GB2223580A - Assay system for fish pathogens - Google Patents

Abstract

The system for the detection and identification of the pathogens, comprises at least two separate culture media, separated from each other, in one container. Each of these contains a different sugar and a pH indicator and optionally means for detecting oxidase activity. Sugars may be replaced by tryptophan, tyrosine or phenylalanine. The identification is made on the basis of fermentation of the sugars or on color formation for the tryptophan etc.

Description

ASSAY SYSTEM FOR FISH PATHOGENS The invention relates to all assay system for the differential diaynosis of fish pathogens based on sugar fermentation which is specific for various pathogens.The assay system conlprises a container in which there are provided at least two separate sterile culture media having different sugar components as well as identification salts sucli as pH indicators and identification Illealls such as tryptophan/phenylalanine fornlalioll. The novel diaynostic system makes possible the early detection and identification of fish pathogens, The determination of their antibiotic sensitivity call be carried oul afterwards. The assay system is suitable for field use and is of a simple and inexpensive nature.

BACKGROUND OF TIIE INVCNTION In recent years fish fanning has become more widespread and since fish under cultivated conditions are found in dense populations, research on fish diseases has so far been mainly directed towards the pathology of the diseases vaccination and prophylactic treatment.

No emphasis has been glade on easy diagnosis followed by targeted treatment and since in recent years prophylactic treatment has been the course of complsints effecting the enviromment thus targeted treatment is becoilliny the method of choice. In salmon, trout and other cold water fish farms the bacteriae of the Vibrionaceae family such as Vibrio anguillarwn, Vibrio salmonticida, V.ordali and Aeromonas salmonicida subsp. salmonicida are highly pathogenic The methods used so far have been by either taking such samples from the fish and sending them to a laboratory for analysis (swab samples of fish usually get smothered by contallinating aquaflora by the tie tliey reach the laboratory), or by sending the fish to the laboratory where they are diagnosed by autopsy and tediouscytological or histological methods. The bacterial growth media used were yenerally not selective and did not inhibit contaminating aquaflora (such as Karel Kovacek et al., Acta vet.Scand 1987, 28 47.54).

The Vibrioaceae family are microorganisllls that ferment different sugars at predetermined conditions in different ways. This, thus enables the particular strain to be easily identified. The media is further rendered selective by the use of high concentrations of salts as well as antifungal agents; all of which hinder theqrowth of contaminatilly aquaflora. The media are provided with pH indicators to ease the reading of the fermentation process.

Further, once the bacteria have grown on the paddle they are tested with commercially available oxidase test system, only bacteria that are oxidase positive are subsequently tested by confirmatory tests. Table I shows the fennentation reactions of sugars of various Vibriaceae and contaminants fouled in the aquaflora, but this is not lillited to the sugars shown nor tlie bacteria species mentioned. There is also a non-fennentative reaction of the fish pathoyens which can be used i.e. the forination of a brown pigment with Phenylalanilie or Tryptophan by Aeromonas salmonicida.

SUMMARY OF THE INVENTION The invention relates to an assay system for the differential diagnosis of fish patlioqens based on sugar fermentation which is specific for various pathogens. The assay system coniprises a contailler ill which there are provided at least two separate sterile media having different sugar components as well as identific ation nleans such as pH indicators and identification means such as tryptophan/phenylalonine formation. The novel diagnostic system makes possible tie early detection and identification of fish pathogens. The deternlination of their antibiotic sensitivity can be carried out subsequently.

Tile assay systeil is suitable for field use and is of a simple and inexpeisive nature.

More specifically, the invention relates to an assay system for the detection and determination of fish pathoyells comprising a container with at least two separate culture media, separated from each other, each of these containing a different sugar and pH indicator, the fenientation of the sugars being indicative of the nature of tie fish pathogen. Preferably, the Kit comprises also means for detecting oxidase activity. Examples of sugars used in the assay are mannitol, sucrose, arabinose, cellobiose, ribose, sorbitol, trehalose, lactose, inositol, salicin or yalactose.

It is advantayeous to use a culture medium containing a high concentration of salts and an antifungal agent, which hinder development of contaminatiny aquaflora. Furthermore, there is used a medium devoid of sugar, but containing phenylalanine or tryptophan for the detection of Aeroinonas salminicida by color formation.

The separate culture media can be provided at two or more surfaces of a paddle suspended from the cap of a vial.

The types of media formulae used are shown below as examples only, where the sugar components can be interchanged, where various different pH indicators can be used and different peptones can also be used.

EXAMPLE 1 In quantities of grams/Litre of distilled water NaCl 15 - 70 g Peptone or Tryptone 2 g Yeast Extract 2 9 Agar 15 g 1 N NaOH 7 ml Matinitol 2 - 12 gr Bromthmol Blue 10 - 30 mg Alnphotericin-B 15 - 30 mg Cycloheximide 25 - 200 mg Water 1 liter Sterilized at 121 0C for 20 minutes and then poured into paddles pH 7.3 +/- 0,3 Mannitol positive will be yellow and negative blue/yreen.

EXAMPLE 2 In quantities of grams/liter of distilled H2O NaCl 15 - 70 g.

Pel)tone or Tryptone 2 9 Yeast Extract 2 g Aqar 15 g i N NaOH 1 ml Sucrose 2 - 12 y Phenol Red 5 - 30 mg Amphotericine-B 30 - 60 mg Cyclohexineide 25 - 200 mg Water 1 liter Sterilized at 121 C for 20 minutes and then poured into paddles pH 7.3 +/- 0.3 Sucrose positive will be yellow and negative red/pink EXAMPLE 3 In quantities grams/liter of distilled H20 NaCl 15 - 70 g Pep tone or Tryptone 2 9 Yeast Extract 2 g Agar 15 9 Bile Salts 5 - 12 y N NaOH 1 ml Arabinose 2 - 12 y neutral Red 10 - 30 mg Ailpioteri ci n-B 30 - 60 nig Cycloheximide 25 -200 mg Water 1 liter Arabinose positive will be yellow and negative red.

EXAMPLE 4 In quantities grams/liter of distilled H20 : NaCl 15 - 70 g Peptone/Tryptone or equivalents 2 g Yeast Extract 2 9 Agar 15 g 1 N NaOH 1 ml Phenylalanine/Tryptophan 0.2 - 4 g Amphotericin 30 - 60 mg Cycloheximide 25 - 200 mg Water 1 liter Sterilize at 121 C for 20 minutes then pour agar into suitable container pH 7.3 @/- 0.3 Pasitive reaction shows brown pigment and negative reaction has no @o@our change.

The formulations are indicative only and the sugars can be interchanged as well as the pH indicators Lxample 4 is without sugar. Other sugars and pH indicators may he used as described as well as different nutritive agents, pH indicators that, may be used are: alizarin, M-dinitrobenzolene urea, brilliant yellow, phenol red, neutral red, m-Niteophenol, cresol red and others. Nutritive agents can be all types of peptones, from all sources, yeast extracts, beef@extracts and others.

USE SAMPLING 1. Open the container and remove the paddle by holding cap in one hand, or expose agar surfaces if not in paddle form. Do not touch paddle or agar surface with hands or fingers.

2. Take sample from recommended place of the fish and with sterile swab/loop inoculate on all stdes by applying gentle pressure and spreading the sample by a zig-zay movement down the length and breadth of the agar surface.

3. Replace the paddle in the container or cover surface with lid DO NOT CLOSE TIG@LY TO ENABLE FREE EXCNANGE OF ATMOSPHERE.

4. Incubate at room temperature, 20 C for Vibrio anguilarum and Aeromonas salmonichla for 40-48 hours and at 15 C for Vibrio salmonicida for up to 4 days. Vibrio anguilarum and Aeromonas salmonicida will also grow at 15 C but slower RESULIS : With the following tables, analyse the results, If the sugar fermentation fits the pattern confirm results by using the oxidase test.

TABLL 2 (see Examples 1 and 2) :

Sucrose Mannitol Oxidase Temperature Time Va + + + 20 C 48 hours Vs - 4 1500 4 days As - + + 20 C 48 hours E. coli different + - 20 C 24 to 48 hours V. ordalif + + + 20 C 48 hours TABLE 3 (see Examples 2 and 3)

Arabinose Sucrose Oxidase Temperature Time Va - + + 20 C 48 hours Vs + - + 15 C 4 days As + - + 20 C 48 hours E. coli + different - 20 C 24 to 48 hours V. ordalii - + + 20 C 48 hours fital TABLE 4 (see Example 3 and Table 1 )

Arabinose Cellobiose Oxidase Temperature Time Va - + + 20 C 48 hours Vs - + + 15 C 4 days As + - + 20 C 48 hours E. coli + - - 20 C 24 to 48 hours V. ordalii - - + 20 C 48 hours TABLE 5 (see Examples 2 and 4

Sucrose Colour forme Oxidase Temperature Time with Phenylalnine Va + - + 20 C 48 hours Vs - - + 15 C 4 days As - + + 20 C 48 hours E. coli different - - 20 C 24 to 48 hours V. ordalii + - + 20 C 24 to 48 hours Table 6. Example using multi well or surfaces on paddles.

Tryptothan Phenyl Sucrose Mannitol Arabinose Cellobiose alanine Oxidase Temperature Time V. anguilarum + + - + - + 20 C 48 hours V. salmonicida - + - + - + 15 C 4 days A. salmonicida - + + - + + 20 C 48 hours E. coli d + + - - - 20 C 48 hours V. ordalii + + - - - + 20 C 48 hours d = different TABLE 1 - Characteristics of Racterial Reactions

FERMENTATION REACTIONS AND OXIDASE TEST FOR VIBRIACEAE AND CONTAMINATING AQUAFLORA Microorganism V. anguillarium + + - + - + + - - - + + - + V. ordalii + + - + - + - - - - + - - + V. salmonicida d + - + - - + - - - + + - + V. marinus + - - + - - - - - - - - + V. logeii + + d d - - - - + + - + V. fisheri + + - + - - d - d + + - + Aeromonas + + - + + d - + + d - + hydrophila A. salmonicida + - - - + + - - d + - - + subsp. achromogenes A. salmonicida + + - - + + - - d + - - + subsp. achromogenes A. salmonicida + + - - - + - - + + - - + subsp. achromogenes E. coli + + + + d + + - d - - Pseudomonas - - - - - - - - - - - - - + chlororaphis Pseudomonas - - - - - - - - - - - - - + florescens Staph. epidermis + - d + - d - - d = different

Claims (10)

1. CLAIMS 1. An assay system for the detection and identification of fish pathogens which comprises a container which contains at least two culture media, separated froiii each other, each of which contains a different sugar and pH indicator, and which contains optionally means for detectiny oxidase activity of cultivated pathogens or where a culture medium is devoid of sugar and contains instead tryptophan tyrosine or pheiiylalatiine for tlie detection of specific pathoyens by color foniatioti, fermentation of a specific sugar and color formation being indicative of tie ideitity of specific fish pathogens.
2. 2. An assay system according to claim 1, where means are provided for detecting oxidase activity of pathogens grown on a culture medium to deteniiiiie whether such pathogen is oxidase positive or negative.
3. 3. An assay system according to claim 1, for the detection of Aeromonas salmonicida, where one culture medium contains instead of a sugar a eieiaber of the group consisting of phenylalanine, tryptophan and tyrosiiie, the presence being detected by formation of a brown color
4. 4. An assay according to claim 1, where the sugars are selected from mannitol, sucrose, arabinose, cellobiose, ribose, sorbitol trehalose, lactose, inositol, salicin and galactose.
5. 5. An assay systeiii according to any of claims 1 to 4, containing phenylainine or tryptophan or tyrosirie or a mixture of the three for the detection of Aeromonas salironicida by brown colour formation
6. 6.An assay system according to claiiii 1, for the detection of the fish pathogens Vibrio anguillaruiii, Vibrio salmonicida and Aeromonas salmonicida, where a combination of amino acid formation with sucrose fermentation identifies each of these, Vibrio anguillarum being sucrose positive and melanine negative, Vibrio salinonicida being sucrose and iiielaiiiine negative and Aeromonas salmonicida being sucrose negative and melanine positive
7. 7. An assay system according to any of claims 1 to 3, containing a high concentration of salts and an antifungal agent, which hinder development of contaminating aquaflora
8. 8.An assay systems according to any of clailns 1 to 7 where the separate culture media are provided at two or more surfaces of a paddle suspended from tlie cap of a vial
9. 9 An assay system according to any of claims 1 to 7, where different culture media are provided in a plurality of wells in a suitable culture plate.
10. 10. A selective assay system for the differential identification of fish pathoyeiis by determination of their fermentation of different sugars, by their reaction with phenylalanine or tryptophan or tyrosine and by establishing their oxidase activity, substantially as hereinbefore described and with reference to any of the examples.