapid-Pp kit, testing for Pasteurella piscicida and the Aquarapid-Yr kit, testing for Yersinia ruckeri, whereas the DIAG-XOTICS KwiK-Dtect kits test for the infectious pancreatic necrosis virus -IPNV.

 

        

What is a rapid diagnostic kit?

The rapid diagnostic kits provide in a self-contained package all materials and reagents that are necessary to sample and quickly test in the field a large number of dead, moribund or even seemingly healthy fish, be they tiny larvae, fry, or larger specimen. The testing process follows a specific protocol described in easy to follow user instructions and the results are read within one to three hours.

The tests utilise the principles of the ELISA (Enzyme Linked Immunosorbent Assay) technique. Continuous stringent field and laboratory testing of these kits in different locations has not documented any false positive reactions.

Their sensitivity is remarkable. In field trials on several Greek fish farms they have proven adequately sensitive with sampled tissue in the range of 0.1g to 0.2g. This may be crucially important when testing tiny fish larvae. The latter may be squashed/homogenised and then sampled, or in the cases where the fish are suitably larger, a target organ may be isolated and used, for example, the minuscule spleen of a 1g sea-bream. From larger fish, parts of an organ, usually the kidney or material from an external lesion may be sampled and tested.

The results become visible by the eventual colour change of the positive tests. All tests are compared with positive and negative controls. The intensity of the colour change is directly proportional to the degree of infection of each particular sample tested.

 

        

Major benefits of rapid pathogen detection

Lesions on very small larvae are often unclear hence the rapid confirmation of a suspect diagnosis is important in order to apply the appropriate management measures and treatment. In the case of acute pasteurellosis in sea-bream mortality may rise to more than 70% among the 40-60 days old juvenile fish. Several days would be needed to confirm the diagnosis by means of classic bacteriology delaying the necessary action to control the outbreak and reduce losses.

The direct detection of bacterial antigens from fish tissues with diagnostic kits provides convenience for the on-site testing of sick fish.

Screening fish populations against certain diseases by testing statistically significant samples has become a practical possibility. The in vitro diagnostic kits are straightforward in their application and do not require equipped laboratories. By testing sufficiently large random samples of fish, the health status of fish batches may be assessed.

Each kit provides usually for 40 or 50 samples/tests. Samples may be pooled; hence a suitably large number of fish may be tested in a matter of hours.

These diagnostic tools ultimately alleviate guess-work and much of the uncertainty shrouding the day-to-day health management of fish stocks. Spotting disease carriers early, especially when used together with a well planned vaccination policy, is expected to reduce antibiotic use, prevent mortalities and improve the quality of the fish and the profits of the farm.

The diagnostic kits may be applied on several fish on site. No equipped laboratory is required. Testing may be performed by farm staff.

Distinct colour change depicts the positive samples and compares to the positive control (bottom right). Colour intensity relates to the amount of bacterial antigen present in the sample.

 

    

Benefits realised by the Greek fish farmers

Some of the beneficial applications of the rapid diagnostic kits on Greek farms comprise:

Rapid detection of pathogens at the first sign of ill-health, such as the loss of appetite, prior to the establishment of mortalities.

Differential diagnosis of diseases producing confusing external symptoms and/or internal signs.

Assessing whether to apply or not prophylactic antibiotic treatments or vaccination.

Quality control of hatchery fry (detection of latent carriers).

Prevention of importing harmful diseases through infected eggs or fish.

Screening of fertilised eggs prior to hatching.

Testing live prey cultures for the presence of certain obligate bacterial pathogens.

 

        

VETCARE Ô
VETERINARY SERVICES TO AQUACULTURE AND
DISTRIBUTION OF FISH HEALTH PRODUCTS